15. BOKU-Symposium TIERERNÄHRUNG · Effect of an algae-clay mix on the use by broiler chickens of...

Tagungsband

15. BOKU-Symposium TIERERNÄHRUNG

Verarbeitung von Futtermitteln für die Mischfutterherstellung

07. April 2016, Wien

Institut für Tierernährung, Tierische Lebensmittel und Ernährungsphysiologie Department für Agrarbiotechnologie

BOKU – University of Natural Resources and Life Sciences, Vienna Universität für Bodenkultur Wien

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Seite II 15. BOKU-Symposium Tierernährung 2016

Tagungsband:

15. BOKU-Symposium TIERERNÄHRUNG

Verarbeitung von Futtermitteln für die Mischfutterherstellung

07. April 2016, Wien

Herausgeber: Christiane Schwarz, Margit Kraft, Julia Braach, Martin Gierus Institut für Tierernährung, Tierische Lebensmittel und Ernährungsphysiologie Department für Agrarbiotechnologie Universität für Bodenkultur Wien Muthgasse 11, A-1190 Wien www.boku.ac.at/tte-symposium tte(at)boku.ac.at Eigenverlag: Institut für Tierernährung, Tierische Lebensmittel und Ernährungsphysiologie ISBN 978-3-900932-33-6 Für den Inhalt der Beiträge sind allein die Autoren verantwortlich.

Inhaltsverzeichnis

15. BOKU-Symposium Tierernährung 2016 Seite III

Inhaltsverzeichnis

Übersichtsvorträge / Keynote speakers Grain processing for ruminants – improving health, performance, carcass quality, and meat composition JS Drouillard 1 Enzyme und Futtermitteltechnologie - Grenzen und Möglichkeiten von Enzymen als Zusatz-stoffe GM Gübitz, S Weiß, D Ribitsch 6 Verfahrenstechnische Möglichkeiten der Hygienisierung von Mischfutter JT Bohlmann 10 Einsatz von hydrothermischen Verfahren sowie von organischen Säuren bei Broilern - Effekte auf die Leistung und die Stoffwechselphysiologie J Zentek, A Mader, F Goodarzi Boorojeni 15 Challenges and solutions in fish feed processing - chasing for new ingredients O Kraugerud, D Miladinovic 21 Partikelgröße im Mischfutter - Bedeutung für Gesundheit und Leistung im Schweine- bzw. Geflügelbestand J Kamphues 25

Kurzvorträge / Oral presentation Effects of the Lactobacillus sakei KTU05-6 supplement on milk production and ruminal pro-cesses in feeding dairy cows V Krungleviciute, E Bartkiene, R Zelvyte, I Monkeviciene, J Kantautaite, R Stankevicius, A Sederevičius, D Starevicius, G Juodeikiene 38 Die Auswirkungen von melassierten Trockenschnitzeln als Ersatz für Mais in konzentratrei-chen Milchkuhrationen auf die Fermentation in vitro (RUSITEC) M Münnich, A Khol-Parisini, F Klevenhusen, E Humer, Q Zebeli 44 Effects of extruded lupins (Lupinus spp.), faba beans (Vicia faba) and peas (Pisum sativum) on dairy cow’s milk sensory properties I Kudlinskiene, Q Zebeli, R Gruzauskas, R Stankevicius, A Miezeliene,GA Lencikiene, M Ots 49 Reproduktionsleistung trächtiger Sauen bei unterschiedlicher Eisenversorgung M Buffler, C Becker, W Windisch 55 Effect of an algae-clay based biocatalyst on ileal digestibility performance of growing pigs MG Suarez, M Gallissot 59 Impact of a dietary probiotic supplementation (Enterococcus faecium DSM 7134) on the performance of fattening pigs K Reckmann, R Hartwigsen, G Thaller, M Rimbach 64 Einfluss einer druckhydrothermischen Behandlung der Einzelkomponente Mais auf die Ver- daulichkeit ausgewählter Nährstoffe sowie zootechnische Leistungen bei Broilern unterschiedlichen Alters R Puntigam, K Schedle, C Schwarz, P Hechenberger, J Eipper, M Gierus 69 Absorption of thymol as main compound of thyme essential oil in broiler chickens V Oceľová, R Chizzola, J Pisarčíková, O Ivanišinová, Š Faix, I Plachá 74

Inhaltsverzeichnis

Seite IV 15. BOKU-Symposium Tierernährung 2016

Poster

Futtermittel und Fütterung / Feedstuff and feeding 1. Milk replacer’s with extruded soy and maize use in calves’ nutrition

I Kudlinskiene, R Stankevicius, J Cernauskiene, J Klementaviciute, G Stanyte, G Dovidai-tiene, R Gruzauskas 79

2. Ersatz von Sojaextraktionsschrot durch Rapsextraktionsschrot und Rapskuchen bei Milchkühen auf die Milchleistung sowie Milchinhaltsstoffe M Leithner, K Schedle, E Schneeberger, M Gierus 84

3. Apparent and standardised ileal digestibility of crude protein and amino acids in distillers dried grains with solubles for pigs S Nitrayová, M Brestenský, J Heger, P Patráš 88

4. Processing some poultry diets using non conventional feedstuffs under the Egyptian desert conditions: A case study R Khidr 92

5. Effect of dietary supplementation of grape seed extract on the growth performance, lipid profile, antioxidant status and immune response of broiler chickens M Farahat, F Abdallah, H Ali, A Hernandez-Santana 94

6. The influence of feeding wheat with blue aleurone on intestinal microflora of broilers O Šťastník, L Detvanová, F Karásek, H Štenclová, L Kalhotka, L Pavlata, E Mrkvicová, P Doležal 99

7. Nutritional value and digestible energy of different genotypes of barley in the horses and rabbits nutrition V Kliseviciute, R Gruzauskas, V Sasyte, A Raceviciute-Stupeliene, G Svirmickas 103

8. Quality of rabbit’s meat after feeding GM maize MON 88034 x NK 603 M Polačiková, M Chrenková, Ľ Chrastinová, Z Formelová, Ľ Ondruška, K Zaujec 108

9. Vergleich zwischen zwei unterschiedlichen Fütterungsstrategien mit gleichem Fütte-rungsniveau bei Karpfen (Cyprinus carpio) in Bezug auf Leistungsparameter und Wachs-tumshomogenität AM Greiling, K Lübke, H Wedekind 113

10. Determination of polyphenol and crude nutrient content and nutrient digestibility of dried and ensiled white and red grape pomace cultivars A Winkler, F Weber, R Ringseis, K Eder, G Dusel 117

11. The influence of solid state and submerged fermentation with Pediococcus pentosaceus KTU05-8, KTU05-9, and KTU05-10 strains on the free amino acids profile in lupine seeds V Starkute, E Bartkiene, V Bartkevics, D Zadeike, G Juodeikiene 124

12. Silage fermentation quality in grass silages inoculated with Lactobacillus kefiri alone or a formulation containing L. kefiri J Kesselring, G Boeck, K Schoendorfer, T Hoeger, G Schatzmayr 129

Futterzusatzstoffe / Feed Additives

13. Untersuchungen zum Einsatz von Lebendhefe in der Milchviehfütterung T Ettle, A Obermaier 134

Inhaltsverzeichnis

15. BOKU-Symposium Tierernährung 2016 Seite V

14. Effekte einer Amylase und Protease Supplementierung auf den ruminalen Abbau von Stärke, Protein und Faser bei trockenstehenden Holstein Kühen M Deml, C Bolduan, W Windisch 138

15. Effect of a live yeast, Saccharomyces cerevisiae I-1077, used as a feed additive for rumi-nants, on in situ ruminal digestibility of alfalfa hay and fibre-associated microorganisms F Chaucheyras-Durand, A Ameilbonne, P Mosoni, E Forano 142

16. Einfluss mittelkettiger Fettsäuren auf die Entwicklung schweinespezifischer pathogener Keime in vitro M Hovenjürgen, D Schulze Schwering, JB Reeken 145

17. Effects of a hydrolysed yeast (Kluyveromyces fragilis) on feed intake and zootechnical performance in weaned piglets B Keimer, A Schlagheck, R Hartwigsen, G Thaller 150

18. Prüfung eines Cnicin-haltigen Extraktes aus Cnicus benedictus als Tränkwasserzusatz zur Stabilisierung der Darmgesundheit bei Absatzferkeln AC Baur, H Kluge, G Horn, J Kalbitz, A Breitenstein, GI Stangl 154

19. Effect of an algae-clay mix on the use by broiler chickens of a diet containing corn DDGS MG Suarez, M Gallissot 158

20. The energy effect of guanidino acetic acid in phytase-supplemented broiler diets A Ion, M Müller, L Stef 162

21. Single and combined effects of mannanoligosaccharides and dietary endo-1,4-β-xylanase and endo-1,3/1,4-β-glucanase on the performance and some intestinal function of laying hens S Alijosius, R Gruzauskas, V Kliseviciute, V Sasyte, A Raceviciute-Stupeliene, A Dauksiene 167

22. Effect of citrus extract and saponins plant extract on broiler zootechnical performances: comparative study to synthetic growth promoters MA Benarbia, A Caussilas, JM Garcia, P Chicoteau 172

23. Effect of lycopene on egg quality parameters and fatty acids composition in the egg yolk, using rapeseed and linseed oil in laying hens nutrition V Buckiuniene, R Gruzauskas, A Bajorinaite, A Raceviciute-Stupeliene, V Kliseviciute, V Sasyte, A Dauksiene, S Alijosius, G Svirmickas 176

24. The effect of dietary supplementation with butyric acid or sodium butyrate on egg pro-duction and physiological parameters in laying hens A Sobczak, A Drażbo, M Hanuszewska, K Kozłowski 181

25. Effect of dietary mannan oligosaccharides and organic acids on common carp health and productivity M Paleckaitis, I Kudlinskiene, A Pockevicius, R Gruzauskas 184

26. Positive effects of a flavonoid-rich phytogenic product on growth, survival, and bacterial infection of Pacific White Shrimp M Korzekwa, T Wilke, S Brenner 189

Weitere Themen aus der Tierernährung / Further aspects in animal nutrition 27. Carcass traits and meat quality analysis of pigs for fattening in their rations used differ-

ent forms of selenium A Raceviciute-Stupeliene, R Gruzauskas, V Kliseviciute, V Sasyte, S Bliznikas 194

Inhaltsverzeichnis

Seite VI 15. BOKU-Symposium Tierernährung 2016

28. Fatty acid profile of pork from a local Prestice Black-Pied pigs and commercial hybrid pigs E Václavková, J Bělková, M Rozkot, S Kuchařová, J Lipenský 198

29. Zum Einfluss von Störfaktoren auf zootechnische Parameter bei Mastschweinen – ein Beitrag zur Tierwohlforschung P Loibl, W Preißinger, G Propstmeier, S Scherb, W Windisch 202

30. Minischweine und eine spezielle Diät für die Erforschung des metabolischen Syndroms M Rozkot, P Klein, P Daněk, E Václavková, J Bělková, J Lipenský 207

31. Pellets oder schrotförmiges Futter an Abrufstationen für Mastschweine - Auswirkungen auf Mast- und Schlachtleistung W Preißinger, G Propstmeier, S Scherb 210

32. Zearalenone adsorption to lignocellulose binders – Differences in vitro and in vivo G Bichl, H Schwartz-Zimmermann, S Fruhauf, K Schedle, S Masching, D Schatzmayr, F Berthiller 214

33. Analysis of the effectiveness of fibre analysis techniques at the determination of the properties of lignocellulose sources and their corresponding effects on broiler production characteristic J Morton, A Kroismayr, N Puvača 218

34. The effects of inorganic and organic zinc dietary supplementation on performance of rabbits and meat mineral elements content Ľ Chrastinová, K Čobanová, M Chrenková, M Poláčiková, Z Formelová, A Lauková, O Bučko, Ľ Grešáková, M Rajský, Ľ Ondruška 223

35. Gesamtelementgehalte und deren Querbeziehungen in handelsüblichem Mischfutter für Rinder, Kälber, Schafe, Pferde, Schweine, Ferkel, Geflügel und Wild M Sager 227

36. Impacts of the fusarium mycotoxin deoxynivalenol on animal health and production K Ghareeb, WA Awad, J Böhm 230

Autorenverzeichnis 235 Sponsoren 238

Drouillard: Grain processing for ruminants: improving health, performance, carcass quality, and meat composition

15. BOKU-Symposium Tierernährung 2016 Seite 1

Grain processing for ruminants: improving health, performance, carcass quality, and meat composition

Jim Drouillard Department of Animal Sciences and Industry, Kansas State University, USA

Introduction Cereal grains normally are incorporated into diets of high-producing ruminant animals to increase energy intake, with the ultimate goal of improving milk yield, growth performance, or carcass value. Starch is the principal energy component of cereal grains, consisting of mostly linear (amylose) and highly branched-chain (amylopectin) glucose polymers. The relative proportions of amylose and amy-lopectin are influenced by plant species and also cultivar within a species, and can have important implications for digestion and processing. Starches within cereal grains are surrounded by a protein matrix, and the degree of organization of this matrix influences susceptibility of starch to digestion by microbial or mammalian enzymes. Moreover, grain kernels are characterized by presence of protective outer pericarp, the chemical structure of which varies considerable by grain species, thus giving rise to species differences in resistance to digestion. Digestion of grains, and thus animal performance, can be improved markedly by disrupting physicochemical barriers, and grain processing methods vary greatly with respect to their ability to enhance utilization of grains. This paper will discuss the struc-tural characteristics of grains that influence their susceptibility to digestion, and the importance of cereal grain processing with respect to its impact on digestion, cattle health, performance, carcass quality, and meat composition.

Grain Kernel Structure and Composition Excellent discussions of caryopsis morphology of cereals have been presented by Hoseney (1986), McAllister et al. (2006), and Serna-Saldavar (2010). All grain kernels are characterized by the presence of a multi-layered pericarp consisting of struc-tural carbohydrates, lignin, protein, and sometimes waxy sub-stances, all of which serve as protective barriers to ensure survival of the dormant embryo found within the germ fraction embedded therein. The precise chemical make-up of the peri-carp differs substantially among plant species. For example, the waxy cuticle found within sorghum grain, and to a lesser extent corn, renders these seeds more resistant to penetration by moisture, whereas wheat kernels more readily assimilate water. Oats and barley are characterized by an additional fi-brous husk that is extensively lignified, making these grains relatively impervious to attack by digestive enzymes. Disruption of the husk and pericarp is thus a key objective in processing grains for feeding to ruminants. The protective barrier functions of the husk and pericarprender whole grains relatively resistant to digestion when fed in the unprocessed form. Physical disruption of these structures through mastication or other forms of com-minution is thus essential for promoting optimal utilization of grains.For cattle, size reduction through

Figure 1: Illustration of the structuralcomponents of a corn kernel. Featuresare similar among grains, though thechemical composition and relative pro-portions of each component may vary asa function of plant species or cultivar


Seite 2 15. BOKU-Symposium Tierernährung 2016

mastication alone normally is insufficient to achieve extensive digestion of whole grains. Mechanical forms of processing, such as cutting, rolling, and grinding; hydrothermal processes such as extrusion, expanding, and steam flaking; or chemical processes such as alkaline treatment or acid treatment, are thus employed to increase susceptibility to enzymatic hydrolysis. These processes effectively increase rate and extent of digestion by enzymes of microbial or mammalian origin. The single largest fraction of the kernel, the endosperm, consists of starch granules that are organized to varying degrees within a protein matrix. As with the pericarp, texture of the grain endosperm varies as a function of plant species. Moreover, the endosperm of grains can be organized into distinct re-gions (Figure 1, above) of floury (amorphous) and corneous (vitreous) starch that vary in terms of their susceptibility to digestion.Vitreous forms of starch are characterized by closely packed starch granules within a highly organized protein matrix, thus yielding a dense, hard, translucent appear-ance. Amorphous regions are relatively soft, floury, contain less protein, and are relatively opaque in appearance. Amorphous starch is more susceptible to digestion compared to vitreous starch, owing in part to its less organized protein structure.Consequently, grains characterized by greater proportions of floury and lesser proportions corneous endosperm generally are more readily digested. The starch within cereals is comprised of varying proportions of amylose and amylopectin. Amylose is essentially linear in structure, consisting of glucose subunits connected by -1,4 glycosidic bonds with relatively few branch points. Amylopectin is a highly branched structure consisting of the combination of linear segments in -1,4 linkages with branch points of -1,6 glycosidic linkages. Amylose has a degree of polymerization ranging from 1,500 to 6,000 glucose subunits, while amylopectin ranges from 300,000 to 3,000,000 (Zobel, 1988). Feed grains typically contain amylose and amylopectin in a 1:3 ratio, though high amylose and high amylopectin cultivars have been developed for some species of grain (Thomas and Atwell, 2010). The linear chains within amylose and amylopectin are capable of forming helical structures. Moreover, proportions of -1,4 and -1,6 affect starch struc-ture.Amorphous regions are typified by increases in -1, glycosidic linkages, whereas the more tightly packed (or pseudo-crystalline) formations are characterized by greater proportions of -1,4 glycosidic bonds(Gallant et al. 1997). The pseudo-crystalline regions generally are more resistant to digestion by carbohydrase enzymes compared to amorphous forms of starch due to their dense structure. As indi-cated previously, grain cultivars vary with respect to amylose and amylopectin content, which can contribute to inherent differences in digestibility of these grains.

Grain Processing Grain processing generally is aimed at reducing particle size by mechanical means, effectively disrupt-ing the pericarp and exposing starches embedded within the protein matrix of the endosperm. By increasing the exposed surface area of the endosperm, additional sites are created for actions of di-gestive enzymes, potentially improving both rate and extent of starch digestion. Grains can be pro-cessed by rolling or grinding, both of which are processes commonly employed within the commercial feed industry. Thermal processes, such as micronizing or popping, also are used, but these are far less common in comparison to hydrothermal methods such as pelleting, extrusion, expansion, or steam flaking. Optimal processing of grains for ruminants must account for a wide range of factors, including input costs for comminution of grains, energy expenditure for hydrothermal processes, en-ergy expenditure for drying, impact on gastrointestinal tract passage rates, implications for rate and extent of ruminal and post-ruminal digestion of starch, influence on rate and extent of ruminal and post-ruminal digestion of protein, and the collective effects of these changes on animal health, animal performance, and carcass characteristics. Cold processing methods such as rolling and grinding rely solely on particle size reduction to improve digestion of grains. Maximizing energy yield from cereal grains [usually] is consistent with maximizing ruminal digestion of starch, as ruminants have limited capacity for starch digestion in the small intes-tine. Decreases in particle size no doubt increase surface area and susceptibility of grains to enzymatic



hydrolysis. Figure 2 illustrates relative rates and ex-tents of microbial fermentation of different particle size fractions from dry rolled corn using an in vitro system inoculated with ruminal contents from a grain-fed steer. Progressive decreases in particle size are associ-ated with more rapid and more extensive rates of di-gestion. Similar differences are noted for dry rolled wheat and sorghum, and differences among particle sizes are perhaps most pronounced with sorghum. These findings are corroborated by observations with in situ methods in which grains of varying particle sizes are suspended in the rumen of cattle within dacron bags. Grains processed to smaller mean geometric diameters are lost from in situ bags at faster rates and overall disappearance is more extensive in comparison to larger particles. Kessen (1997) included sorghum grain processed to mean geometric particle diameters of 2,000, 1,500, or 1,000 microns in diets of growing cattle (40% concentrate, 60% roughage) and observed linear improvement in starch digestion (82.3, 88.3, and 94.4%, respectively) and feed:gain ratio (7.69, 7.58, and 7.25, respectively) as particle size decreased. In a study with finishing cattle fed diets containing 6% roughage and 94% concentrate, however, Drouillard et al. (1998) observed gains of 1.62 vs. 1.83 kg/day and feed:gain of 6.26 vs. 5.71, when corn was dry rolled to mean geometric particle sizes of 2,000 and 3,800 microns, respectively. Differences were in large part attributable to decreased intake of the finely processed diet (9.96 vs 10.41 kg/day for fine and coarse, respectively). These data suggest that finishing diets are less forgiving in comparison to diets containing higher concentrations of roughages. It is important to recognize that small particles also can be fermented too rapidly by microbes within the rumen, resulting in accumulation of organic acids and depression of ruminal pH.Ultimately, this may increase incidence of subacute or acute ruminal acidosis and related maladies, such as bloat, liver abscesses, endotoxemia, and laminitis. When grain particles are too small, they also can pass from the rumen prematurely, and when presented in excess post-ruminally can overwhelm capacity for digestion of starch in the small intestine. Starch that escapes digestion in the rumen and small intes-tine eventually appears in the hind gut where it is subject to a secondary microbial fermenta-tion.Carbohydrate overload in the hindgut can induce hind-gut acidosis, as well as other aforementioned metabolic dysfunctions. Premature escape of small particles from the rumen can be partially overcome by feeding more roughage or by feeding roughages with greater particle size, ef-fectively entrapping the grain particles within a floating mat layer formed by the roughages, thereby increasing retention time and assuring more complete ruminal digestion. Excessive particle size reduc-tion of grains also can lead to reduced feed intake, which may be attributable to excessive fermenta-tion rates and(or) poor palatability of small, floury particles that can be rejected by livestock. The ideal particle size must therefore take several factors into consideration, including fermentability of the grain, the proportion of concentrates fed, the amount and particle size of roughage in the diet, and desirability to the animal being fed. Pelleting consists of a combination of particle size reduction (i.e., grinding) and hydrothermal pro-cessing, in which grains are treated briefly with steam and then forced under mechanical pressure through a die opening to cause particle agglomeration. Pellet quality, or durability, is inversely related to particle size of the grain, such that grains with smaller particle sizes form pellets that are more resistant to mechanical damage during storage, transport, and blending with other feed ingredi-ents.Pelleting can improve palatability of finely ground grains through agglomeration of fine particles that otherwise would be rejected by livestock. I spite of their agglomerated nature, however, pelleted concentrates quickly disintegrate into small particles when masticated and mixed with fluid contents of the rumen. Consequently, pelleted feeds often are subject to rapid rates of fermentation as described previously.



Steam flaking is one of processing methods commonly employed in preparing processed grains for ruminants, especially beef and dairy cattle. During this process, small amounts of water, normally ranging from 0 to 6% by weight, are combined with grains and allowed to steep for a period of sever-al minutes to several hours. Surfactants often are added to facilitate moisture assimilation by the grain. The pre-conditioned grain is then transferred to a steam chest, where live steam is injected under atmospheric conditions. Steam conditioning times vary considerably for different types of grains, ranging from as short as 15-20 minutes for wheat to an hour or more for grain sorghum. Dif-ferences in conditioning times largely reflect dissimilarities in characteristics of the grain pericarp, which in the cases of sorghum or corn are far more impervious to moisture in comparison to barley or wheat. After conditioning, the moistened, partially cooked kernels are compressed between corrugat-ed steel rolls to flatten the kernels. The rollers in flaking mills operate at the similar speeds, thus avoiding the shearing action characteristic of dry rolling. The process yields flattened kernels or “flakes”, effectively increasing surface area without fracturing the grain into small pieces.Flaking im-proves energetic feed value of corn by 18 or more (Zinn et al., 2002), and sorghum has been ob-served to respond even more favorably than corn to steam flaking (Chen et al., 1994). During the steam flaking process, starch is at least partially gelatinized, though the limited amounts of available moisture likely restricts the extent of gelatinization. Gelatinization constitutes a loss of crys-talline structure, which renders the starch more susceptible to enzymatic digestion. Linear segments of starch molecules can realign as the grain is cooled, forming helices with organized chemical struc-tures that are less susceptible to enzymatic digestion, which is a process known as retrogradation (staling). As retrogrades form, the grain becomes less pliable and is subject to fracture during han-dling, mixing, and transport. Starches also can form retrogrades with lipids, which also can adversely affect starch availability. In addition to effects on starch, hydrothermal processes, including flaking, can decrease ruminal availability of protein. Changes in ruminal degradation can be exploited in the case of high-producing dairy cattle, but in the case of growing and finishing ruminants it is mostly perceived as a negative effect, requiring that diets be supplemented with sources of ruminally de-gradable protein in order to optimize ruminal function. Current research in our laboratory is focused on additives that can retard starch retrogradation and partially inhibit formation of protein-carbohydrate complexes that affect protein availability. Extrusion constitutes one of the more rigorous forms of grain processing, combining both mechanical and hydrothermal elements to effect changes in starch and protein structure.Extruders are character-ized by high mechanical shear, which effectively reduce grains to very small particle sizes.Extrusion also subjects grains to intense heat and pressure, resulting in extensive gelatinization of starch.Moisture is flashed as steam from the extrudate as it exits the die or gap, and the resulting product normally is then cooled and dried. In the absence of free moisture, starch migration is less, which restricts retrograde formation to some degree. Several studies have been conducted in our research facilities to assess extrusion as an alternative to steam flaking (Depenbusch, 2009). Generally speaking, extrusion processing of total mixed rations yielded efficiencies in gain superior to those achieved with flaking. However, due to the extensive comminution of particles during extrusion processing, greater amounts of roughage were needed to maintain digestive health. In addition to extrusion, flaking, grinding, rolling, and pelleting, we have investigated alternative forms of processing in an effort to decrease energy requirements for processing. We have evaluated, on a very limited scale, grains that have been treated with high-intensity laser beams designed to score the pericarp and expose the endosperm within. Other methods evaluated in pilot experiments or small animal feeding trials include mechanical scarification to increase moisture assimilation, pericarp removal by alkaline treatment with sodium hydroxide and chemical lime, and enzymatic treatment. Much additional research is needed before these process can be applied effectively at a commercial scale.



In summary, grain processing is critically important for improving nutritional value of feed grains for ruminants. Attaining optimal digestion normally is consistent with maximizing ruminal digestion of cereal starches, though some hydrothermal processes also can adversely impact other nutrients—most notably protein, altering both site and or extent of digestion.

References Chen, K.H., J.T. Huber, C.B. Theurer, R.S. Swingle, J. Simas, S.C. Chan, Z.Wu, and J.L. Sullivan. 1994. Effect of steam flaking of corn and sorghum grains on performance of lactating cows. J. Dairy Sci. 77:1038-43.

Depenbusch, B.E. 2009. Extrusion Processing of Feedlot Diets.Doctoral Dissertation. Kansas State University, Manhattan.

Drouillard, J.S. 1998, Molasses-fat blend as an energy source and conditioning agent in feedlot diets. Cattlemen’s Day Report of Progress. Pp 79-82, SRP 804. Kansas State University AgriculturalExperiment Station and Cooperative Extension Service, Manhattan.

Gallant, D.J., B. Bouchet, and P.M. Baldwin. 1997. Microscopy of starch: Evidence of a new level of granule organization. Carbohydrate Polymers 32:177-191.

Hoseney, R.C. 1986. Principles of Cereal Science and Technology. Amer. Assn. Cereal Chem. St. Paul, MN.

Hoseney, R.C., H.C. Davis, and L.B. Harbers. 1974. Pericarp and endosperm structure of sorghum grain shown by scanning electron microscopy. Amer. Assn. Cereal Chem. 51:552-558.

Jampala, B., W.L. Rooney, G.C. Peterson, S. Bean, and D.B. Hays. 2012. Estimating the relative effects of the endosperm traits of waxy and high protein digestibility on yield in grain sorghum. Field Crops Research 139:57–62.

Kessen, T.J. 1997. Effects of Particle Size and Enzymatic Treatment of Dry-rolled Grain Sorghum on Digestion and Performance of Growing Calves.M.S, Thesis.Kansas State University, Manhattan.

McAllister, T.A., D. J. Gibb, K. A. Beauchemin and Y. Wang. 2006. Starch type, structure and ruminal digestion. Proceedings of the Cattle Grain Processing Symposium.Stillwater, Oklahoma.

Serna-Saldavar, S. O. 2010. Cereal Grains: Properties, Processing, and Nutritional Attributes. CRC Press. Taylor and Francis Group, LLC. Boca Raton, FL.ISBN 13:978-1-4398-8209-2.

Thomas, D. J. and W.T. Atwell. Starches. Amer. Assn. of Cereal Chem. Int. http://dx.doi.org/10.1094/1891127012.fm.ISBN: 1-891127-01-2

Zobel, H. F. 1988. Molecules to granules: A comprehensive starch review. Starch/Staerke 40:44-50.

Zinn, R.A., F. N.Owens, and R.A. Ware.2002.Flaking corn: processing mechanics, quality standards, and impacts on energetic availability and performance of feedlot cattle. J.Anim. Sci. 80:145-56.

Corresponding author Jim Drouillard Department of Animal Sciences and Industry Kansas State University, USA E-mail: jdrouill(at)ksu.edu

Gübitz et al.: Enzyme und Futtermitteltechnologie - Grenzen und Möglichkeiten von Enzymen als Zusatzstoffe


Enzyme und Futtermitteltechnologie - Grenzen und Möglichkei-ten von Enzymen als Zusatzstoffe

Georg M. Gübitz1,2, Stefan Weiß2 und Doris Ribitsch1,2 1 Institut für Umweltbiotechnologie, Department für Agrobiotechnologie IFA Tulln, Universität für Bodenkultur Wien, AT 2 ACIB GmbH, Graz, AT

Abstract Feed enzymes have made their way as important additives in feed-stock. Despite the availability of a number of commercial preparations, there is still a need for improvement of enzymes. This concerns their stability and activity at extreme conditions regarding temperature and pH value / proteolytic degradation during processing (pelleting) and during their action in the gastrointestinal tract, respec-tively. Here we compare two routes towards better enzymes, namely based on screening for suitable candidates in nature and based on genetic improvement of existing enzymes. Potential sources for microbial enzyme producers studied included rumen liquid or plant (e.g. soya) pathogenic organisms. From these sources relevant enzymes can be either directly isolated (purified) or identified based on sequence similarities and subsequently recombinantly produced. Genetic improvement of the resulting or existing enzymes is used to improve stabilities, reduce proteolytic degradation and to tune sub-strate specificities including sorption characteristics. Modern techniques ranging from microbial popu-lation analysis (imaging techniques like FISH-CLSM and genetic tools) to in-silico screening and enzyme engineering are discussed.

Einleitung Die kontinuierliche Steigerung der Weltbevölkerung hat dazu geführt, dass sich der Fleischkonsum in den letzten 50 Jahren vervierfacht hat. Etwa 40-60 % der Produktionskosten in der Tierhaltung entfal-len auf die Fütterung. Der Tierernährung kommt damit eine entscheidende Bedeutung für die Wirt-schaftlichkeit dieses Produktionszweiges zu [1]. Der Einsatz von Futtermittelenzymen hat in den letzten Jahren dazu geführt, dass bei gleicher Futtermenge eine bessere Wachstumsleistung und Fut-terverwertung erreicht wurde. Die Zugabe von Enzymen hat vor allem bei der Fütterung von mono-gastrischen Tieren wir Geflügel oder Schweinen große Bedeutung erlangt, da diesen die Alloenzyme der Rumen Mikroflora fehlen und daher komplexe Futtermittel nur unvollständig verdauen können. Der Zusatz von exogenen Enzyme dient hier als Ergänzung von körpereigenen Enzymen bei subopti-maler Eigenenzymsynthese oder als Zufuhr von Enzymen, die vom Tier nicht gebildet werden [2]. Zusätzlich können zugeführte Enzyme und mikroporöse Mineralien auch antinutritive Faktoren abbau-en (Proteasen) und immobilisieren oder wie kürzlich gezeigt sogar Pilzgifte in Futtermitteln unschäd-lich machen [3]. Futtermittelenzyme sind die am meisten erforschten Futtermittelzusätze und daher in großer Vielfalt kommerziell erhältlich. Dennoch herrscht ein großer Bedarf an neuen Enzymen, die besser an die Bedingungen des tierischen Verdauungstraktes, an die Bedingungen der Futtermittel-produktion (z.B. Dampf-Konditionierung und Pelletierung) sowie insbesondere an das umzusetzende Substrat angepasst sind. So wurden beispielsweise Proteasen für Waschmittel hinsichtlich des Abbaus von Proteinen aus Milch und Ei optimiert / selektiert [4] und sind nicht notwendigerweise ebenso aktiv auf Sojaproteinen. In gleicher Weise haben wir und andere Forschergruppen schon vor mehr als zwei Jahrzenten Xylanasen, Mannanasen, Pectinasen und Endoglucanses sowie auch Enzyme die Brücken zu Lignin spalten können (Ferulasäure-Esterasen) für den Einsatz in der Zellstoff- und Textilindustrie



entwickelt [5-9]. Auch bei diesen Enzymen besteht Bedarf an Optimierung hinsichtlich des Einsatzes in Futtermitteln. Die Entwicklung neuer Enzyme kann grundsätzlich auf zwei Arten erfolgen: durch Suche nach neuen Enzymen sowie durch Modifizierung von bereits existierenden Enzymen. Beispielsweise für den Abbau von Polysacchariden im Verdauungstrakt sind überwiegend Enzyme der dort vorhandenen Mikroorga-nismen verantwortlich [10]. Andererseits werden Polyssacharide auch in der Umwelt von Mikroorga-nismen abgebaut. In laufenden Forschungsarbeiten suchen wir z.B. sowohl in Rumenflüssigkeit sowie unter Soya-pathogenen Mikroorganismen nach neuen, Polymer-abbauenden Enzymen. Dabei können die Enzyme entweder direkt isoliert werden oder aufgrund von Ähnlichkeiten mit bekannten Enzymen identifiziert (in-silco screening) und sodann rekombinant produziert werden. Der zweite Weg zu neuen Enzymen führt über die Verbesserung der Eigenschaften existierender Enzyme mit Hilfe moderner molekularbiologischer Methoden. Diese erlauben sowohl die Erhöhung der Stabilität unter extremen Bedingungen bei der Verarbeitung und im Verdauungstrakt (pH Wert, Temperatur) sowie auch die Anpassung an die umzusetzenden Substrate. Letzteres betrifft nicht nur die Art der chemischen Bin-dungen sondern insbesondere auch effiziente Adsorption und Desorption an die polymeren Substrate wie wir in diesem Beitrag zeigen möchten [11]. Dazu sind detaillierte Studien der Substrat/Enzym Interaktionen notwendig unter Berücksichtigung von in Zukunft verstärkt eingesetzter Futtermittel wie z.B. DDGS. Genauso, müssen Wechselwirkungen und potentielle Synergien der unterschiedlichen En-zyme besser untersucht und genutzt werden.

Material und Methoden Mikrobielle hydrolytisch/fermentative Mischpopulationen auf lignozellulosereichen Substraten (z.B. Grassilage) wurden mittels konfokaler Laserraster Mikroskopie (CLSM) spezifisch visualisiert, aber auch mittels Elektronenmikroskopie hinsichtlich ihrer Morphologie und Fähigkeit zur Biofilmbildung unter-sucht und mittels 16S-basierter Metagenomanalyse in ihrer Zusammensetzung charakterisiert, um letztlich potenzielle mikrobielle Enzymquellen natürlicher Konsortien des Verdauungstraktes von z.B. Mastvieh zu erschließen.Eine Identifizierung und Charakterisierung extrahierter Enzyme wurde mittels LC-MS/MS Analysen vorgenommen. Aktive Proteinbanden wurden durch native Gel-auftrennungen isoliert und einem Trypsin-Verdau zur weiteren Separation mittels nano-HPLC (Agilent 1200 System mit Zorbax 300SB-C18 & Zorbax 300SB-C18 nano-Säulen) zugeführt. Die anschließende Ionisierung und Sequenzierungsanalyse erfolgte in einem Thermo LTQ-FT Massenspektrometer. Neue Enzyme wurden zudem basierend auf bekannten Sequenzen mittels in-silico Suche in Sequenz-Datenbanken identifiziert. Selektionierte Gene wurden hinsichtlich ihrer natürlichen Expression (intra- oder extrazellulär) analysiert und die Nukleotidsequenzen codon-optimiert. Die synthetischen Gene wurden rekombinant in E.coli oder Pichia pastoris exprimiert und die Expression durch Variation der Promotoren und Expressionbedingungen optimiert. Nach chromatogrpahischer Reinigung wurde die Aktivität der Enzyme sowohl auf kurzzeitigen Modellsubstraten wie auch auf unlöslichen (Bio)Polymeren bestimmt und hinsichtlich T-optimum, pH-Optimum und Stabilität untersucht. Für die Analyse der Reaktionsprodukte wurden photometrische Methoden und chromatographische Techniken eingesetzt. Für das Einbringen von zusätzlichen Disulfidbrücken zur Verbesserung der Thermostabilität wurde die Software DiSuP eingesetzt, die geometische Beschränkungen wie C-C-Distanzen, Cβ-Cβ-Distanzen, dihedrale Winkel χ1 und χ3von Seitenketten und Energieberechnungen zur Voraussage von S-S-Brücken verwendet [12] Engineering von Glykosilierungsstellen an der Enzymoberfläche zur Stabilisierung gegen proteolyti-schen Verdau erfolgt nach Analyse der Enzymstruktur nach geeigneten Aminosäurepositionen mittels gerichteter Mutagenese und Expression im Hefepilz Pichia pastoris [13].



Die Konstruktion der Enzyme mit Polymer-Bindungsdomänen erfolgte durch C-terminale Fusion von natürlichen Bindungsdomänen der Cellobiohydrolase I aus Hypocrea jecorina (CBM) und der Polyhyd-roxyalkanoate depolymerase aus Alcaligenes faecalis (PBM) [11].

Ergebnisse und Diskussion Eine Reihe von Methoden zur Charakterisierung funktioneller mikrobieller Gemeinschaften wurde entwickelt, um deren Aktivität auf bestimmten Substraten zu verfolgen (Bild 1). Sekretierte Enzyme konnten zudem anhand von chemischen Aktivitätstests nachgewiesen und anschließend extrahiert, isoliert und mittels Peptidsequenzierungsanalysen detailliert charakterisiert werden. So wurden Exocellobiohydrolasen der Klasse II, Amylasen, Hemicellulasen wie beispielsweise die 1,4-β-Mannanohydrolase, aber auch verschiedene Zucker-hydrolysierende Enzyme je nach Futtermittel, Temperatur und Inkubationszeitraum identifiziert werden. Neben der Un-tersuchung von mikrobiellen Populationen wurden auch ge-zielt potentielle Produzenten von Futtermittelenzymen ausgewählt. So ist der Pilz Phytophthora sojae als Erreger der Wurzelfäule von Sojabohne bekannt. Das gram-negative Bak-terium Xanthomonas axonopodis pv. glycines wiederum führt bei Sojapflanzen zur Pustelbildung. Beide Stämme sind Produ-zenten von extrazellulären Proteasen. Die in-silico Analyse der verfügbaren Genome hat zur Auswahl von Serinproteasen

geführt, die zunächst für die heterologe Expression in ihrer Nukleotidsequenz optimiert und anschlie-ßend in E.coli und Pichia pastoris exprimiert wurden.

Die Stabilität, insbesondere die Thermostabilität, des Enzyms im Verarbeitungsprozeß sowie im tierischen Verdauungstrakts ist ein entscheidendes Kriterium für die Anwendung als Futtermittelzusatz. Zur Verbesse-rung der Stabilität wurden anhand der gelösten 3D Strukturen zusätzliche Disulfidbrücken berechnet und mittels gerichteter Mutagenese in das Enzym einge-

baut. Disulfidbrücken werden von der Natur wie „Klammern“ in Proteine eingebaut, um die Struktur zu fixieren und vor Entfaltung zu schützen. Ein ähnlicher

Effekt kann durch den gezielte Einbau von Glykosilierungsstellen erreicht werden. Die Glykosilierung wird auch in der Natur verwendet, um Proteine gegen Denaturierung zu stabilisieren und vor proteoly-tischem Abbau zu schützen. In Hinblick auf einen effizienteren Substratumsatz wurden auch Methoden entwickelt um die Adsorption und Desorption der Enzyme auf den unlöslichen Fasern zu verbessern. Dazu wurden einerseits die Oberflächeneigenschaften der Enzyme verändert sowie spezielle Bindemo-dule an die Enzyme fusioniert. Um die Enzyme auch für eine industrielle Verwertung zugänglich zu machen, ist eine effiziente und kostengünstige Produktion notwendig. Für die Produktionsentwicklung von industriellen Enzymen wurde eine Expressionsplattform mit den Expressionswirten E.coli und Pichia pastoris entwickelt. Die Enzyme können nun durch Optimierung der Gensequenz, Auswahl des Expressionssystems sowie Op-timierung der Expressionsbedingungen auch für die Produktion im großen Maßstab entwickelt werden.

Bild 1: CLSM Analyse von hydrolyti-schen Populationen auf Grassilagefa-sern

Bild 2: Eine Esterase fusioniert an ein Cellulo-sebindemodul



Schlussfolgerungen Futtermittelenzyme werden seit Jahren erfolgreich eingesetzt. Dennoch können mit modernen bio-technologischen und molekularbiologischen Methoden bessere Enzyme entwickelt werden sowohl hinsichtlich Stabilität bei der Futtermittelerzeugung sowie auch hinsichtlich höherer Aktivität und Stabi-lität im Tier.

Danksagung Diese Arbeit wurde unterstützt vom Österreichischen BMWFW, BMVIT, SFG, Standortagentur Tirol, Niederösterreichische Landesregierung und der Business Agency Vienna über das Österreichische FFG-COMET- Funding Program.

Literatur [1] Munir K and Maqsood S (2013) A review on role of exogenous enzyme supplementation in poultry production. Emir. J. Food Agric. 25 (1): 66-80.

[2] Ravindran V and Son JH (2011) Feed Enzyme Technology: Present Status and Future Developments. Rec. Pat. Food, Nutr. Agric. 3:102-109.

[3] Heinl, S., Hartinger, D., Thamhesl, M., Vekiru, E., Krska, R., Schatzmayr, G., Moll, W.D. and Grabherr, R. (2010). "Degradation of fumonisin B1 by the consecutive action of two bacterial enzymes". J. Biotechnol.145: 120–129

[4] Proteasepaper

[5] Klug-Santner BG, Schnitzhofer W, Vrsanska M, Weber J, Agrawal PB, Nierstrasz VA, Guebitz GM. Purification and characteri-zation of a new bioscouring pectate lyase from Bacillus pumilus BK2. Journal of Biotechnology 2006, 121:390-401.

[6] Rumbold K, Biely P, Mastihubova M, Gudelj M, Gübitz GM, Robra K-H, Prior B. Purification and properties of a feruloyl ester-ase involved in lignocellulose degradation by Aureobasidium pullulans. Appl Environ Microbiol 2003, 69:5622-5626.

[7] Schmidt A, Gübitz GM, Kratky C. Xylan binding subsite mapping in the xylanase from Penicillium simplicissimum using xy-looligosachharides as cryo-protectant. Biochemistry 1999, 38:2403-2412.

[8] Mansfield SD, Saddler JN, Gübitz GM. Characterisation of endoglucanases from the brown-rot fungi Gloeophyllum sepiarium and Gloeophyllum trabeum. Enzyme Microb Technol 1998, 23:133-140.

[9] Gübitz GM, Hayn M, Sommerauer M, Steiner W. Mannan Degrading Enzymes from Sclerotium rolfsii: Characterization and Synergism of 2 Endo Mannanases and a ・-Mannosidase. Biores Technol 1996, 58:127-135.

[10] El Kaoutari A, Armougom F, Gordon JI, Raoult D and Henrissat B (2013) The abundance and variety of carbohydrate-active enzymes in the human gut microbiota. Nature Rev. Microbiol. 11: 497–504

[11] Ribitsch D, Orcal Yebra A, Zitzenbacher S, Nowitsch S, Steinkellner G, Greimel K, Doliska A, Oberdorfer G, Gruber CC, Gruber K, Schwab H, Stana-Kleinschek K, Herrero Acero E, and Guebitz GM (2013) Fusion of Binding Domains to Thermobifida cellulosilytica Cutinase to Tune Sorption Characteristics and Enhancing PET Hydrolysis. Biomacromolecules 14 (6): 1769–1776.

[12] Dombkowski, Alan A., and Gordon M. Crippen. Disulfide recognition in an optimized threading potential. Protein engineering 13.10 (2000): 679-689.].

[13] Shental-Bechor, Dalit, and Yaakov Levy. Effect of glycosylation on protein folding: a close look at thermodynamic stabilization. Proceedings of the National Academy of Sciences 105.24 (2008): 8256-8261.

Autorenanschrift Georg M. Gübitz Konrad-Lorenzstrasse 20 3430 Tulln an der Donau E-mail: guebitz(at)boku.ac.at

Bohlmann: Verfahrenstechnische Möglichkeiten der Hygienisierung von Mischfutter


Verfahrenstechnische Möglichkeiten der Hygienisierung von Mischfutter

Janine T. Bohlmann Forschungsinstitut Futtermitteltechnik der IFF, DE

Abstract The compound feed industry plays an important role in assuring quality and hygienic soundness of food from animal origin. To fulfil both legal requirements and market demands, compound feed un-dergoes hydrothermal and/or chemical treatment. Aim of hygienisation processes is to reduce the germ number by at least three decimal powers. A long-term conditioning process with temperature levels above 81 °C and residence times up to 4 min as well as the expanding of mesh feed (T > 110 °C, p 40 bar) have been established as effective and reliable hygienisation processes. Organic acids fulfil the task of preserving a once achieved hygienic level. How far a combination of organic acid sup-ply and hydrothermal treatment may lead to an optimum hygienisation process, is currently under investigation.

Einleitung Hygiene ist ein grundlegendes Problem bei der gewerblichen Mischfutterherstellung. Futtermittelher-steller bilden in der Erzeugerkette von Lebensmitteln tierischen Ursprungs die Schnittstelle zwischen der Rohwarenseite und der Tierhaltung und haben eine Wächterfunktion inne. Über Rohwaren können neben physikalischen und chemischen Verunreinigungen auch Schadkeime in das Futter eingetragen werden. Der Futtermittelverderb wird in der Regel durch bestimmte Hefen und Schimmelpilze, die darüber hinaus Giftstoffe (Mykotoxine) bilden können, eingeleitet; bei steigender Feuchtigkeit im Fut-termittel kommen Bakterien als Verderbniserreger hinzu. Ein zu hoher Gehalt an Bakterien und Schimmelpilzen im Futtermittel kann sich ungünstig auf die tierische Leistung auswirken und sogar zu Erkrankungen bzw. zum Tod von Tieren führen [1]. Um den Anforderungen des Marktes und den gesetzlichen Vorgaben gerecht zu werden, wird Mischfutter daher in der Regel einer Hygienisierung unterzogen.

Abbildung 1: Der Mischfutterhersteller innerhalb der Erzeugerkette von Lebensmitteln tierischen Ursprungs



Mikrobiologischer Hintergrund Unter der Hygienisierung versteht man eine Reduzierung der im Medium enthaltenen Mikroorganis-men um mindestens 3 Zehnerpotenzen, das heißt, von ursprünglich 1.000 vermehrungsfähigen Kei-men soll nicht mehr als ein Keim überleben. Die Reduktion der Keimzahl hängt von den Umgebungsbedingungen wie Temperatur, pH-Wert und Feuchtegehalt des Futtermittels ab und ist für jede Spezies unterschiedlich. Allgemein lässt sie sich durch eine Reaktionskinetik erster Ordnung be-schreiben:

Hierbei sind N0 die Ausgangskeimzahl, N die Anzahl lebensfähiger Organismen zum Zeitpunkt t und k die spezifische Inaktivierungskonstante. Die Zeitdauer, die zur Verringerung um eine Zehnerpotenz, also zur Abtötung von 90 % der aktuell vitalen Keime notwendig ist, wird als Dezimalreduktionswert oder kurz D-Wert bezeichnet. Abbildung 2 zeigt Absterberaten von Staphylococcus aureus bei verschiedenen Temperaturen mit den zugehörigen D-Werten nach [2]. Wie man sieht, ist der D-Wert stark temperaturabhängig und mehr als verzehnfacht sich für die dargestellte Spezies, wenn die Temperatur 65 °C ( ) statt 71 °C ( ) beträgt. In der Literatur werden D-Werte beispielsweise für Salmonellen bei einer Tem-peratur von 65,6 °C mit Werten von 1,2 bis 15 s angegeben [2]. Eine Reduktion um 3 Zehnerpoten-zen erfordert demnach eine Behandlungszeit zwischen 3,6 und 45 s.

Abbildung 2: Absterberaten von Staphylococcus aureus bei verschiedenen Temperaturen und zugehörige D-Werte nach [2]

Verfahren zur Hygienisierung von Mischfutter Eine Hygienisierung von Futtermitteln ist grundsätzlich auf thermischem Wege, durch Strahlung oder durch eine chemische Behandlung möglich. Auf Strahlung basierende Verfahren sind in der Futtermit-telindustrie nicht etabliert und bislang nur für Einzelfuttermittel vorstellbar. Im Folgenden sollen daher ausschließlich die thermische und chemische Hygienisierung von Mischfuttermitteln diskutiert werden. Konventionelle (Kurzzeit-) Konditionierung: Eine konventionelle Pelletierung, die aus den Pro-zessschritten Kurzzeitkonditionieren, Pressen und Kühlen besteht, vermag infolge der Temperaturer-

Nln — = – k · t . N0

(D71 ≈ 1,5 S)(D65 ≈ 20 S)



höhung im Konditioneur gekoppelt mit dem zusätzlichen Energieeintrag während des Pelletiervorgan-ges in der Presse die Gesamtkeimzahl zu reduzieren. In Abbildung 3 sind das konventionelle Verfah-rensschema sowie die Veränderungen von Temperatur, Feuchtigkeit und spezifischem Volumen längs des Prozesses qualitativ dargestellt. Gegenüber Nestern von Enterobakterien ist die hygienisierende Wirkung jedoch nicht zuverlässig. Während des Konditionierens ist das Temperaturniveau – üblicherweise 60 - 85 °C – bei einer Ver-weilzeit von nur 10 - 20 s nicht ausreichend und bewirkt eine Reduzierung der Keimzahl nur um etwa 2 Zehnerpotenzen [3]. Die zusätzliche Temperaturerhöhung während des Pressvorganges, die durch die Reibung des Materials in den Presskanälen hervorgerufen wird, wirkt hauptsächlich in den äußeren Schichten des Pellets und kann solche Nester ebenfalls nicht zuverlässig reduzieren. Nicht zuletzt ha-ben die nationalen Programme skandinavischer Staaten gezeigt, dass die bloße Kombination von Kurz-zeitkonditionieren und Pelletieren keine sichere Form der Hygienisierung darstellt [4-6].

Abbildung 3: Konventionelles Pelletieren: Verfahrensschema, Konditioneur und Zustandsänderungen relevanter Parameter

Zur Erfüllung der hygienischen Anforderungen bei der Herstellung von Nutztierfutter haben sich die Langzeit- und Druckkonditionierung etabliert, bei denen das Futter entweder direkt vor dem Pelletie-ren hygienisiert oder in einem gemeinsamen Schritt hygienisiert und pelletiert wird. Beim Langzeit-konditionieren wird die Futtermischung durch Zufuhr von Sattdampf auf Temperaturen bis zu 95 °C erhitzt, die Verweilzeit beträgt ca. 2 bis 4 min. Für den Hygienisierungsprozess werden unterschiedlich konzipierte Konditioneure verwendet, denen die Pelletpresse nachgeschaltet werden kann. Bei der Druckkonditionierung durchläuft das vorkonditionierte Futter einen Expander, in dem es sich aufgrund des mechanischen Energieeintrages auf etwa 100 – 130 °C erwärmt und einem Druck von 40 bar ausgesetzt wird. Beim Austritt bewirkt der schlagartig abfallende Druck das Expandieren des Futters sowie das Verdampfen eines Teils des enthaltenen Wassers. Das Verfahren hat trotz der geringen Verweilzeiten um 10 s eine sicher hygienisierende Wirkung gegenüber Bakterien. Darüber hinaus bewirkt es eine signifikante Erhöhung des Stärkeaufschlusses, eine verbesserte Aufnahme von Fett und Melasse in die Futterstruktur sowie die Agglomeration des Futters, das im direkten Anschluss mit einer Presse zu Pellets verarbeitet werden kann. Säureadditive: In der Mischfutterherstellung werden organische Säuren ‒ meist Ameisensäure und Propionsäure ‒ wenn, dann dem Endprodukt nach dem Pelletieren zugegeben, um den durch die hyd-



rothermisch-mechanischen Prozesse erreichten Hygienezustand zu konservieren. Ameisensäure wirkt vorrangig gegen Bakterien und wird zur Reduzierung der Keimbelastung im Futter eingesetzt. Propi-onsäure hingegen wirkt hauptsächlich gegenüber Pilzen und wird zur Verhinderung der Kontamination mit Schimmel bzw. zur Reduktion des Schimmelwachstums eingesetzt. Zu beachten ist hierbei der hohe Bedarf von 3 - 4 % Propionsäure, der notwendig ist, um eine deutliche Keimzahlreduzierung von beispielsweise 105 bis 107 nach 5 Tagen zu erzielen [6].

Zusammenfassung Bei den hydrothermischen Verfahren sind Temperatur und Verweilzeit entscheidend für die hygienisie-rende Wirkung. Sie findet hauptsächlich während der Konditionierung statt, der Pressvorgang beim Pelletieren selbst trägt nicht mehr signifikant zur Hygienisierung bei.

Abbildung 4: Verfahren zur Hygienisierung von Mischfutter

Als wirksame Verfahren zur sicheren, standardmäßig anwendbaren Hygienisierung haben sich die Langzeitkonditionierung (meist mit anschließendem Pelletieren, aber auch zur Mehlfutter-Behandlung) sowie die Druckkonditionierung erwiesen, vgl. die Übersicht in Abbildung 4. Organische Säuren wer-den eher konservierend, d. h. einen bereits erreichten Hygienestatus erhaltend, eingesetzt. Inwieweit eine Kombination von Säure und hydrothermischer Behandlung zu einem energetisch und ökonomisch optimierten Verfahren entwickelt werden kann, ist zurzeit Gegenstand laufender Forschungsprojekte an der IFF.

Literatur [1] Kersten, J., Rhode, H.-R., Nef, E.: Mischfutterherstellung, Verlag Agrimedia GmbH & Co. KG, Clenze (2010)

[2] Sinell, H.-J. (Hrsg.): Einführung in die Lebensmittelhygiene, Georg Thieme Verlag (2004)

[3] Mc Capes, R.H.; Ekperigin, H.E.; Cameron, W.J., Richie, W.L.; Slagter, J; Stangeland, V.; Nagaraja, K.V.: Effect of a newpel-leting process on the level of contamination of poultry mash by Escherichia coli and Salmonella, Avian Diseases, 33 (1), S. 103-111 (1989)

[4] Danske Slagterier: Danish Qualitätssicherungsgarantie, Integrierte Qualitätssicherung über alle Stufen der dänischen Schweinefleischproduktion, (Anlage 6: Produktion von salmonellenfreiem Futter gemäß Verordnung Nr. 677 des Pflan-zendirektoriats vom 25. August 1997), Kopenhagen (2002)

[5] Wierup, M., Widell, S.: Estimation of costs for control of Salmonella in high-risk feed materials and compound feed, Infection Ecology and Epidemiology 2014, 4: 23496 – http://dx.doi.org/10.3402/iee.v4.23496



[6] Löwe, R.: Untersuchungen zur hygienisierenden Wirkung unterschiedlicher Konditionierverfahren für die Mischfutter-herstellung in klein- und mittelständischen Unternehmen. Schlussbericht zum AiF-Forschungsvorhaben Nr. 11847, Hg. IFF-Forschungsinstitut Futtermitteltechnik, Braunschweig-Thune (2000)

Autorenanschrift Dr.-Ing. habil. Janine T. Bohlmann Institutsdirektorin Forschungsinstitut Futtermitteltechnik der IFF e. V. Frickenmühle 1A D-38110 Braunschweig E-Mail: jt.bohlmann(at)iff-braunschweig.de

Zentek et al.: Einsatz von hydrothermischen Verfahren sowie von organischen Säuren bei Broilern - Effekte auf die Leistung und die Stoffwechselphysiologie


Einsatz von hydrothermischen Verfahren sowie von organischen Säuren bei Broilern - Effekte auf die Leistung und die Stoff-wechselphysiologie

Jürgen Zentek1, Anneluise Mader1,2 und Farshad Goodarzi Boorojeni1

1 Institut für Tierernährung, Freie Universität Berlin, Berlin, DE 2 Bundesinstitut für Risikobewertung, Berlin, DE

Abstract Several studies have shown the potential of thermal processes in the reduction of microbial contami-nations of feedstuffs. However, feed treated with heat is at the risk for recontamination, especially during the cooling process. Organic acids are often used in feed production to reduce bacterial con-taminations and to prevent recontamination and both strategies may act synergistically. Thermal pro-cessing of feed and the inclusion of organic acids can improve the stability and hygiene of feed, might alter the chemical and physical characteristics of its constitutive ingredients, improve the nutritional value of animal feeds, have beneficial effects on the gastrointestinal function and may affect the mi-crobial status of gastrointestinal tract. The project “SiLeBAT” was performed to study the impact of different combinations of hydrothermal treatments including pelleting, long-term conditioning and expansion at two different temperatures (110°C and 130°C) without or with a blend of formic and propionic acid at three inclusion levels (0, 0.75 and 1.5%). Measurement included feed microbiology, performance of broilers, nutrient digestibility and bacterial composition and activity in the gastrointes-tinal tract of broiler chicks. Data show that the combination of short term hydrothermal treatments with organic acids can be used without negative effects on performance and digestive function.

Einleitung Die Technologie der Herstellung von Geflügelfutter umfasst eine breite Palette von hydrothermischen Verfahren wie Konditionieren, Pelletieren, Extrusion und Expansion. Es ist gut dokumentiert, dass während der thermischen und hydrothermalen Verarbeitungsprozesse chemische und physikalische Veränderungen in der Futtermatrix stattfinden, die Auswirkungen auf die Hygiene und physikalischen Eigenschaften des Futters, auf den Verdauungsprozess, insbesondere die Nährstoffverdaulichkeit so-wie auf die Darmmorphologie, -physiologie und intestinale Mikrobiota haben können. Während hydro-thermische Prozesse effektive Strategien für die Aufbereitung und Dekontamination von Mischfuttermitteln sind, sind die Wirkungen im Tier häufig weniger eindeutig einzuschätzen. Dieses umfasst die Auswirkungen der unterschiedlichen Verarbeitungstechnologien auf die Stärke- und Pro-teinverdaulichkeit, das Zusammenspiel zwischen hydrothermischen Prozessvariablen mit Nicht-Stärke-Polysacchariden wie auch mögliche Interaktionen mit Proteinen und Aminosäuren sowie anderen Nährstofffraktionen. Das Ausmaß der Effekte, z.B. von Stärkeaufschluss und Proteindenaturierung während der hydrothermischen Verfahren, folgt nicht immer dem gleichen Muster, daher erklären sich auch widersprüchliche Ergebnisse in der Literatur. Organische Säuren werden als Futterzusatzstoffe zur Konservierung von Futtermitteln sowie wegen ihrer beschriebenen positiven Wirkungen im tieri-schen Organismus in der Broilerernährung regelmäßig eingesetzt. Ihre möglichen Interaktionen mit hydrothermischen Behandlungsverfahren wurden bislang nur wenig beachtet. Daher soll in der vorlie-genden Arbeit ihr Einsatz beim Geflügel dargestellt und hinsichtlich der möglichen Interaktionen disku-tiert werden. Viele hier vorgestellten eigenen Ergebnisse beruhen auf den Untersuchungen im Rahmen des vom Bundesministerium für Bildung und Forschung geförderten Verbundprojekts zur



Sicherstellung der Lebensmittelversorgung der Bevölkerung für den Fall von bio- und agroterroristi-schen Schadenslagen („SiLeBAT“) erarbeitet. Der Hauptaspekt soll dabei auf die Wirkungen im Tier gelegt werden. Eine detaillierte Übersichtsarbeit zu hydrothermischen Verfahren ist derzeit in Vorbe-reitung. Weiterführende Literatur kann gern von den Verfassern angefordert werden.

Hydrothermische Verfahren und Stärke- und Proteinverdaulich-keit Die Haupteffekte hydrothermischer Verfahren werden insbesondere im Sinne eines besseren Auf-schlussgrades der Stärke gesehen. Die technologischen Effekte auf die Gelatinisierung von Stärke wurde in mehreren Übersichtsarbeiten umfassend dargestellt (Svihus et al., 2005). Durch die hydro-thermischen Verfahren kommt es im Prozess zu einer Schwellung der Stärkegranula sowie zu Verän-derungen der intermolekularen Bindungen, dadurch tritt schließlich der Verlust der kristallinen Struktur ein. Während im Zuge des Pelletierungsprozesses nur geringe Veränderungen zu beobachten sind, sind die Effekte von Expandierung und Extrudierung deutlich ausgeprägter (Thomas et al., 1999; Masoero et al., 2005). Eine positive Wirkung auf die scheinbare Verdaulichkeit der Stärke beim Geflü-gel wird einerseits durch die Effekte auf die Stärkegelatinisierung, andererseits aber auch durch die Inaktivierung von α-Amylaseinhibitoren erklärt (Zimonja et al., 2008; Abdollahi et al., 2010). Durch die Extrusion bzw. Expansion kommt es auch zu einer Reduktion von antinutritiven Faktoren wie Phytin-säure, Tanninen und Polyphenolen (Alonso et al., 1998; Alonso et al., 2000; Singh et al., 2007). Ne-ben diesen potenziell verdaulichkeitssteigernd wirkenden Prozessen sind auch gegenläufige Reaktionen beschrieben, insbesondere durch eine Zunahme der Digestaviskosität sowie durch eine Retrogradierung der Stärke, besonders im Rahmen von Expansions- und Extrusionsprozessen (Asp and Bjorck, 1989; Htoon et al., 2009; Svihus, 2011) . Das Ausmaß der Retrogradierung hängt unter anderem von der Prozesstemperatur, dem Feuchtigkeitsgehalt sowie dem Anteil an Amylose und Amy-lopektin ab (Svihus et al., 2005). Neben den Effekten auf die scheinbare Verdaulichkeit der Stärke stehen auch die Eiweißverwertung sowie die Verdaulichkeit der Aminosäuren im Zentrum vieler Arbeiten. Die mit hydrothermischen Pro-zessen verbundenen hohen Temperaturen, Scherkräfte, Druckbedingungen sowie der Dampfzusatz gelten als wichtige Faktoren zur Beeinflussung der Proteinstruktur und –verdaulichkeit (Camire, 1991; Ludikhuyze et al., 2003). Die thermischen Einwirkungen führen zu einer Verschiebung der Peptidket-ten, einer Veränderung der dreidimensionalen Proteinstruktur, sie induzieren neue Bindungsstrukturen und führen somit zu einer Denaturierung (Davis and Williams, 1998; Svihus and Zimonja, 2011). Dadurch werden funktionelle Eigenschaften sowie die Verdaulichkeit des Futterproteins beeinflusst, auch durch die Inaktivierung von Enzyminhibitoren (Svihus and Zimonja, 2011) und die Veränderung antigener Eigenschaften, die prinzipiell zu einer besseren Verträglichkeit z.B. von Leguminosen führen könnte (Davis and Williams, 1998). Eine hohe Proteinlöslichkeit wird als wesentliche Voraussetzung für den Verdauungsprozess gesehen (Cowieson and Ravindran, 2008). Bei Fütterung pelletierter Diäten war ein positiver Zusammenhang zwischen der Löslichkeit des Proteins und der Stickstoffretention bei Broilern festzustellen (Selle et al., 2012). Daraus lässt sich prinzipiell ableiten, dass eine verbesserte Proteinverdaulichkeit nicht zwangsläufig im Einklang mit der Denaturierung im Laufe einer hydrother-mischen Prozessierung steht. Positiv kann sich die Denaturierung von Proteaseinhibitoren in pflanzli-chen Futtermitteln auswirken. Interessanterweise konnte dargestellt werden, dass die Verwendung von Nicht-Stärke-Polysaccharid-abbauenden Enzymen als Futterzusatzstoffe effizienter wird, wenn das Futter zuvor hydrothermisch prozessiert wurde (De Vries et al., 2012). Bei Einsatz sehr hoher Tempe-raturen ist das Risiko einer verminderten Proteinverdaulichkeit zu sehen (Cheftel, 1986). Die Endpro-dukte von Maillard-Reaktionen sind enzymatisch nicht abbaubar, wodurch eine Reduktion der Protein-, Aminosäuren- und Kohlenhydratverdaulichkeit resultiert (Hendriks et al., 1994). Hitzelabile Aminosäu-ren, insbesondere Zystein, Lysin, Arginin, Threonin und Serin können unter den entsprechenden Tem-peraturbedingungen eine verminderte Verfügbarkeit haben (Papadopoulos, 1989; Camire et al., 1990). Andererseits führt wiederum die Deaktivierung von antinutritiven Faktoren zu einer höheren



Aminosäurenverdaulichkeit (Karr-Lilienthal et al., 2006). In diesem Zusammenhang ist der Hinweis angebracht, dass die normalen Prozessbedingungen im Zuge der Pelletierung kein hohes Risiko für die Bildung von Maillard-Produkten darstellen, zumindestens liegen keine entsprechenden Hinweise vor. Bei näherer Untersuchung einzelner Studien zeigen sich hinsichtlich der hydrothermischen Effekte negative Auswirkungen der Extrusion auf die ileale Verdaulichkeit von Aminosäuren aus Erbsen (Al-Marzooqi and Wiseman, 2009), entsprechende Hinweise ergaben sich auch für Aminosäuren aus Soja-protein (Powell et al., 2011). Andere Studien zeigten demgegenüber positive Wirkungen durch hydro-thermische Verfahren. Ganz offensichtlich sind für die Bewertung futtermittelspezifische Aspekte, zum Beispiel der Gehalt an Nicht-Stärke-Polysacchariden, von entscheidender Bedeutung.

Effekte organischer Säuren als Futterzusatzstoffe beim Broiler Kurzkettige organische Säuren und ihre Salze werden als Futterzusatzstoffe verwendet, da sie einer-seits konservierende Wirkungen im Futter entfalten, wobei die Wirkungen von der Säure bzw. der eingesetzten Kombination, der Dosierung und der Persistenz des Zusatzes im Futter auch bei ungüns-tigen Verarbeitungsbedingungen abhängen (Hinton and Linton, 1988; Rouse et al., 1988). Im Gastro-intestinaltrakt von Broilern konnten günstige Effekte auf die intestinale Mikrobiota gezeigt werden, z.B. reduzierte Keimgehalte im Kropf und spezifisch gegen pathogene Keime und Zoonosenerreger wie Campylobacter jejuni, Escherichia coli und Salmonella spp. (Izat et al., 1990; Hadorn et al., 2001). Positive Effekte auf den Verdauungsprozess sind verschiedentlich beschrieben, mechanistisch werden in diesem Zusammenhang eine erhöhte Nährstoffverfügbarkeit im Dünndarm (Dibner and Buttin, 2002) und eine verminderte mikrobielle Gallensäuredekonjugation diskutiert (Klaver and Van der Meer, 1993; Engberg et al., 2002). Organische Säuren werden individuell oder oft auch in Mischungen eingesetzt. Neben der antimikrobiellen Wirkung können sie den pH-Wert in der Digesta beeinflussen, weiterhin werden Effekte auf die Aktivität der Verdauungsenzyme, die intestinale Mikrobiota und die Darmschleimhaut diskutiert (Jongbloed et al., 2000). Aufgrund von interferierenden Variablen wie der Pufferkapazität der Futterkomponenten, der endogenen Enzymproduktion und des Hygienestatus in den Betrieben sind die Effekte organsicher Säuren beim Geflügel eher schwer vorhersagbar (Dibner and Buttin, 2002).

Untersuchungen im Rahmen des Projekts „SiLeBAT“ beim Broiler Im Projekt „SiLeBAT“ wurden Fragen der Sicherung von Warenketten bearbeitet, ein Teilaspekt war die Betrachtung möglicher mikrobieller Kontaminationen in Futtermitteln für Broiler. Zu dem Einfluss von verschiedenen hydrothermischer Maßnahmen auf den Hygienestatus von Futtermitteln sowie den Futterwert und den sich daraus ergebenden Konsequenzen für die Tierernährung liegen vergleichbar wenige Erkenntnisse vor. Daher wurde mittels eines umfassenden Versuchsdesigns ein Tierversuch, welchem ein Technikumsversuch vorausging, durchgeführt, um mögliche Effekte thermischer und chemischer Behandlungsverfahren auf die Futtermittel direkt und indirekt nach Verfütterung an Broiler und deren Einfluss auf zootechnische und ernährungsphysiologische Parameter zu untersuchen. Für den Technikumsversuch wurde konventionelles Broilerfutter mit zwei für die Tierernährung zugelasse-nen Probiotika beimpft, welche Surrogate für Salmonella spp. beziehungsweise Bacillus anthracis dar-stellten. Das Futter wurde in einem 3 x 4 faktoriellen Ansatz mittels Zusatz einer organischen Säuremischung (0, 0,75 und 1,5 %) und Hitzebehandlung (Pelletieren bei 70 °C, Langzeitkonditionie-rung bei 85 °C und anschließender Pelletieren, Expandieren bei 110°C, Expandieren bei 130°C) pro-zessiert. Der Einfluss auf die Keimzahl aller Futtermittelgruppen wurde erhoben. Für den Tierversuch wurde das Broilerfutter auf die gleiche Weise behandelt und an Broiler (Cobb) über eine Mastperiode von 35 Tagen verfüttert (12 Gruppen). Die Futtermittel wurden auf ihre Inhaltsstoffe und mikrobiolo-gischen Status untersucht. Leistungsparameter wurden wöchentlich erhoben. Am Tag der Schlachtung wurden Proben zur Bestimmung der Effekte auf die Organgewichte, den pH der Digesta, die intestina-



le Mikrobiota und bakteriell gebildete Metaboliten untersucht. Ergänzt wurden diese Daten durch die Messung der ilealen Nährstoffverdaulichkeit und intestinaler transportphysiologischer Parameter. Fol-gende Ergebnisse wurden zusammenfassend erzielt:

Im Rahmen des Technikumsversuches wurde durch die thermischen Verfahren und den Säu-renzusatz eine Reduktion der vegetativen Keime, jedoch nicht der sporenbildenden Bazillen festgestellt.

Die Verfütterung der thermisch und mit organischen Säuren behandelten Futtermittel hatte keine Effekte auf die Lebendmasse der Broiler und den Futteraufwand.

Es konnten einige Effekte auf die relativen Organgewichte (Kropf, Drüsenmagen, Muskel-magen, Zwölffingerdarm, Ileum und Zäkum) festgestellt werden (Goodarzi Boroojeni et al., 2014a).

Die scheinbare ileale- und Gesamtverdaulichkeit des Rohproteins und einzelner Aminosäuren war leicht reduziert, wenn das Futter einer thermischen Langzeitbehandlung unterzogen wur-de.

Die Behandlung der Futtermittel hatte keinen Einfluss auf die untersuchten transportphysiolo-gischen Eigenschaften des Jejunums (Ruhnke et al., 2015).

Zusätzlich wurden zahlreiche Effekte bei der Bakterienzusammensetzung sowie auf die mikro-biellen Metabolite in Kropf, Muskelmagen, Ileum und Zäkum festgestellt (Goodarzi Boroojeni et al., 2014b).

Die scheinbare Verdaulichkeit von Mineralstoffen und Spurenelementen wurde durch die thermische Langzeitbehandlung des Futters teilweise negativ beeinflusst, die Knochenminera-lisierung erwies sich jedoch als unbeeinflusst (Hafeez et al., 2014).

In der Zusammenfassung zeigen die Daten, dass eine kurze Wärmebehandlung in Kombination mit organischen Säuren zur Hygienisierung von Broiler-Futtermitteln ohne negative Effekte auf die Leis-tung eingesetzt werden kann.

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Zimonja, O., Hetland, H., Lazarevic, N., Edvardsen, D., Svihus, B., 2008. Effects of fibre content in pelleted wheat and oats diets on technical pellet quality and nutritional value for broiler chickens. Can. J. Anim. Sci. 88, 613-622.

Autorenanschrift Jürgen Zentek Institut für Tierernährung, Freie Universität Berlin, Königin-Luise-Str. 49, 14195 Berlin E-Mail: zentek.juergen(at)vetmed.fu-berlin.de

Kraugerud and Miladinovic: Challenges and solutions in fish feed processing - chasing for new ingredients


Challenges and solutions in fish feed processing - chasing for new ingredients

Olav Fjeld Kraugerud and Dejan Miladinovic Centre for Feed Technology (FôrTek) – Norwegian University of Life Sciences (NMBU), NO

Abstract The paper will go through the flow for fish feed processing. Bottlenecks and possible sites for changes will be pinpointed, especially with regard to the inclusion of ingredients which are not added as meal. Both aspects regarding practical issues such as feed plant design, and nutritional aspects are consid-ered. Potential new ingredients will be discussed, with respect to both their novelty, and possible use in the future.

Strategies to ensure deliverability The increased demand for high quality food, implicitly leads to the fact that an ever more important resource for protein-supply to humans in the future will be intensive farming of aquatic species. More-over, intensive aquaculture will require compound feed. FAO predicted that the human population will grow very fast, with of 9.1 billion people until year 2050 (FAO, 2009). At present, the modern feed diet for the aquatic species includes 50% less fishmeal when compared to 20 years ago (Ytrestøyl et al., 2015). The aquaculture and the related feed industry must document its impact on the environment by using sustainable ingredient resources. The awareness of consumers as well as safety and security of the food, leads to such action regarding ingredients. One main argument against sustainability of the fish farming is its dependence on the fishmeal. The fish feed industry and intensive aquaculture need more than ever the ideal novel ingredients for the future. In order to call biomass a feed ingredient it would need severe upgrading regarding nutritional quality, and undoubtedly, approval as feed ingre-dient. First, such ingredients must be safe for fish and humans. Secondly, it must be without any anti-nutritional factors, and third it would need to have taste and flavor that is accepted by the fish. Over-all, there is a need of a reliable supply of these new raw materials. A decade ago, our present University was putting a lot of efforts into the search for alternatives to fishmeal, and developed a lot of know-how related to ingredients and processing, primarily ingredients of plant origin. Then the research and the research groups were branded under the name “Aquacul-ture Protein Centre”. For instance, the idea of pretreating individual ingredients the way that opti-mized its nutritional value was developed successfully at that time, instead of processing all ingredients the same. In figure 1 the pretreatment of soybean meal with inclusion of phytase is shown, as phytic acid has to be reduced before feeding, due to the low body temperature for salmon-ids. Much of that processing was performed at the Centre for Feed Technology.



Figure 1: The results shown here indicate that at an incubation time of 10 minutes in the conditioner, availability of phosphorous can be increased profoundly (Figure from Denstadli et al., 2006)

One ingredient that we investigated thoroughly was krill meal. Krill meal is considered a sustainable ingredient, when harvested according to the advices from Commission for the Conservation of Antarc-tic Marine Living Resources (CCAMLR). Krill has been recorded to result in very good fish performance. However, krill meal has a lipid content which affects the processing. In figure 2, it is shown how the chemical composition in the mash changes when adding increasing levels of krill (Kraugerud et al., 2009). A change in lipid level in the mash may affect degree of starch gelatinization, specific mechani-cal energy in the extruder, and at the end of the extruder; the expansion of the diet. Hence, prior to processing, such changes in recipe must be considered up front processing.

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0 123 257 383 598 752

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Figure 2: Chemical composition in mash (feed prior extrusion) for different diets as an effect of increasing krill inclusion (Kraugerud et al., 2009)



A new research Centre, Foods of Norway, at the Norwegian University of Life Sciences (NMBU) is a good example how science and technology can contribute developing better and more sustainable protein resources. Question asked prior establishing such a Centre for Research-based Innovation as Foods of Norway, was based on how to develop novel feed ingredients with unconventional approach, technology and raw materials. Foods of Norway will have a period of time of 8 years and total budget of 192 million Norwegian kroner (~20 mil. Euro). Goal for Foods of Norway is to develop high-quality feed ingredients from natural bio-resources that are not suitable for direct human consumption, such as forest biomass and seaweed. The aim of Foods of Norway also is to improve the efficiency of feed ingredients and hence the feeds by novel feed-processing technology, advanced genomic analysis and identification of different selection criteria for aquatic farmed animals. Foods of Norway, with support from Centre for Feed Technology (FôrTek), aim to improve the nutritional value of novel feed ingredi-ents by using a new approach towards feed processing and technology. When including ingredients with a relatively low content of protein, e.g. seaweed meal, this often leads to the inclusion of other more pure protein sources. One such protein source is wheat gluten. The higher the inclusion rates of such “pure” ingredients are, the more important it is to perform thorough studies of its properties. As seen in the study by Storebakken et al. (2015) the specific mechanical energy (SME) was reduced with increased inclusion (0%; 6.72%; 13.46%) of hydrolyzed wheat gluten (HWG), see table 1. The content of fishmeal in the diets was 64.1%; 55.1%; and 46.2% respectively. In table 1 it is also seen other main observations from the feed process. Although SME decreases, expansion of the pellets slightly increases. This is in contrast to conventional results. A positive correlation of increased HWG levels and lipid loss for the diets is also seen.

Table 1: Effect of increased inclusion of hydrolyzed wheat gluten (HWG) in the diets on selected parameters

Inclusion of HWG (%) Observations 0 6,72 13,46 SME (Wh kg -1) 66 59 48 Expansion (%) * 28 33 34 Lipid loss (%) ** 1.7 2.3 3.4

* 10 measurements per diet ** 2 measurements per diet

The linear regression for apparent digestibility of nitrogen with inclusion of HWG was y=98.6 + 0.0060xHWG with p<0.001. As a protein source, HWG had comparable nutritive value as high-quality fishmeal. Data growth performance will also be presented. We at the Centre for Feed Technology believe that the approach of tailor made treatment of the most important ingredients at the feed plant will be an area of increased importance. This does not de-pendent if the ingredients are in the form of liquid or the more traditional dry meal. This specialized treatment combined with the coming pool of new ingredients will be a major step when it comes to reduce the ecological footprint from the feed industry and ensure future deliverability.

About the Centre The Centre for Feed Technology is a department at the Norwegian University of Life Sciences. The Centre’s strength is bridging principle to practice. From the client’s seminal concept to the Centre’s deliverable product, experienced professionals analyze each step. State-of-the-art equipment supple-ments our know-how in feed technology. The Centre serves the international feed industry by carrying out researches in all areas of fish feed, pet food and animal feed. The production lines are based on twin screw extrusion and pelleting (ring die and flat die) process, utilized by the present-day feed industry. This enables variation of process conditions and ingredients in the production of all kinds of fish and animal feed as well as the pet food. To ensure our competitiveness one of our employees is



now part of PhD project, which takes into consideration utilization of various enzymes and their com-bination for influencing better rheological and tribological characteristics of novel protein-based feed ingredients. One aim in this project is lowering the electrical consumption during the extrusion pro-cess. Another important mission given to the Centre is training graduate students in the MSc in Feed Manufacturing Technology at the Department for Animal and Aquacultural Sciences. Graduates pro-vide feed companies with qualified personnel well-balanced in theory and practice. Teaching and training is partly based on lectures, but a considerable part is offered as demonstrations and training in groups, such that the candidates are getting a lot of hands-on knowledge.

References Denstadli, Vegard; Vestre, Rotsukhon; Svihus, Birger; Skrede, Anders; and Storebakken, Trond; 2006. Phytate Degradation in a Mixture of Ground Wheat and Ground Defatted Soybeans during Feed Processing: Effects of Temperature, Moisture Level, and Retention Time in Small- and Medium-Scale Incubation Systems. Journal of Agricultural and Food Chemistry, 54, 5887-5893, DOI: 10.1021/jf0605906

FAO, 2009. How to Feed the World in 2050, http://www.fao.org/fileadmin/templates/wsfs/docs/expert_paper/How to_Feed_the_World_in_2050.pdf

Kraugerud, Olav Fjeld; Shearer, Karl D.; Øverland, Margareth; Storebakken, Trond. 2009. Feed processing of krill based diets – the challenges of dynamic processing inputs and responses, World Aquaculture Society, September 2009, Veracruz, Mexico.

Storebakken, Trond; Zhang, Yuexing; Ma, Jingjing; Øverland, Margareth; Mydland, Liv Torunn; Kraugerud, Olav Fjeld; Apper, Emanuelle; Feneuil, Aurélien. 2015. Feed technological and nutritional properties of hydrolyzed wheat gluten when used as a main source of protein in extruded diets for rainbow trout (Oncorhynchus mykiss), Aquaculture 448, 214–218, doi:10.1016/j.aquaculture.2015.05.029

Ytrestøyl, Trine; Aas, Turid Synnøve; Åsgård, Torbjørn; 2015. Utilisation of feed resources in production of Atlantic salmon (Salmo salar) in Norway, Aquaculture 448, 365–374, doi:10.1016/j.aquaculture.2015.06.023

Corresponding author PhD Olav Fjeld Kraugerud Center for Feed Technology (Fôrtek) Section Manager Husdyrfagbygget, H U137 E-mail:olav.kraugerud(at)nmbu.no

Kamphues: Partikelgröße im Mischfutter - Bedeutung für Gesundheit und Leistung im Schweine- bzw. Geflügelbestand


Partikelgröße im Mischfutter - Bedeutung für Gesundheit und Leistung im Schweine- bzw. Geflügelbestand

Josef Kamphues Institut für Tierernährung, Stiftung Tierärztliche Hochschule Hannover, DE

Abstract The physical form of diets for pigs and poultry is determined mainly by the mass/amounts/proportions/fractions of particles with different sizes. These characteristics depend predominantly on the diminution/grinding of native ingredients and further processes like compaction or granulation. The intensity of grinding, the technique (hammer vs. roller mill) and different forms of hydrothermal treatment and compaction are on debate since decades. In pigs it is – without any doubt – necessary to have a minimum grinding to achieve a high precaecal digestibility of nutrients but in poultry the activity of the gizzard results in an intended diminution of cereals and further ingre-dients. Recent experimental studies indicate that especially protein rich ingredients may need a higher grinding intensity to maximize precaecal digestibility of protein. In pigs and poultry there are clinically relevant diseases (pigs: gastric ulcers; chickens: dilatation of the proventriculus) in cases of a very high grinding intensity followed by pelleting the fine mixtures. In both species the physical form of diets has further marked effects on the mass, morphology and architecture of the stomach, the gut, and the pancreas as well. Finally it has to be underlined that diverse processes along the GIT are in-fluenced by diets’ physical form, beginning with the ingestion behaviour, continued by the passage rate of the ingesta up to the excretion/production and composition of faeces. Special emphasis is giv-en to the welcome side effects of a coarse structure of diets; i. e. reducing the prevalence of Salmo-nella spp. (zoonotic pathogen) in swine and the translocation rate of pathogens into the animals (on herd/group levels). The starch based stimulated production of butyrate by the intestinal flora might be one reason – but not the only one – that result in benefits for the gut health in pigs as well as in poul-try.

Einleitung Mischfuttermittel können auf vielfältigste Weise Einfluss auf die Leistung und Gesundheit von Nutztie-ren nehmen: Die Qualität des Futters ist eben eine Resultante unterschiedlicher Einflussgrößen, die von der „Schmackhaftigkeit“ über die chemische Zusammensetzung bis zum mikrobiologisch-hygienischen Status reichen (KAMPHUES et al. 2014). Im nachfolgenden Beitrag geht es um eine wei-tere Facette von Qualitätskriterien, nämlich die „physikalische Struktur“ eines Mischfutters für Schwei-ne und Geflügel, die ganz wesentlich, aber eben nicht ausschließlich durch die Verteilung von Partikel- oder Korngrößen (Massenanteile unterschiedlich großer Partikelfraktionen) bestimmt bzw. zu charak-terisieren ist. Ein ganzer Workshop auf der GfE-Jahrestagung (Göttingen 2011) war dieser Thematik gewidmet, entsprechende Beiträge finden sich in den Proceedings dieses Kongresses. In diversen Untersuchungs- und Forschungseinrichtungen sind seit dieser Zeit weitere experimentelle Studien durchgeführt wor-den, mit teils unterschiedlicher Ausrichtung und Fokussierung. Dabei standen Fragen zur möglichen Beeinflussung der Leistung (Futteraufnahme, Nährstoffverdaulichkeit und Futteraufwand) und/oder die Gesundheit (insbesondere das Risiko für die Entwicklung von Zoonose-Erregern wie Salmonellen) im Zentrum wissenschaftlicher Ausführungen oder von Diskussionen bei Mischfutterproduzenten und Tierhaltern. Vor diesem Hintergrund ist eine Einschätzung des Status quo vorgenommen worden, die



Frage der Priorität (Leistung vs. Gesundheit) hat dabei nicht zuletzt eine politische Dimension (s. Dis-kussionen/Vorgaben zum Thema „Tierwohl“).

Die Mischfutterstruktur Bei Mischfuttermitteln für Schweine und Geflügel geht es zunächst, d. h. primär um die Partikelgrö-ßenverteilung, da nahezu alle Komponenten vor dem Mischen vermahlen werden, um einen möglichst hohen Nutzen für das Tier (Verdaulichkeit) zu erzielen. Der Vermahlungsgrad von Komponenten im Mischfutter für Schweine war schon vor Jahrzehnten Ge-genstand diverser Untersuchungen. Hierbei standen die möglichen Effekte auf die Verdaulichkeit (WEISTHOFF 1990; praecaecal bzw. über den gesamten Verdauungstrakt) im Vordergrund des Inte-resses. Die Struktur eines Mischfutters ist aber nicht nur durch die Vermahlung bestimmt (s. Übersicht 1), sondern ist als Resultante verschiedener Einflussfaktoren zu verstehen.

Struktur des Futters bei

der Aufnahme durch das Tier

- Partikelgrößen-Verteilung -

ART/ANTEIL von Komponenten

- pflanzenanatomische Einflüsse

- Struktur der Ergänzungen (Min.stoffe etc.)

- bestimmte Nebenprodukte

ANGEBOTSFORM

- als Trockenfutter (Förderungstechnik)

- als Flüssigfutter (Standzeit/Rühreffekte)

- als Breifutter (Quellung)

KONFEKTIONIERUNG

- Pelletierung (an sich)

- Pellet-Durchmesser („Strukturverlust“)

- Nachträgliche Bröselung ( “ )

VERMAHLUNG

- Art der Zerkleinerung (Zertrümmern)

- Siebloch-Durchmesser (HM)

- nachgeschaltete Siebung

Struktur des Futters bei

der Aufnahme durch das Tier

- Partikelgrößen-Verteilung -

ART/ANTEIL von Komponenten

- pflanzenanatomische Einflüsse

- Struktur der Ergänzungen (Min.stoffe etc.)

- bestimmte Nebenprodukte

ANGEBOTSFORM

- als Trockenfutter (Förderungstechnik)

- als Flüssigfutter (Standzeit/Rühreffekte)

- als Breifutter (Quellung)

KONFEKTIONIERUNG

- Pelletierung (an sich)

- Pellet-Durchmesser („Strukturverlust“)

- Nachträgliche Bröselung ( “ )

VERMAHLUNG

- Art der Zerkleinerung (Zertrümmern)

- Siebloch-Durchmesser (HM)

- nachgeschaltete Siebung

Übersicht 1: Einflüsse auf die „Struktur“ eines Mischfutters für Schweine (KAMPHUES 2009)

Die Art der Vermahlung (Hammermühle, Walzenstuhl) war und ist von erheblicher Bedeutung für die Struktur, d. h. die Partikelgrößenverteilung im vermahlenen Material (ARLINGHAUS et al. 2013). Die Prallzertrümmerung (Prinzip der Hammermühlenvermahlung) liefert dabei allgemein ein sehr breites Band an Partikelgrößen, während die Walzenstuhlvermahlung bezüglich der Entstehung von feinsten Partikeln weniger Risiken birgt. Auch die Kombination unterschiedlicher Vermahlungstechniken (Stufe 1: Keilscheibenzerkleinerung; Stufe 2: nur abgesiebte Grobanteile werden mittels Hammermühle nachvermahlen) ist möglich und führt insgesamt zu einem engeren Band/Spektrum an Partikelgrößen (BORGELT 2015). Unbestreitbar hat auch die Konfektionierung des Mischfutters Auswirkungen auf die Struktur. Die Nachzerkleinerung beim Pelletieren („sekundäre Vermahlung“) hängt dabei von ver-schiedenen Einflussfaktoren ab. Neben der Konditionierung ist auch die Abnutzung der Koller von Be-deutung, so dass bei stärker abgenutzter Technik die Nachzerkleinerung auch allgemein deutlicher ausfällt. Nicht zuletzt ist zu erwähnen, dass auch die Art des Angebots (in der Schweinefütterung z. B. Standzeit und Rühren des Flüssigfutters vor der Zuteilung/dem Transport zum Trog) zu einer „feine-ren“ Struktur im Futter führen kann. In der Geflügelfütterung sind weitere bzw. besondere Praktiken bekannt und etabliert, die im Misch-futter zu sehr unterschiedlichen „Korngrößen“ führen. So wird vor dem Pelletieren gern und häufig unvermahlenes Getreide (5/10/>20 %!) in die Mischungen gegeben, so dass beim nachfolgenden Pelletieren zwar auch noch eine gewisse Zerkleinerung stattfindet, aber dennoch gröbere Strukturen (halbe, geviertelte Körner) erhalten bleiben.



Interesse verdient des Weiteren, dass bei der Vermahlung auch nach den Rohwaren unterschieden wird. Dabei sollten beispielsweise für das Getreide eher gröbere Vermahlungen angestrebt werden und die proteinreichen Komponenten (z. B. Sojavollfettbohnen, Sojaextraktionsschrot, Ackerbohnen und Erbsen) eher feiner vermahlen sein (KLUTH 2011). Neueste experimentelle Studien zur „Feinst-vermahlung“ von Sojaextraktionsschrot lassen zumindest im Vergleich zur herkömmlichen Partikel-struktur von Sojaextraktionsschrot einige Vorteile in der Verdaulichkeit erkennen (HEUERMANN et al. 2016). In dem Bemühen um „Struktur“ im Mischfutter für Geflügel finden auch verstärkt wieder Kom-ponenten Verwendung, die über längere Zeit eher verpönt waren (Hafer, Haferspelzen/-schälkleie, Gerste, Produkte aus nicht entschälter Saat). Was eine nähere, d. h. quantitative Charakterisierung der Struktur im Mischfutter angeht, so liegt hierzu eine umfassende Übersicht vor (WOLF et al. 2012), in der die Technik der Siebanalyse an sich, aber auch die Darstellung der Ergebnisse und ihre Bedeutungerfasst wurden. Eine Analyse der Futterstruktur schrotförmiger Mischfutter erfolgt in der Regel mittels trockener Sie-banalyse. Dabei ist folgendes Vorgehen üblich: Die schrotförmige Probe (50 g) wird auf einen Sieb-turm (Siebe mit unterschiedlichen Sieblochgrößen) gegeben, dann von Hand oder auf einem Schüttler bewegt, und zwar bis zur Massenkonstanz auf den jeweiligen Sieben (oder mit definierter Zeitvorga-be). Die Fraktionen werden gewogen und dann als Anteile der insgesamt ausgewogenen Masse ange-geben. Mischfuttermittel in pelletierter / gebröselter Form können leider noch nicht gemäß einer offiziellen, in den Verbandsvorschriften der VDLUFA angegebenen Methode analysiert werden, da es eine solche nicht gibt. Derartige Proben müssen mittels nasser Siebanalyse auf ihre Partikelgrößenverteilung untersucht werden. Etwa 50 g einer Mischfutterprobe werden in ein Becherglas gegeben und verblei-ben bei Raumtemperatur mit 1000 ml Wasser (ca. 30 °C) für eine Stunde (nach ca. 30 Minuten einmal umrühren). Danach wird die Futter-Wasser-Suspension auf das oberste Sieb eines zuvor im Trocken-schrank bei 103 °C auf Gewichtskonstanz gebrachten, in Exsikkatoren abgekühlten und anschließend gewogenen Siebturmes gegeben. Es werden Siebe mit diversen Sieblochgrößen (3,15 bis 0,2 mm) verwendet. Der Siebturm wird auf einen Auffangboden mit Auslauf gestellt, anschließend wird die Probe mit 10 l kaltem, destillierten Wasser durchgespült (mittels Spritzpistole, die an ein Dest.-Leitungssystem mit ~ 1 bar Wasserdruck angeschlossen ist). Dann ist im Allgemeinen keine Trübung mehr im ablaufenden Wasser erkennbar. Nach Trocknung bei 103 °C wird der Siebturm in den Exsik-kator gestellt und ausgewogen. Die Berechnung der prozentualen Anteile der einzelnen Siebfraktionen wird unter Berücksichtigung des Trockensubstanzgehaltes im analysierten Futter durchgeführt. Alle durch den Waschvorgang herausgelösten, d. h. wasserlöslichen Substanzen werden der Fraktion <0,2 mm zugerechnet. Hierbei werden die Siebe nicht bewegt, so dass nur das zugeführte Wasser den Transport der Partikel im Siebturm bewirkt. Bei den unterschiedlichen Möglichkeiten zur Darstellung von Ergebnissen der Siebanalyse ist je nach Zielsetzung zu differenzieren: Geht es dabei eher um die Beschreibung der „mittleren“ Partikelgröße (z. B. über den GMD oder dMEAN) eines Mischfutters (vgl. dazu WOLF et al. 2012) oder um die Cha-rakterisierung von Anteilen besonders grober oder besonders feiner Partikel, bei deren Überschreiten „nachteilige Effekte“ auftreten (Einbußen in der Verdaulichkeit? Höhere Frequenz von Magenulzera bei Schweinen? Drüsenmagendilatation beim Geflügel?). Je nach primärer Intention sind dann unter-schiedliche Parameter und Richtwerte zu empfehlen (KAMPHUES et al. 2014).

Verdaulichkeit des Futters Wie eingangs erwähnt, wird die Notwendigkeit einer Zerkleinerung des Futters mittels Vermahlung u. a. mit der dadurch günstigeren Verdaulichkeit des Futters begründet, was für das Schwein auch prin-zipiell zutrifft, beim Geflügel aber kritisch hinterfragt werden darf. Mit dem Muskelmagen verfügen Hühner, Puten, aber auch andere granivore Vögel über eine besondere anatomische Einrichtung, die auch bei Aufnahme intakter Getreidekörner höchste Verdaulichkeitswerte ermöglicht, wie es von BANK



et al. (2010) auch in einer neueren Arbeit wieder gezeigt werden konnte. Wenn überhaupt, so waren hier allenfalls für Maiskörner signifikant günstigere Verdaulichkeitswerte (organische Substanz und Stärke) nach der Zerkleinerung zu beobachten, während beispielsweise beim Weizen das nati-ve/unbehandelte Korn zu höchsten Werten führte (im Vergleich: ganz/gebrochen/vermahlen). Bei der Frage nach der Bedeutung der Vermahlung für die Verdaulichkeit sind weitere Differenzierun-gen unabdingbar, nämlich nach dem

- Niveau der Zerkleinerung/Intensität der Vermahlung, - Versorgungsniveau (auf Erhaltung bzw. bei ad lib.-Fütterung), - Gesamtverdaulichkeit bzw. praecaecale Verdaulichkeit, - Art der Komponenten (Getreide/Leguminosen), - Einflüsse zusätzlicher Bearbeitungsverfahren (Pelletieren etc.).

In den beiden Übersichten von WOLF et al. (2012) sowie ARLINGHAUS et al. (2013) werden diverse in diesem Zusammenhang zu berücksichtigende Einflussfaktoren näher behandelt. Zur Bedeutung der Vermahlungsintensität und der Mischfutterkonfektionierung für die Verdaulichkeit des Futters wurden am hiesigen Institut mit Absetzferkeln und mit Broilern entsprechende Verdaulich-keitsstudien durchgeführt, und zwar unter den Bedingungen einer ad-libitum-Fütterung, da diese das übliche Versorgungsniveau unter Praxisbedingungen repräsentiert.

Tabelle 1: Zum Einfluss der Futterstruktur auf die Verdaulichkeit von Nährstoffen bei jungen Schweinen (20 – 25 kg; H: Hammermühle, W: Walzenstuhl; nach ARLINGHAUS et al. 2013)

Futter PF SG PG EG - Vermahlung fein (H) grob (W) grob (W) grob (W) - Verpressung pelletiert schrotförmig pelletiert extrudiert

Partikel (%) > 1,0 mm 8,97 45,8 41,6 29,3 < 2,0 mm 42,4 27,2 32,7 43,0

Verdaulichkeit (%) über den gesamten Verdauungstrakt - org. Substanz 85,5 85,7 85,5 85,0 (± 1,72) (± 1,10) (± 1,65) (± 1,17) - Rohprotein 80,9ab 81,8a 79,0b 79,2b (± 2,90) (± 2,32) (± 3,12) (± 3,10)

Verdaulichkeit (%) im Dünndarmbereich (vor dem Blinddarm)1 - org. Substanz 70,9 70,3 69,6

nicht geprüft - Rohprotein 78,7 77,4 75,0 - Stärke 96,5a 96,2a 94,8b

1)ileumfistulierte Mastschweine, 70-75 kg KM

Bei ganz erheblichen Unterschieden in den Partikelfraktionen > 1 mm bzw. < 0,2 mm ergaben sich unter den Bedingungen der ad-libitum-Fütterung abgesetzter Ferkel (KM 20 – 25 kg) keine signifikan-ten Unterschiede für die Verdaulichkeit von organischer Substanz und Rohprotein (Pellet fein vs. Schrot grob), andererseits aber signifikante niedrigere Werte für das Rohprotein, wenn grob vermah-lenes Mischfutter zusätzlich pelletiert oder extrudiert wurde. Besondere Erwähnung verdient die prae-caecale (pc) Verdaulichkeit, die allerdings unter den Bedingungen einer restriktiven Fütterung bei Tieren im Endmastalter (70 – 75 kg KM) geprüft wurde. Das pelletierte Mischfutter aus feinvermahle-nen Komponenten war auch hinsichtlich der pc Verdaulichkeit nicht vom grob vermahlenen Mischfut-ter (als Schrot angeboten) zu unterscheiden. Hinsichtlich der pc Verdaulichkeit kam es bei der Stärke zu leichten, aber signifikanten Einbußen, wenn das grob vermahlene Mischfutter in pelletierter Form zum Einsatz kam. Die Untersuchungsergebnisse von ARLINGHAUS et al. (2013) sprechen eindeutig dafür, dass unter den derzeit üblichen Produktionsbedingungen des Mischfutters für Schweine die Vermahlungsintensität deutlich zurückgenommen werden kann, und zwar ohne Einbußen in der Ver-daulichkeit hinnehmen zu müssen. Dieses steht allerdings im Gegensatz zu Schlussfolgerungen von



LIERMANN et al. (2015), die bei einem Vergleich einer Vermahlung in der Hammermühle mit einem 6 bzw. 3 mm Sieb Vorteile in der Verdaulichkeit für das feiner vermahlene Mischfutter sahen. Vergleicht man aber die Ergebnisse einmal kritisch (Tabelle 2), so sind trotz der enormen Unterschiede in den Korngrößen beim Angebot des Futters als Schrot bzw. als Pellet nicht ein einziges Mal signifikante Unterschiede in der Verdaulichkeit aufgetreten, zumindest was die org. Substanz, das Rohprotein und die NfE-Fraktion betrifft.

Tabelle 2: Zum Einfluss der Vermahlungsintensität auf die Verdaulichkeit ausgewählter Rohnährstoffe bei Mast-schweinen (Anfangs-/Mittelmast) nach LIERMANN et al. (2015)

Angebotsform Schrot Pellet Vermahlung fein grob fein grob Siebdurchmesser (mm) 3 6 3 6 % > 1 14,8 63,8 - - % < 0,125 15,0 3,7 - - oS 86,4abc 85,3abc 86,5abc 84,9bc

Rp 87,4ab 84,5b 86,6ab 85,0b

NfE 90,7 90,1 90,6 89,3

oS 86,0ab 85,5ab 85,8ab 85,4ab

Rp 83,6a 81,9ab 83,5a 84,0a

NfE 91,2ab 90,8ab 90,8ab 90,3ab

Vor dem Hintergrund möglicher Energieeinsparungen für die Zerkleinerung von Einzelfuttermitteln in der Mischfutterproduktion verdient auch die zweistufige Vermahlung Erwähnung, wobei auf der 1. Stufe eine Zerkleinerung mittels Walzenstuhl oder Keilscheibenzerkleinerung erfolgt und in der 2. Stu-fe die abgesiebten Grobanteile einer Nachvermahlung in der Hammermühle unterzogen werden. Trotz der gröberen Struktur des Mischfutters aus der zweistufigen Vermahlung (im Vergleich zum Hammer-mühlen-vermahlenen Futter) unterschieden sich die Verdaulichkeitswerte für die org. Substanz, Roh-protein und Stärke nicht signifikant (BORGELT et al. 2015), der Zerkleinerungsprozess ermöglichte jedoch erhebliche Kosteneinsparungen von ca. 30 %. Bei dem Trend zum Einsatz von Grobfuttermitteln (z. B. Maissilage) als Faser-liefernde Komponente in der Flüssigfütterung von Schweinen (insbesondere für tragende Sauen) verdient gerade hier die Ver-daulichkeit in Abhängigkeit von der Vermahlung Erwähnung (HOHMEIER und KAMPHUES 2015). Un-abhängig von Art und Intensität der Zerkleinerung (Vermusen nach Wässern/Feuchtvermahlung im Cracker/Häckselung) war die Verdaulichkeit der org. Substanz nicht unterschiedlich (57 – 60 %). In der Broilerfütterung werden pelletierte Mischfuttermittel standardmäßig eingesetzt. Diese Pellets können dabei aus unterschiedlich intensiv behandelten/zerkleinerten Ausgangskomponenten erstellt werden. Neben der üblichen Hammermühle (H) sind auch der Walzenstuhl (W) und weitere Technolo-gien (Expandate/Extrudate) im Einsatz, die dann allesamt zu einem Mischfutter führen, das ad libitum angeboten wird. Vor diesem Hintergrund wurden bei einer ad-libitum-Fütterung Verdaulichkeitsunter-suchungen durchgeführt, die schließlich von SANDER et al. (2013) noch um entsprechende Studien zur pc Verdaulichkeit ergänzt wurden (Tabelle 3). Trotz erheblicher Unterschiede in der mittleren Partikelgröße (GMD von 244 bis 480 m!) waren keine signifikanten Unterschiede in der Verdaulichkeit der organischen Substanz nachweisbar; auch die Stärkeverdaulichkeit im Dünndarmbereich bzw. über den gesamten Verdauungstrakt war nicht auffäl-lig unterschiedlich. Andererseits sprechen die Zahlen schon eindeutig für Effekte der Vermahlungsin-tensität auf die pc Verdaulichkeit von Rohprotein und Aminosäuren (hier nur Lys und Met aufgeführt). Die Ergebnisse stützen somit die von KLUTH (2011) getroffene Schlussfolgerung, nach der beim Ge-flügel eine intensivere Vermahlung der Protein-liefernden Komponenten angeraten ist, während beim Getreide diesbezüglich keine so gravierenden Unterschiede zu erwarten sind.



Tabelle 3: Einfluss der Vermahlung/Partikelgrößenverteilung und Konfektionierung auf die Gesamtverdaulichkeit sowie die praecaecale Verdaulichkeit von Nährstoffen bei Broilern

Mischfutter 1 2 3 4 Vermahlung Konfektionierung

fein, H Pellet

grob, W Pellet

fein, H +1) Pellet

grob, W Extrudat

Partikelgrößen, % > 1 mm < 0,2 mm GMD, m

17,1 41,3 316

45,5 30,9 468

40,8 30,6 480

14,1 47,2 244

Verdaulichkeit2), % - org. Substanz - Stärke

68,0 91,4

68,1 93,9

68,9 90,8

69,3 95,3

pc Verdaulichkeit3), % - Stärke - Rohprotein - Lysin - Methionin

94,1 80,9 82,4 90,6

95,9 77,8 80,2 88,0

95,1 76,7 79,2 86,3

96,3 85,7 86,6 92,2

1) 22 % ganze/intakte Weizenkörner vor dem Pelletieren dem vermahlenen Mischfutter zugesetzt;

2) am Lebenstag 27 – 35; 3) am Lebenstag 21 – 27

Vor diesem Hintergrund werden die Untersuchungsergebnisse von HEUERMANN et al. (2016) ver-ständlich, nach denen bei einer eher knappen Rp- und AS-Versorgung Broiler mit signifikant höheren Leistungen reagierten, wenn anstelle eines üblichen Sojaextraktionsschrotes eines aus einer „Feinst-vermahlung“ (100 % der Partikel < 60 m!) zum Einsatz kam. Im Zusammenhang mit der Frage nach der Futtermittelstruktur und Futterverdaulichkeit verdient Er-wähnung, dass rein methodisch-versuchstechnisch bedingt der Einsatz eines schrotförmigen Mischfut-ters mit größeren Unsicherheiten behaftet ist. Der nicht selten zu beobachtende nachteilige Effekt eines gröberen schrotförmigen Futters auf den Futteraufwand (z. B. in Mastversuchen) ist evtl. mit erhebliche höheren Futterverlusten (Staub, Verluste auf dem Boden etc.) verbunden. Werden unterschiedlich intensiv vermahlene Mischfutter vergleichend geprüft (betrifft Schweine und Geflügel), so ist immer die Frage nach der Höhe der Futteraufnahme zu stellen. Nach einer umfassen-den Auswertung einer Vielzahl an Fütterungsversuchen mit Schweinen ist bei gröberem Futter fast durchgängig der Trend zu einer leicht höheren Futteraufnahme zu verzeichnen, wodurch tendenziell die Leistung gefördert werden dürfte (ARLINGHAUS et al. 2013, LIERMANN et al. 2015), trotz der vermutlich höheren Futterverluste und einer tendenziell niedrigeren Verdaulichkeit. Werden in der Geflügelfütterung im Mischfutter „gröbere“ Strukturen angestrebt, z. B. durch die Ein-arbeitung ganzer Körner in ein pelletiertes Mischfutter, so ist nach Erfahrungen in der Praxis eher ein Trend zu einer leicht niedrigeren Futteraufnahme zu beobachten. Dieses Phänomen steht vermutlich im Zusammenhang mit einem Aspekt, der bisher nicht näher im Fokus stand, aber größeres Interesse verdiente, nämlich die Frage nach der Passagegeschwindigkeit des Chymus durch den Magen, Dünn- und Dickdarm. Die Auswirkungen der Futterstruktur in diesen drei Lokalisationen sind weder gleichar-tig noch gleichgerichtet. Die feine Vermahlung führt zu einer schnelleren Magenpassage, aber zu einer eher protahierten Passage des Dickdarms. Das „grobe“ Futter hat hingegen eher die umgekehrte Wir-kung, zumindest beim Schwein.

Die Gesundheit des Magen-Darm-Trakts Mit nachfolgender Übersicht soll zunächst einmal verdeutlicht werden, welche Faktoren insgesamt zu berücksichtigen sind, wenn die Gesundheit des Magen-Darm-Trakts näher charakterisiert werden soll. Dabei bestehen diverse Interaktionen und Abhängigkeiten, so dass ein insgesamt sehr komplexes Phänomen näher zu behandeln ist.



Gastrointestinalflora-- exogene Keime- Eu-/Dysbiose

Primärfunktionen- Verdauung- Absorption- Exkretion

Immunologische Funktion- Kontakt zu Immunogenen- zelluläre Abwehr- Antikörpersekretion

Gesundheit desMagen-Darm-Traktes

Anatomische / histologischeEntwicklung / Integrität

Gastrointestinalflora- residente Flora- exogene Keime- Eu-/Dysbiose

Primärfunktionen des GIT- Verdauung- Absorption- Exkretion

Immunologische Funktionen- Kontakt zu Immunogenen- zelluläre Abwehr- Antikörpersekretion


Gastrointestinalflora-- exogene Keime- Eu-/Dysbiose

Primärfunktionen- Verdauung- Absorption- Exkretion

Immunologische Funktion- Kontakt zu Immunogenen- zelluläre Abwehr- Antikörpersekretion


Anatomische / histologischeEntwicklung / Integrität

Gastrointestinalflora- residente Flora- exogene Keime- Eu-/Dysbiose

Primärfunktionen des GIT- Verdauung- Absorption- Exkretion

Immunologische Funktionen- Kontakt zu Immunogenen- zelluläre Abwehr- Antikörpersekretion


Übersicht 2: Die Gesundheit des Magen-Darm-Traktes - Facetten, die einer näheren Charakterisierung dienen

(KAMPHUES 2009)

Bezüglich der Gesundheit des Magen-Darm-Traktes in Abhängigkeit von der Mischfutterstruktur zählen die Magenulzera vermutlich zu den bekanntesten Störungen. Wie mit der Arbeit von GROSSE LIESNER (2008) eindeutig zu belegen (s. Abbildung 1), steigen die Risiken für die Entwicklung von Magenulzera mit zunehmender Intensität der Futtermittelbearbeitung und Feinheit des Futters. Auch in den neue-ren Arbeiten von LIERMANN et al. (2015) wird ausdrücklich hervorgehoben, dass mit der Pelletierung an sich eine erhöhte Disposition für die Entwicklung von Alterationen in der Pars nonglandularis am Mageneingang verbunden ist. In den Arbeiten zur zweistufigen Vermahlung war mit der gröberen Struktur des Mischfutters (im Vergleich zur Hammermühlenvermahlung) ein sehr günstiger Effekt auf die Magengesundheit (Integrität der Schleimhaut) verbunden (BORGELT et al. 2015).

0,0

grob sehr grob grob fein sehr fein

schrotförmig pelletiert pelletiert pelletiert pelletiert

histologischer Score

makroskopischer Score

18,8 35,7 50,8 54,2 56,0

56,6

35,2

40,0 44,8 42,0

24,4 29,12,11,19,2

0%

20%

40%

60%

80%

100%

[Mas

sen

-%] ≥ 2,0 mm

≥ 0,4 < 2,0 mm

< 0,4 mm

Abbildung 1: Effekte der Futterkonfektionierung (Schrot/Pellet) sowie der Vermahlungsintensität

(Partikelgrößenverteilung) auf die Integrität der Magenschleimhaut von Absetzferkeln (nach GROSSE LIESNER 2008)

Weniger bekannt (zumindest in den meisten Lehrbüchern zur Tierernährung) sind die bei Masthüh-nern durch eine sehr feine Futterstruktur ausgelösten Veränderungen, die als „Drüsenmagen-Dilatation“ klinische Bedeutung haben. Hierbei kommt es – insbesondere bei Einsatz pelletierter Misch-futter auf der Basis feinstvermahlener Komponenten – zu einer derartigen Ausdehnung des Organs mit Verlust des typischen Isthmus am Übergang zum Muskelmagen, so dass bei Volumenzunahme



dieses veränderten Organs ein Verenden unter dem Bild des Herz-Kreislaufversagens typisch ist, ins-besondere in der letzten Mastwoche. Diese Störungen konnten in hiesigen Untersuchungen (WITTE 2012) mit dem feinpelletierten Alleinfutter in erheblicher Prävalenz beobachtet werden.

Tabelle 4: Prävalenz und Ausprägung von Drüsenmagen-Dilatationen (PD) nach WITTE (2012)

Mischfutter Pellet fein Pellet grob Pellet mit Korn Extrudat sezierte Tiere

total (n) 75 75 75 75 d 35 (n) 48 52 50 52

Tiere mit PD

(n) 8 1 1 1 (%)1) (10,7/16,7) (1,33/1,92) (1,33/2,00) (1,33/1,92)

Grad der PD

ggr. 2 1 1 1 mgr. 3 0 0 0 hgr. 3 0 0 0

1)Bezogen auf die Gesamtzahl der Versuchstiere/Tiere, die in der letzten Versuchswoche seziert wurden

Neben diesen klinisch auffälligen Störungen in Abhängigkeit von der Mischfutterstruktur ist auch von Interesse, dass die relative Organmasse (Bezug auf die Körpermasse) von Magen, Muskelmagen und – noch erstaunlicher – des Pankreas durch die Mischfutterstruktur beeinflusst, d. h. mit bestimmt wer-den, wie es u. a. von BETSCHER (2010), ARLINGHAUS (2013), WITTE (2012) und VON UND ZUR MÜHLEN (2015) beobachtet wurde. Die Funktion des Magens als Barriere für oral aufgenommene Keime wird ebenfalls durch die Mischfut-terstruktur mitbestimmt. Nur bei einer gröberen Mischfutterstruktur kommt es zu einer gewissen Schichtung des Mageninhalts von Schweinen, und damit zu einer Ausbildung von erheblichen pH-Wert-Gradienten, (hohe pH-Werte im Kardia-Drüsenteil, tiefste pH-Werte im Fundusdrüsenbereich; s. MOESSELER et al. 2014). Auf histologischer Ebene erwies sich – neben den Speicheldrüsen – die Ausbildung des terminalen Ileums (Ileo-Caecal-Klappe) als ganz wesentlich von der Futterstruktur beeinflussbar (CAPPAI et al. 2015). Schließlich hatte – wie die experimentellen Arbeiten von BETSCHER (2010) an Absetzferkeln zeigten – die Mischfutterstruktur erhebliche Effekte auf den Anteil proliferativer Zellen in der Schleim-haut von Magen und Caecum sowie auf den Anteil apoptotischer Zellen (an den Epithelzellen insge-samt) im Duodenum. Unter dem Aspekt von Haftung und Vermehrung diverser Infektionserreger verdient Erwähnung, dass der Anteil von Becherzellen mit sauren Muzinen in der Schleimhaut von Ileum und Caecum signifikant höher war, wenn die jungen Mastschweine ein grobes Schrot anstelle von Pellets aus fein gemahlenen Komponenten erhielten. Wie zuvor im Kapitel „Verdaulichkeit“ bereits angesprochen, ist mit einer gröberen Vermahlung – zu-mindest beim Schwein – eine gewisse Verlagerung von Verdauungs- und Absorptinsprozessen in den Dickdarm verbunden, d. h. dass unter diesen Bedingungen auch ein größerer Anteil von Stärke den Dickdarm erreicht, was aus rein tierernährerischer/energetischer Sicht einen Nachteil bedeutet, aus diätetischer, infektionsmedizinischer Sicht aber geradezu erwünscht sein kann (s. Mechanismen der Salmonellen-Vorbeuge). Neben der Absorption ist eine normale Elimination des Unverdaulichen ebenso eine essentielle Aufga-be des Verdauungstraktes. Wie von WARZECHA (2006) beschrieben, ist mit einer groben Vermahlung des Mischfutters eine eher forcierte Chymuspassage im Dickdarm verbunden (niedrigere TS-Gehalte, geringere Härte des Kotes), was insbesondere aus diätetischer Sicht in der Fütterung tragender Sauen Interesse verdient, da hier Verstopfung und damit verbundene Störungen keine Seltenheit sind. Die Mischfutterstruktur - bestimmt durch Intensität der Vermahlung und anschließende Kompaktie-rung - hat auch eine erhebliche Bedeutung für die Flora im Verdauungstrakt von Schweinen, und auch beim Geflügel. So hat die bei gröberer Futterstruktur im Magen von Schweinen typische „Schichtung“ und damit geringere Durchsetzung des Chymus mit Salzsäure im Kardiadrüsenteil des Magens eine



Förderung grampositiver Keime zur Folge (BULLERMANN 2011), währen im terminalen Ileum gramne-gative Keime wie E. coli eher zurückgedrängt werden (MIKKELSEN 2004). Ein forcierter Einstrom von Getreidestärke in den Dickdarm fördert des Weiteren die Bildung von Propion- und Buttersäure im Chymus, mit entsprechenden erwünschten Auswirkungen auf den Stoffwechsel von Salmonellen (s. u. a. LAWHON et al. 2002). Weniger auffällig waren die Auswirkungen einer gröberen Mischfutterstruktur auf die Darmflora bei Masthähnchen (ÜFFING 2012), evtl. auch dadurch bedingt, dass - infolge der Muskelmagenaktivität - die Partikelgrößenverteilung im Chymus am Ende des Dünndarms oder im Dickdarm nicht so stark differiert wie im Futter. Nach diversen experimentellen Untersuchungen wie auch größeren Feldstudien ist die gröbere Misch-futterstruktur – insbesondere der Verzicht auf die Pelletierung – eine diätetische Maßnahme zur Re-duktion der Salmonellen-Prävalenz in Schweinebeständen (VISSCHER et al. 2009). Auch in neueren Untersuchungen zu den Auswirkungen der Mischfutterstruktur bei Masthähnchen (RATERT et al. 2013) unter den Bedingungen einer künstlichen Salmonelleninfektion, erwies sich das pelletierte Mischfutter mit eingearbeiteten ganzen Weizenkörnern in der Infektion und Translokalisation (Über-gang der Salmonellen in den Tierkörper, d. h. Lymphknoten und Leber) als die diätetisch günstigste und wirksamste Mischfutterkonfektionierung (Abb. 2).

Abbildung 2: Anteil Salmonellen-positiver Proben der Kontakttiere nach einer experimentellen Infektion von

Broilern mit SalmonellaEnteritidis (nach Ratert et al. 2015)

In weiteren Untersuchungen zur Barrierefunktion des Magens bzw. des Mageninhalts beim Schwein in Abhängigkeit von der Mischfutterstruktur wurde Salmonella Derby in vitro dem Mageninhalt (aus dem Fundusbereich bei der Sektion entnommen) zugesetzt, dieser inkubiert und nachfolgend die Entwick-lung der Salmonellen-Keimzahl bestimmt (KOOP 2013). Während im Mageninhalt der mit grobem Schrot versorgten Tiere bereits 3 Minuten nach der Inkubation keine Salmonellen mehr nachweisbar waren, blieben im Mageninhalt der mit feinvermahlenem und pelletiertem Futter versorgten Tiere noch Salmonellen nachweisbar, hier kam es nach 2 Stunden bereits wieder zu einem Anstieg der Keimzahlen. Bei prinzipiell ähnlichem Vorgehen (VON UND ZUR MÜHLEN 2015) ergaben sich für bestimmte, als hochpotente Durchfallerreger bekannte E.-coli-Keime keinerlei vergleichbare Effekte, d. h. diese er-wiesen sich als höchst unempfindlich, was das Milieu im Mageninhalt angeht (sehr pH-tolerant!). Des Weiteren „überwanden“ diese E.-coli-Keime bei oraler Infektion die Magenbarriere unabhängig von der Mischfutterstruktur, so dass die Autoren einer Prophylaxe mittels der Vermahlungsintensität eher skeptisch gegenüberstehen.



Vor dem Hintergrund der beschriebenen Bedeutung der Mischfutterstruktur für die Verdaulichkeit und die Gesundheit des Magen-Darm-Trakts werden Zielkonflikte deutlich: Wann immer es zu einer Über-betonung des einen Zieles kommt, gibt es nachteilige Effekte/Veränderungen bei anderen Zielvorga-ben. In der Praxis, in der auch in Zukunft pelletierte Mischfuttermittel eine erhebliche Bedeutung haben werden – insbesondere wegen ihrer vielfältigen „logistischen“ Vorteile -, stellt sich die Frage nach Ansätzen zur Entschärfung des Problems, die in der nachfolgenden Übersicht zusammengestellt sind (Übersicht 3).

Übersicht 3: Möglichkeiten einer verstärkten Berücksichtigung/Nutzung des Faktors STRUKTUR im Futter von Schweinen und Geflügel

Maßnahmen/Möglichkeiten Schweine Geflügel Auswahl der Getreideart Spelz- statt Nachtgetreide evtl. Hafer/-schalen (Weizen/Mais)

Einsatz intakten Getreides (ganzes Korn) - verbreitet (evtl. kombinierte Fütterung)

Art der Vermahlung von Getreide Walzenstuhl u. ä. statt Hammermühle Intensität der Vermahlung Sieblochdurchmesser/Drehzahl/Walzenabstand

Verzicht auf Pelletieren (Kompaktierung) Risiko der Entmischung bei Legehennen üblich, sonst selten

Ergänzung mit „Struktur“-Einzel-FM (Non-cereal-ingredients) Schalen-/Spelzen-/Lignin-reiche Einzel-FM

Limitierung feinstpartikulärer Komponenten Nebenprodukte der Bäckerei/Altbrot/ähnliche Komponenten Partikuläre Lignocellulose Ansätze, aber Effekte noch offen Extra-Angebot von „Grundfutter“1) Maisganzpflanzensilage u. ä. 1)eher unter ethologischen Aspekten aktuell in der Diskussion

Zusammenfassung Ähnlich zur Wiederkäuerfütterung, in der ein Bedarf an „physikalisch wirksamer Struktur/Faser“ abso-lut unstrittig ist, muss man auch in der Fütterung von Schweinen und Geflügel von einem Bedarf an „Partikelgrößen“ ausgehen. Wird dieser Parameter nicht genügend berücksichtigt, kommt es zu diver-sen nachteiligen Effekten auf die Gesundheit des Magen-Darmtraktes, die bis zu sehr spezifischen Krankheitsbildern führen können. Andererseits ist eine gewisse Zerkleinerung der hier üblichen Kom-ponenten (Getreide und diverse proteinliefernde Futtermittel) unabdingbar, nicht zuletzt aus pragma-tischen und logistischen Gründen. Auch wenn das Geflügel sehr wohl natives Getreide sehr effizient nutzen kann, so stellt sich auch hier die Frage nach der Versorgung mit „Minorkomponenten“ (Mine-ralstoffe, Vitamine, andere Zusatzstoffe), die eben nicht mit ganzem Getreide eine „stabile“ Mischung ergeben. Schrotförmige Mischfutter haben auch ihre spezifischen Risiken (s. früheres Vorkommen von Entmischungsproblemen); es wäre evtl. aber schon viel erreicht, wenn man dem Grundsatz folgte: Nur so fein vermahlen, wie unbedingt für eine hohe Verdaulichkeit erforderlich und nicht nach dem Motto: So fein wie möglich – für eine maximale Verdaulichkeit! Die derzeitigen Bemühungen um Tier-wohl, Nachhaltigkeit und auch Lebensmittelsicherheit stehen diesem veralteten Motto diametral ent-gegen.

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Autorenanschrift Prof. Dr. Josef Kamphues Stiftung Tierärztliche Hochschule Hannover Institut für Tierernährung Bischofsholer Damm 15 D-30173 Hannover E-Mail: josef.kamphues(at)tiho-hannover.de

Krungleviciute et al.: Effects of the Lactobacillus sakei KTU05-6 supplement on milk production and ruminal processes in feeding dairy cows


Effects of the Lactobacillus sakei KTU05-6 supplement on milk production and ruminal processes in feeding dairy cows

Vita Krungleviciute2, Elena Bartkiene2, Rasa Zelvyte1, Ingrida Monkeviciene1, Jone Kantautaite1, Rolandas Stankevicius3, Antanas Sederevičius1, Danielius Starevicius4 and Grazina Juodeikiene5

1 Department of Anatomy and Physiology, Research Center of Digestive Physiology and Pathology, Veterinary Academy, Lithuanian University of Health Sciences; Kaunas, LT 2 Department of the Food Safety and Quality, Veterinary Academy, Lithuanian University of Health Sciences; Kaunas, LT 3 Department of Animal Nutrition, Veterinary Academy, Lithuanian University of Health Sciences, Kaunas, LT 4 Department of Non-Infectious Diseases, Veterinary Academy, Lithuanian University of Health Sciences, Kaunas, LT 5 Department of Food Research and Technology, Kaunas University of Technology, Kaunas, LT

Abstract The aim of this study was to evaluate the effects of the Lactobacillus sakei KTU05-6 supplement on milk production and ruminal processes in feeding dairy cows. Trial and control groups received identi-cal diets, however the trial group received also 100 g of the supplement per head daily (109colony-forming units (CFU) of L. sakei/head/d) during 65 days. It was found that at the end of the experi-ment milk yield and content of milk fat, protein, lactose and urea in the trial group did not differ sig-nificantly compared to the control group (P> 0.05). At the end of the experiment ruminal pH, total and individual volatile fatty acids, total nitrogen, ammonia nitrogen, D(-)-lactic acid, reduction activity of bacteria, glucose fermentation reaction, protozoa number, total count of lactobacilliand enterobac-teria in trial group had no significant difference (P> 0.05) from those characteristics in the control group. We conclude that 109 CFU/head/dLactobacillus sakei KTU05-6 may not be beneficial for dairy cows, as positive effect on the milk yield and milk composition, on the activity of the ruminal fermen-tation and microorganisms of rumen fluid was not observed in the studied animals.

Introduction Lactic acid bacteria (LAB) including bacteriocin-like inhibitory substances (BLIS) producing strains are generally recognized as safe (GRAS) (Mayo et al., 2010). Because of their antimicrobial effect, BLIS, or their producing organisms, have a potential use as modifiers of the ecosystem in the gastrointesti-nal tract (Seo et al., 2010) and stimulators of animal production (Yasuda et al., 2007). Gomez-Basauri et al. (2001) evaluated the effect of a supplement containing LABon dry matter intake, milk yield and milk component concentration. However, the effect of Lactobacillus strains is variable even within a same species (Mayo et al., 2010). Recently the positive effect of Lactobacillus sakei (L. sakei) on the treatment of humans and laborato-ry animals was defined (Kim et al., 2013). L. sakei KTU05-6 produced BLIS show wide-ranging antimi-



crobial activities against gram positive and gram negative strains (Cizeikiene et al., 2013). However, no studies were conducted to evaluate possible effect of L. sakei feeding on ruminants. The aim of this study was to evaluate the effects of the Lactobacillus sakei KTU05-6 supplement on milk production and ruminal processes in feeding dairy cows.

Material and methods Preparation of supplement with high amount of L. sakei KTU05-6 Extrusion of rye wholemeal was used for flour thermal treatment with the intention to reduce the level of microbiological pollution.Extruded rye wholemeal produced by a single-screw extruder (Ustukiu malunas Ltd, Lithuania) was used as the fermentation medium for Lactobacillus sakei KTU05-6. L. sakei were isolated from rye sourdoughs by proving additionally their bacteriocin activity (Digaitiene et al., 2012).Strains were stored at -80oC in a Microbank system (PRO-LAB DIAGNOSTICS) and propa-gated in MRS broth (CM 0359, Oxoid Ltd, Hampshire, UK) at 30oC for 48 hours with the addition of 40 mM fructose and 20 mM maltose prior to be used. The bacteria were diluted with a physiological saline to a concentration of 108 CFU/mL before the experiment. The fermented product (65 % moisture content) has been prepared using 3.0 kg of extruded rye whole meal flour and 4.5 L of water. LAB cell suspension (50 mL) containing about 108 CFU/mLwas added, followed by fermentation for 24 h at temperature optimal for the strain. The final colony num-ber in the fermented product was on average 109 CFU/g. Extruded rye wholemeal with high amount of LAB was divided in to the 100 g pieces and used for cow feeding. New portion of fermented sup-plement was preparing every 7 days, because LAB count in high moisture extruded rye flour during the 7 days period (storage temperature +4ºC) was stabile (109 CFU/g). Experimental animals and diets Effects of L. sakei KTU05-6 supplement have been investigated on 40 mid-lactation dairy cows divided into two groups each containing 20 cows: control (C) and trial (T). Animals were cared according to the Requirements for keeping, maintenance and use of animals intended for experimental and other scientific purposes. The experiment was performed in the winter at a farm of BlackWhite Holstein dairy cattle. Distribution of the cows into groups was based on parity, body weight and stage of the lactation cycle. Both groups received identical diet. The diet was balanced according to the animal requirements in all nutrition parameters (Jeroch et al., 2004). However the trial group also received 100 g per head ofthesupplement daily during 65 days (109 CFU of L. sakei/head/d). The supplement was fed individually. Water was available ad libitum. Milk samples collection and analyses Milk samples from all experimental cows (n=40) were collected and daily milk yields were recorded during control milking three times: first – at the beginning of the experiment, second – in the middle of the experiment and third – at the end of the experiment. Milk yield adjusted to 4 percent fat (fat-corrected milk; FCM) was calculated with the following equation: 4% FCM = 0.4×actual milk yield (in kg/d)+15×milk fat (in kg/d). The following parameters of milk were measured: milk fat, milk protein, milk lactose, milk urea by the equipment „LactoScope FTIR” (FT1.0. 2001; Delta Instruments, Hol-land). Sample collection and analyses of rumen fluid During the experiments rumen fluid samples were collected two times: first – at the beginning of the experiment and second – at the end. The activity of ruminal fermentation and the microbiology of rumen fluid were investigated from the rumen fluid.



The following parameters of the ruminal fermentation were studied: pH, total volatile fatty acids (VFA), total nitrogen (N), ammonia nitrogen (NH3-N), L(+)- and D(-)-lactic acid. Ruminal pH was measured immediately after sampling, using a hand held pH-meter (Horiba - Twin pH, Spectrum Technologies). Total VFA was defined by rumen fluid distillation in a Marcgamus apparatus according to the method of Pustovoj (1978). Total N was analysed by Kjeldahl procedure, NH3-N – by titrimetric method with the preliminary distillation. The concentrations of L(+)- and D(-)-lactic acid were determined by an enzyme test kit (R-biopharm AG - Roche, Darmstadt, Germany), as reported elsewhere (De Lima et al., 2009). The following parameters of the microbiology of rumen fluid were studied: glucose fermentation reaction (GF), reduction activity of bacteria (RA), protozoa number, total count of aerobic and facultative anaerobic microorganisms (TCM), total count of lactobacilli (TCL) and enterobacteria (TCE). GF reaction and RA of bacteria were evaluated according to the methods described by Bakunas (2004). Protozoa number was counted in a Fuks-Rozental chamber according to Schultz (1971). Total count of aerobic and facultative anaerobic microorganisms was determined on plate count agar (CM0325, Oxoid, UK) after incubation at 30°C for 72 hours. Analysis of viable LAB in rumen fluid samples was performed under anaerobic conditions using the atmosphere generation system AnaeroGen (Oxoid, Basingstoke, UK). For the detection of total number of enterobacteria the diluted samples were placed on MacConkey agar (CM0007, Oxoid, UK).The number of microorganisms was calculated and expressed as log10 of colony-forming units per milliliter (log CFU/mL).

Statistical analyses The data of the experiments were evaluated statistically by a statistic package R 2.2.0 and presented as mean ± standard error of the mean (mean ± SEM). The significance of arithmetic differences (P) was defined according to the Student t-test. All statistical analyses were considered significantly different at the P< 0.05 level.

Results The effect of the L. sakei supplement on the milk production and composition is presented in Table 1. At the beginning of the experiment no statistically significant differences were observed in any of the evaluated production parameters between the two groups (P> 0.1). During the experiment milk yield declined steadily in the control group. The same situation was with the trial group. Milk yield adjusted to 4 percent fat (fat-corrected milk) at the end of the experiment decreased in both groups. The dif-ferences in milk yield during the experiment were found statistically not significant (P> 0.05).

Table 1: Milk production and composition (mean±SEM)

Milk parameters Beginning of the experiment Middle of the experiment End of the experiment C group T group C group T group C group T group

Yield, kg/d 19.54±1.15 17.78±1.51 18.2±0.94 17.46±1.31 16.44±0.99 15.72±1.33 4 FCM 21.03±1.29 19.16±1.38 19.63±0.70 19.97±1.54 17.81±0.85 17.7±1.16 Fat, % 4.53±0.18 4.64±0.33 4.58±0.17 5.03±0.41 4.63±0.21 4.97±0.32 Protein, % 3.11±0.15 3.39±0.18 3.45±0.17 3.70±0.18 3.45±0.14 3.78±0.21 Lactose, % 4.46±0.09 4.59±0.06 4.49±0.07 4.61±0.07 4.46±0.06 4.60±0.04 Urea, mg/dL 17.1±1.64 18.1±1.8 10.2±0.70 11.4±1.51 17.2±1.56 16.4±1.36

During the experiment, a statistically significant change in the percentage of milk fat, protein and lactose content was not found in any group (P> 0.05). At the end of the experiment, milk urea content in the trial group did not differ significantly compared to the control group (P> 0.05).



The effect of the L. sakei supplement on the ruminal fermentation and the microbiology of rumen fluid is shown in Table 2. At the beginning of the experiment no statistically significant differences were observed in any of the evaluated parameters between the two groups (P> 0.1). All investigated rumi-nal fermentation and microbiological parameters were in physiological ranges both before and after the treatment. At the end of the experiment ruminal pH, total VFA, total N and NH3-N, D(-)-lactic acid, reduction activity of bacteria, glucose fermentation reaction, protozoa number, TCL and TCE in trial group had no significant differences (P> 0.05) from those characteristics in control group and in trial group at the beginning of the experiment, but L(+)-lactic acid and TCM were different. At the end of the exper-iment L(+)-lactic acid in trial group decreased by 0.28 mmol/L (P< 0.05). Though this parameter decreased in the control group these data were not statistically significant (P> 0.05). At the end of the experiment total count of aerobic and facultative anaerobic microorganisms in the trial group increased by 1.25 log CFU/mL (P< 0.05) in compare with the trial group at the beginning of the experiment and 0.64 log CFU/mL (P> 0.05) in compare with the control group.

Table 2: The activity of ruminal fermentation (mean±SEM)

Parameters Beginning of the experiment End of the experiment C groupa T groupb C groupc T groupd

pH 6.62±0.06 6.40±0.23 6.21±0.29 6.35±0.18 Total VFA, mmol/L 117.0±5.00 120.0±0.05 143.3±6.67 137.5±4.79

Relative proportion of VFA, % Total N, mg/dL 75.25±7.35 61.95±8.75 77.47±11.32 70.52±9.39 NH3-N, mg/dL 7.17±1.35 7.17±0.79 6.50±0.36 7.28±1.19 L(+)-lactic acid, mmol/L 0.49±0.15 0.38±0.05 0.18±0.09 0.10±0.04bd D(-)-lactic acid, mmol/L <0.001 <0.001 <0.001 <0.001 Significant differences between groups b and d – P< 0.05

Conclusion We conclude that Lactobacillus sakei KTU05-6 supplementation by 109 CFU/head/d may not be benefi-cial as probiotic bacteria for dairy cows, as positive effect on the milk yield and milk components, on the activity of the ruminal fermentation and microorganisms of rumen fluid was not observed.

References Bakunas J. (2004). Galviju stemples ir prieskrandzio ligos (in Lithuanian). Terra Publika, Kaunas, Lithuania, pp. 20–65.

Cizeikiene D., Juodeikiene G., Paskevicius A., Bartkiene E. (2013). Antimicrobial activity of lactic acid bacteria against pathogenic and spoilage microorganism isolated from food and their control in wheat bread. Food Control, 31: 539–545.

De Lima C.J.B., Coelho L.F., Blanco K.C.,Contiero J. (2009). Response surface optimization of D-(-)-lactic acid production by Lactobacillus SMI8 using corn steep liquor and yeast autolysate as an alternative nitrogen source. Afr. J. Biotechnol., 8: 5842–5846.

Digaitiene A., Hansen A.S., Juodeikiene G., Eidukonyte D.,Josephsen J. (2012). Lactic acid bacteria isolated from rye sourdouhs produce bacteriocin-like inhibitory substances active against Bacillus subtilis and fungy. J. Appl. Microbiol., 112: 732–742.

Erwin E.G., Marco G.J., Emery E.M. (1961). Volatile Fatty acid analysis of blood and rumen fluid by gas chromatography. J. Dairy Sci., 44: 1768.

Jeroch H., Seskeviciene J., Kulpys J. (2004). Zemes ukio gyvuliu ir pauksciu mitybos fiziologines reikmes (in Lithuanian). Naujasis lankas, Kaunas, Lithuania, 160 pp.

Jouany J.P., Morgavi D.P. (2007). Use of ‘natural’ products as alternatives to antibiotic feed additives in ruminant production. Animal, 10(1): 1443–466.



Kim J.Y., Park B.K., Park H.J., Park Y.H., Kim B.O., Pyo S. (2013). Atopic dermatitis-mitigating effects of new Lactobacillus strain, Lactobacillus sakei probio 65 isolated from Kimchi.J. Appl. Microbiol., 115(2): 517–26.

Mayo B., Aleksandrzak-Piekarczyk T., Fernández M., Kowalczyk M., Álvarez-Martín P., Bardowski J. (2010). Updates in the metabolism of lactic acid bacteria. In: Biotechnology of lactic acid bacteria: Novel applications, Mozzi F., Raya R.R., Vignolo G.M. (eds.). Wiley-Blackwell, Iowa, USA, pp. 3–33.

Pustovoj V.K. (1978). Gazohromatograficheskoje opredielienije zirnyh kislot v kormah i biologicheskih substratah selskohozeistvenyh zyvotnyh (in Russian). Mietodychestkije rekomendacii. Borovsk, Russia, pp. 3–8.

Schultz J. A. (1971). Lehrbuch der Rinderkrankheiten (in German). Leipzig, I, pp. 209–243.

Seo J.K., Kim S.-W., Kim M.H., Upadhaya S.D., Kam D.K., Ha J.K. (2010): Direct-fed Microbials for Ruminant Animals. Asian Austral. J. Anim., 23(12): 1657–1667.

Yasuda K., Hashikawa S., Sakamoto H., Tomita Y., Shibata S., Fukata T. (2007). A new synbiotic consisting of Lactobacillus casei subsp. casei and dextran improves milk production in Holstein dairy cows. J. Vet. Med. Sci., 69: 205–208.

Corresponding author Vita Krungleviciute Department of the Food Safety and Quality, Veterinary Academy Lithuanian University of Health Sciences; Tilzes str. 18, LT-47181, Kaunas E-mail: Vitakrungleviciute(at)gmail.com

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Münnich et al.: Die Auswirkungen von melassierten Trockenschnitzeln als Ersatz für Mais in konzentratreichen Milchkuhrationen auf die Fermentation in vitro (RUSITEC)


Die Auswirkungen von melassierten Trockenschnitzeln als Er-satz für Mais in konzentratreichen Milchkuhrationen auf die Fermentation in vitro (RUSITEC)

Matthias Münnich, Annabella Khol-Parisini, Fenja Klevenhusen, Elke Humer und Qendrim Zebeli Institut für Tierernährung und funktionelle Pflanzenstoffe, Veterinärmedizinische Universität Wien, AT

Abstract A rumen simulation technique (RUSITEC) was used to evaluate the effect of pelleted beet pulp (BP) on fermentation parameters, focusing on ruminal digestibility and short chain fatty acid (SCFA) pro-duction. Six diets, in which ground maize was partially or totally substituted by the same amount of beet pulp, were tested. The resulting diets contained 0% (0% BP), 8% (8% BP), 16% (16% BP), 24% (24% BP), 32% (32% BP) or 40% (40% BP) of beet pulp, respectively. For all diets there was a 48:52 forage to concentrate ratio. Three runs with 12 vessels each were conducted, allowing six inde-pendent measurements for each diet. Neutral detergent fiber (NDF) digestibility was increased by the inclusion of beet pulp (p<0.001). Total SCFA and acetate production were not affected significantly by the treatment. The amount of propionate in the fluid increased (p<0.001), while n-Butyrate was de-creased (p<0.001) by the inclusion of beet pulp into the diet. The experiment showed that with re-gard to fermentation parameters beet pulp can replace high amounts of maize in the diet for dairy cows and thereby possibly improve the rumen environment and the sustainability of the diet.

Einleitung In der Fütterung hochleistender Milchkühe werden große Mengen Getreide eingesetzt, um dem Ener-giebedarf der Tiere gerecht zu werden und eine hohe Milchleistung zu ermöglichen. Der damit ver-bundene hohe Stärkegehalt der Ration und ein Mangel an Struktur im Futter führen bei Kühen jedoch häufig zu einer subakuten Pansenazidose (SARA) (Zebeli et al., 2008), die schwerwiegende Erkran-kungen wie Leberabszesse und Lahmheiten, aber auch eine Verringerung der Futteraufnahme und der Milchleistung mit sich bringt. Zudem ist diese Fütterungsstrategie nicht nachhaltig, da die Verfütterung von Getreide an Milchkühe in Konkurrenz zur menschlichen Ernährung steht (Cassidy et al., 2013). Trockenschnitzel könnten eine Alternative zur Verfütterung von Getreide darstellen. Da sie keine Stär-ke enthalten und reich an Neutraler Deternz Faser (NDF) und Pektin sind, haben sie, anders als Ge-treide, keinen negativen Effekt auf die Pansengesundheit. Zudem sind Trockenschnitzel als Nebenprodukt der Zuckerindustrie für den Menschen als Nahrung nicht verwertbar. Ihre Verfütterung zur Milchproduktion würde somit netto zu einem Nahrungsmittelgewinn führen, im Gegensatz zum Nahrungsmittelverlust, wie er bei der Verfütterung von Getreide auftritt. Mit unserer Studie soll gezeigt werden wie sich unterschiedliche Mengen Trockenschnitzel als Ersatz für Mais auf das Pansenmilieu auswirken.



Material und Methoden

Sechs verschiedene Rationen wurden mittels der Pansen-Simulations-Technik (RUSITEC) getestet. Alle Rationen wiesen ein Grundfutter zu Kraftfutter Verhältnis von 48:52 auf. Das Grundfutter bestand dabei je zur Hälfte aus Maissilage und Grassilage. Der Kraftfutteranteil setze sich, für die Ration 0% BP, aus 40% Mais, 10% Rapsextraktionsschrot, 0,9% Futterkalk, 0,8% Harnstoff und 0,3% eines kommerziell erhältlichen Mineral- und Vitamin-Futters (RINDAVIT TMR 11 ASS-Co+ ATG, H. Wilhelm Schaumann GmbH & Co KG, Brunn am Gebirge; Austria) zusammen. Die fünf weiteren Rationen ent-hielten jeweils 8%, 16%, 24%, 32% und 40% Trockenschnitzel (8%BP, 16%BP, 24%BP, 32%BP, 40%BP), welche die jeweils gleiche Menge (TS) Mais ersetzten (Abb. 1).

Abbildung 1: Trockenschnitzel- und Maisanteile in den Rationen

Das Experiment bestand aus drei Durchläufen mit jeweils 12 Fermentern. Jede Ration konnte somit sechsmal unabhängig voneinander getestet werden. Der Versuch wurde entsprechend der Studie von Klevenhusen et al. (2015) durchgeführt. Das Futter befand sich in Nylonbeuteln mit einer Porenweite von 150 µm. Jeder Futterbeutel wurde nach 48 stündiger Inkubation ersetzt. Nach einer fünftägigen Adaptationsphase folgte eine, ebenfalls fünftägige, Probennahmephase. Während der Probennahmephase wurden täglich Proben der Fermenterflüssigkeit sowie die unverdau-ten Futterreste bei -20°C eingefroren. Die Proben der Fermenterflüssigkeit wurden mittels Gaschroma-tographie auf ihren Gehalt an flüchtigen Fettsäuren (FFS) untersucht. Unverdaute Futterreste wurden auf ihren Gehalt an organischer Masse (OM), Rohprotein (XP), NDF und Nicht Faser Kohlenhydraten (NFC) untersucht und anschließend die Verdaulichkeit von OM, XP, NDF und NFC berechnet.

Ergebnisse Der Gehalt an NFC fiel durch den Einsatz von Trockenschnitzeln deutlich von 50,3% (0% BP) auf 38,3% (40% BP) ab (Abb. 2). Im Gegensatz dazu stieg der Gehalt an NDF in der Ration von 26,1% (0% BP) auf 35,3% (40% BP). Der Gehalt an XP wurde kaum beeinflusst. Die Verdaulichkeit von OM und XP lag bei durchschnittlich 78,9% und 88,1% und wurde durch die verschiedenen Rationen nicht beeinflusst. Die NDF-Verdaulichkeit stieg mit dem Einsatz von Trocken-schnitzeln von 46,6% auf 57,8% an (p<0,001). Auch die Verdaulichkeit der NFC-Fraktion konnte durch den Einsatz von Trockenschnitzeln leicht verbessert werden (p=0,01).



Abbildung 2: Rationsanalyse Abbildung 3: Verdaulichkeit

Die Gesamtmenge an FFS in der Fermenterflüssigkeit wurde durch den Einsatz von Trockenschnitzeln nicht signifikant beeinflusst und lag durchschnittlich bei 136,0 mmol/l. Die Menge an Azetat zeigte nur eine Tendenz zu einer Steigerung (p<0,1). Sie stieg von 64,7 mmol/l (0% BP) auf 72,3 mmol/l (40% BP). Die Konzentration von Propionat stieg durch den Einsatz von Trockenschnitzeln von 22,3 mmol/l (0% BP) auf 29,7 mmol/l (40% BP) an (p<0,001). Die Konzentration von n-Butyrat verhielt sich ge-gensätzlich und fiel von 28,2 mmol/l (0% BP) auf 21,8 mmol/l (40% BP) ab (p<0,001). Entsprechend den Veränderungen in der Konzentration von Azetat und Propionat sank das Azetat-Propionat-Verhältnis von 3,13 (0% BP) auf 2,52 (40% BP) (p<0,001).

Diskussion Die OM- und XP-Verdaulichkeit in diesem Experiment waren deutlich höher als in vergleichbaren Stu-dien (Boguhn et al., 2010, Mojtahedi and Mesgaran, 2011, Zhao et al., 2013). Ein Grund dafür kann in der relativ großen Porenweite (150 µm) der Futterbeutel im gegenständigen Versuch sein. Diese er-möglichte einerseits eine bessere Erreichbarkeit des Futters für die Mikroben, insbesondere die Proto-zoen, was eine bessere Verdaulichkeit ermöglichte. Andererseits wurde aber auch mehr feinen Futterpartikeln ermöglicht unverdaut den Futterbeutel zu verlassen. Trotz des gestiegenen NDF-Anteils und der Verringerung des NFC-Gehalts in der Ration wurde die OM-Verdaulichkeit nicht durch den Einsatz von Trockenschnitzeln beeinflusst. Dies zeigt, in Zusammenhang mit der gesteigerten NDF-Verdaulichkeit, dass die Faser aus Trockenschnitzeln, durch ihre hohe Verdaulichkeit im Vergleich zu Mais, einen geringeren NFC-Anteil ausgleichen kann.

Unbenannt-1 1 21.03.16 11:28



Die nur geringen Unterschiede in der Verdaulichkeit spiegeln sich in der Menge an gebildeten FFS wieder. In Übereinstimmung mit Studien von Zhao et al. (2013) und Shahmoradi et al. (2015) wurde die Gesamtmenge an gebildeten FFS nicht signifikant beeinflusst. Das Verhältnis der einzelnen FFS zu einander wurde hingegen deutlich verändert. So ist die Tendenz der Azetatkonzentration zu einer Erhöhung mit der vermehrten Verdauung von NDF zu erklären und ist im Einklang mit anderen Stu-dien (Mojtahedi and Mesgaran, 2011, Zhao et al., 2013). Während in den genannten Studien die Kon-zentration an Propionat jedoch sinkt und die Konzentration an n-Butyrat stets steigt, zeigten diese beiden FFS in unserem Versuch ein gegenteiliges Verhalten. Dies ist besonders erstaunlich, da der Gehalt an Stärke, als Hauptsubstrat für die Propionatbildung durch den Ersatz von Mais durch Tro-ckenschnitzel von 40,0% auf 11,1% sank.

Schlussfolgerungen Die Ergebnisse unseres Versuchs zeigen, dass Trockenschnitzel die Fermentation im Pansen in vitro nicht negativ beeinflussen und somit eine vielversprechende Alternative zu Mais als Futtermittel für Wiederkäuer ist. In vivo Studien sind nötig, um die Fragestellung eines Ersatzes von Mais durch Tro-ckenschnitzel abschließend zu klären.

Danksagung Dieses Projekt wurde durchgeführt im Rahmen des Förderungsprogramms Sparkling Science, gefördert vom Bundesministerium für Wissenschaft, Forschung und Wirtschaft.

Literatur Boguhn, J., H. Kluth, M. Bulang, T. Engelhard, and M. Rodehutscord. 2010. Effects of pressed beet pulp silage inclusion in maize-based rations on performance of high-yielding dairy cows and parameters of rumen fermentation. animal 4(01):30-39.

Cassidy, E. S., P. C. West, J. S. Gerber, and J. A. Foley. 2013. Redefining agricultural yields: from tonnes to people nourished per hectare. Environmental Research Letters 8(3):034015.

Klevenhusen, F., K. Deckardt, Ö. Sizmaz, S. Wimmer, A. Muro-Reyes, R. Khiaosa-Ard, R. Chizzola, and Q. Zebeli. 2015. Effects of black seed oil and Ferula elaeochytris supplementation on ruminal fermentation as tested in vitro with the rumen simulation technique (Rusitec). Animal Production Science 55(6):736-744.

Mojtahedi, M. and M. D. Mesgaran. 2011. Effects of the inclusion of dried molassed sugar beet pulp in a low-forage diet on the digestive process and blood biochemical parameters of Holstein steers. Livestock Science 141(2):95-103.

Shahmoradi, A., M. Alikhani, A. Riasi, G. Ghorbani, and M. Ghaffari. 2015. Effects of partial replacement of barley grain with beet pulp on performance, ruminal fermentation and plasma concentration of metabolites in transition dairy cows. Journal of animal physiology and animal nutrition.

Zebeli, Q., J. Dijkstra, M. Tafaj, H. Steingass, B. Ametaj, and W. Drochner. 2008. Modeling the adequacy of dietary fiber in dairy cows based on the responses of ruminal pH and milk fat production to composition of the diet. Journal of dairy science 91(5):2046-2066.

Zhao, X., C. Liu, Y. Liu, C. Li, and J. Yao. 2013. Effects of replacing dietary starch with neutral detergent–soluble fibre on ruminal fermentation, microbial synthesis and populations of ruminal cellulolytic bacteria using the rumen simulation technique (RUSITEC). Journal of animal physiology and animal nutrition 97(6):1161-1169.

Autorenanschrift Matthias Münnich

Institut für Tierernährung und funktionelle Pflanzenstoffe Veterinärmedizinische Universität Wien E-Mail: Matthias.Muennich(at)vetmeduni.ac.at

Kudlinskiene et al.: Effects of extruded lupins (Lupinus spp.), faba beans (Vicia faba) and peas (Pisum sativum) on dairy cow’s milk sensory properties


Effects of extruded lupins (Lupinus spp.), faba beans (Vicia fa-ba) and peas (Pisum sativum) on dairy cow’s milk sensory prop-erties

Ieva Kudlinskiene1, Qendrim Zebeli2, Romas Gruzauskas1, Rolandas Stankevicius1, Aldona Miezeliene3, Gitana Alencikiene3 and Meelis Ots4 1 Lithuanian University of Health Sciences, Veterinary Academy, Kaunas, LT 2 University of Veterinary Medicine in Vienna, AT 3 Kaunas University of Technology, Food Institute, Kaunas, LT 4 Estonian University of Life Sciences, Tartu, EST

Abstract The aim of our research was to determine the influence of extruded lupins (Lupinus spp.), faba beans (Vicia faba) and peas (Pisum sativum) on dairy cow’s milk sensory properties. For the trial 40 Lithua-nian Black-and-White cows with analogous characteristics were selected and randomly allocated into 4 groups (control and experimental‘s), 10 animals each. The control group was fed a conventional diet consisting mainly of grass silage, ground barley grain, enriched extruded full fat soybean, and mineral premix. The experimental groups were fed a similar diet, but instead of 1.5 kg of soybean meal, the cows were given the same amount of the extruded lupins, faba beans and peas. Milk sensory analysis was performed in the sensory analysis laboratory of Kaunas University of Technology Food institute established according to ISO 8589 requirements. The results of this study shown that soybeans re-placement with lupins (Lupinus spp.), faba beans (Vicia faba) and peas (Pisum sativum) in dairy cows rations, had no negative influense on milk sensory parameters. No difference among groups were identified regarding odour‘s intensity with all showing an apparent milk‘s specific odour. After 2nd and 3rd months of feeding extruded lupins and soybeans as well as extruded peas and soybeans, milk became light and obtained a non-specific bitter and dry alike milk savour.

Introduction Since 2001 the European Commission banned the use of meat and bone meal and its byproducts in diets for livestock animals (EC directive 999/2001) in order to assure consumer safety on animal products. Consequently, soybean meal became the most utilised protein source in the intensive livestock systems. Soybean meal solvent extract (s.e.) is a by-product of oil industry, where soybean seeds are treated with organic solvents (e.g. hexane) and subsequently with high temperature. For this reason soybean meal has been banned in the organic livestock (EC directive 2092/1991; EC directive 834/2007). Thus, the search for alternative protein sources has led to an increasing interest in the use of grain legumes, as they supply the important source of plant protein. The most commonly in dairy cows nutrition used legume grains are peas (Pisum sativum), faba beans (Vicia faba), and lupins (Lupinus spp.), it is characterised by high energy density allowed to the high protein, starch and/or fat concentrations, as more than sufficient is their calcium concentration. Within the grain legumes, lupins have higher amounts of crude protein (324–381 g/kg dry matter), compared to faba beans (301g/kg dry matter) and peas (246 g/kg dry matter) (Degussa, 2006). Jezierny et al. (2007) reported similar contents of crude protein in different batches of lupins, faba beans and peas averaging 387, 308 and 249 g/kg dry matter, respectively. Many studies had been done in order to



investigate legume grains effects on dairy cows performance and milk composition, nevertheless, there haven‘t been done many researches in pursuance to investigate its effects on milk sensory profiles. So the aim of our research was to determine the influence of extruded lupins’ fodder beans and peas on dairy cow’s milk sensory properties.

Materials and methods Dairy cows feeding trial. For the trial, 40 Lithuanian Black-and-White cows with analogous characteristics were selected. The animals selected were divided in 4 groups (control and experimental‘s), 10 animals each. The trial lasted for 90 days. Raw material‘s for the trial was extruded by SC „Kauno Grūdai“. The control group was fed a conventional diet consisting mainly of grass silage, barley grain, enriched extruded full fat soybean flour, and mineral premix. The experi-mental groups were fed a similar diet, but instead of 1.5 kg of soybean meal, the cows were given the same amount of the extruded lupins, faba beans and peas. Feeding scheme is provided in Table 1.

Table 1: Feeding scheme in the experimental period

Group Number of cows, n Feeding characteristics

Control 10 Farm diet + extruded full fat soybean (1,5 kg) Experimental 1 10 Farm diet + extruded lupins (1,5 kg), extruded full fat soybean (300 g) Experimental 2 10 Farm diet + extruded faba beans (1,5 kg), extruded full fat soybean (400 g) Experimental 3 10 Farm diet + extruded peas (1,5 kg), extruded full fat soybean (700 g)

Energy and nutritional values of the diets were calculated with the feeding software HYBRIMIN® Fut-ter 2008. Milk samples for the sensory analysis were taken 3 times: after 1st month of the trial, after 2nd month of the trial and at the end of the trial.

Sensory analysis. A quantitative descriptive analysis (QDA) was carried out for the assessment of the sensory properties, and sensory profiles were created for each prepared beverage. Sensory asses-sors were staff of Kaunas University of Technology Food institute. A total group of 5 trained assessors (female, ages 20–60 years old) having work experience in evaluation of various food products not less than 20 hours and trained according ISO 8586 were selected. All training and data collection sessions were held in the sensory analysis laboratory of Kaunas University of Technology Food institute estab-lished according to ISO 8589 requirements. For the development of the sensory profiles, a full balanced randomized sample presentation plan with two repetitions was applied. Panel responses were collected using a computerized program (Fizz, Bio-systems, France). A 15 cm line scales with 1 cm indented anchors (left – “low intensity/absent,” right – “high intensity”) were used to evaluate each sensory attribute. Scales were presented for each sample on a single screen for evaluation of the attributes of odour, texture and taste. Assessors were asked to rinse their mouth and palate with water and white bread before testing each presented sam-ple. Before evaluation raw milk samples were pasteurised at the temperature 74 ±2ºC, then cooled down to the temperature 16±2 ºC. Then samples (approximately 20 mL) were presented to the panel in 30 mL plastic cups, coded with three digital numbers. Statistical analysis. Data were analysed by ANOVA, if significant interactions were determined, multiple comparisons were made. The differences were classified by a Duncan multiple comparison test (P<0.05). SPSS software, version 15.0 (Chicago, IL, USA, 2006) was used for the statistical anal-ysis of the data.Statistical analysis of medical data was performed using SPSS (Statistics Base 19.0).



Dependent t-test for paired samples was carried out to examine the differences between the baseline and 21-day follow-up results. The significance level of P≤0.05 was used for all the analyses.

Results After first month of the research results of milk sensory parameters (Table 2) had shown that milk samples had no difference from each other by odour‘s intensity, every sample had an apparent milk‘s specific odour. All the samples were pasteurised by the same conditions, yet 1st experimental group‘s cow‘s (it‘s ration was supplemented with extruded lupins and extruded soybeans) milk samples had a more intense typical pasteurised milk‘s odour. Applied feeds had no negative influence on milk‘s odour, there were no lateral, atypical odour. 3rd experimental group‘s cows (it‘s ration was supplemented with extruded peas and soybeans) milk‘s samples had a more intense sense of mouthcoating. All samples had equal richness of the taste.

Table 2: Milk sensory parameters after 1st month of the trial

Parameter

Group Control

(Farm diet + extrud-ed soybean)

Experimental 1 (Farm diet + ex-

truded lupins, extruded soybean)


truded faba beans, extruded soybean)

Experimental 3 (Farm diet + ex-truded peas, ex-truded soybean)

Overall odour 11,50 a 11,00 a 11,10 a 10,80 a

Pasteurisation odour 8,25 b 8,00 b 5,63 a 6,36 a

Natural milk odour 7,20 a 7,90 a 8,40 a 8,50 a

Atypical odour 1,00 a 1,40 a 1,00 a 1,00 a

Mouthcoating 8,80 b 8,40 b 9,20 b 6,25 a

Overall taste 12,10 a 10,90 a 12,00 a 10,90 a

Richness of the taste 11,88 a 10,20 a 10,80 a 10,10 a

Acid taste 1,70 a 1,80 a 1,70 a 1,70 a

Sweet taste 8,30 a 8,20 a 8,40 a 7,70 a

Pasteurised milk taste 6,90 a 7,67 a 7,88 a 6,88 a

Natural milk taste 9,60 a 8,80 a 9,40 a 9,80 a

Nonfresh milk taste 1,10 a 1,10 a 1,30 a 1,20 a

Atypical taste 1,10 a 1,50 a 1,20 a 1,50 a

Aftertaste 7,30 a 7,20 a 7,50 a 7,00 a

a, b -Means in the same row with no common superscript differ significantly (P < 0.05)

After the second month of feeding sensory milk evaluation‘s results (Table 3) had shown that milk samples had no difference from each other by odour‘s intensity, every sample had an apparent milk‘s specific odour. All the samples were pasteurised by the same conditions, therefore all samples distinguished by specific pasteurised milk‘s odour. Applied feeds had no negative influence on milk‘s odour, there were no lateral, atypical odour. All the samples had intense overall taste, it distinguished by having sweet and natural milk‘s taste. Sensory parameter‘s intensity of all samples was identical. 1st and 3rd experimental group‘s cows milk samples had a light, non-specific bitter and dry alike milk savour.



Table 3: Milk sensory parameters after 2nd month of the trial

Parameter

Group

Control (Farm diet + ex-truded soybeans)


truded lupins, extruded soy-

beans)


truded faba beans, extruded soy-

beans)

Experimental 3 (Farm diet + ex-truded peas, ex-truded soybeans)

Overall odour 10.3 a 8.9 a 9.1 a 8.7 a Pasteurisation odour 6.5 a 5.7 a 5.2 a 5.9 a Natural milk odour 7.8 a 8.4 a 8.2 a 8.8 a Atypical odour 1.0 a 1.1 a 1.2 a 1.7 a Mouthcoating 7.5 a 7.2 a 6.2 a 6.4 a Overall taste 11.6 a 11.0 a 10.4 a 10.1 a Richness of the taste 10.9 a 10.9 a 10.7 a 10.8 a Acid taste 1.3 a 1.3 a 1.5 a 1.6 a Sweet taste 8.1 a 7.7 a 7.9 a 7.8 a Pasteurised milk taste 7.7 a 7.3 a 6.8 a 6.9 a Natural milk taste 8.2 a 6.9 a 8.0 a 7.3 a Nonfresh milk taste 1.2 a 1.4 a 1.5 a 1.5 a Atypical taste 1.3 a 2.8 b 1.3 a 3.1 b Aftertaste 7.6 a 8.1 a 7.1 a 7.4 a

Table 4: Milk sensory parameters after 3rd month of the trial

Parameter

Group

Control (Farm diet + extruded soy-

beans)

Experimental 1 (Farm diet + extrud-ed lupins, extruded

soybeans)


truded faba beans, extruded soy-

beans)

Experimental 3 (Farm diet + ex-truded peas, ex-truded soybeans)

Overall odour 12,5 a 12.1 a 12.4 a 12.5 a Pasteurisation odour 9.9 a 9.0 a 9.0 a 9.4 a Natural milk odour 10.3 a 10.2 a 10.6 a 10.7 a Atypical odour 1.0 a 1.0 a 1.0 a 1.5 a Mouthcoating 7.5 a 7.7 a 7.7 a 7.4 a Overall taste 12.2 a 12.3 a 12.4 a 12.4 a Richness of the taste 11.7 a 10.2 a 11.0 a 10.7 a Acid taste 1.1 a 1.2 a 1.2 a 1.2 a Sweet taste 8.2 a 7.4 a 8.4 a 7.8 a Pasteurised milk taste 8.0 a 7.6 a 8.0 a 7.8 a Natural milk taste 10.6 a 10.7 a 11.0 a 10.6 a Nonfresh milk taste 1.2 a 1.2 a 1.1 a 1.2 a Atypical taste 1.0 a 1.9 b 1.0 a 1.9 b Aftertaste 8.1 a 7.7 a 8.3 a 8.1 a a, b -Means in the same row with no common superscript differ significantly (P < 0.05)

In the end of the research sensory milk evaluation‘s results (Table 4) had shown that milk samples had no difference from each other by odour‘s intensity, every sample had an apparent milk‘s specific odour. All samples distinguished by specific pasteurised milk‘s odour. Applied feeds had no negative influence on milk‘s odour, there were no lateral, atypical odour. Sensory parameter‘s intensity of all samples was identical. 1st and 3rd experimental group‘s cows milk samples i.q. after the second month of feeding had a light, non-specific bitter and dry alike milk savour.

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Conclusion The results of this study shown that soybeans replacement with lupins (Lupinus spp.), faba beans (Vicia faba) and peas (Pisum sativum) in dairy cows rations, had no negative influence on milk sensory parameters. All groups (control and experimentals) milk samples had no difference from each other by odour‘s intensity, every sample had an apparent milk‘s specific odour. After 2nd and 3rd months of feeding 1st (fed extruded lupins and soybeans) and 3rd (fed extruded peas and soybeans) experimental group‘s cows milk samples had a light, non-specific bitter and dry alike milk savour.

References Council Regulation (EEC) No 2092/91 of 24 June 1991 on organic production of agricultural products and indications referring thereto on agricultural products and foodstuffs L 198 , 22/07/1991 P. 0001 – 0015

Degussa, (2006). The amino acid composition of feedstuffs. 5th rev. ed., Degussa AG, Feed Additives, Hanau, Germany.

Council Regulation (EC) No 834/2007 on organic production and labelling of organic products and repealing Regulation (EEC) No 2092/91 L 189/1- L 189/23.

European Commission directive, 999/2001. Regulation (EC) No. 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies. Official Journal of the European Communities L 147/1–L 147/40.

Jezierny, D., Mosenthin, R., Eklund, M. & Rademacher, M., (2007). Determination of standardized ileal digestibilities of crude protein and amino acids in legume seeds for growing pigs. Proceedings of the 16th International Science Symposium on Nutrition of Domestic Animals, Radenci, pp. 198–203.

Corresponding author Ieva Kudlinskiene Lithuanian University of Health Sciences, Veterinary Academy Kaunas, Lithuania E-mail: ieva.kudlinskiene(at)lsmuni.lt

Buffler et al.: Reproduktionsleistung trächtiger Sauen bei unterschiedlicher Eisenversorgung


Reproduktionsleistung trächtiger Sauen bei unterschiedlicher Eisenversorgung

Marzell Buffler, Christiane Becker und Wilhelm Windisch Lehrstuhl für Tierernährung, Hans-Eisenmann-Zentrum Wissenschaftszentrum Wei-henstephan, TU München, DE

Abstract According to NRC (1), recommendations for iron supply in pregnant and lactating sows is 80-100 mg/kg diet. This value exists for more than 35 years and was evaluated for the last time in 1985 (3). However, within the last decades reproductive performance of sows has increased significantly by breeding progress (2) making it questionable, if recommendations are still valid. In this study the in-fluence of differential iron supply to sows during gestation on reproductive performance should be assessed. 20 multiparous sows were divided into two groups supplied with common diets from insem-ination to farrowing. Iron content in the control group was according to actual recommendations (115 mg/kg DM) and high iron group was additionally supplemented with 146 mg FeSO4/kg diet to a total iron content of 261 mg/kg DM. After farrowing, litter size, number of piglets born alive and piglet weights were recorded. Litter size was significantly different between the two feeding groups. In av-erage, sows from the low iron group received 3 piglets less than sows fed with high iron diet. Con-comitantly, number of piglets born alive was decreased by 29% in low iron group compared to high iron group. The massive decline in reproductive performance confirms the assumption of an increased iron demand during pregnancy due to improved performances following breeding processes. Subse-quently, iron requirements of pregnant and lactating sows have to be reevaluated and, in conse-quence, recommendations have to be adjusted for modern pig lines.

Einleitung Eine bedarfsgerechte Zufuhr des essentiellen Spurenelements Eisen im Bereich der landwirt-schaftlichen Schweineproduktion kann nur bei adulten Tieren mit niedriger Leistung durch die nativen Gehalte in praxisüblichen Futtermischungen gedeckt werden. Sobald jedoch besondere physiologische Anforderungen an die Tiere gestellt werden, erhöht sich deren Bedarf dramatisch. Dies betrifft vor allem Zuchtsauen während Trächtigkeit und Laktation (3). Während in zahlreichen Studien Möglichkei-ten zur Prophylaxe eines Eisenmangels bei neugeborenen Ferkeln untersucht sind (4), wurde die Ver-sorgung der Muttersau bisher immer vernachlässigt. Die wenigen Untersuchungen und die daraus abgeleiteten Versorgungsempfehlungen stammen allesamt aus den Jahren vor 1985 (5), sodass der enorme Zuchtfortschritt in der Schweineproduktion während der letzten 30 Jahren darin nicht berück-sichtigt wird (2). Hinzu kommt, dass innerhalb dieser Studien einer adäquat versorgten Kontrollgruppe zumeist extreme eisendefiziente bzw. eisenüberladene Behandlungen gegenübergestellt wurden, die sich außerhalb des physiologischen Rahmens befinden, wodurch pathologische Effekte den tatsächli-chen Einfluss der Eisenversorgung auf die Reproduktionsleistung überlagern könnten. In dieser Studie sollten die derzeitig gültige Bedarfsempfehlung von 80-100mg Fe/kg (1) Futtertro-ckenmasse an einer modernen Sauenlinie mit aktuell praxisnahen Reproduktionsleistungen überprüft werden. Dazu wurden zwei identische Rationen eingesetzt, die sich in ihren Eisengehalten unterschie-den (Bedarfsempfehlung vs. praxisüblich eingesetzt).



Material und Methoden Im Versuch wurden 20 Sauen (DL x Pie) in zwei Gruppen gleicher mittlerer Trächtigkeitsnummer und bisheriger durchschnittlicher Wurfgrößen eingeteilt und ab der Besamung restriktiv mit isoenergeti-schen und isonitrogenen Rationen unterschiedlichen Eisengehaltes auf Basis von Mais und Sojaextrak-tionsschrot bis zur Abferkelung gefüttert (Tag 1-84: 2,5 kg; Tag 85-115: 3,2 kg). Die Basisration der Kontrollgruppe entsprach in ihrer Nährstoffzusammensetzung den Empfehlungen der GFE (2006) und enthielt einen nativen Eisengehalt von 115 mg/kg. Für die Hoch-Eisen-Gruppe wurde eine identische Ration mit Fe(II)Sulfat auf einen Gesamteisengehalt von 261 mg/kg aufgewertet. Die Eisenbestim-mung in den Rationen erfolgte mittels Atomabsorptionsspektrometrie (AAS) Unmittelbar nach der Geburt wurden Ferkelzahl und Anzahl lebend geborener Ferkel bestimmt und alle Tiere gewogen. Von den Sauen wurden am Tag des Abferkelns Kotproben gewonnen und die Eisengehalte in der Trockenmasse bestimmt (AAS). Die Berechnung der scheinbaren Eisenverdaulich-keit erfolgte auf Basis von Lignin als nativer interner Marker. Dieser wurde im Rahmen eines Stan-dardverfahrens nach VDLUFA bestimmt (Van Soest, 1967).

Ergebnisse Beide Sauengruppen zeigten über die gesamte Trächtigkeit eine vollständige Futteraufnahme, wodurch die tägliche Menge aufgenommenen Eisens in der Hoch-Eisen-Gruppe um das Doppelte hö-her lag als bei der Kontrolle (Tabelle 1). Ein signifikanter Unterschied war bei den Eisengehalten (565 mg Fe/kg TM vs. 1195 mg Fe/kg TM) im Kot (p=0,004), sowie der täglich ausgeschiedenen Menge Eisen (295 mg vs. 710 mg) erkennen. Die scheinbaren Eisenverdaulichkeiten von 10,6% in der Kon-trollgruppe und 5,3% in der Hocheisengruppe unterschieden sich nicht signifikant.

Tabelle 1: Eisenbilanz der trächtigen Sauen Kontrolle Hoch Fe SEM1 p-Wert2

Futter Eisengehalt (mg/kg) 115 261 Tägliche Eisenaufnahme (mg) Tag 1-84 257 587 Tag 85-115 329 751 Kot Eisengehalt (mg/kg) 565a 1195b 161 0,004 Tägliche Eisenexkretion Kot (mg) 295a 710b 83 0,0004 Scheinbare Eisenretention (mg/Tag)3 34 41 75 0,931 Scheinbare Eisenverdaulichkeit (%) 10,6 5,31 15,1 0,733 1SEM= Standard errors of Means, 2p-Werte ≤ 0,05 deuten auf signifikante Effekte hin, 3endogene und renale Eisenverluste vernachlässigbar gering

Tabelle 2: Wurfgrößen und Geburtsgewichte bei unterschiedlicher Eisenversorgung der Muttersau

Kontrolle Hoch Fe SEM p-Wert Wurfgröße 9,3a 12,1b 0,8 0,017 Lebend geborene Ferkel 8,6a 11,1b 0,83 0,041 Ferkelgewicht 1,51 1,57 0,08 0,41 Wurfgewicht 15,5a 19,8b 1,6 0,015 1SEM= Standard errors of Means, 2p-Werte ≤ 0,05 deuten auf signifikante Effekte hin

Ein signifikanter Unterschied zwischen den beiden Gruppen zeigte sich bei den Wurfgrößen. Dabei brachten die Tiere der Kontrollgruppe im Schnitt 2,8 Ferkel weniger zur Welt als die Hoch-Eisen-Gruppe (Tabelle 2).



Ebenso unterschied sich die Anzahl lebend geborener Ferkel in einer ähnlichen Größenordnung, wobei in der Kontrollgruppe 29% weniger lebende Ferkel geboren wurden. Während die Wurfgewichte bei der Hoch-Eisen-Gruppe deutlich höher waren als bei der Kontrollgruppe, konnten beim mittleren Fer-kelgewicht keine Unterschiede gezeigt werden.

Diskussion In der aktuellen Studie lagen beide Rationen über den von der NRC (1) ausgesprochenen Empfehlun-gen für Eisen (80-100 mg/kg). Der von Roth-Maier et al. (5) ermittelte Bedarf von 140 mg wurde bei einer vorgelegten Futtermenge von 2,5 kg bei niedertragenden und 3,2 kg bei hochtragenden Sauen ausreichend gedeckt. Ausgehend von einer Verwertung von 20% und dem angeführten Bruttobedarf (5), ergibt sich ein errechneter Nettobedarf von 28 mg/Tag. Allerdings konnten auch schon deutlich höhere scheinbare Eisenretentionen bei Jungsauen gezeigt werden (6). Auch in der aktuellen Studie wird der angenommene Bedarf um 21% bzw. 48% überschritten. Da die Homöostase des potentiell hochtoxischen Eisens ausschließlich über die Absorption reguliert wird, ist eine erhöhte Aufnahme wie sie hier in der scheinbaren Eisenretention dargestellt werden kann, ein deutliches Zeichen für den gesteigerten Bedarf in modernen Sauenlinien. Aufgrund der stark reduzierten Absorption von Spuren-elementen während der letzten 10 Trächtigkeitstage (7), ist zu früheren Zeitpunkten der Gravidität eine noch höhere Eisenaufnahme, als hier am Tag der Abferkelung ermittelt, anzunehmen. Diese Ergebnisse werden durch die signifikanten Unterschiede in den Reproduktionsleistungen beider Gruppen manifestiert. Ein Einbruch der Wurfgrößen um 30% bei der den Empfehlungen entsprechen-den Versorgung mit Eisen gegenüber den supplementierten Sauen ist von entscheidender Bedeutung. Diese Ergebnisse decken sich mit Daten von Venn et al. (8) wo größere Wurfzahlen durch eine Eisen-supplementierung von trächtigen Sauen erzielt werden konnten. Ähnliche Effekte zeigt der Einsatz von organischen gegenüber anorganischen Eisenverbindungen bei identischem Eisengehalt (9), was auf eine verbesserte Bioverfügbarkeit zurückzuführen ist. Eine Supplementierung mit Eisen zu einem spä-teren Zeitpunkt der Trächtigkeit, unabhängig von der Applikationsform (oral vs. parenteral) liefert keine eindeutigen Ergebnisse. Während in mehreren Studien keine Unterschiede in der Ferkelzahl erzielt wurden (10), konnten Petrichev et al. (11) positive Effekte auf die Wurfgrößen durch die Verab-reichung von Eisen-Methionat zeigen. Der Anteil totgeborener Ferkel in dieser Untersuchung ist in beiden Gruppen identisch (8%). Sowohl die fetale Sterblichkeit als auch die Ferkelsterblichkeit post partum wird, wie bei McGowan et al. (12) gezeigt, durch keine der beiden Eisenversorgungsstufen verändert. Keine Unterschiede ergeben sich bei den mittleren Ferkelgewichten beider Versorgungsgruppen. Dies deckt sich mit Daten von Kirchgessner et al. (13) wo zusätzliche Eisengaben bei der Muttersau keinen Einfluss auf das Gewicht der Nachkommen zeigen konnten. Andere Studien weisen dagegen auf höhe-re Geburtsgewichte bei neugeborenen Ferkeln nach Eisensupplementierung der Muttersau hin (11).

Schlussfolgerung Zahlreiche Studien haben den Einfluss der Eisenversorgung bei trächtigen Sauen auf ihre Nachkom-men untersucht. Insgesamt wurden dabei nur geringe Effekte auf die Eisenmitgift der Ferkel oder auf den Eisengehalt der Sauenmilch gezeigt werden. Daher erschien eine Anpassung der Bedarfsempfeh-lung, trotz stetig steigender Reproduktionsleistungen nicht nötig. In dieser Untersuchung konnte je-doch gezeigt werden, dass der Eisenbedarf möglicherweise die derzeit gültigen Empfehlungen übersteigt und eine erhöhte Eisenversorgung zu verbesserten Reproduktionsleistungen führen kann. Um dies zu evaluieren ist jedoch noch eine Vielzahl weitreichender Studien zur Bedarfsableitung nötig, um die Empfehlungen den aktuellen Bedürfnissen der Tiere anpassen zu können.



Danksagung Mit freundlicher Unterstützung der Bayerischen Arbeitsgemeinschaft Tierernährung e.V. und der Hildegard Grunow Stiftung für Ernährungsforschung.

Literatur 1.Research CN. Nutrient Requirements of Swine: Eleventh Revised Edition. Washington, DC: The National Academies Press; 2012.

2.Kim S, Wu G, Baker D. Amino acid nutrition of breeding sows during gestation and lactation. Pig News Info CABI. 2005;26:89N-99N.

3.Ball RO, Samuel R, Moehn S. Nutrient requirements of prolific sows. Advances in Pork Production. 2008;19:223-36.

4.Lipinski P, Starzynski RR, Canonne-Hergaux F, Tudek B, Olinski R, Kowalczyk P, et al. Benefits and risks of iron supplementation in anemic neonatal pigs. The American journal of pathology. 2010;177(3):1233-43. doi: 10.2353/ajpath.2010.091020.

5.Roth-Maier DA, Kirchgessner M, Spoerl R. Fe balances of pregnant and lactating breeding sows by different alimentary iron administration. Zentralblatt fur Veterinarmedizin Reihe A. 1985;32(10):739-51.

6.Cao J, Chavez ER. Comparative trace mineral nutritional balance of first-litter gilts under two dietary levels of copper intake. Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements. 1995;9(2):102-11.

7.Kirchgessner M, Sporl R, Rothmaier DA. Fecal excretion and apparent absorption of copper, zinc, nickel and manganese of nongravid and gravid sows with different dietary supply of trace elements. Zeitschrift Fur Tierphysiologie Tierernahrung Und Futtermittelkunde-Journal of Animal Physiology and Animal Nutrition. 1980;44(2):98-111.

8.Venn JA, McCance RA, Widdowson EM. Iron metabolism in piglet anaemia. J Comp Pathol Ther. 1947;57(4):314-25.

9.Peters JC, Mahan DC. Effects of dietary organic and inorganic trace mineral levels on sow reproductive performances and daily mineral intakes over six parities. Journal of animal science. 2008;86(9):2247-60. doi: 10.2527/jas.2007- 0431.

10.Bradley NS, Heigenhauser GJ, Roy BD, Staples EM, Inglis JG, LeBlanc PJ, et al. The acute effects of differential dietary fatty acids on human skeletal muscle pyruvate dehydrogenase activity. Journal of applied physiology. 2008;104(1):1-9. doi:10.1152/japplphysiol.00636.2007.

11.Petrichev MH, Bambova M. The effects of oral administration of iron methionate to pregnant sows and their litters. Folia Vetereinaria. 2005;49(3):125-8.

12.McGowan JP, Crichton A. Iron Deficiency in Pigs: Biochem J. 1924;18(1):265-72.

13.Kirchgessner M, Pallauf J. Zum Einfluss zusätzlicher Eisengaben an Muttersauen ante partum als Anämieprophylaxe bei Saugferkeln. Züchtungskunde. 1973;45:245-8.

Autorenanschrift Marzell Buffler Lehrstuhl für Tierernährung Liesel-Beckmann-Straße 2 85354 Freising E-Mail: marzell.buffler(at)wzw.tum.de

Suarez and Gallissot: Effect of an algae-clay based biocatalyst on ileal digestibility performance of growing pigs


Effect of an algae-clay based biocatalyst on ileal digestibility performance of growing pigs

Maria Garcia Suarez and Marie Gallissot Olmix SA, Brehan, FR

Abstract The aim of this study was to evaluate the effect of supplementing an algae-clay mix on ileal digestibil-ity performance of growing pigs. Five ileorectal-anastomosed pigs (average weight 30kg) were placed in individual cages. Three weeks after surgery, the pigs received three consecutive diets: the standard diet (control), the test diet supplemented with 0.1% of algae-clay mix (T+), and a low protein and low energy diet (LP-LE) used to estimate the endogenous losses. Ileal digestive utilization coefficient (CUDi) and standard-ized digestive utilization coefficient (CUDs) werecalculated for dry matter (DM), mineral matter (MM), organic matter (OM), nitrogen (N), crude fibre (CF), NDF/ADF, gross energy (GE) and amino acids (AA). Results were submitted to analysis of variance. An increase of ileal digestibility of energy (+105 kcal, P=0.02) and an improvement in ileal protein (+3.6%, P=0.11) and amino acids utilization was observed in the algae-clay mix supplemented diet, including essential amino acids such as lysine (+3.6%, P=0.05) and threonine (+5.3%, P=0.04), providing more nutrients to be absorbed at the small intestinal level. Results of this study show that the algae-clay mix increases ileal digestibility of energy and essential amino acids such as lysine and threonine which are among the first limiting factors in feed formulas.

Introduction Maximizing feed efficiency depends primarily on the quality of both nutrients and the digestive pro-cess. Natural enzymatic digestion in the small intestine is a key factor for an optimal nutrient utiliza-tion. In the last years, several studies pointed out the ability of clays to increase the activity of digestive enzymes in the small intestine via the formation of stable clay-enzyme complexes (Cabezas et al., 1991; Paolo et al., 1999; Reichardt, 2008; Habold et al., 2009; Xia et al., 2004) thus improving nutrient digestibility. Furthermore, the presence of metallic ions in the clay may contribute to the acti-vation of some enzymes, through their action as cofactors (Niederhoffer, 2000; Reichardt, 2008; Habold et al., 2009). Olmix developed a product based on the synergy of clay (montmorillonite) and algae extracts (Ulva sp and Solieria chordalis), which contain diverse metallic ions that are required cofactors for enzymatic activation. The objective of the present study was to evaluate the effect of this algae-clay mix (MFeed+) on the ileal digestibility performance of growing pigs.

Materials and methods The study was monitored at INRA Saint-Gilles (French National Institute for Agronomic Research). In order to evaluate the ileal digestibility performance of the animals, 5 pigs (average weight 30 kg) underwent ileorectal anastomosis (removal of the large intestine) and were placed in individual cages. Three weeks after surgery, the pigs received the three consecutive diets of the study (Table 1), fol-lowing a Latin square model: the standard diet (control), the standard diet supplemented with 0.1%



of algae-clay mix (test) and a low protein and low energy diet (LP-LE), used to estimate endogenous losses. None of the studied diets contained exogenous enzymes. Chemical composition (%, DM) and essential amino acids (%, DM) are shown in Table 2 and Table 3 respectively. Each diet was fed dur-ing one week, including 4 days of acclimation and 3 days of trial measurements. Animals were fed at 8 am and 3:30 pm every day. Feeding was adapted per pig depending on their daily intake (175g/kg BW0.60).

Table 1: Composition of the diets (%)

Standard Algae-clay mix LP-LE Composition (%) Wheat 23.661 23.635 Corn 23.661 23.635 Barley 23.661 23.635 Soybean meal 15.224 15.208 Wheat bran 2.380 2.378 Sugarcane molasses 2.856 2.853 Sunflower oil 0.952 0.951 L-lysine-HCl 0.276 0.276 L-threonine 0.093 0.093 L-tryptophan 0.015 0.015 DL-methionine 0.031 0.031 Corn starch 83.810 Sugar 5.000 Cellulose 4.000 Algae-clay mix - 0.100 Vitamins and trace elements 7.190 7.190 7.190

Table 2: Chemical Composition (%) DM

Mineral matter 8.6 Organic matter 91.4 Proteins (N X 6,25) 16.2 Fat 2.8 Starch 50.2 NDF 11.8 ADF 3.6 ADL 0.59 Crude fibre 3.46 Crude energy (MJ/Kg DM) 17.44

Table 3: Essential amino acids, (%) DM

Lysine 1.01 Threonine 0.67 Methionine 0.28 Tryptophan 0.16 Valine 0.78 Isoleucine 0.63 Leucine 1.19 Histidine 0.38 Phenylalanine 0.74 Arginine 0.90 ∑ Essential AA 6.76



Refused feed and ileal juices were weighed. Dry matter (DM), mineral matter (MM), organic matter (OM), nitrogen (N), gross energy (GE) and amino acids (AA) contents of ileal juices were measured. Endogenous losses of the animals were measured with the LP-LE diet. From these data, ileal and standardized digestive utilization coefficients (CUD) were calculated for DM, OM, N, CF (crude fibre), NDF/ADF (neutral detergent fibre/acid detergent fibre), GE and AA. Variations due to the different diets in the Ileal and Standardized CUD were calculated using ANOVA test. PRO MIXED SAS software was used to conduct all statistical analysis with a significant level of 5%.

Results When compared to the standard diet, algae-clay mix diet presented significantly increased Apparent Ileal Digestive Utilization Coefficient (%) of gross energy (GE), dry matter (DM), organic matter (OM), crude fibre (CF) and acid detergent fibre (ADF) respectively (+3.4%, P ≤ 0.05), (+3.4%, P ≤ 0.01), (+3.1%, P ≤ 0.01), (+72.3%, P ≤ 0.05) and (+45.2%, P ≤ 0.05) (see Table 4 and Figure 1). Algae-clay mix diet showed significantly increased Standardize Ileal Digestive Utilization Coefficient (%) for ∑ Non-essential Amino acids (+3.8%, P ≤ 0.01), Lysine (+3.6%, P ≤ 0.01) andThreonine (+5.3%, P ≤ 0.01) (see Table 5 and Figure 2).

Table 4: Apparent Ileal Digestive Utilization Coefficient (CUDi), %

Standard Algae-clay mix P-value1 DM 68.4b 70.7a 0.01 OM 71.2b 73.4a 0.01 NDF 23.1 29.6 0.06 ADF 7.3b 13.3a 0.03 Crude Fibre 4.0b 14.4a 0.02 Energy 70.7b 73.1a 0.02 1 ANOVA analysis a,b Different superscripts on a same line indicate a significant difference

*** P ≤ 0.01; *P ≤ 0.05; †P ≤ 0.10.

Figure 1: Apparent Ileal Digestive Utilization Coefficient (CUDi), %



Table 5: Standardize Ileal Digestive Utilization Coefficient (CUDs), %

Standard Algae-clay mix P-value2 Proteins 74.7 77.4 0.11 Lysine 81.0b 83.9a 0.05 Threonine 74.9b 78.9a 0.04 Methionine 80.8 82.9 0.25 Tryptophan 66.6 70.9 0.18 Valine 75.7 78.4 0.15 Isoleucine 79.2 81.3 0.19 Leucine 80.3 82.5 0.13 Histidine 78.7 80.4 0.28 Phenylalanine 81.5 83.1 0.24 Arginine 82.9 84.9 0.17 Σ essential AA 79.3 81.8 0.12 Cystine 72,7b 77,7 0.04 Tyrosine 81,7 83,2 0.30 Alanine 71,2 75,0 0.12 Aspartic acid 74,2b 78,3a 0.03 Glutamic acid 85,0b 87,4a 0.03 Glycine 68,9 73,4 0.06 Serine 75,7 79,0 0.66 Proline 83,9 85,9 0.09 Σ non-essential AA 79,2b 82,2a 0.05 1 ANOVA analysis a,b Different superscripts on a same line indicate a significant difference

*** P ≤ 0.01; *P ≤ 0.05

Figure 2: Standardized digestibility of amino acids, %

Discussion The study shows a positive effect of the algae-clay mix supplementation in terms of improved of di-gestibility of energy and protein. Reichardt, 2008 and Habold et al., 2009 described the ability of clays to enhance the contact between digestive enzymes and nutrients, and underlined the presence of enzymatic cofactors in clays through metallic ions. These allow an increased enzymatic activity.Metallic ions such as zinc and copper have the capacity to activate specific enzymes (Niederhoffer, 2000; Jondreville et al., 2002; Williams, 1960). The presence of montmorillonite clay and Ulva sp and So-



lieria chordalis macroalgae in the tested mix, both significant sources of metallic ions (Kim, 2012), have favoured the activity of digestive enzymes and thus contributed to the increased digestibility.

Conclusion Thanks to the biocatalytic network provided by Olmix Exfoliated Algo-clay (OEA)technologyin MFeed+, enzymatic hydrolysis was increased in pigs supplemented with MFeed+. This resulted in an increased ileal digestibility of energy (+105 kcal, P=0.02), providing more nutrients to be absorbed at the small intestinal level. MFeed+ also improved the ileal utilization of proteins (+3.6%, P=0.11) and amino acids, including essential amino acids such as lysine (+3.6%, P=0.05) and threonine (+5.3%, P=0.04). Therefore, MFeed+ contributes to a better use of the diet.

References Cabezas M.J., Salvador D. and Sinisterra J.V., J. Chem. Tech. Biotechnob 1991, 52, 265-274.

Habold C., Reichardt F., Le Maho Y., Angel F., Liewig N., Lignot J.H., Oudar H., 2009. Brit J Nutr, (102), 249-257.

Kim I.H., Li J., Anim Feed Sci Technol. 2013; 184:100–4. doi: 10.1016.

Niederhoffer E.C., 2000. Southern Illinois Univ.

Paolo, P., G. Martellia, L. Sardia, and F. Escribano, 1999. Anim. Feed Sci. Technol. 79:155–162.

Reichardt F., 2008. In : thesis (Université Louis Pasteur Strasbourg).

Xia M.S., Hu C.H., and Xu Z.R., 2004 Poultry Science 83:1868–1875

Corresponding author Maria Garcia Suarez Olmix SA ZA du Haut du Bois 56580 Brehan, France E-mail: animalcare.ts(at)olmix.com

Reckmann et al.: Impact of a dietary probiotic supplementation (Enterococcus faecium DSM 7134) on the performance of fattening pigs


Impact of a dietary probiotic supplementation (Enterococcus faecium DSM 7134) on the performance of fattening pigs

Karoline Reckmann1, Rudolf Hartwigsen2, Georg Thaller2 and Martin Rimbach3

1 Provita Supplements GmbH, Pinneberg, DE 2 Institute of Animal Breeding and Husbandry, Kiel University, Kiel, DE 3 ISF GmbH, Wahlstedt, DE

Abstract High zootechnical performances in modern pig production require a sound intestinal tract of the ani-mals. A dietary probiotic supplementation has a regulatory and stabilising function in pig intestines. The objective of the study was to identify the effects of a supplementation of fattening feeds with the probiotic Enterococcus faecium DSM 7134 on the performance of fattening pigs. In a university trial, 144 fattening pigs were divided into a control (no probiotic) and a treatment group (supplemented with E. faecium). Results indicate that the average daily weight gain increased significantly by 5.6 % during the total fattening stage. Feed intake did not vary considerably between animal groups. Thus, feed conversion among pigs was found to be significantly better (-5.8%) when adding E. faecium to the diet. With almost same slaughter weights, pigs of the two trial groups showed virtually no differences in the lean-meat content. It can be concluded that the pigs utilised the nutrients, especially the amino acids, more eficiently when receiving the dietary probiotic supplemen-tation.

Introduction High performances of fattening pigs might be associated with health problems which mainly affect the pigs’ intestine. Superior performance levels are accompanied by a great nutrient consumption in the gut and require high capacities of the intestine. Thus, they are only feasible with a sound intestinal tract of the animals. Probiotic bacterial cultures are key elements of feeding concepts in pig production by having a regula-tory and stabilising function in pig intestines. Main impacts are the inhibition of pathogenic germs and the stimulation of desired bacteria in the intestinal flora. Since it is commonly known that the microbi-ota are strongly related to the immune status in the gut (Simon, 2010), a healthy immune system and optimal performances of the animals can be concluded. The lactic acid bacteria faced in the present study (Enterococcus faecium DSM 7134 by Lactosan Aus-tria) is licensed throughout the EU for, amongst others, the application in all stages of pig produc-tion.The objective of the study was to identify the effects of a supplementation of fattening feeds with this probiotic gut flora stabiliser on the performance of fattening pigs.

Material and methods The effect of a dietary probiotic supplementation on the performance of fattening pigs was assessed in a trial by the University of Kiel. 144 fattening pigs (Dan-Genetics x Pietrain) were allocated into a stable with 72 pens containing 2 animals of the same gender per pen. Pigs were divided into two

Reckmann et al.: Impact of a dietary probiotic supplementation (Enterococcus faecium DSM 7134) on the per-formance of fattening pigs


groups (control and treatment), kept on plan floor with straw litter. The duration of the trial was 77 days, raising the pigs from approx. 30 kg to 117 kg of live weight (LW). Animals in the control group received a basal fattening diet composed of soya, corn, wheat, rye and mineral feed (Table 1). It was formulated according to the nutrient requirements for fattening pigs as recommended by the Society of Nutrition Physiology (2006). The basal diet of the treatment group was supplemented with the probiotic additive Enterococcus faecium DSM 7134 (Lactosan GmbH & Co. KG, Austria) with an inclusion rate of 0.2 x 109 CFU per kg of feed. Probiotic contents of the supple-mented feeds were confirmed in feed analyses. Two-phase feeding of the pigs was divided into phase I (up to 77 kg LW) and phase II (from 78 kg LW). Pigs were weighed individually at the beginning of the trial and then weekly until the end of the trial. Thus, 72 observations of body weight were gathered per stage and trial group. Feed parameters were documented per pen weekly, resulting in 36 observations per stage and trial group. Slaughter data were recorded individually at slaughter (72 observations per group).

Table 1: Ingredients and nutrient levels of fattening diets

Fattening feed I (30-77 kg)

Fattening feed II (78-117 kg)

Trial group Control group Treatment group Control group Treatment group Ingredients Wheat [%] 29.5 29.5 20.0 20.0 Barley [%] 25.5 25.5 33.0 33.0 Rye [%] 20.0 20.0 27.0 27.0 Soybean meal [%] (CP 44%) 19.0 19.0 13.0 13.0 Soybean oil [%] 1.5 1.5 0.5 0.5 Oat bran [%] 1.0 1.0 3.0 3.0 Mineral feed [%] 3.0 3.0 3.0 3.0 Feed acid [%] 0.5 0.5 0.5 0.5 E. faecium - + - + Nutrient levels Energy (ME) [MJ/kg] 13.2 12.7 Crude protein [%] 17.2 15.2 Lysine [%] 1.11 0.97 Calcium [%] 0.70 0.70 Phosphor [%] 0.45 0.45

Results and discussion The supplementation of E. faecium to the diet of fattening pigs was proven to have beneficial effects on their performance levels. The average daily live weight gain increased significantly by 5.6 % when adding E. faecium to the diet of fattening pigs compared to control animals, as shown in Table 2. This effect was more distinct in the fattening phase II (78-117 kg) with an increased daily LW gain of 10.9 % compared to a rela-tive difference of 1.4 % between treatment and control groups in phase I (Figure 1). Results empha-size that daily LW gains of treatment groups surpass those values of the control group at each point of weighing.



Table 2: Results of main performance parameters in the total fattening stage with and without a probiotic sup-plementation

Total fattening stage Control group Treatment group Relative difference Significance level

SD

SD [%] p

Daily live weight gain [g] 1,025 ± 111

1,082 ± 86 + 5.6 ≤ 0.001

Daily feed intake [g] 2,640 ± 0.26

2,620 ± 0.21 - 0.8 -

Feed conversion 1: [g/g] 2.58 ± 0.13

2.43 ± 0.15 - 5.8 ≤ 0.01

Lean meat content [%] 57.5 ± 2.4

57.5 ± 2.3 ±0 -

Feed intake did not vary considerably between animal groups (Table 2). Moreover, feed intake in the treatment group was even slightly lower, concluding a high feed efficiency of these animals. They realized higher weight gains with the recorded feed intake. Thus, adding Enterococcus faecium DSM 7134 to the diet was associated with an improved feed conversion of the pigs. Feed conversion among fattening pigs was found to be significantly better (- 5.8 %) with probiotic treatment than in control animals (Table 2). The most vigorous effect occurred in the fattening stage II with highly sig-nificant differences between control (1 : 3.00) and treatment groups (1 : 2.76). One possible explana-tion might be the ability of the probiotic to colonize in the digestive tract of the host, thereby enhancing its promoting effects on functional properties of pigs intestines.

Figure 1: Mean daily weight gains of pigs in the different fattening stages with and without a dietary

supplementation of Enterococcus faecium DSM 7134 (*p ≤ 0.05; **p ≤ 0.001)

With almost the same slaughter weights, pigs of the two trial groups showed virtually no differences in the lean-meat content. Considering the favourable daily LW gains, growth rate of the lean-meat was better in the treatment group (475 g) compared to control animals (453 g). It can be concluded that the pigs utilised the nutrients more efficiently to produce lean-meat and thus, did not become fatty. The higher growth rate might be the consequence of an improved protein retention resulting from an increased amino acid utilisation due to the dietary probiotic supplementation, as reported by Simon (2010).

*

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Conclusion High zootechnical performances in modern pig production increasingly require a sound intestinal tract of the animals. The effects of probiotics are complex comprising various factors such as stabilisation of gut flora with decreased gastrointestinal disorders and activation of immune systems. The results of the present study clearly indicate positive effects on the performance level of fattening pigs due to a dietary supplementation with Enterococcus faecium DSM 7134. Growth performance was improved by up to 5.6 %. The supplementation of probiotics stimulates the nutrient utilisation, result-ing in a more effective feed conversion (- 5.8 %).

References GfE, 2006: Empfehlungen zur Energie- und Nährstoffversorgung von Schweinen. Gesellschaft für Ernährungsphysiologie. DLG-Verlag, Frankfurt am Main.

Simon, O. 2010: An interdisciplinary study on the mode of action of probiotics in pigs. Journal of Animal and Feed Sciences 19, 230-243.

Corresponding author Dr. Karoline Reckmann Provita Supplements GmbH An der Muehlenau 4, D-25421 Pinneberg E-mail: karoline.reckmann(at)provita-supplements.de

Puntigam et al.: Einfluss einer druckhydrothermischen Behandlung der Einzelkomponente Mais auf die Verdaulichkeit ausgewählter Nährstoffe sowie zootechnische Leistungen bei Broilern unterschiedlichen Alters


Einfluss einer druckhydrothermischen Behandlung der Einzel-komponente Mais auf die Verdaulichkeit ausgewählter Nährstoffe sowie zootechnische Leistungen bei Broilern unterschiedlichen Alters

Reinhard Puntigam, Karl Schedle, Christiane Schwarz, Peter Hechenberger, Jens Eipper und Martin Gierus Institut für Tierernährung, Tierische Lebensmittel und Ernährungsphysiologie (TTE), Universität für Bodenkultur Wien, AT

Abstract The effects of short- and long term conditioning and expanded maize on nutrient digestibility and growth performance were determined using 384 broiler chicks. The four treatments with corn were used in a corn-soybean meal based diet with untreated (C), short (SC)- (60 sec.) and long (LC)- term conditioned (1080 sec.) and afterwards expanded (~45 kWh/t, OEK 15, A. Kahl, GE) maize of the same batch. Treatment 4 (LC+AA) received diets with corn processed as treatment 3, but including additionally 10% lysine (as reference amino acid to maintain the ideal protein), based on literature reports that ME increases of up to 10% after intensive expander treatment. Feeding was divided into three phases to come up with nutrient and energy requirements during fattening. It was a starter (day 1-8, 12.35 MJ AMEN/kg), a grower (day 9-22, 12.80 MJ AMEN/kg) and a finisher (day 23-38, 12.7 MJ AMEN/kg) phase. The hydrothermal treatment of the single component corn resulted in higher daily weight gain and grain/feed ratio in the grower- as well the finisher phase. With p<0.10, higher amounts of AMEN due to the higher digestibilities of DM and starch were meas-ured in the grower-phase. Despite numerical decrease of abdominal fat due to the supplementation of amino acids in LC+AA, the amount of breast meat didn't change. Considering nutrient digestibility, growth performance and carcass characteristics the hydrothermal treatment of maize is an effective option of feed treatment.

Einleitung Die Verdaulichkeit der Nährstoffe spiegelt sich überwiegend in der Höhe des Gehaltes an umsetzbarer Energie (ME), dem energetischen Bewertungsmaßstab beim Geflügel, wieder (Kluth und Bormann, 2014). Neben der reduzierten Partikelgröße (Kilburn und Edwards, 2001; Peron et al. 2005) und dem Einsatz von Enzymen (Jiang et al. 2008), stellt vor allem die Anwendung von high temperature short time (HTST) Technologien eine Möglichkeit der Nutzungseffizienzsteigerung von Futtermitteln dar. Speziell die Verarbeitung von Einzelkomponenten bietet die Möglichkeit sehr hohe Energieeinträge (kWh/t) anzuwenden, um die chemische und die physikalische Beschaffenheit des Futtermittels zu beeinflussen. Neben der Ruptur von Faserbestandteilen ist vor allem die Gelatinisierung der Stärke zu nennen. Auf Basis dessen wird den Verdauungsenzymen die Zugänglichkeit, speziell der Amylase, erleichtert und somit die Verdaulichkeit gesteigert (Moritz et al. 2005; Gracia et al. 2003). Dies bietet den Vorteil, dass ein möglicher limitierter Energiegewinn auf Basis reduzierter Sekretion der Amylase, speziell innerhalb der ersten Lebenswoche, abgeschwächt werden kann. Sowohl Jimenez-Moreno et al. (2009) als auch Gracia et al. (2009) konnten unter Einsatz von druckhydrothermisch behandeltem Mais einen signifikanten linearen Anstieg der AMEN vom 4., 8., 13. bis hin zum 20. Lebenstag beim Broiler im Vergleich zum nicht-verarbeiteten Mais bei gleichem Alter feststellen. In genannten For-



schungsarbeiten wurde ein möglicher Anstieg von ca. 10% der AMEN pro kg druckhydrothermisch behandeltem Mais nachgewiesen. Dieser macht jedoch eine Anpassung des Lysin : Energieverhältnis-ses erforderlich, um das genetische Leistungspotential der Broiler optimal auszunutzen. Aufbauend darauf wird die Hypothese gestellt, dass auf Grund der druckhydrothermischen Behandlung der Ein-zelkomponente Mais der Gehalt an AMEN in MJ/ kg Trockenmasse um 10% steigt und eine Anpassung des Lys : Energieverhältnisses, in gesteigerten Leistungen, unabhängig vom Alter der Broiler, resul-tiert.

Material und Methoden Für den vorliegenden Fütterungsversuch wurden 384 gesunde Eintagsküken (Ross 308, Lebendge-wicht: 44 ± 0,14g) beiden Geschlechtes, in 24 Boxen zu je 16 Tieren, in der Geflügelversuchsstation Wimitz (Äußere Wimitz, A-9311 Kraig) randomisiert aufgestallt (Tabelle 1). Die Versuchsdauer er-streckte sich über einen Zeitraum von 38 Tagen, wobei eine praxisübliche 3-Phasenfuttermischung (Starterfutter (1.-8. Masttag): 12,35 MJ AMEN/kg TM, Growerfutter (9.-22. Masttag): 12,80 MJ AMEN/kg TM und Finisherfutter (23.-38. Masttag): 12,70 MJ AMEN/kg TM) zum Einsatz kam. C wies im Gegensatz zu den Versuchsgruppen SC (kurzzeitkonditioniert und expandiert), sowie LC und LC+AA (langzeitkonditioniert und expandiert) unbehandelten Mais auf. In Versuchsgruppe LC+AA wurde un-ter Annahme eines 10%-igen Anstieges des ME-Gehaltes, verursacht durch die druckhydrothermische Behandlung des Maises, eine Anpassung des Aminosäuren : Energieverhältnisses, zur Aufrechterhal-tung des Idealproteins, vorgenommen. Unabhängig von der Mais-Verarbeitung, wurde in allen Ratio-nen der gleiche Anteil an Mais eingemischt. Für die Zerkleinerung, des sowohl unbehandelten, als auch behandelten Maises (Amandus Kahl GmbH & Co KG, OEK 15.2) kam eine Hammermühle (Loch-sieb: 2 mm) zum Einsatz. Neben der Ermittlung der Praecaecalen- und Gesamttrakterdaulichkeiten (sowohl nach der Grower-, als auch Finisherphase, d.h. am 23. Tag und 38. Masttag) wurden Mast- und Schlachtleistungsparameter, sowie Teilstückgewichte erhoben.

Tabelle 1: Versuchsdesign

Versuchsgruppe C SC LC LC+AA Mais unbehandelt expandiert expandiert expandiert Vorkonditionierdauer - 60 sec 1080 sec 1080 sec Spez. Energieeintrag - Expander - 45 kWh/t 46 kWh/t 46 kWh/t Aminosäuren-Zulage normal normal normal erhöht 1) Tiere, n 16 16 16 16 Boxen, n 6 6 6 6 1) unter Annahme einer Steigerung der ME um 10 %

Ergebnisse und Diskussion Eine Steigerung der praecaecalen Stärkeverdaulichkeit innerhalb der Growerphase wurde beobachtet (Tabelle 2). Zudem konnte anhand der Verdaulichkeitsergebnisse nachgewiesen werden, dass die druckhydrothermische Behandlung des Maises zu einem tendenziellen Anstieg des Gehaltes an AMEN resultierte (FG 1 vs. FG 2: +4,99%, FG 3: +9,67%, FG 4: +7,11%). Der hohe AMEN-Gehalt konnte in der finisher-Phase (FG 1 vs. FG 2: +0,25%, FG 3: +2,58%, FG 4: +3,62%), trotz tendenziellem An-stieg der Fettverdaulichkeit, nicht bestätigt werden. Darauf basierend lassen sich Nutzeffizienzsteige-rungen pro kg Mais (TM) wie in Tabelle 3 dargestellt, errechnen. Die dargestellten Steigerungen der AMEN konnte ebenfalls bei Lundblad et al., (2011) nachgewiesen werden. Zurückgeführt wird dies in der Studie auf die geteigerte Zugänglichkeit der Verdauungsenzy-me,als Folge der druckhydrothermischen Behandlung.Die Mastleistungsergebnisse (Tabelle 4) wiesen beim Broiler, sowohl in der Starter-, als auch in Growerphase eine höheren täglichen LM-Zunahme,



wie auch des Futteraufwandes aus. Ebenso konnte im Bereich der Schlachtleistung eine Steigerung der Ausschlachtung auf Grund der druckhydrothermischen Futtermittelbehandlung beobachtet wer-den. Betrachtet man den relativen Anteil des Abdominalfettes, so wird ersichtlich, dass dieser mit zu-nehmender Behandlungsintensität ansteigt und mittels adäquater Aminosäurenergänzung (LC+AA) verringert werden kann. Jedoch spiegelte sich diese Ergänzung nicht in einem erhöhten Brustgewicht wieder.

Tabelle 2: Ergebnisse der praecaecalen und Gesamttraktverdaulichkeit beim Broiler

praecaecale Verdaulichkeit in % grower- Phase finisher- Phase

C SC LC LC+AA SEM p C SC LC LC+AA SEM p TM 66,80(b) 69,36(ab) 70,08(a) 69,50(ab) 0,529 (*) 72,72b 74,29a 73,54ab 74,30a 0,260 * XP 72,81 72,26 70,09 68,21 1,158 n.s. 82,52 82,80 83,71 84,80 0,919 n.s. XS 96,94b 97,29a 97,31a 97,27a 0,050 * 97,36b 97,71a 97,68ab 97,75a 0,187 *

Gesamttraktverdaulichkeit in % TM 71,89b 72,9ab 73,88a 71,57b 0,299 * 73,88 73,74 72,64 73,84 0,307 n.s. OM 75,44b 76,46ab 77,44a 75,34b 0,277 * 76,61 76,62 75,01 76,31 0,306 n.s. XS 97,79(b) 98,04(a) 98,20(a) 98,11(a) 0,048 (*) 98,36b 98,60a 98,67a 98,65a 0,038 * GXL 76,94 79,22 79,39 78,57 0,640 n.s. 84,38(b) 87,00(a) 86,00(ab) 86,85(a) 0,395 (*) AMEN 10,65(b) 11,18(a) 11,68(a) 11,41(a) 1,647 (*) 11,26 11,29 11,55 11,67 1,463 n.s. n.s. nicht signifikant; (*) Tendenz, <0,1; * signifikant, <0,05

Tabelle 3: Nutzeffizienzsteigerung von Mais/kg TM

Tabelle 4: Mast- und Schlachtleistungen, sowie Teilstückgewichte der Broiler

Mast- und Schlachtleistungen Versuchsgruppen C SC LC LC+AA SEM p-Wert

Tägliche Futteraufnahme (g/d) Starterphase 26 27 27 26 0,26 n.s. Growerphase 88 87 88 88 0,84 n.s. Tägliche Zunahmen (g/d) Starterphase 15b 17a 17a 17a 0,27 * Growerphase 53b 57ab 59a 61a 0,99 * Futteraufwand (kg/kg) Starterphase 1,77a 1,54b 1,58b 1,52b 0,02 * Growerphase 1,65a 1,52ab 1,50b 1,43b 0,02 * Ohne Darm Ware warm (g) 1898a 1964a 1973a 2007a 22,46 n.s. Grillfertige Ware (g) 1688a 1758a 1759a 1800a 20,62 n.s. Ausschlachtung (%) 79,69b 80,67a 80,95a 80,73a 0,09 ** Gewichte in % der grillfertigen Ware Brustgewicht 31,80a 31,41a 32,50a 32,36a 0,59 n.s. Abdominalfett 1,90b 2,22a 2,27a 2,10ab 0,60 * n.s. nicht signifikant; (*) Tendenz, <0,1; * signifikant, <0,05; ** hoch signifikant, <0,001

grower-Phase finisher- Phase MaisAMEN

(MJ /kg TM) Relative Steigerung (%) Mais AMEN (MJ /kg TM) RelativeSteigerung (%)

C 14,89 100,0 14,89 100,0 SC 16,32 109,6 14,95 100,4 LC 17,66 118,6 15,56 104,5 LC+AA 16,93 113,8 15,83 106,3



Schlussfolgerung Mit dargestelltem Fütterungsversuch konnte nachgewiesen werden, dass der erwartete Energiege-winn, auf Basis einer gesteigerten Verdaulichkeit durch die druckhydrothermische Behandlung der Einzelkomponente Mais, ausschließlich in der Growerphase erzielt werden konnte. Dies lässt den Schluss zu, dass eine mangelnde Amylaseaktivität vermutlich durch eine starke Oberflächenvergröße-rung im Zuge der Futtermittelbehandlung kompensiert werden konnte. Der Nutzung von HTST Tech-nologien unter Einsatz von sehr hohem spezifischem Energieeintrag zur Behandlung von Einzelkomponenten sollte daher speziell in der Growerphase ihren Einsatz finden.

Literatur

Gracia, M. i., Aranibar, M. J., Lazzaro, R., Medel, P. und Mateos G. G. (2003): α-Amylase supplementation of broiler diets based on corn. Poultry Science 82, 436-442.

Gracia, M.I., Lazaro, R., Latorre, M.A., Medel, P. Aranibar, M.J., Jimenez-Moreno, E., Mateos, G.G. (2009): Influence of enzyme supplementation of diets and cooking–flaking of maize on digestive traits and growth performance of broilers from 1 to 21 days of age. Animal Feed Science and Technology 150, 303–315.

Jimenez-Moreno , E., Gonzalez-Alvarado, J. M., Lazaro, R. and Mateos, G. G. (2009): Effects of type of cereal, heat processing of the cereal, and fiber inclusion in the diet on gizzard pH and nutrient utilization in broilers at different ages. Poultry Science 88, 1925–1933.

Kilburn, J. and Edwards, Jr. H. M. (2001): The response of broilers to the feeding of mash or pelleted diets containing maize of varying paticle sizes. British. Poultry Science 42, 484-492.

Kluth, H. und Bormann, T. (2014): Herausforderungen in der Geflügelfütterung: Aktuelle Erkenntnisse zur Verdaulichkeit von Stärke und Aminosäuren. 13. BOKU-Symposium- Tagungsband, Wien.

Kraler M., Gierus M. (2015): Einfluss druckhydrothermischer Behandlung von Mais auf die enzymatische in vitro Verdaulichkeit und Stärkeaufschluss. 14. BOKU-Symposium- Tagungsband, Wien.

Lundblad, K. K., Issa, S., Hancock, J. D., Behnkec, K. C., McKinney, L. J., Alavic, S., Prestløkkena, E. (2011): Effects of steam conditioning at low and high temperature, expander conditioning and extruder processing prior to pelleting on growth performance and nutrient digestibility in nursery pigs and broiler chickens. Animal Feed Science and Technology 169, 208-217.

Moritz, J. S., Parsons, A. S., and Buchanan, N. P (2005): Effect of Gelatinizing Dietary Starch Through Feed Processing on Zeroto Three-Week Broiler Performance and Metabolism. Journal of Applied Poultry Research14, 47–54.

Peron, A., Bastianelli, D., Oury, F.-X., Gomez, J. and Carree. B. (2005): Effects of food deprivation and particle size of ground wheat on digestibility of food components in broilers fed on a pelleted diet. British. Poultry Science 46, 223-230.

Svihus, B., Uhlen, A. K. and O. M. Harstad. (2005): Effect of starch granule structure, associated components and processing on nutritive value of cereal starch: A review. Animal Feed Science and Technology 122, 303–320.

Zhengyu Jiang, Yanmin Zhou, Fuzeng Lu, Zhaoyu Han and Tian Wang (2008): Effects of Different Levels of Supplementary Alpha-amylase on Digestive Enzyme Activities and Pancreatic Amylase mRNA Expression of Young Broilers. Asian-Aust. Journal of Animal Science Vol. 21, No. 1 : 97 – 102.

Autorenanschrift Ansprechpartner: Univ. Prof. Dr. Martin Gierus Universität für Bodenkultur Wien Institut für Tierernährung, Tierische Lebensmittel und Ernährungsphysiologie (TTE), Muthgasse 11, A-1190 Wien E-mail:martin.gierus(at)boku.ac.at

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Oceľová et al.: Absorption of thymol as main compound of thyme essential oil in broiler chickens


Absorption of thymol as main compound of thyme essential oil in broiler chickens

Vladimíra Oceľová1, Remigius Chizzola2, Jana Pisarčíková1, Oksana Ivanišinová1, Štefan Faix1 and Iveta Plachá1

1 Institute of Animal Physiology, SAS, Košice, SK 2 Institute of Animal Nutrition and Functional Plant Compounds, University of Veterinary Medicine Vienna, AT

Abstract According to the fact that prophylactic use of antibiotic growth promoters for poultry production is forbidden since 1.1.2006, the development of phytogenic feed additives can be suitable for poultry production. Our research was focused on the detection of tymol, the main compound of Thymus vul-garis essential oil (EO) in intestinal gut content, plasma and liver of broiler chickens fed a diet with different EO concentrations (0.01, 0.05, 0.1% w/w).Samples were analysed by solid-phase microex-traction (SPME) followed by gas chromatography coupled to mass spectrometry. Significantly higher thymol levels in colon in comparison to duodenum after addition 0.05% and 0.1% of thyme EO were observed, what could indicate lower thymol absorption or higher thymol elimination in these experi-mental groups.

Introduction Beneficial impact of phytogenic feed additives on animal health is an actual topic for the scientific research nowadays. Essential oils (EO) could be deemed as one of the alternatives as feed supple-ment to improve health and performance parameters in animal production. Essential oil is a complex mixture of volatile compounds produced by plants and obtained by distillation. Numerous studies have already confirmed the beneficial effects of EOs but there are still some unanswered questions con-cerning the mode of action, metabolic pathway and optimal dosage of phytogenic additives in poultry. It is suggested that thymol as main compound of thyme EO is absorbed in cranial parts of gastrointes-tinal tract (Michiels et al., 2008) and transported by blood to target tissues. The concentration of thy-mol as bioactive compound in the blood is an important factor influencing its effectiveness in the tissue. Because of lack of information related to EO compounds absorption in animals and especially in poultry, we examined concentrations of thymol in plasma and chyme in cranial part of small intestine (duodenum) and colon after the addition of different EO concentrations to the diet.

Materials and methods Our experiment was carried out on 32 one-day old broiler chickens (ROSS 308) divided randomly to 4 experimental groups (n=8) with increasing concentrations of thyme EO in basal diet (BD): 0.01%, 0.05%, 0.1% w/w and a control group with BD without supplementation of thyme EO. Broiler chick-ens were reared under the standard conditions required for the current breed. Water and feed were provided ad libitum. Feed intake, weight gain and feed to gain ratio were recorded once a week. Chickens were slaughtered after 4 weeks experimental period and samples of gut content, liver and plasma were collected. Quantitative analysis of the thymol in used EO was performed by high perfor-mance liquid chromatography (HPLC). Thymol represented 47.85 % of the examined EO. The main



compound thymol derived from thyme EO in the gut content, plasma and liver of the animals was analysed using solid-phase microextraction (SPME) followed by gas chromatography coupled to mass spectrometry. Prior to the analysis, the addition of the enzyme β-glucuronidase was needed to release metabolised thymol from chemical bonds in plasma and liver samples. Samples for GC/MS analysis were pooled from two animals resulting in 4 samples for each group (n=4). Identification of thymol was proved by specific mass spectra and Kovats index. Quantification was calculated by plotting the peak area ratios sample peak to internal standard peak (o-cresol) against thymol concentration. Sam-ples from control group were used for generating of calibration curve by adding known thymol amounts. Differences between thymol content in duodenum and colon as well as between plasma and liver were analysed by unpaired t-test. The data presented are the mean values ± SD, probability values of less than 0.05 were considered significant.

Results According to our results, the growth performance of broiler chickens was not affected by any concen-tration of thyme EO in the diet. When comparing thymol content in plasma and liver, its concentra-tions in plasma were significantly higher in comparison to liver in all experimental groups p<0.05, Figure 1). Thymol concentrations in colon gut content were significantly higher in comparison to thymol concen-trations in duodenum gut content (p<0.05) when 0.05% and 0.1% of thyme EO was added to the diet (Figure 2).

0.01% 0.05% 0.1%0

200

400

600

800

1000liverplasma

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Figure 1. Thymol content in liver and plasma.

Thyme essential oil concentration in feed.

Thym

ol co

nten

t in

liver

(ng.

g-1 )

and

plas

ma

(ng.

ml-1

)



Discussion Plasma concentration of thymol as bioactive plant compound is a key indicator of its absorption and distribution in animal organism. Effective absorption of thymol from digestive tract in our experiment was proved by its concentrations in plasma. Interestlingly, thymol levels in plasma were significantly higher than thymol amounts in liver. Probably, this results could be due to the intensive thymol bio-transformation in the liver and consequently fast elimination from this organ could occur. Significantly higher activity of biotransformation enzymes in the liver in relation to the thyme and its compounds (thymol and carvacrol) in mice (Sasaki et al.,2005) could promote this suggestion. Thymol amounts in duodenum could represent thymol molecules which were not absorbed by intesti-nal mucosa or thymol molecules absorbed into enterocytes and transported back to the intestinal lu-men by efflux transporters. These transporters are localised in enterocytes of intestinal epithelium (Chan et al., 2004) and theoretically could cause elimination of thymol from the enterocytes. Activity of efflux transporters in enterocytes can influence intestinal absorption of bioactive compounds and drugs (Chan et al., 2004). Thymol concentration in colon gut content could also origin from non-absorbed thymol molecules, but presence of thymol absorbed into enterocytes, metabolized by liver and excreted in bile could be also hypothesised. According to the literature, thymol excretion in bile was observed by Michiels (et al., 2008) in pigs after single dose of orally administred thymol. There-fore, biliary excretion of thymol metabolism and elimination cannot be ignored. Intestinal absorption of drugs and bioactive compounds is a complex mechanism involving physiology of individual intestinal segments, therefore it would be suitable to analyse thymol concentrations in all individual intestinal parts and more detailed information according to thymol absorption could be gained.

Conclusion In relation to our results, we can suggest absorption of thymol in digestive system and its transport within the organism by blood. Liver as the main biotransformation organ contained significantly lower thymol levels than plasma, what could be influenced by intensive thymol biotransformation in liver and fast elimination from this organ. Significantly higher thymol levels in colon in comparison to duodenum in our experiment could suggest lower thymol absorption after higher thyme essential oil addition and/or hypothesised increased excretion of thymol in bile.

0

20000

40000

60000

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100000 duodenumcolon

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**

0.01% 0.05% 0.1%

Thyme essential oil concentration in feed.

Figure 2. Thymol content in duodenum and colon.

Thym

ol c

onte

nt (n

g.g-1

DM)

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Acknowledgement The research was supported by the Slovak Scientific Agency VEGA project 2/0078/16.

References Chan, L.M.S., Lowes, S., Hirst, B.H.: The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability. European Journal of Pharmaceutical Sciences, 2004, Vol. 21, 25–51

Michiels, J., Missotten, J., Dierick, N., Fremaut, D., Maene, P., De Smet, S.: In vitro degradation and in vivo passage kinetics of carvacrol, thymol, eugenol and trans-cinnamaldehyde along the gastrointestinal tract of piglets. Journal of the Science of Food and Agriculture, 2008, Vol. 88, 2371–2381

Sasaki K, Wada K, Tanaka Y, Yoshimura T, Matuoka K, Anno T.: Thyme (Thymus vulgaris L.) leaves and its constituents increase the activities of xenobiotic-metabolizing enzymes in mouse liver. Journal of Medicinal Food, 2005, Vol. 8, 184-189.

Corresponding author Vladimíra Oceľová, DVM Institute of Animal Physiology Slovak Academy of Sciences Šoltésovej 4-6 040 01 Košice , Slovak Republic E-mail: ocelova(at)saske.sk

Kudlinskiene et al.: Milk replacer’s with extruded soy and maize use in calves’ nutrition


Milk replacer’s with extruded soy and maize use in calves’ nutri-tion

Ieva Kudlinskiene, Rolandas Stankevicius, Janina Cernauskiene, Jolita Klementaviciute, Guoda Stanyte, Gintare Dovidaitiene and Romas Gruzauskas Lithuanian University of Health Sciences, Veterinary Academy, Kaunas, LT

Abstract Objectives of the work: To investigate the effect of the milk replacer with extruded soy and maize on calves’ health, growth rate and feed consumption.Twelve Lithuanian black/white breed calves were randomly divided into 2 groups (control and experimental), each group containing 6 animals. The calves from the control group were fed with milk replacer “X”, and the experimental group with milk replacer with extruded components. The results indicated that calves, which were fed milk replacer with extruded soy and corn received 16.7 percent less metabolic energy and 8.45 percent less fat, but 5.26 percent more proteins comparedwith the control group. The calves in the experimental group consumed 6.25 kg or 13.62% more combinated feed comparedwith the control group. During the test period, calves’ growth was intensive in both control and experimental groups. During the 62-day test period, average daily weight gain of the calves in the experimental group, which were fed milk replac-er with extruded components, was 995 g, which is 6.2 percent bigger than that of the control group, which were given to drink milk substitute "X". In total, during the test period, weight change of one calf in the control group was 61.67 kg, which is 5.82 percent bigger than that of the control group.

Introduction Calf management programs have traditionally focused on strategies that restrict the amount of milk or milk replacer offered to the calf, to encourage grain intake in an effort to accelerate weaning, reduce the potential for scours and other illness, and reduce the cost of feeding and management. The scien-tists have focused on emergent feeding strategies that facilitate early weaning and transitioning from liquid to solid feed (Baldwin et al., 2004). One of effective strategies is using milk replacer (MR) in-stead of whole milk for young calves, which are usually made up of ingredients such as skim milk powder (60–75 percent), vegetable or animal fat (15–25 percent), butter milk powder, whey protein (5–10 percent), soy lecithin (1–2 percent) and vitamin-mineral premix. A small proportion of other ingredients like glucose, non-milk protein and cereal flour can also be used (FAO, 2011). Objectives of the work: To investigate the effect of the milk replacer with extruded soy and maize on calves’ health, growth rate and feed consumption.

Materials and methods A new milk replacer for calves with extruded components was developed in JSC “Naujasis Nevėžis“, Lithuania. This milk replacer is made from vegetable and animal origin’s components, and its’ compo-sition is: whey powder, sweet whey fat powder, extruded maize, extruded soy, soy protein isolate, dextrose, wheat starch, dried brewer's yeast, wheat gluten and premix. It is designed for calves from 3 to 12 weeks of age.



The research was carried out in Lukšiai Agricultural Company (Lauciškiai farm) with a view to investi-gate the efficiency and influence of this milk replacer on calves health, growth rate and feed con-sumption. Twelve Lithuanian black/white breed calves were randomly divided into 2 groups (control and experimental), each group containing 6 animals. The tests began when the calves were 20 days old and lasted for 62 days, until the calves were 82 days old. The calves from the control group were fed with milk replacer “X”, and the experimental group with milk replacer which contains of extruded corn and soy. Both groups of calves were given starter com-pound feed, haylage and water ad libitum (Table 1).During the trial, all the consumed feeds were rec-orded, by carrying out control feeding every week.

Table 1: Feeding scheme in the experimental period

Group Calves number per group

Calves age, days Feeding characteristic

Control 6 20 – 68 3 litres of milk replacer „X“ + starter combinated feed, hay-

lage and water ad libitum

69 - 82 2 litres of milk replacer „X“ + starter combinated feed, hay-lage and water ad libitum

Experimental 6 20 – 68 3 litres of milk replacer with extruded maize and soy +

starter combinated feed, haylage and water ad libitum

69 - 82 2 litres of milk replacer with extruded maize and soy + starter combinated feed, haylage and water ad libitum

Milk replacers were prepared in accordance with the recommendations: 1 kg of milk replacer powder was dissolved in 8 liters of water to obtain a diluted 9 liters of prepared milk replacer. Milk replacer powder was added into 1/3 of water (43 - 45 ° C) and was mixed until smooth.Then the remaining part of the water was added(42 ° C) and mixed thoroughly. Milk replacers were fed immediately after preparation - 38 - 40 ° C.

Table 2: Chemical composition of control and experimental groups milk replacers, their energy and nutritional values

Parameter Control Milk replacer „X“

Experimental Milk replacer with extruded components

Dry matter, pct. 95,15 95,02 In 1 kg dry matter: Crude protein, pct. 21 22 Crude fat, pct. 16,5 15 Crude fiber, pct. 0,05 0,5 Crude ash, pct. 9,5 7 Energy, MJ 18 15,25 Vit. A, IU 50 000 40 000 Vit. D, IU 5000 7500

Both milkreplacers had enough protein, fat and other nutrients which ensure intensive calves growth. Milk replacer with extruded maize and soy compared to milk replacer "X" has more protein - 1 per cent, and 2,500 IU of vitamin D, as well as 0.45 percent higher fiber content, but lower by 1.5 percent fat, 2.5 percent ash and 2.75 MJ metabolism energy (Table 2). Calves’ weight was determined by weighing them individually as follows: at the beginning of the test, that is at the age of 20 days; and after 31 day (this means in the middle of the test period) and in the end of test. The data was processed using STATISTIKA forWindows (version 7; Stat Soft. Inc. Tulsa, OK, USA) andpresented as arithmetic mean (x) and its‘ error (SE). The differences were significant at P < 0.05.



Results Calves growth speed and health wellness depends on many factors, but the factor with greatest im-pact is feeds and feeding. Results of control and experimental groups feeds consumption, it’s energy and nutritional values aregiven in Table 3 and Table 4.

Table 3: Results of controlgroup feeds consumption, it’s energy and nutritional values

Parameters Feeding days Feeds and nutrients consump-tion in 62 days per calve: 20-68 69-82

Milk replacer „X“ consumption per day (1 calve), kg: 6 4 344 Compound feed consumpion per day (1 calve), kg 0,57 0,88 39,63 DM, kg 1,25 1,28 77,87 ME, MJ 19,3 18,01 1178,5 Crude protein, kg 0,25 0,26 15,66 Crude fat, kg 0,13 0,27 10,42 Crude fiber, g 30,38 50,25 2352

Table 4: Results of experimentalgroup feeds consumption, it’s energy and nutritional values

Parameters Feeding days Feeds and nutrients consump-tion in 62 days per calve: 20-68 69-82

Milk replacer with extruded maize and soy consump-tion per day (1 calve), kg: 6 4 344 Compound feed consumpion per day (1 calve), kg 0,67 0,97 45,88 DM, kg 1,34 1,36 83,37 ME, MJ 18,11 17,43 1113,32 Crude protein, kg 0,28 0,27 17,13 Crude fat, kg 0,12 0,21 8,96 Crude fiber, g 34,75 57,6 2555

Calves in the experimental and control groups were given the same amount of milk replacers, but the experimental group consumed 6.25 kg or 13.62% more combinated feed compared with the control group.Calves, that were fed milk replacer with extruded components received 16.7 percent less meta-bolic energy and 8.45 percent less fat, but 5.26 percent more proteins compared with the control group.

Table 5: The results of calves growth speed

Parameter Group Control Experimental

Calves weight, kg: In the begining of the trial (20 d.) 46,42±0,70 48,08±0,79 In the middle of the trial (61 d.) 70,2±1,08 73,5±0,90 Average weight gain per day, g (1st period) 766±0,04 820±0,03 In the end of the trial (82d.) 104,5±1,16 109,7±1,27 Average weight gain per day, g (2nd period) 1108±0,03 1169±0,05 Average weight gain per day, g 937±0,04 995±0,05

During the test period, calves’ growth was intensive in both control and experimental groups.During the 62-day test period, average daily weight gain of the calves in the experimental group, which were fed milk replacer with extruded maize and soy, was 995 g, which is 6.2 percent more than that of the control group, which were given milk substitute "X". In total, during the test period, weight change of one calf in the control group was 61.67 kg, which is 5.82 percent bigger than that of the control group.



Conclusion The results indicated that dry milk replacer for calves with extruded maize and soy produced by JSC “Naujasis Nevėžis” meets all zootechnical requirements for calves’ milk replacers, ensures intensive growth of calves and has no negative influence on their health.

References Baldwin, R. L. VI, K. R. McLeod, J. L. Klotz, and R. N. Heitmann. (2004). Rumen development, intestinal growth and hepatic metabo-lism in the pre- and postweaning ruminant. J. Dairy Sci. 87(E. Suppl.):E55–E65.

FAO. 2011. Rearing young ruminants on milk replacers and starter feeds. FAO Animal Production and Health Manual No. 13. Rome.

Corresponding author Ieva Kudlinskiene Lithuanian University of Health Sciences, Veterinary Academy Kaunas, Lithuania E-Mail: ieva.kudlinskiene(at)lsmuni.lt

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Leithner et al.: Ersatz von Sojaextraktionsschrot durch Rapsextraktionsschrot und Rapskuchen bei Milchkühen auf die Milchleistung sowie Milchinhaltsstoffe


Ersatz von Sojaextraktionsschrot durch Rapsextraktionsschrot und Rapskuchen bei Milchkühen auf die Milchleistung sowie Milchinhaltsstoffe

Markus Leithner1, Karl Schedle1, Eduard Schneeberger2 und Martin Gierus1 1 Institut für Tierernährung, Tierische Lebensmittel und Ernährungsphysiologie (TTE), Universität für Bodenkultur Wien, AT 2 Garant Tiernahrung, Pöchlarn, AT

Abstract The experiment was conducted with the support of three dairy farms located in Austria. The total sample of 68 dairy cows was composed of Fleckvieh and Holstein cattle. Pairs were formed with cows of each breed based on milk yield, milk fat, milk protein and days of lactation and equally divided into a soybean-fed and a rapeseed-fed group. The time frame for the experiment was seven weeks and was divided into a one-week adaption period and a six-week-long data collection period. The dairy cows were fed ad libitum with a partly mixed ration based on grass and maize silage. Con-centrate feeds were supplied separately by concentrate feeders containing either soybean meal (SBM) or rape seed meal (RSM) and rape seed cake (RC) as the primary source of protein. The concentrates were calculated to be equal in NEL (7.3 MJ/kg FM) and XP (190 g/kg FM). The substitution of SBM with RSM and RC did not negatively affect milk yield and composition of the main constituents. The milk yield of the SBM-group and the RSM+RC group reached 27.4 and 27.7 ECM kg/d with 3.96 and 4.02% milk fat and 3.50 and 3.46% milk protein content, respectively. The level of milk urea content was lower for the RSM+RC group with 17.7 mg/dl compared to 19.3 mg/dl for the soybean-fed group (P<0.05). The present results showed that SBM can be fully replaced by RS+RC in concentrate feeds for dairy cows. Precondition is an exact knowledge of the nutrient con-tents of the feed to calculate nutrient balanced diets.

Einleitung Die Milcherzeugung ist der wichtigste Produktionszweig innerhalb der österreichischen Landwirtschaft, der Anteil am landwirtschaftlichen Produktionswert betrug 2013 circa 16,6% beziehungsweise 1,2 Mrd. Euro. Im Vergleich zum Vorjahr stieg die Anzahl der Milchkühe um 1,2% an, die Betriebszahl nahm hingegen um 2,9% ab. Die Rohmilcherzeugung betrug 3.393.057 Tonnen, davon wurden 86,46% an die österreichischen Molkereien angeliefert. Im Durchschnitt lag die Milchleistung der Kon-trollbetriebe bei 7.200 kg je Kuh mit 4,14 % Milchfett und 3,40 % Milcheiweiß (BMLFUW 2014). Die Milchproduktion in Österreich ist seit Jahresbeginn 2010 zu 100% deklariert gentechnikfrei, dem-entsprechend sind die Milchlieferanten nach den Qualitätsvorgaben des Österreichischen Lebensmit-telcodex dazu verpflichtet, auf den Einsatz von gentechnisch veränderten Futtermitteln zu verzichten. In der konventionellen Milchviehfütterung hat die Gentechnikfreiheit in erster Linie Auswirkungen auf den Einsatz von Sojaextraktionsschrot, der vielfach eine integrale Rationskomponente zur Proteiner-gänzung darstellt. Die vorliegende Arbeit soll auf Grundlage von Milchleistungsparametern untersuchen, inwieweit So-jaextraktionsschrot durch Rapsextraktionsschrot und -kuchen im Leistungskraftfutter von Milchkühen bei unterschiedlichen Grundfutterausgangsbedingungen ersetzt werden kann.



Material und Methoden Der Versuch umfasste insgesamt 68 Kühe der Rassen Fleckvieh (FV) und Holstein Friesian (HF), da-runter waren 18 Erst- und 50 Mehrkalbskühe. Die Versuchstiere wurden auf drei einzelnen Betrieben innerhalb der jeweiligen Rasse (Fleckvieh oder Holstein Friesian) und Laktation (Erst- oder Mehrkalbs-kühe) nach den Kriterien Milchleistung (Milchmenge) und Milchinhaltsstoffe (Milchfett- und Milchei-weißgehalt) unter Berücksichtigung des Laktationsstandes (Tag der Laktation) zu Paaren zusammengefasst und gleichmäßig auf die Versuchsgruppen (Soja- oder Rapsgruppe) aufgeteilt. Die Unterschiede zwischen den beiden Versuchsgruppen beziehen sich auf die Hauptproteinträger Sojaex-traktionsschrot beziehungsweise Rapsextraktionsschrot und -kuchen im Leistungskraftfutter (LKF). Die aufgewertete Grundfutterration wurde als Mischration ad libitum angeboten und basierte auf unter-schiedlichen Anteilen von Gras- und Maissilage. Die Grundfutterzusammensetzung war in der Vor- und Hauptperiode auf den jeweiligen Betrieben für beide Versuchsvarianten identisch.

Tabelle 1: Zusammensetzung und Inhaltsstoffe der Leistungskraftfutter

Leistungskraftfutter (LKF) Einheit Sojamischung Rapsmischung

(Ang

aben

in F

M)

Körnermais % 35,00 30,00

Weizen % 14,12 13,00

Zusa

mm

ense

tzun

g Gerste % 10,00 8,50 Sojaextraktionsschrot HP NON-GMO % 23,83 - Rapsextraktionsschrot % - 30,89 Rapskuchen Danubia % - 10,00 Weizenkleie % 9,80 - Melasse % 4,00 4,00 Futterfett % 0,70 2,04 Calciumcarbonat % 1,70 0,90 Monocalciumphosphat % 0,20 -

Viehsalz % 0,40 0,42

Vormischung % 0,25 0,25

(Ang

aben

je k

g FM

)

NEL MJ 7,3 7,3

In

halts

stof

fe XP g 190 190

nXP g 166 167 RNB g 3,9 3,9 UDP % 32 34 XF g 35 58

XL g 32 48

In der statistischen Auswertung wurden insgesamt 32 Tierpaare (64 Tiere) miteinbezogen. Die statis-tische Auswertung erfolgte mit dem Statistikprogramm SAS Enterprise Guide 6.1, hierbei wurden die Prozedur GLM und der Mittelwertvergleich nach dem Tukey-Kramer-Verfahren angewendet. Der mehr-faktoriellen Varianzanalyse wurde nachfolgendes Modell zugrunde gelegt: Yij = μ + αi + βj + εij wobei: Yij = Beobachtungswert; μ = Mittelwert; αi = fixer Effekt der i-ten Ver-suchsgruppe (i = 1, 2); βj = fixer Effekt des j-ten Tierpaares (j = 1,…, 32); εij = zufälliger Restfehler.

Ergebnisse und Diskussion Die Kraftfutteraufnahme der Versuchsgruppen war in allen Versuchsabschnitten nahezu identisch und nahm im Verlauf des Versuches sukzessive ab (Tabelle 2).



Tabelle 2: Ergebnisse zur Kraftfutteraufnahme (kg FM/Tag)

Sojagruppe Rapsgruppe SEM p-Wert Versuchsabschnitt 1 4,36 4,37 0,31 0,9053 Versuchsabschnitt 2 4,21 4,21 0,29 0,9764 Versuchsabschnitt 3 4,08 4,06 0,31 0,8760 Ø Kraftfutteraufnahme 4,19 4,19 0,17 0,9978

Die energiekorrigierte Milchmenge (ECM) nahm im Verlauf der Hauptperiode in beiden Versuchsgrup-pen ab. Die Versuchstiere der Soja- und Rapsvariante lagen mit einer täglichen energiekorrigierter Milchmenge von 27,4 und 27,7 kg auf einem insgesamt mittleren Leistungsniveau. Die Ergebnisse von Ettle et al. (2013) decken sich mit den Beobachtungen dieser Untersuchung. Auch in einem Versuch von Kluth et al. (2005) wurde der vollständige Ersatz von Soja- durch Rapsextraktionsschrot in der Fütterung der Hochleistungskuh untersucht. Insgesamt konnte die RES-Gruppe mit 40,5 kg eine signifikant höhere Milchleistung als die SES-Gruppe mit 40 kg pro Tag erzielen. Im Zusammenhang mit den Ergebnissen der Milchleistung verwiesen Kluth et al. (2005) auf eine erhöhte Körperfettmobilisation der RES-Gruppe im Vergleich zu der SES-Gruppe, die durch Mes-sung der Rückenfettdicke und des Milchacetongehalts ermittelt werden konnte. Kluth et al. (2005) konnten aus dem Versuch eine Einsatzempfehlung von bis zu 4 kg RES je Kuh und Tag ableiten. In der Studie von Pries et al. (2012) wurde an drei Versuchsanstalten ein Vergleich von Raps- und So-jaextraktionsschrot bei unterschiedlichen Grundfutterrationen durchgeführt. In der grasbetonten Rati-on erzielte die Variante RES im Vergleich zu den Varianten SES + RES bzw. SES eine signifikant höhere Milchmenge, in der ausgeglichenen und maisbetonten Ration traten keine Unterschiede zwi-schen den Varianten auf. Im Versuch von Pries et al. (2012) wurden teilweise Mengen von bis zu 5,2 kg RES je Tier und Tag aufgenommen. Die prozentualen Gehalte an Milchfett waren im Versuchsverlauf in den Gruppen relativ konstant und zwischen den Gruppen mit 3,96 und 4,02% bei Soja- bzw. Rapsgruppe relativ ähnlich. Die Ausführun-gen von Ettle et al. (2013) bestätigen die Ergebnisse in Bezug auf den Milchfettgehalt, der mittlere Milchfettgehalt lag im Versuchszeitraum von 12 Wochen bei 3,90 % in der SES-Gruppe und bei 3,78 % in der RES-Gruppe. Auch die prozentualen Gehalte an Milcheiweiß waren zwischen der Soja- und Rapsgruppe über den gesamten Versuchszeitraum mit 3,50 und 3,46 % relativ konstant und vergleichbar. In der Studie von Pries et al. (2012) war der Milcheiweißgehalt in % in der gras- und maisbetonten Grundration bei den Versuchsvarianten (RES bzw. RES + HS) jeweils niedriger als bei den Kontrollvarianten (SES bzw. SES + RES). Im Gegensatz dazu lag der Milcheiweißgehalt in kg der Versuchsgruppen unabhängig von der Grundfutterration (grasbetont, maisbetont oder ausgeglichen) über oder auf demselben Niveau der Kontrollgruppen. Die Ursache hierfür ist auf die höhere Milchleistung zurückzuführen. Dagegen lag in allen Versuchsabschnitten der Milchharnstoffgehalt der Sojagruppe über dem der Rapsgruppe. In der Auswertung über den gesamten Versuchszeitraum lag der Milchharnstoffgehalt in der Sojagruppe bei 19,3 mg/dl und in der Rapsgruppe bei 17,7 mg/dl. Die Milchharnstoffgehalte lagen zwar im physiologischen Bereich, aber insgesamt im unteren Drittel der Empfehlungen – nach Spie-kers et al. (2009) sind in der Stallfütterung Werte von 20 bis 25 mg/dl anzustreben. Steingass et al. (2010) führten vergleichende Untersuchungen mit Rapsextraktionsschrot (ungeschützt und geschützt) und Sojaextraktionsschrot durch, die verabreichten Rationen waren nach Energie und Rohprotein aus-geglichen. Im Versuch waren die Milchharnstoffgehalte in den RES-Gruppen mit 21,8 mg/dl (unge-schützt) und 21,2 mg/dl (geschützt) signifikant niedriger als in der SES-Gruppe mit 26,6 mg/dl. Die Differenzen im Harnstoffgehalt der Milch konnten durch die Unterschiede in der ruminalen Abbaubar-keit des Rohproteins erklärt werden. In ergänzenden Untersuchungen zum Proteinabbau wurden mit-tels in situ Methoden bei einer Passagerate von 8 % je h für RES ungeschützt, RES geschützt und SES Werte von 57, 41 und 70 % ermittelt.



Die Referenzstudien bestätigen demnach die vorliegenden Befunde, dass Sojaextraktionsschrot in der Proteinergänzung von Milchkühen unabhängig

• vom Milchleistungsniveau (niedrig, mittel oder hoch), • vom Laktationsstadium (frischmelkend oder altmelkend) und • von der Grundfutterration (grasbetont, maisbetont oder ausgeglichen)

vollständig durch Rapsextraktionsschrot – mit oder ohne Rapskuchen – ersetzt werden kann.

Schlussfolgerung Im Allgemeinen kann aus den vorliegenden Versuchsergebnissen abgeleitet werden, dass Sojaextrak-tionsschrot unter Beachtung der unterschiedlichen Energie- und Proteinwerte vollständig durch Raps-extraktionsschrot und -kuchen im Leistungskraftfutter von Milchkühen ersetzt werden kann.

Literatur BMLFUW – Bundesministerium für Land- und Forstwirtschaft, Umwelt und Wasserwirtschaft (2014): Grüner Bericht 2014 – Bericht über die Situation der österreichischen Land- und Forstwirtschaft. 55. Auflage, Wien: Selbstverlag.

Ettle, T.; Obermaier, A.; Aichner, V.; Spiekers, H. und Windisch, W. (2013): Untersuchungen zum Austausch von Soja- durch Rapsextraktionsschrot beim Milchvieh. In: Verband der Landwirtschaftskammern – VLK (Hrsg.): Forum angewandte Forschung in der Rinder- und Schweinefütterung. Bonn: Selbstverlag, 62-65.

Kluth, H.; Engelhard, T. und Rodehutscord, M. (2005): Zum Ersatz von Sojaextraktionsschrot durch Rapsextraktionsschrot in der Fütterung der Hochleistungskuh. Züchtungskunde 77 (1), 58-70.

Pries, M.; Mahlkow-Nerge, K.; Engelhard, T.; Meyer, A. und Steingass, H. (2012): Einsatz von Raps- und Sojaextraktionsschrot in der Fütterung von Kühen mit hoher Milchleistung und unterschiedlichen Anteilen an Maissilage in der Grobfutterration – Teil 2: Fütterungsversuche. In: Verband der Landwirtschaftskammern – VLK (Hrsg.): Forum angewandte Forschung in der Rinder- und Schweinefütterung. Bonn: Selbstverlag, 45-48.

Spiekers, H.; Nußbaum, H. und Potthast, V. (2009): Erfolgreiche Milchviehfütterung. 5. erweiterte und aktualisierte Auflage, Frankfurt am Main: DLG-Verlag.

Steingass, H.; Kneer, G.; Essig-Kozo, C. und Koch, C. (2010). Aktuelle Untersuchungen zum Proteinwert von Raps-Nebenprodukten und deren Einsatz in Rationen für Milchkühe. In: (Hrsg.): Proceedings of the 19. International Scientific Sym-posium on Nutrition of Farm Animals. Radenci: Selbstverlag, 177-184.

Autorenanschrift M. Leithner, K. Schedle, M. Gierus Institut für Tierernährung, Tierische Lebensmittel und Ernährungsphysiologie (TTE) Universität für Bodenkultur Wien Muthgasse 11, A-1190 Wien, Austria E-Mail: martin.gierus(at)boku.ac.at E. Schneeberger Garant-Tiernahrung Gesellschaft m.b.H. Raiffeisenstr. 3, A-3380 Pöchlarn, Austria E-Mail: schneeberger(at)garant.co.at www.garant.co.at

Nitrayová et al.: Apparent and standardised ileal digestibility of crude protein and amino acids in distillers dried grains with solubles for pigs


Apparent and standardised ileal digestibility of crude protein and amino acids in distillers dried grains with solubles for pigs

Soňa Nitrayová, Matej Brestenský, Jaroslav Heger and Peter Patráš National Agricultural and Food Centre, Research Institute of Animal Production Nitra, Institute of Nutrition, SK

Abstract Chemical composition, apparent (AID) and standardised (SID) ileal digestibility of crude protein (CP) and amino acids (AA) were evaluated in 2 samples of dried distillers grains with solubles (DDGS). The cereal sources used for ethanol production were wheat + barley (WB diet) and maize (M diet). The content of lysine, fat, starch, NDF, ADF and phosphorus was lower for the wheat/barley DDGS than for that from maize. The concentration of CP and lysine in tested DDGS was 364.3 and 7.05 g/kg DM for wheat/barley and 305.3 and 7.17 g/kg DM for maize DDGS. The diets were fed to six gilts fitted with an ileal T-cannula. Experimental periods comprised of a 6-d preliminary period followed by a 1-d collection period during which samples of ileal digesta were col-lected. The experimental data were subjected to ANOVA and when significant value for treatment effect (P<0.05) was observed, the differences between means were assessed using Fisher's LSD pro-cedure. Using acid insoluble ash as a marker the AID and SID of CP and AA were calculated. The AID and SID of CP was greater (P<0.001) in maize DDGS (68.53 and 75.08%) compared with wheat/barley DDGS (62.30 and 67.40%).The mean AID of AA was higher in M (71.49%) compared to WB (61.57%) sam-ple. Digestibility of lysine (AID and SID) in WB sample (43.17 and 51.60 %) was lower (P<0.001) compared to maize DDGS (55.35 and 63.90%). Nutritional value of DDGS varies among sources and the protein quality of DDGS is low despite rela-tively high CP content.

Introduction Distiller’s dried grains with solubles (DDGS) have become more available as an ingredient for animal feed following an increase in ethanol production and for that reason the nutritional value of ethanol industry byproducts is evaluated. Ethanol may be produced from maize, wheat, barley, sorghum or other crops. In general, DDGS has higher concentrations of nutrients such as protein, fat, vitamins, minerals, and fibre than its parent grain. Experiments conducted on pigs so far suggest that the nutritional value of DDGS is highly vari-able, presumably due to different raw materials used, methods of fermentation, drying temperatures or the proportion of solubles in the final product (Stein et al., 2006; Widyaratne and Zijlstra, 2007; Curry et al., 2014). In pigs, the high fibre content may reduce nutrient digestibility, thus reducing the digestibility and availability of energy (Urriola et al., 2010). Therefore, the aim of the present study was to determine chemical composition and the apparent and standardized ileal crude protein and amino acids digestibilities in tested samples of DDGS.



Material and methods The characteristics and chemical composition of samples of DDGS are given in Table 1. Two semipurified diets (WB and M) were formulated with DDGS being the only source of AA and CP in the diets containing distillers grains (56.6 %), maize starch (20 %), sucrose (20 %) and minerals and vitamins (2.4 %). Celite (1 %) was added to the diets as a source of acid insoluble ash (AIA). All diets were fed twice daily at 0630 and 1600 h in two equal meals at a daily rate of 90 g DM/kg0.75. Water was available ad libitum. The experimental procedures were reviewed and approved by the Animal Care Committee of the Re-search Institute of Animal Production Nitra (Slovakia). Six Large white gilts of the Institute herd fitted with an ileal T-cannula (average BW 39.9 ± 1.9 kg) were used in a 2 period changeover trial with 2 diets. Each experimental period lasted 7 d: 6-d adaptation and 1-d ileal digesta collection. Ileal digesta was collected into plastic bags attached to the cannula continuously during 24 hours. Digesta samples were acidified with 6M H2SO4 to pH 3.5 andimmediately frozen at –20°C to prevent bacterial degrada-tion of AA in the digesta. After that the samples were lyophilized, ground to pass a 0.5 mm screen and stored for subsequent analysis. The samples of diets and ileal digesta were analyzed for dry matter (DM), total N, fat, ash, NDF, ADF, P and AIA in accordance with standard methods of AOAC (1990). AA contents after the acid hydrolysis with 6M-HCl and methionine and cysteine after the oxidative hydrolysis were determined using auto-matic amino acids analyzer AAA 400 (Ingos, Praha). The apparent (AID) and standardized ileal digestibilities (SID) of CP and AA were calculated using the method of Stein et al. (2006) and AIA as a marker. The endogenous AA losses in ileal digesta for cal-culation SID were values published by Jansman et al. (2002) as suggested by Stein et al. (2007), which were estimated by using a protein-free diet. The experimental data were subjected to ANOVA using Statgraphic Plus package (version 3.1., Statis-tical Graphics Corp., Rockville, MD, USA). When a significant F-value for treatment means (P<0.05) was observed, the differences between means were assessed using Fisher's LSD procedure.

Results and discussion Overall for the measured chemical characteristics, their contents were lower for the wheat/barley DDGS than the maize DDGS, except for dry mater, crude protein and ash (Table 1).

Table 1: Characteristics and chemical composition of distillers grains (g/kg DM)

Sample Product Dry matter Crude protein Lysine Fat Ash Starch NDF ADF P (Cereal source) WB DDGS (wheat, barley) 928.5 364.3 7.05 43.7 56.6 23.3 366.0 148.1 9.43 M DDGS (maize) 894.6 305.3 7.17 122.8 50.8 38.7 375.6 166.7 9.55

The concentration of CP was about 19.3% higher in WB sample, by reason that CP content in maize is generally lower. Considerable difference was observed in the concentration of fat. The fat in M sample was approximately 2.8 times higher than in WB sample.The content of phosphorus was about equal among the DDGS. Similar to CP content, the amount of total and non-essential AA were higher for wheat/barley DDGS than maize DDGS (Table 2), but the content of essential AA and lysine as the first-limiting AA was higher for maize DDGS.Similar findings were published by Widyaratne and Zijlstra (2007). The total amount of AA was about 20% higher for WB sample than M sample. The difference in the content of essential AA was lower (5.66%).



Table 2: Amino acid composition of distillers grains (g/kg DM)

Our results (Table 3) indicated that the AID and SID of CP was greater (P<0.001) in maize DDGS (68.53 and 75.08%) compared with wheat/barley DDGS (62.30 and 67.40%). These values were comparable with data reported by Pedersen et al. (2007).

Table 3: Apparent and standardized ileal digestibility of AA and N (%) 1

Apparent ileal digestibility Standardized ileal digestibility Item Diet

P-value Diet

Pooled SEM P-value WB2 M3 WB2 M3 Crude protein 62.30 68.53 0.000 67.40 75.08 0.86 0.000 Aspartic acid 48.18 68.59 0.003 54.62 74.79 1.51 0.000 Threonine 57.31 65.70 0.000 64.27 72.92 1.58 0.002 Serine 65.01 76.01 0.001 70.09 81.81 1.24 0.000 Glutamic acid 77.60 82.52 0.138 79.29 85.87 0.79 0.000 Proline 51.35 37.44 0.885 58.56 48.83 5.91 0.288 Glycine 42.51 41.88 0.000 51.75 54.08 2.98 0.591 Alanine 59.08 80.59 0.000 65.48 84.65 1.20 0.000 Valine 62.38 75.15 0.000 67.02 80.41 1.01 0.000 Isoleucine 63.14 77.07 0.000 67.31 82.15 0.80 0.000 Leucine 69.66 86.02 0.000 73.30 88.56 0.60 0.000 Tyrosine 65.92 81.23 0.601 73.51 87.94 1.43 0.000 Phenylalanine 73.04 74.53 0.000 76.29 78.47 1.96 0.446 Histidine 57.06 71.17 0.001 60.92 75.04 0.98 0.000 Lysine 43.17 55.35 0.001 51.60 63.90 2.00 0.000 Arginine 72.51 79.41 0.000 76.72 84.02 1.19 0.001 Cysteine 59.62 76.37 0.000 65.03 81.64 1.37 0.000 Methionine 79.12 86.27 0.000 82.03 89.96 0.66 0.000 1 Values are means of 6 observations per treatment, 2 WB - wheat, barley DDGS,3 M - maize DDGS

The mean AID and SID of AA was lower in WB sample (61.57 and 66.93%) compared to M sample (71.49 and 77.35%) The measured digestibilities for AA in the present study are comparable with the

Amino acid DDGS (wheat/barley) DDGS (maize) Essential Arginine 14.27 13.47 Histidine 6.69 6.90 Isoleucine 11.57 9.80 Leucine 22.45 33.13 Lysine 7.05 7.17 Methionine 5.95 4.84 Phenylalanine 16.11 13.70 Tyrosine 6.67 7.78 Threonine 10.45 10.40 Valine 14.54 13.26 Total essential AA 115.75 120.45 Non-essential Alanine 11.48 18.68 Aspartic acid 17.26 18.49 Cysteine 4.97 5.25 Glutamic acid 84.01 43.76 Glycine 13.38 10.47 Proline 31.82 20.77 Serine 14.18 12.81 Total non-essential AA 177.11 130.22 Total AA 292.86 250.67



range of coefficients measured for maize DDGS (Fastinger et al., 2006; Stein et al., 2006) and wheat DDGS (Nyachoti et al., 2005). However, the SID of the most of AA in DDGS obtained in the present experiment is greater than those given by NRC (NRC, 2012). Digestibility of lysine (AID and SID) in WB sample (43.17 and 51.60%) is lower compared to maize DDGS (55.35 and 63.90%), the differences being significant (P < 0.001). Despite a relatively high CP content, the protein quality of tested DDGS is low, which is undoubtedly due to the low content and digestibility of lysine as a first limiting AA.

Conclusion Nutritional value of DDGS vary among sources and the protein quality of DDGS is low despite relative-ly high CP content which is undoubtedly due to the low content and digestibility of lysine as a first limiting AA in tested DDGS.

Acknowledgements This article was written during realization of the project "ZDRAVIE no. 26220220176" supported by the Operational Programme Research and Development funded from the European Regional Development Fund.

References AOAC. 1990. Official Methods of Analysis. 15th ed. Assoc. Off. Anal. Chem., Arlington, VA

Curry, S. M., J. K. Htoo, H. V. Masey O'Neill, and H. H. Stein. 2014. Digestibility of amino acids in distillers dried grains with solubles produced in Europe from wheat, maize, or mixtures of wheat and maize and fed to growing pigs. J. Anim. Sci 92: 643

Fastinger, N. D. and Mahan, D. C. 2006. Determination of the ileal amino acid and energy digestibilities of corn distillers dried grains with solubles using grower-finisher pigs. J. Anim. Sci. 84: 1722–1728.

Jansman, A. J. M., W. Smink, P. van Leeuwen, and M. Rademacher. 2002. Evaluation through literature data of the amount and amino acid composition of basal endogenous crude protein at the terminal ileum of pigs. Anim. Feed Sci. Technol 98:49-60.

NRC. 2012. Nutrient Requirements of Swine, 11th rev. ed. National Academy Press, Washington

Pedersen, C., M. G. Boersma, H. H. Stein. 2007. Digestibility of energy and phosphorus in ten samples of distillers dried grains with solubles fed to growing pigs. J. Anim. Sci. 85: 1168-1176.

Stein, H. H., M. L. Gibson, C. Pedersen, M. G. Boersma. 2006. Amino acid and energy digestibility in ten samples of distillers dried grain with solubles fed to growing pigs. J. Anim. Sci. 84: 853-860.

Stein, H. H., M. F. Fuller, P. J. Moughan, B. Sève, R. Mosenthin, A. J. M. Jansman, J. A. Fernández, and C. F. M. de Lange. 2007. Definition of apparent, true, and standardized ileal digestibility of amino acids in pigs. Livest. Sci. 109:282-285.

Nyachoti, C. M., House, J. D., Slominski, B. A. and Seddon, I.R. 2005. Energy and nutrient digestibilities in wheat dried distillers’ grains with solubles fed to growing pigs. J. Sci. Food Agric.85: 2581–2586.

Urriola,P. E., G. C. Shurson, H. H. Stein. 2010. Digestibility of dietary fiber in distillers coproducts fed to growing pigs. J. Anim. Sci. 88: 2372-81

Widyaratne, G. P., and R. T. Zijlstra. 2007. Nutritional value of wheat and corn distillers’ dried grain with solubles: Digestibility and digestible contents of energy, amino acids and phosphorus, nutrient excretion and growth performance of grower-finisher pigs. Can. J. Anim. Sci. 87:103–114.

Corresponding author

Soňa Nitrayová National Agricultural and Food Center, Research Institute of Animal Production Nitra, Institute of Nutrition, Hlohovecká 2, 951 41 Lužianky, Slovakia E-mail: nitrayova(at)vuzv.sk

Khidr et al.: Processing some poultry diets using non conventional feedstuffs under the Egyptian desert conditions: A case study


Processing some poultry diets using non conventional feedstuffs under the Egyptian desert conditions: A case study

Raafat Khidr Animal and Poultry Production Division, Desert Research Center, Ministry of Agricul-ture and Land Reclamation, EG

Abstract Egypt, as one of the developing countries faces a pronounced deficiency in poultry feedstuffs. The feed cost represents approimately 75 % of the total production cost. This could interpret that the most conventional feedstuffs (corn and soya bean) are imported with higher prices. In addition, there is a competition between human and animal consumption concerning their materials. Therefore, the future development of poultry production in Egypt, particularly under the desert and newly reclaimed areas, could depends partially on the availability of some conventional feedstuffs (date stone meal, olive cake and desert shrubs and tropical legumes) under such conditions. Research works revealed that processing diets in a pelleted form in ducklings containing some of non conventional feedstuffs increased live body weight, weight gain, feed intake and improved feed conversion values when com-pared to mash or crumble form. Moreover, the lowest consumption of water was recorded by birds fed pelleted diets under the desert conditions. The pellets groups showed the best net return and the highest value of economic efficiency among all experimental groups. Processing diets in a pelleted form had some positive effects in alleviating some of anti nutritional factors might be found in some of non conventional feedstuffs used in poultry feeding under the desert conditions. Enhanced perfor-mance of processing diets in pelleted form may be due to increased digestibility, decreased ingredient segregation, reduction of energy during prehension and increased palatability. The effects of physical form, including some of non conventional feedstuffs on the performance of birds are discussed.

Corresponding author Dr. Raafat Khidr Professor of Poultry Nutrition Animal and Poultry Production Division, Desert Research Center, Ministry of Agriculture and Land Reclamation, Egypt E-Mail: raafatkhidr(at)yahoo.com

Unbenannt-1 1 21.03.16 11:29

Farahat et al.: Effect of dietary supplementation of grape seed extract on the growth performance, lipid profile, antioxidant status and immune response of broiler chickens


Effect of dietary supplementation of grape seed extract on the growth performance, lipid profile, antioxidant status and im-mune response of broiler chickens

Mahmoud Farahat1, Fatma Abdallah2, Haitham Ali3 and Aaron Hernandez-Santana4 1 Department of Nutrition and Clinical Nutrition, College of Veterinary Medicine, Zaga- zig University, Zagazig, EG 2 Department of Virology, College of Veterinary Medicine, Zagazig University, Zagazig, EG 3 Department of Biochemistry, College of Veterinary Medicine, Zagazig University, Zagazig, EG 4 Plantextrakt GmbH & Co. KG, Vestenbergsgreuth, DE

Abstract This experiment investigated the effect of supplementing broiler diets with grape seed extract (GSE) as a natural antioxidant at levels of 125, 250, 500, 1000, and 2000 ppm on the growth performance, serum lipid profile, liver reduced glutathione, thigh muscle malondialdehyde, and humoral immune response against Newcastle disease virus vaccines. The results of broilers fed on diet supplemented by GSE were compared to those fed on the basal diet (control) or the basal diet supplemented by butylated hydroxytoluene as a synthetic antioxidant (BHT, 125 ppm). No significant differences were observed in the growth performance, percent livability, total lipid, high and very low density lipopro-teins when the use of GSE or BHT were compared to the control. Total cholesterol and low-density lipoprotein were significantly decreased after dietary intake of GSE compared to BHT. The reduced glutathione level in liver tissues was significantly increased by inclusion of GSE but not by BHT. Inclu-sion of GSE or BHT decreased significantly the malondialdehyde level found in meat tissue. The anti-body titer against Newcastle disease virus vaccines was significantly elevated in 28 and 35 day old broilers fed with a diet supplemented with GSE or BHT, the former providing a higher response. It can be concluded that GSE can be used as an effective natural antioxidant and immunostimulant agent in broiler chicken diets, and that 125-250 ppm can be considered as the optimum dosage.

Introduction With the ongoing intensification of poultry industry, broilers are exposed to several stressors that in-crease the production of reactive oxygen species (ROS). Excess ROS cause injury to the cellular com-ponents and adversely affect growth performance, decrease immune response, increase lipid peroxidation and lower meat quality (Panda and Cherian, 2014). The adverse effect of ROS can be minimized via supplementation of chickens with an adequate supply of antioxidants (Grashorn, 2007). Synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and ethoxyquin are commonly used in poultry diets. However, their use is debated due to their carcino-genic nature (Niki et al., 1991). To meet consumer’s demands, researchers are in a continuous search for natural antioxidants to replace synthetic ones. Grape seed extracts (GSE) are known to contain polyphenols with high antioxidant capacity (e.g. gallic acid; the monomeric flavan-3-ols catechin, epi-catechin, gallocatechin, epigallocatechin and epicatechin 3-O-gallate; procyanidin dimers, trimers and more highly polymerized procyanidins; Shi et al., 2003). Dietary GSE has been found to possess anti-



oxidant and immunostimulant properties (Bagchi et al., 2000; Katiyar, 2015) and enhances the oxida-tive stability of meat (Mitsumoto et al., 2005).

Materials and methods This study investigated the influence of dietary supplementation of broiler chickens from 0-42 days of age with different levels of GSE on the growth performance, serum lipid profile, liver reduced glutathi-one, thigh muscle malondialdehyde, and humoral immune response against Newcastle disease virus vaccines. A total of 245 one day old, unsexed chicks (Ross 308 strain) were obtained from a commer-cial hatchery. On arrival, they were weighed, banded, and randomly distributed into 7 dietary treat-ments; each treatment included 5 pens with 7 chicks per pen. Chicks were vaccinated against Newcastle disease virus (Hitchner B1 strain at day 7 via eye-drop, inactivated oil adjuvant clone 30 strain at day 15 via s/c injection of 0.25 mL per chick, and LaSota strain at day 17 via eye-drop). Diets were based on corn-soy-gluten, and were formulated to meet or exceed the nutrient requirements of broilers as set by the National Research Council (NRC, 1994). Feeding program was divided into 3 phases: starter (0-21 days of age; 23% CP and 3000 Kcal/kg diet), grower (22-35 days of age; 21% CP and 3100 Kcal/kg diet), and finisher (36-42 days of age; 19% CP and 3200 Kcal/kg diet). Dietary treatments were as followings: treatment 1, basal diet (control); treatment 2, basal diet plus 125 ppm BHT; treatments 3, 4, 5, 6 and 7 were fed basal diets plus 125, 250, 500, 1000 and 2000 ppm of GSE respectively. The GSE was a water-extracted powder obtained from Plantextrakt GmbH & Co. KG, Germany. Its total polyphenol content was > 40%, of which > 5% were monomeric flavan-3-ols and > 30% procyanidins. Bird weights and feed consumed were measured for each feeding phase to calculate the average body weight, average daily gain, average daily feed intake, and feed conversion ratio.At day 42, blood samples were collected from the wing vein of eight birds per treatment for measuring serum lipid profile using commercial kits. Samples from the liver and thigh muscle of 8 chickens per treatment were used for the assays of reduced glutathione in liver tissues, and malondialdehyde in meat tissues using commercial kits according to Beutler et al. (1963) and Ohkawa et al. (1979) respectively. Blood samples were collected from eight chickens per treatment at 21, 28, and 35 days of age for measur-ing the antibody titer against Newcastle disease virus vaccines using ELISA kit (BioChek B. V., Reeu-wijjk, Holland). ANOVA was conducted to determine the treatment effect, and LSD was conducted to determine if significant differences exist among treatments at P < 0.05. Contrast testing (control vs. BHT; control vs. GSE; BHT vs. GSE) and polynomial contrasts (linear and quadratic for GSE levels) were also analyzed using Statistix 9 (Analytical Software, 2008).

Table 1: Effect of dietary GSE on the performance of broiler chickens at 42 days of age

Performance parameters (g)

Treatments (ppm)

s.e.m

P-value of contrast

CON 125 BHT

125 GSE

250 GSE

500 GSE

1000 GSE

2000 GSE

CON Vs. BHT

CON Vs. GSE

BHT Vs. GSE

Average body weight 2490 2474 2602 2511 2378 2510 2489 94.0 (-) (-) (-)

Average daily gain 58.3 57.9 60.9 58.8 55.6 58.8 58.3 2.23 (-) (-) (-)

Average daily feed intake 104 103 107 104 101 100 105 1.67 (-) (-) (-)

Feed conversion ratio 1.79 1.79 1.75 1.77 1.81 1.80 1.80 0.03 (-) (-) (-)

CON = control; BHT = butylated hydroxytoluene; GSE = grape seed extract; s.e.m = standard error of the mean; (-) = no significant effect (P>0.05);Linear and quadratic response because of the GSE in the diet was not significant (P>0.05)



Results Supplementation of broiler diets with GSE or BHT did not result in significant differences in the meas-ured growth performance parameters (Table 1). No significant difference was observed among treat-ments in the livability percentage. The total lipid, high-density and very low-density lipoprotein levels were not significantly different among treatments. Dietary inclusion of GSE resulted in significant de-creases in the total cholesterol (P<0.01) and low-density lipoprotein (P<0.05) levels by up to 35% and 57% respectively compared to inclusion of BHT (Table 2).

Table 2: Effect of dietary GSE on the serum lipid profile of broiler chickens at 42 days of age (mg/dL)

Lipid profile

Treatments (ppm)

s.e.m

P-value of contrast

CON 125 BHT

125 GSE

250 GSE

500 GSE

1000 GSE

2000 GSE

CON Vs. BHT

CONVs. GSE

BHT Vs. GSE

Total lipid 301 344 254 318 322 250 318 21.8 (-) (-) (-) Total choles-terol

185ab 201a 145cd 179abc 176abc 131d 154bcd 13.1 (-) (-) 0.01

High-density lipoprotein

67.8 64.6 67.8 65.1 60.3 66.7 65.9 7.0 (-) (-) (-)

Low density lipoprotein

101ab 118a 64.4bc 96.7ab 99.2ab 50.2c 71.7bc 13.2 (-) (-) 0.05

Very low-density lipo-protein

16.9 18.6 12.7 17.1 16.2 14.2 16.2 1.4 (-) (-) (-)

CON = control; BHT = butylated hydroxytoluene; GSE = grape seed extract; s.e.m = standard error of the mean; (-) = no significant effect (P>0.05); a,b,c Means in a row with different superscripts differ significantly (P<0.05); Linear and quadratic response for GSE was not significant.

Inclusion of GSE resulted in a significant (P<0.01) increase in the reduced glutathione level by up to 60% compared to the control. Reduced glutathione of broilers fed on BHT diet was not significantly different from those fed on the GSE or control diets. The highest values were found in broilers fed on diets containing 125, 250 and 500 ppm GSE. Supplementation with GSE and BHT resulted in a signifi-cant (P<0.01) decrease of the lipid peroxidation marker malondialdehyde in the broiler’s meat by lev-els up to 36.7 and 73.3% respectively compared to the control. The best results were observed in broilers fed on diets containing 125 ppm BHT followed by those fed on diets containing GSE (Table 3).

Table 3: Effect of dietary GSE on the liver glutathione reduced and meat malondialdehyde of broilers

Antioxidant parameters

Treatments (ppm)

s.e.m

P-value of contrast CON 125

BHT 125 GSE

250 GSE

500 GSE

1000 GSE

2000 GSE

CON Vs. BHT

CONVs. GSE

BHT Vs. GSE

Glutatione reduced (mg/g)

3.4c 4.1bc 5.1ab 5.5a 5.4ab 4.6ab 4.9ab 0.45 (-) 0.01 (-)

Malondialdehyde (nmol/g)

14.8a 9.3b 9.9b 11.0b 9.8b 9.4b 11.8ab 1.26 0.01 0.01 (-)

CON = control; BHT = butylated hydroxytoluene; GSE = grape seed extract; s.e.m = standard error of the mean; (-) = no significant effect (P>0.05); a,b,c Means in a row with different superscripts differ significantly (P<0.05); Linear and quadratic response for GSE was not significant.



Intake of GSE resulted in a significant increase in the antibody titer against Newcastle disease virus vaccines by levels up to 57% (at 28 days of age; P<0.01) and 76% (at 35 days of age; P<0.05) com-pared to the control. Inclusion of BHT resulted in significant (P<0.05) increases in the titer by 35% and 50% at 28 and 35 days of age respectively compared to the control. Best results were reported in broilers fed on diets with 125-500 ppm GSE (Table 4).

Table 4: Effect of dietary GSE on the antibody titer against Newcastle disease virus vaccines in broiler chicken

Titer range

Treatments (ppm) s.e.m

P-value of contrast CON 125

BHT 125 GSE

250 GSE

500 GSE

1000 GSE

2000 GSE

CON Vs. BHT

CON Vs. GSE

BHT Vs. GSE

21 days of age 1391 1401 1479 1617 1504 1280 1322 107 (-) (-) (-) 28 days of age 2100c 2836ab 3106ab 2919ab 3300a 2588abc 2441bc 236 0.05 0.01 (-) 35 days of age 2541c 3828ab 4051ab 3950ab 4478a 2891bc 3243bc 390 0.05 0.05 (-) CON = control; BHT = butylated hydroxytoluene; GSE = grape seed extract; s.e.m = standard error of the mean; (-) = no significant effect (P>0.05); a,b,c Means in a row with different superscripts differ significantly (P<0.05); Linear response for GSE was not significant (P>0.05); Quadratic response for GSE was highly significant at 28 and 35 days of age (P<0.01).

Discussion Although not many studies have been carried out with grape seed and broilers, some positive results have been observed regarding growth performance (Wang et al., 2008 utilizing 5 to 80 ppm of GSE). Nevertheless, high dosages may be detrimental to the growth performance (Viveros et al. (2011) / 7200 ppm; Chamorro et al. (2013) / 5000 ppm). The variation in response to inclusion of GSE in dif-ferent trials could be attributed to the difference in concentration and profile of polyphenols and pro-anthocyanidins in the tested products. The extraction solvent has an impact on the phytochemical profile and the biological activity of the resulting extract; a water based extract should contain princi-pally monomeric flavonoids, dimer and trimer procyanidins, while organic extraction solvents will also tend to extract higher molecular weight procyanidins (Khanal et al., 2009). Highly polymerized procy-anidins are thought to be less bioavailable than their monomeric constituents (Tsang et al., 2005). The levels of total cholesterol and low-density lipoprotein were decreased by inclusion of GSE, which complies with the findings of El-Damrawy (2014). Supplementation of broiler diets with GSE resulted in a significant increase in the level of reduced glutathione (an important cellular antioxidant) in liver tissue, which is considered as an indicator of the antioxidant status of birds (Wang et al., 2010). This agrees with the findings of El-Damrawy (2014) and Wang et al. (2008). Dietary GSE can delay the lipid peroxidation of thigh meat as indicated by decreased malondialdehyde level in muscle tissue. Brenes et al. (2010) revealed that GSE polyphenols can be absorbed at sufficient levels to enhance the antioxidant status in chicken tissue. Supplementation of broiler chicken diets with GSE resulted in a significant increase in the antibody titer against Newcastle disease virus vaccines. To the best of the authors’ knowledge, no studies have been published regarding the ability of GSE to enhance the hu-moral immune response of broilers. The immunostimulatory function of GSE is attributed to its antiox-idant and free radical scavenging properties that increase the integrity and proliferation of B-lymphocytes and enhances its differentiation into antibodies producing plasma cells (Hamilos et al., 1989). In conclusion, GSE can be used as a feed supplement in broiler chickens from 0-42 days of age to enhance their antioxidant status, immune response and meat quality. Additionally, it can replace syn-thetic antioxidants (e.g. BHT), and be utilized to increase vaccination effectiveness. Levels ranging from 125-250 ppm were found to be the optimum levels for the GSE used in this study.



References Chamorro S, Viveros A, Centeno C, Romero C, Arija I and Brenes A 2013. Effects of dietary grape seed extract on growth performance, amino acid digestibility and plasma lipids and mineral content in broiler chicks. Animal 7, 555-561.

Hamilos DL, Zelarney P and Mascali JJ 1989. Lymphocyte proliferation in glutathione-depleted lymphocytes: direct relationship between glutathione availability and the proliferative response. Immunopharmacology 18, 223-235.

Khanal RC, Howard LR and Prior RL 2009. Procyanidin composition of selected fruits and fruit byproducts is affected by extraction method and variety. Journal of Agriculture and Food Chemistry 57, 8839-8843.

Niki E, Yamamoto Y, Komuro E and Sato K 1991. Membrane damage due to lipid oxidation. American Journal of Clinical Nutrition 53, 201S–205S.

Shi J, Yu J, Pohorly JE and Kakuda Y 2003. Polyphenolics in grape seeds—biochemistry and functionality. Journal of Medicinal Food 6, 291-299.


Mahmoud Farahat Department of Nutrition and Clinical Nutrition, College of Veterinary Medicine, Zagazig University, Zagazig, 44511,Egypt E-mail: drmahmoud3d(at)yahoo.com

Šťastník et al.: The influence of feeding wheat with blue aleurone on intestinal microflora of broilers


The influence of feeding wheat with blue aleurone on intestinal microflora of broilers

Ondřej Šťastník1, Lenka Detvanová2, Filip Karásek1, Hana Štenclová1, Libor Kalhotka2, Leoš Pavlata1, Eva Mrkvicová1 and Petr Doležal1

1 Department of Animal Nutrition and Forage Production, Mendel University in Brno, CZ 2 Department of Agrochemistry, Soil Sciences, Microbiology and Plant Nutrition, Men- del University in Brno, CZ

Abstract The aim of this study was to evaluate the effect of feeding of wheat with blue aleurone layer with higher content of anthocyanins to bodyweight and microbial colonization in gut of broiler chickens. Sixty chickens were divided into two equal groups: Control and Experimental. The experimental group received feed mixture containing 38.2% of wheat with blue aleurone layer “UC 66049” with content of cyaniding-3-glucoside 47.64 mg/kg. Control group received the same amount of common wheat “Vánek” with content of cyaniding-3-glucoside 5.10 mg/kg. The feeding wheat with blue aleurone did not affect bodyweight and gut microbial colonization of broilers.

Introduction The purple colour of wheat is caused by anthocyanins accumulated in the pericarp, while the blue colour is generated by anthocyanins in the aleurone layer of the wheat grain (Zeven, 1991). It is well known that herbal anthocyanins are functioning as antioxidants and they have antibacterial and anti-carcinogenic effects (Abdel-Aal and Hucl, 1999). Cyanidin 3-glucoside is the most abundant anthocya-nin represented approximately 31% of the total anthocyanins (Abdel-Aal et al., 2006). It is speculate that the antiinflammatory effect of anthocyanins involves mechanisms related to their intestinal func-tions and their interaction with the intestinal microbiota. Polyphenols might promote intestinal coloni-zation by specific groups of bacteria because the microbiota plays an important role in the metabolism of these compounds in the gastrointestinal tract (Espley et al., 2014; Neyrinck et al., 2013). Different polyphenols have been suggested to affect the relative viability of colonic bacterial groups (Selma et al., 2009; Parkar et al., 2013). Polyphenols should be considered to have a prebiotic action (Faria et al., 2014).

Material and methods The experiment was performed with 60 cockerels of Ross 308 hybrid which were fattened on conven-tional deep litter system. Wood shavings were used as bedding material. The adaptive period when the chickens received a commercial feed mixture BR1 was carried out to day 12 of the chicken’s age. The trial was conducted from day 13 to day 37 of chicken’s age. Room temperature and humidity were controlled. Lighting system was 16 hours light and 8 hours dark. Cockerels were divided into two equal groups Control and Experimental (UC). The experimental group received feed mixture contain-ing 38.2% of wheat with blue aleurone layer cultivar “UC 66049” with 47.64 mg/kg cyaniding-3-glucoside. The feed mixture of control group contained 38.2% of common wheat cultivar “Vánek” with 5.10 mg/kg cyaniding-3-glucoside. The composition of used feed mixtures is shown in Table 1. Table



2 shows chemical composition of used diets. The chickens were fed ad-libitum. During the trial, health status was evaluated daily, whereas feed intake and live weight were measured every week. Mean feed consumption per one chicken was calculated.

Table 1: Composition of feed mixtures (g/kg)

Component Control UC Wheat 382 382 Maize 243 273 Soybean extruded 190 190 Soybean meal 105 95 Premix* 30 30 Rapeseed oil 20 20 Wheat gluten 19 0 Monocalciumphosphate 7 7 Limestone (CaCO3) 4 4 * lysine 60.0 g; methionine 75.0 g; methionine + cysteine 75.0 g; calcium 195.0 g; phosphorus 55.0 g; sodium 46.0 g; copper 4.0 mg; zinc 3.70 mg; tocopherol 1.50 mg; biotin 6.0 mg per kg. Retinol 450 IU; calciferol 166.70 IU

Table 2: Chemical composition of used diets (as fed)

Control UC Dry Matter (g/kg) 880 880 Crude Protein (g/kg) 187.4 190.3 Crude Fat (g/kg) 71.2 71.7 Crude Fibre (g/kg) 28.7 26.7 Crude Ash (g/kg) 51.8 55.7

At the end of experiment 6 birds were selected randomly from each group and slaughtered. Microbial analyses were carried out at the 37th days of broilers age. Fresh digesta were obtained from the lower ileum. The digesta were diluted into a fluid state with saline solution. Weighed digesta (1 g) was ho-mogenised in centrifuged tube. Into Petri dishes 1 ml of sample was inoculated. Five groups of micro-organisms were determined. The Escherichia coli (E. coli) on Rapid Ecoli 2 agar (Biorad, USA) for 24 hrs at 37˚C. Enterococci (ENT) on Slanetz-Bartley agar (Merck, Germany) for 72 hrs at 37˚C. Lacto-bacilli (LBC) cultivated anaerobically on MRS agar (Biokar Diagnostics, France) for 48 hrs at 37˚C. After cultivation, counts of microorganisms were expressed as log CFU/g (Colony Forming Unit). The data were processed by Microsoft Excel (Microsoft, USA) and Statistica version 12.0 (StatSoft, CZ). We used one-way analysis (ANOVA). To ensure evidential differences Scheffe´s test was applied at P < 0.05.

Results and discussion Table 3: Average live weight (mean ± standard error of the mean) at the end of trial (g)

Control (n = 30) UC (n = 30)

2,352 ± 34.4 2,232 ± 47.4

The average body weight of broilers was higher in control group compare to experimental group at the day of slaughter (Table 3). The results were not statistically significant (P > 0.05). In our earlier experiments with wheats with different colour of grain (Karásek et al., 2014; Šťastník et al., 2014) performance parameters of broilers were not affected too. According to the technological procedure for ROSS 308, the average body weight of cockerels should be 2,493 g at 37 days of age (Aviagen



Group, 2014). Our results are only slightly lower compare to this technological procedure. This can be caused by frequent handling of chickens during the trial.

Table 4: Mean number of microorganism (CFU/g)

Group n E. coli (107) ENT (103) Lactobacillus (104) Mean ± standard error

Control 6 5.61 ± 0.308 3.21 ± 0.192 8.39 ± 0.197 UC 6 5.87 ± 0.324 2.82 ± 0.292 8.23 ± 0.270

Table 4 shows the mean content of microorganism in gut of broiler chickens. On behalf of each bacte-rial species were not detected statistically significant differences (P > 0.05) between the groups at 37th day of age. Based on our results we can conclude that the higher content of cyanidin-3-glucoside not affect monitored microbiological parameters of intestinal contents. Jakubcová et al., (2014) in experiment with supplementation of chamomile extract found that lactic acid bacteria was higher in the group without chamomile extract. On the other hand, Lichovnikova et al., (2014) observed in their trial with added red grape pomace (Vitis Vinifera L.) that this had a posi-tive effect on the content of Lactobacillus.

Conclusion The feeding of wheat with blue aleurone layer did not affect on body weight or gut microbial coloniza-tion of chickens.

Acknowledgments The project was supported by the project by NAZV QJ 1510206.

References Abdel-Aal, E.-S.M., Hucl, P. (1999): A rapid method for quantifying total anthocyanins in blue aleurone and purple pericarp wheats. Cereal Chemistry 76, 350–354.

Abdel-Aal, E.-S.M., Young, J.C., Rabalski, I. (2006): Anthocyanin composition in black, blue, pink, purple, and red cereal grains. Journal of Agricultural and Food Chemistry 54, 4696–4704.

Aviagen Group (2014): Technological procedure for broiler Ross [online]. Aviagen Group 2014. Available from: http://en.aviagen.com/ross-308

Espley R.V., Butts C.A., Laing W.A., Martell S., Smith H., McGhie T.K. (2014): Dietary flavonoids from modified apple reduce inflammation markers and modulate gut microbiota in mice. Journal of Nutrition 144(2), 146–54.

Faria, A., Fernandes, I., Norberto, S., Mateus, N., & Calhau, C. (2014): Interplay between anthocyanins and gut microbiota. Journal of Agricultural and Food Chemistry 62(29), 6898–6902.

Jakubcová, Z., Kalhotka, L., Dostálová, L., Detvanová, L., Šťastník, O., Mrkvicová, E., Mrázková, E., Zeman, L. (2014): Effect of chamomille extract on gut microflora in broiler chickens. In 13. BOKU-Symposium Tierernährung. 1. vyd. Wien: Institut für Tierernährung 284–286. ISBN 978-3-900932-16-9.

Karásek, F., Mrkvicová, E., Šťastník, O., Mrázková, E., Trojan, V., Vyhnánek, T., Jakubcová, Z., Hodulíková, L., Štiasna, K., Presinszká, M., Dostálová, Y., Janečková, M., Hřivna, L., Pavlata, L., Doležal, P.(2014): The effect of colored wheat feeding on broiler chickens performance parameters and antioxidant activity measured in the liver tissue. In NutriNet 2014. 3. vyd. Košice: Publishing Centre of UVMF in Košice, 68–71.

Lichovnikova, M., Kalhotka, L., Adam, V., Klejdus, B., Anderle, V. (2015): The effects of red grape pomace inclusion in grower diet on amino acid digestibility, intestinal microflora, and sera and liver antioxidant activity in broilers. Turkish Journal of Veterinary and Animal Sciences 39(4), 406–412.



Neyrinck A.M., Van Hée V.F., Bindels L.B., De Backer F., Cani P.D., Delzenne N.M. (2013): Polyphenol-rich extract of pomegranate peel alleviates tissue inflammation and hypercholesterolaemia in high-fat diet-induced obese mice: potential implication of the gut microbiota. British Journal of Nutrition 109(5):802–809.

Parkar, S. G., Trower, T. M., Stevenson, D. E. (2013): Fecal microbial metabolism of polyphenols and its effects on human gut microbiota. Anaerobe 23, 12−19.

Selma, M. V., Espin, J. C., Tomas-Barberan, F. A. (2009): Interaction between phenolics and gut microbiota: role in human health. Journal of Agriculture and Food Chemistry 57, 6485−6501.

Štastník, O., Mrkvicová, E., Karásek, F., Trojan, V., Vyhnánek, T., Hřivna, L., Jakubcová, Z. (2014): The influence of colored wheat feeding on broiler chickens performance parameters. Proceedings of the International PhD Student Conference MendelNet. Brno: Mendel University in Brno, 196-198.

Zeven, A.C. (1991): Wheat with purple and blue grains: a review. Euphytica 56, 243–258.

Corresponding author Ing. Ondřej Šťastník Department of Animal Nutrition and Forage Production Mendel University in Brno Zemedelska 1, 613 00, Brno, Czech Republic E-mail: ondrej.stastnik(at)mendelu.cz

Kliseviciute et al.: Nutritional value and digestible energy of different genotypes of barley in the horses and rabbits nutrition


Nutritional value and digestible energy of different genotypes of barley in the horses and rabbits nutrition

Vilma Kliseviciute1, Romas Gruzauskas1, Vilma Sasyte1, Asta Raceviciute-Stupeliene1 and Gintautas Juozas Svirmickas2 1 Institute of Animal Rearing Technology, Lithuanian University of Health Sciences Veterinary Academy, Kaunas, LT 2 Institute of Animal Sciences, Lithuanian University of Health Sciences Veterinary Academy, Kaunas, LT

Abstract Barley is one of the main sources of protein for the nutrition of humans and livestock. They has been used in the feeding of adult monogastrics and ruminants, all of which have an important digestible capacity.Barley is less used in European rabbit and horses diets though it can substitute wheat in weaning diets as it contains more fibre and less protein. The aim of this study was to evaluate the nutritive value and content of digestive energy for rabbits and horse of different barley varieties grown in Lithuania. Eleven different genotypes of barley, with their know growth conditions were ana-lysed by the following methods: chemical analyses (dry matter, crude protein, crude fat, crude ash) were determined according to Pašarų tyrimo metodai (2003), starch – by Near infra-red reflectance spectroscopy (NIRS); rabbits digestible energy calculated by Perez et al. (1998) method; horses di-gestible energy calculated by Pagan (1998) method. The results showed that the average of values was: dry matter – 94.29%, crude protein – 11.11%. crude fat – 1.24%, crude ash – 2.06%, starch – 59.83%, crude fiber – 4.45%, ADL – 1.40%, ADF – 6.12%, NDF – 18.39%. Digestible energy for rab-bits – 13.07 MJ/kg DM, for horses – 14.92 MJ/kg DM.

Introduction The barley (Hordeum vulgare L.) is the fourth most produced cereal in the world after wheat, maize and rice. It is mainly used as animal feed but there is a growing interest in it for human food. In general, barley has been used in the feeding of adult monogastrics and ruminants, all of which have an important digestible capacity (Andersson et al., 1999). The major components of barley are starch, dietary fibre, and crude protein, constituting: 60, 20, and 12% of dry matter, respectively (Oscarsson et al., 1996). The energy content of barley is slightly lower than the energy value of corn and of wheat, partially due to its higher fiber content (neutral detergent fiber, or NDF, and acid detergent fiber, or ADF). The crude protein content of barley is higher than in corn and similar to wheat and oats, but lower than in field peas (Rowe et al., 2001). Cereal grains are fed to all classes of domestic livestock and the digestibility of starch in pigs, poultry, sheep and cattle has been intensively studied. Much is known about the site of starch digestion in these animals and published results indicate that starch digestibility varies considerably both between and within grain type. Energetic concentration is one of the main variables in the formulation of diet for growing rabbit. Dietary carbohydrates, especially starch, are the main sources of energy for mono gastric animals to maximize growth during the short rearing period (Leng, 2008).



Cereal grain is an important feed ingredient for most intensively managed horses and although cereals provide a valuable source of digestible energy their feeding to horses is always associated with some risk. The danger in feeding cereal grain to horses lies in the risk of incomplete digestion of starch in the small intestine and the possibility that significant amounts of starch can pass through to the caecum and colon. Starch entering the hind gut is fermented very much more quickly than roughage and this rapid fermentation leads to accumulation of acidic end products and low pH (Juliand et al., 2006). Barley may be fed to horses as the only grain in the ration. Barley is higher in DE than oats, but lower than corn. It is intermediate between corn and oats with respect to energy content. The aim of this study was to evaluate the nutritive value and content of digestive energy for rabbits and horse of different barley varieties grown in Lithuania.

Materials and methods Samples. Clean anduncontaminated samples of eleven cultivars of barley (Luoke, Aura DS, Alisa DS (8611), Arka DS (8056-2), Barke, Sebastian, Beatrix, Publican, Quench, NFC Tipple, Keops)were taken for nutritional value analyses. All cultivars were grown in experimental fields of Institute of Agriculture at LRCAF (Lithuania). The fertilization of barley was NPK 17-10-14 + 11S 300 kg/ha, additional – ammonium nitrate 100 kg/ha. Chemical analyses of barley samples. Different genotypes of barley, with their growth conditions known were analysed by the following methods: dry matter was determined as the difference be-tween moist and dry grain, drying for 3h at 105°C (Pašarų tyrimo metodai, 2003).Crude protein was examined following Kjeldahl analysis method, determining nitrogen concentration in a sample (Pašarų tyrimo metodai, 2003).Crude fat was calculated after samples had been extruded with ether (Pašarų tyrimo metodai, 2003).Crude ash was calculated on the basis of sample residue after its organic mat-ters were incinerated at the temperature of 550°C (Pašarų tyrimo metodai, 2003). Starch analysis. Near infra-red reflectance spectroscopy (NIRS) analysis. Barley samples reflectance spectra were measured by means of NIRS using a Foss-Tecator equipment DS2500 on milled grain samples. NIRS calibration models for starch and moisture content were developed using software The ISIscan Nova (Germany). Fibre analysis. Neutral detergent fibre (NDF), Acid detergent fibre (ADF) and Acid detergent lignin (ADL) were analysed using the Fibertec™ 2021/2023 FiberCap™ system (FOSS Analytical AB, Sweden) according to the manufacturer’s instructions with modifications adapted from Goering and Van Soest (1970) and Kitcherside et al. (2000). Rabbits digestible energy (DE) calculated by Perez et al. (1998) method:

DE (MJ/kg DM) = 13.94 – 0.196 CF (Perez et al., 1998) Horses digestible energy (DE) calculated by Pagan (1998) method:

DE (MJ/kg DM) = 8.86 + 0.05097 × Crude protein (g/kg) – 0.0392 × ADF – 0.0160 × (NDF-ADF) + 0.197 × Crude fat + 0.085 × (100-Crude protein-NDF-Crude fat-Crude ash) – 0.110 × Crude ash

Statistical analysis. The results of the experiment were analyzed using the 1-way ANOVA test, and significant differences between groups were determined by Duncan’s multiple range test. Statistica 8.0. for Windows TM software was used. Differences were considered significant at P<0.05.



Results and discussion The results of present study clear demonstrated that the highest constituent in barley varieties were starch (55.92-62.26 % DM), followed crude protein (9.44-13.57 % DM). The remaining components of barley grain (crude fat and crude ash) were minor constituents (Table 1).

Table 1: Chemical composition of barley grown in Lithuania, %

Total of Samples = 11 Dry matter Crude protein Crude fat Crude ash Starch Average 94.29 11.11 1.24 2.06 59.83 Minimum 93.53 9.44 1.14 1.71 55.92 Maximum 95.21 13.57 1.40 2.45 62.26

The results of the present study for composition of barley grain (Table 1) are in agreement and com-parable to those reported by Grove et al. (2003), Baik and Ullrich (2008). However other researches (Makeri et al., 2013) determined higher content of chemical components than presented in our stud-ies. Nutritional components of barley are generally reported as averages; however, the barley may differ greatly in chemical composition due to genetic and environment factors. Metayer et al. (1993) found a big variability in chemical composition of different barley varieties. Making feed formulations for rabbits and horses, it is important to know not only the amount of crude fiber, but concentrations of NDF, ADF, ADL as well. So in present studies there are presented separat-ed data of crude fiber composition.

Table 2: The amount of crude fibre and its composition of barley grown in Lithuania, %

Total of Samples = 11 Crude fiber ADL ADF NDF Average 4.45 1.40 6.12 18.39 Minimum 3.90 1.20 5.48 16.10 Maximum 5.27 1.54 7.34 23.99

Concentration of NDF is considerably important, because the amount of metabolisable energy in cere-al depends on its concentration. After examination of fiber content and its components in different genotypes of barley (Table 2), the highest amount was determined in Luoke variety (5.27% DM); the lowest – in Alisa DS variety (3.90% DM). There were no significant differences in the ADL content be-tween barley varieties. Analyzing the content of ADF, statistically significant differences were found between barley varieties Alisa DS (5.48% DM) and Luoke (7.34% DM). There were no significant dif-ferences (P>0.05) in the ADF content of the other varieties. By analysing the NDF content there were significant differences (P<0.05) between the Luoke (23.99% DM) and Alisa DS (16.10% DM). Grove et al. (2003) determined that barley NDF is 18.2–22.6%, ADF – 5.1–7.0%, while Žilić et al. (2011) de-termined barley ADF average to be 2.18–2.61%, NDF – 18.58–27.81%. The analysis of digestible energy (DE) of different barley varieties for rabbits showed no statistically differences between the barley varieties. Average of digestible energy of different barley genotypes for rabbits is 13.07 MJ/kg DM. The lower barley digestible energy content for rabbits (12.7 DE MJ/kg) recommended by Sauvant et al. (2004). De Blas and Wiseman (2010) claim that digestible energy of barley for rabbits are 12.90 MJkg-1. By analysing the DE for horses content there were significant differences (P<0.05) between the Luoke and Barke varieties. Average of digestive energy of different barley genotypes for horses is 14.92 MJ/kg DM. Sauvant et al. (2004) claim that digestible energy of barley for horses are from 14.7 MJ/kg to 15.4 MJ/kg.



Table 3: Digestible energy of different barley genotypes for rabbits and horses, MJ/kg DM

Genotypes Digestible energy,MJ/kg DM Rabbits Horses

Luoke 12.91ab 14.31a Aura DS 13.06ab 14.89ab Alisa DS 13.18ab 15.17ab Arka DS 13.12ab 15.03ab Barke 13.12ab 15.13b Sebastian 13.15ab 15.08ab Beatrix 12.98ab 14.76ab Publican 13.10ab 14.87ab Quench 13.10ab 15.04ab NFC Tipple 13.01ab 14.94ab Keops 13.02ab 14.94ab

Conclusions The different barley varieties grown in Lithuania characterized by the high content of crude protein and crude fiber, i.e. 9.44–13.57% and 3.90-5.27% respectively. Digestible energy of different barley genotypes for rabbits depending on the barley genotype compris-es from 12.91 MJ/kg DM to 13.18 MJ/kg DM, for horses - from 14.31 MJ/kg DM to 15.13 MJ/kg DM.

Acknowledgments The study was supported by the ministry of Agriculture of the Republic of Lithuania; project MT 11/30.

References Andersson A.A.M., Elfverson C., Andersson R., Regne´r S. and Åman P. (1999): Chemical and physical characteristics ofdifferent barley samples. Journal of Science of Food and Agriculture. 79, 979–986.

Baik B.K. and Ullrich S.E. (2008): Barley for food: characteristics, improvement, and renewed interest (rev.). Journal of CerealScience. 48, 233–242.

de Blas C. and Wisem J. (2010): Nutrition of the rabbit -- 2nd ed.

Goering H.K. and Van Soest P.J. (1970): Forage Fiber Analysis (Apparatus, Reagents, Procedures, and Some Applications). Agricultural Handbook 379. USA: U.S. Department of Agriculture.

Grove V., Hepton J. and Hunt C. W. (2003): Chemical Composition and Ruminal Fermentability of Barley Grain, Hulls, and Straw as Affected by Planting Date, Irrigation Level, and Variety. Professional Animal Scientist. 19, 273–280.

Julliand V., De Fombelle A. and Varloud M. (2006): Starch digestion in horses: the impact of feed processing. Livestock Science. 100, 44–52.

Kitcherside M.A., Glen E.F. and Webster A.J.F. (2000): FibreCap: an improved method for the rapid analysis of fibre in feeding stuffs. Animal Feed Science andTechnology. 86, 125–132.

Leng R.A. (2008): Digestion in the rabbit –a new look at the effects of their feeding and digestive strategies. In: International Workshop Organic rabbit farming based on forages. 25-27 November , Cantho University, Cantho City, Vietnam.

Makeri M.U., Nkama I. and Badau M.H. (2013): Physico-chemical, malting and biochemical properties of some improved Nigerian barley cultivars and their malts. International Food Research Journal. 20(4), 1563–1568.

Metayer J.P., Grosjean F. and Castaing J. (1993): Study of variability in French cereals.Animal Feed Science and Technology. 43, 87–108.

Oscarsson M., Andersson R., Salomonsson A.C. and Åman P. (1996): Chemical composition of barley samples focusing on dietaryfibre components. Journal of Cereal Science. 24, 161–169.

Pagan J.D. (1998): Measuring the digestible energy content of horse feed. In: J.D. Pagan (ed). Advances in Equine Nutrition. Nottingham University Press, Nottingham, 71–76.



Pašarų tyrimo metodai (2003): Normatyvinių aktų rinkinys. Lietuvos Respublikos žemės ūkio ministerija. P. 304.

Perez J.M., Lamboley B. and Béranger C. (1998): Valeur nutrtitive de différentes luzernes déshydratées utilises seules ou en mélange dans le regime du lapin en croissance. 7émes Journ. Rech. Cunicole Fr. Lyon, ITAVI Ed., Paris, 129–132.

Rowe J., Brown W. and Bird S. (2001): Safe and effective grain feeding for horses. Pub. 01/148. Rural Industries Research and Development Corporation. Kingston, ACT

Sauvant D., Perez J.M. and Tran G. (2004): Tables of composition and nutritional value of feed materials: pigs, poultry, cattle, sheep, goats, rabbits, horses, fish. D. Sauvant, J.M. Perez & G. Tran (Eds). Wageningen Academic Publishers, Wageningen and INRA Editions, Versailles.

Žilić S., Dodig D., Milašinović Šeremešić M., Kandić V., Kostadinović M., Prodanović S. and Savić D. (2011): Small grain cereals protein and dietary fibre content. Genetika. 43 (2), 381–395.

Corresponding author Vilma Kliseviciute Lithuanian University oh Health Sciences Veterinary Academy Kaunas, Lithuania E-Mail: Vilma.Kliseviciute (at)lsmuni.lt

Polačiková et al.: Quality of rabbit’s meat after feeding GM maize MON 88034 x NK 603


Quality of rabbit’s meat after feeding GM maize MON 88034 x NK 603

Mária Polačiková, Mária Chrenková, Ľubica Chrastinová, Zuzana Formelová, Ľubomír Ondruška and Kvetoslav Zaujec National Agricultural and Food Centre, Animal Production Research Centre Nitra, Institute of Nutrition, SK

Abstract Getetically modified (GM) plants are considered to be one of the important tools for sustainable pro-duction of food, feed and raw materials for industry.GM maize MON 89034 × NK603 produces the Cry1A.105 and Cry2Ab2 insecticidal proteins, which impart protection against damage caused by the European corn borer (ECB, Ostrinia nubilalis) and other lepidopteran insect pests and expresses the CP4 EPSPS proteins, derived from Agrobacterium sp. strain CP4, which provides tolerance to glypho-sate (the active ingredient of a broad spectrum Roundup® herbicide). The aim of this work was to determine the effect of GM maize MON 89034 x NK 603 in complete mix-ture for rabbits on growth performance and meat quality. In the experiment we tested genetically modified maize (MON 89034 x NK 603), isogenic maize (DKC 5143) and 3 reference hybrids of maize growing on allotment in CVRV Piešťany. One hundred twenty rabbits (meat line M91 and P91, 5 weeks old) were divided into 5 experimental groups and fed with complete granulated mixtures en-riched with 12 % maize per 100 kg mixture ad libitum with free access to water. During the experi-ment were examined general health status and feed conversion. After death the samples of Musculus longissimusdorsi (MLD) were collected immediately, stored at 5°C for 24h and physico-chemical anal-yses (according to STN 57 0185) were made. Obtained results demonstrate that feeding of transgenic maize had no negative influence on performance, physical and chemical characteristics of meat in MLD muscle and fatty acids profile of rabbit’s meat.

Introduction After wheat and rice, maize is the third most frequently cultivated plant worldwide. Maize use in Eu-rope is dominated by the demand for animal feed. High crop loss for maize has been observed due to the major pest, the European Corn Borer (Ostrinia nubilalis) and other lepidoptera. Soil bacterium Bacillus thuringiensis produces a protein (Cry protein), which leads to the death of the corn borer larvae after intake. In the case of GM maize MON 89034 the Cry1Ab and Cry2Ab2 genes were inte-grated into the plants’ genome leading to the production of this protein. Glyphosate is one of the most widely used herbicides in the world. It is highly effective against the majority of annual and perennial grasses and broad-leaf weeds. Herbicide tolerance has been introduced, through genetic modification, into a number of crops including maize. Thus NK 603 contains a gene for resistance to glyphosate (CP4 EPSPS gene, derived from the soil bacterium Agrobacterium sp.). GM maize MON 89034 × NK603 consists in the combination of traditional breeding of two genetically modified parental inbred lines derived from MON 89034 and NK 603. MON 89034 × NK 603 produces the Cry1A.105 and Cry2Ab2 insecticidal proteins, which impart protection against damage caused by the European corn borer (ECB, Ostrinia nubilalis) and other lepidopteran insect pests and expresses the CP4 EPSPS proteins, derived from Agrobacterium sp. strain CP4, which provides tolerance to glyphosate (the active ingredient of a broad spectrum Roundup® herbicide).



The aim of this work was to determine the effect of GM maize MON 89034 x NK 603 in complete mix-ture for rabbits on growth performance and meat quality.

Material and methods In our experiment we tested genetically modified maize (MON 89034 x NK 603), isogenic maize (DKC 5143) and 3 reference hybrids of maize growing on allotment in CVRV Piešťany. One hundred twenty rabbits (meat line M91 and P91, 5 weeks old) were divided into 5 experimental groups. In the first, second and third experimental group (EG-1, EG-2, EG-3) were tested complete granulated mixtures enriched with 12 % reference hybrids of maize per 100 kg mixture. The rabbits in the 4th group (EG-4) were fed with granulated mixture including 12 % GM maize MON 89034 × NK603 and in the 5rd group (EG-5) with granulated mixture including 12 % isogenic maize (DKC 5143).

Table 1: Ingredients and chemical analysis of the experimental diets for rabbits

Ingredients in % Parameter (g. kg-1) EG-1 EG-2 EG-3 EG-4 EG-5 Lucerne meal 32.0 Dry matter 892.9 893.9 890.5 901.9 894.1 Oats 11.0 Crude protein 137.2 136.2 136.6 137.0 137.2 Barley 7.0 Crude fibre 163.0 170.2 165.9 165.6 169.0 Rape extr. meal 9.0 Fat 35.9 37.3 37.0 37.8 36.6 Wheat bran 8.0 N free extract 489.8 482.8 482.4 492.2 484.3 DDGS 5.0 Organic matter 826.1 826.4 821.9 832.6 827.2 Maize 12.0 Ash 66.9 66.7 68.5 69.3 66.9 Dry malting gems 13.0 Starch 178.2 175.5 181.7 183.5 179.9 Mineral &Vitamins* 1.7 Calcium 6.63 6.68 7.14 6.99 6.99 NaCl 0.3 Phosphorus 4.51 4.59 4.47 4.63 4.42 Limestone, pulverized 1.0 ME (MJ. kg-1) 9.42 9.16 9.42 9.16 8.99 *Provided per kg diet: vit. A 12000 IU; vit.D2 2500 IU; vit. E 20 mg; vit.B1 1.5 mg; vit. B2 7.5 mg; vit. B6 4.5 mg; vit.B 12 30 µg; vit.K 3 mg; nicotic acid 45 mg; folic acid 0.8 mg; biotin 0.08 mg; choline chloride 450 mg; premix minerals: Ca 9.25 g; P 6.2 g; Na 1.6 g; Mg 1.0 g; K 10.8 g; Fe 327.5 mg; Mn 80 mg; Zn 0.7 mg

The experiment lasted 42 days. Rabbits were kept in standard cages under controlled environmental conditions. The rabbits had ad libitum access to feed and water. Body weight and feed intake were measured every week. Rabbits were slaughtered at 77 days of age. The samples of Musculus longissimus dorsi (MLD) were collected immediately after death, stored at 5°C for 24 hours and next physico-chemical analyses were made. Value of pH was assessed by combined glass-gel electrode and apparatus Radelkis 24 hours post mortem. Content of water, proteins and intramuscular fat in muscular substance was analysed by Infratec 1265 apparatus. Water holding capacity was determined by pressure method as described by Grau-Hamm in an apparatus modified by Hašek and Palanská (1976). Energy value in Musculus longissimus dorsi (MLD) was calculated using the following formula: EV (kJ/100 g) = (16.75 x protein content) + (37.68 x fat content). Fatty acid methyl esters (FAME) were prepared using the modified procedure of Juárez et al. (2008) and determined by gas chromatography on GC 6890N (Agilent, Technologies) using wide bore columns with ID of 0.53 mm and a length of 30m with and stationary phase (50 % cyanopropylphenyl-methylpolysiloxane). The relative proportion of each fatty acid was expressed as the relative percentage of the sum of total fatty acids. The samples of individual feeds were analyzed for content of nutrients (Table 1) according to the methods specified in the Journal of the Ministry of Agriculture of The Slovak Republic No. 8, 2010. The results were expressed as mean ± standard deviation (SD); statistical evaluation of the results was performed by the one-way ANOVA and Tukey test for multiple comparisons at the level of significance p≤ 0.05.



Results and discussion In fattening experiment were studied the growth of live weight and consumption of feed mixtures per unit of live weight growth (Table 2). Among the experimental groups no significant difference was noted in feed intake, feed conversion ratio and carcass value. The biophysical and chemical parameters of MLD muscles are presented in table 3. No significant ef-fect in chemical analyses on values of total protein, water and intramuscular fat content was found, only in group EG5 (isogenic maize) was content of fat lower compared to the other groups. There have been many independent animal feeding studies using GE crops (Flachowsky et al., 2012). ). No significant differences in digestibility and animal health as well as no biological relevant unintended effects on performances of animals and composition of food of animal origin were found. Nutritional feeding study on poultry with MON 89034 x NK603 was reported by Taylor et al (2007).

Table 2: Results from fattening experiment

Parameter EG1

EG2

EG3

EG4-MON 89034 x NK603

EG5- DKC 5143

N 24 24 24 24 24 Daily weight gain g/day 32.2 39,0 41.0 34.3 31.4 Feed conversion ratio in g/g 3.2 3.5 3.4 3.4 3.4 Carcass yield in % 57.6 57.8 57.4 57.8 57.3 Mortality 1 4 4 2 1

Table 3: Physico-chemical characteristics of meat (MLD) 24 h post mortem (x±SD)

Parameter (g/100g MLD) EG1

EG2

EG3

EG4-MON 89034 x NK603

EG5- DKC 5143

Content of water 75.8±0.2 75.7±0.2 75.8±0.1 75.7±0.3 75.9±0.2 Total proteins 21.6±0.1 21.8±0.2 21.7±0.5 21.8±0.4 21.5±0.1 Content of fat 1.6±0.2 1.6±0.1 1.5±0.1 1.5±0.1 1.0±0.1 Energetic value (kJ/100g) 421.5±5.8 423.1±5.9 419.3±4.0 422.2±1.8 421.8±9.5 pH24 6.0±0.1 5.93±0.23 5.82±0.13 6.01±0.07 5.85±0.01 Electrical conductivity (μS) 1.4±0.9 2.1±0.8 1.7±0.2 1.0±0.3 1.0±0.1 Colour L 51.6±6.2 50.2±3.0 50.2±2.7 50.0±1.6 49.9±3.2 A 0.8±0.3 1.00±0.7 0.7±0.3 0.8±0.4 0.6±1.2 B 4.6±0.3 7.5±0.2 7.3±0.7 6.4±0.6 6.7±1.2 Water holding capacity 37.9±5.8 42.1±7.9 33.9±4.8 34.6±5.2 36.9±2.8 Ash 1.00±0.1 1.0±0.1 1.0±0.1 1.1±0.1 1.0±0.1

Table 4: Fatty acid profile inmeat (MLD) of rabbits (x±SD)

Parameters (n=6)

EG1

EG2

EG3-

EG4-MON 89034 x NK603

EG5- DKC 5143

Σ SFA (%) 34.6±2.8 38.6±3.9 44.0±1.8 41.6±2.0 40.9±5.6 C16:1n-7 3.3±0.6 4.1±0.8 3.6±0.4 3.1±0.9 3.2±0.7 C18:1n-9 29.3±2.7 26.7±1.3 23.6±0.8 26.3±2.5 26.3±3.5 Σ MUFA (%) 32.7±3.0 30.9±1.4 27.3±0.6 29.4±2.7 29.2±3.5 C18:2n-6 24.3±1.5 24.1±2.2 23.4±1.7 23.6±1.3 24.1±2.9 C18:3n-3 5.9±0.4 5.5±1.3 4.5±0.3 4.6±0.2 4.8±0.8 C20:4n-6 1.2±0.2 1.00±0.3 0.8±0.1 0.8±0.2 0.7±0.3 Σ PUFA (%) 32.6±2.0 30.6±3.7 28.6±1.7 29.0±1.5 29.6±3.6 PUFA/ SFA 0.89 0.79 0.64 0.72 0.72 SFA: saturated fatty acids, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty acids Σ SFA = C10:0, C12:0, C14:0, C16:0, C18:0, C20:0, C22:0; Σ MUFA = C16:1n-7, C18:1n-9 Σ PUFA = C18:2n-6; C18:3n-3; C20:4n-6

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The fatty acids composition in MLD muscles is shown in Table 4. The fatty acid patterns of MLD mus-clesdemonstrated similar proportions at feeding of GM maize and isogenic maize and coincide with the results of Aulrich et al. (2001).

Conclusion Feed intake, feed conversion, carcass yield and meat quality were not different for rabbits fed with GM maizeMON 89034 x NK603 and rabbits fed with isogenic and conventional maize. The diet containing MON 89034 x NK603 was nutritionally equivalent to the diets containing the isogenic or reference maize when fed to rabbits.

Acknowledgement This article was written during realization of the project "BELNUZ 26220120052" supported by the Operational Programme Research and Development funded from the European Regional Development Fund.

References Aulrich, K., Flachovsky, G., Daenicke, R., Halle, I. 2001. Bt- mais in der Tierernährung. Ernährungsforschung, 46, 13-20.

Flachowsky, G., Schafft, H., Meyer, U. 2012. Animal feeding studies for nutritional and safety assessments of feeds from genet-ically modified plants: a review. Journal fur Verbraucherschutz und Lebensmittelsicherheit (Journal of Consumer Protection and Food Safety) 7,179-194

Hašek, A., Palanská, O., 1976. Determination of water holding in the meat by apparatus with constant pressure. Poultry Indus-try, 18, 228-233.

Journal of the Ministry of Agriculture of the Slovak Republic No. 8, 2010.

Juárez, M., Polvillo, O., Conto, M., Ficco, A., Ballico, S., Failla, S., 2008. Comparison of four extraction/methylation analytical methods to measure fatty acid composition by gas chromatography in meat. Journal of Chromatography A., 1190, 327-332

Taylor, M., Lucas, D., Nemeth, M., Davis, S., Hartnell, G. 2007. Comparison of Broiler Performance and Carcass Parameters When Fed Diets Containing Combined Trait Insect-Protected and Glyphosate-Tolerant Corn (MON 89034 × NK603), Control, or Conventional Reference Corn. Poultry Science. 86,1988–1994

Corresponding author Mária Chrenková National Agricultural and Food Centre, Research Institute for Animal Production Nitra, Hlohovecká 2, 951 41 Lužianky, Slovak Republic E-mail: chrenkova(at)cvzv.sk

Greiling et al.: Vergleich zwischen zwei unterschiedlichen Fütterungsstrategien mit gleichem Fütterungsniveau bei Karpfen (Cyprinus carpio) in Bezug auf Leistungsparameter und Wachstumshomogenität


Vergleich zwischen zwei unterschiedlichen Fütterungsstrategien mit gleichem Fütterungsniveau bei Karpfen (Cyprinus carpio) in Bezug auf Leistungsparameter und Wachstumshomogenität

Alexander M. Greiling, Kay Lübke und Helmut Wedekind Bayerische Landesanstalt für Landwirtschaft, Institut für Fischerei, DE

Abstract The aim of this study was to investigate the effect of two different feeding regimes on growth param-eters of common carp reared in a recirculation system over a duration of 8 weeks. The daily ration was set at a feeding level of 1.2 % BW in both treatments and adjusted weekly. In one treatment the feed was offered as a single ration, whereas in the other the feed was offered continuously over a period of 6 hours. The groups fed continuously had a significantly better specific growth rate (0.89 ± 0.07 % fish-1 d-1) and a lower feed conversion ratio (1.15 ± 0.09) when compared to the other groups (0.68 ± 0.05 % fish-1 d-1 and 1.51 ± 0.11, respectively). However the fish in groups fed a single ration exhibited a more homogenous growth as compared to the continuously fed groups.

Einleitung Ein Forschungsschwerpunkt im Bereich Fischfuttermittel ist das Erschließen neuartiger Proteinquellen als Fischmehlersatz, insbesondere für karnivore Spezies wie Lachse und Forellen (Naylor et al. 2009). Hierbei ist der Einsatz alternativer pflanzlicher Rohstoffeein zentraler Aspekt der Forschung (Dalsgaard et al. 2012, Drew 2007, Francis et al. 2001, Gatlin 2007).Weniger untersucht wurde bisher in wie weit verschiedene Fütterungsstrategien, wie die Darbietung und das Fütterungsregime, Einfluss auf das Wachstum und die Futterverwertung nehmen. Hierbei standen in der Regel Fischarten im Vorder-grund, die intensiv gehalten werden und vollständig auf Zufütterung angewiesen sind (bspw. Lachse und Forellen) (Jørgensen & Jobling 1992, Sveier & Lied 1998, Wedekind & Radandt 2000, Yamamoto et al. 2002). Auch in Bezug auf Fischarten die bisher vorwiegend in Teichen produziert wurden und immer häufiger in intensiven Produktionssystemen, wie Kreislaufanlagen, gezüchtet werden ist diese Fragestellung von wachsendem Interesse (Almazán-Rueda et al. 2004).Wenig ist hingegen darüber bekannt wie normalerweise extensiv, auf Basis von Naturnahrung, gezüchtete Spezies, beispielsweise der Karpfen (C. carpio), auf unterschiedliche Fütterungsstrategien unter intensiven Aufzuchtbedingun-gen reagieren. Dabei werden Karpfen in Deutschland teilweise in Kreislaufanlagen vorgestreckt, um der Fraßfeindproblematik in Teichen (z.B. durch Kormorane, Reiher u.ä.) entgegen zu wirken und robuste, größere Satzfische für den Teichbesatz zu produzieren.Der Karpfen ist aufgrund seiner langen Nutzung als Aquakulturart sehr gut untersucht. Trotz des umfassenden wissenschaftlichen Kenntnis-stands zur Ernährung dieser Fischart bestehen, insbesondere in der Aufzucht unter Aquakulturbedin-gungen, Unklarheiten bei der Fütterung mit Trockenmischfuttermitteln. Da Karpfen keinen Magen besitzen wird vermutet, dass ein günstiges Wachstum und eine gute Futterverwertung erreicht wer-den kann, wenn die Futterration verteilt über mehrere Stunden hinweg verabreicht wird. In der expe-rimentellen Untersuchung sollten daher zwei verschiedene Fütterungsstrategien verglichen werden. Die kontinuierliche Futtergabe über Futterautomaten soll einer einmal täglichen Fütterung gegenüber-gestellt und die Auswirkungen auf Wachstum und Futterverwertung untersucht werden.



Material und Methoden Es wurde eine Einzelfütterung mit einer kontinuierlichen Fütterung über sechs Stunden hinweg in ei-nem 8-wöchigen Fütterungsversuch bei Karpfen verglichen. Die tägliche Fütterungsrate betrug 1,2 % des Gesamtgewichts der jeweiligen Versuchsgruppe (= beobachtete maximale Futteraufnahme bei einmaliger Fütterung bis zur Sättigung). Jede Versuchsgruppe bestand aus 50 Fischen mit einem An-fangsgewicht von 3,8 ± 0,53 g (MW ± STABW). Bei der Einzelfütterung wurde die gesamte Futter-menge täglich um 9:00 Uhr verfüttert. Bei der kontinuierlichen Fütterung wurden mechanische Bandfutterautomaten so eingestellt, dass die tägliche Futtermenge zwischen 9:00 und 15:00 Uhr gleichmäßig verteilt verfüttert wurde. Es wurde ein kommerzielles Fischfuttermittel mit 45 % Rohpro-tein, 18 % Rohfett, 1,7 % Rohfaser, 7,6 % Asche, 1,0 % Gesamt P und einer Gesamtenergie von 21,5 MJ/kg verfüttert. Einmal wöchentlich wurde das Gesamtgewicht der einzelnen Versuchsgruppen be-stimmt. Auf dieser Basis wurde die Futtermenge entsprechend angepasst. Am Wiegetag erfolgte keine Fütterung.Der Versuch wurde unter kontrollierten Bedingungen in sechs gleichartigen geschlossenen Kreislaufsystemen durchgeführt. Diese bestanden jeweils aus zwei übereinander angeordneten 400 l Aquarien. Das obere war die Produktionseinheit, während das untere als Partikel- und Biofilter fungier-te. Die Produktionseinheiten wurden durch eine wasserdurchlässige Trennwand geteilt, sodass in je-dem Kreislauf beide Fütterungsstrategien untersucht werden konnten. Einmal wöchentlich wurde je 25 % des Altwassers durch Frischwasser ersetzt. Die mittlere Wassertemperatur lag zwischen 22,9 °C und 23,6 °C. Versuchsbegleitend erfolgte eine tägliche Messung von Wassertemperatur, Sauerstoff-gehalt und pH-Wert in den Becken. Einmal wöchentlich wurde zudem der Ammonium-, Nitrit- und Nitratgehalt bestimmt. Die entsprechenden Durchschnittswerte sind in Tabelle 4 aufgeführt. Bis auf den pH-Wert lagen alle Durchschnittswerte im optimalen Bereich für die Karpfenerzeugung, der pH-Wert lag knapp außerhalb des Optimums im eingeschränkten oberen Bereich (Schreckenbach et al. 1987).

Tabelle 4: Übersicht zu den Wasserwerten der Versuchseinheiten Versuchseinheit O2

[mg/l] Wassertemperatur

[°C] pH-Wert NH4

[mg/l] NO2

[mg/l] NO3

[mg/l]

1 7,7 ± 0,5 23,1 ± 0,6 8,5 ± 0,2 0 0,01 ± 0,01 47 ± 9 2 7,7 ± 0,6 23,1 ± 0,6 8,6 ± 0,1 0 0,01 ± 0,01 50 ± 13 3 7,8 ± 0,6 22,9 ± 0,6 8,7 ± 0,1 0 0,03 ± 0,07 53 ± 9 4 7,7 ± 0,6 23,3 ± 0,5 8,6 ± 0,1 0 0,01 ± 0,02 50 ± 0 5 7,7 ± 0,6 23,6 ± 0,6 8,6 ± 0,1 0 0,03 ± 0,02 50 ± 13 6 7,7 ± 0,6 23,5 ± 0,7 8,6 ± 0,1 0 0,03 ± 0,07 53 ± 9

Dargestellt sind Mittelwerte und Standardabweichung,Für Sauerstoffgehalt (O2), Wassertemperatur und pH-Wert: n = 56; Für Ammonium (NH4)-, Nitrit (NO2)- und Nitrat (NO3)-Gehalt: n = 8

Die statistische Analyse erfolgte durch einen Mittelwertvergleich der jeweils sechs Gruppen der beiden Behandlungen. Dazu wurde die Normalverteilung der Daten mit dem Shapiro-Wilk-Test und die Vari-anzhomogenität mit dem Levene-Test bestätigt und dann ein T-Test durchgeführt (p < 0,05) (SPSS Statistics 21, Chicago, USA).

Ergebnisse Über die Versuchsdauer von acht Wochen zeigte sich ein deutliches Auseinanderwachsen der beiden Versuchsgruppen. Zum Versuchsstart lag das Gesamtgewicht der Fische mit einmaliger Fütterung bei durchschnittlich 189,8 g und das der Gruppen mit kontinuierlicher Fütterung bei 189,0 g (Abbil-dung 1). Bereits nach 3 Wochen besaßen die Gruppen mit kontinuierlicher Fütterung im Mittel ein um



12,5 g höheres Gesamtgewicht. Diese Tendenz hielt bis zum Versuchsende an. Nach acht Wochen waren die Gruppen mit kontinuierlicher Fütterung durchschnittlich über 30 g schwerer (Abbildung 1).

Abbildung 1: Gewichtsentwicklung der Fische im Versuchszeitraum. Dargestellt sind die Mittelwerte der Gesamtgewichte aller Gruppen beider Behandlungen (n = 6)

Tabelle 5: Vergleich der Leistungsparameter zwischen einmaliger Fütterung (1_F) und Fütterung über 6 Stunden (6_F) bei gleichem Fütterungsniveau bei Karpfen (C. carpio)

Behandlung Startgewicht [g]

Endgewicht [g]

Zuwachs [%]

FQ 1 SWR 2 [% Fisch-1 Tag -1]

1_F 3,8 ± 0,53 5,55 ± 0,96 a 46,5 ± 4,34 a 1,51 ± 0,11 a 0,68 ± 0,05 a 6_F 3,8 ± 0,53 6,20 ± 1,49 b 63,8 ± 7,01 b 1,15 ± 0,09 b 0,89 ± 0,07 b

1 FQ (Futterquotient) = g Zuwachs / g gefressenes Futter; 2 SWR (Spezifische Wachstumsrate) [% Fisch-1 Tag-1] = 100 x (ln Endgewicht - ln Endgewicht) / Tage. Dargestellt sind Mittelwerte und Standardabweichung der beiden Behandlungen (n = 6), Hochbuchstaben innerhalb einer Spalte zeigen signifikante Unterschiede (p < 0,05)

Demzufolge war auch das Durchschnittsgewicht der Karpfen bei der einmaligen Fütterung mit 5,55 ± 0,96 g signifikant geringer (T=5,347; df=10; p<0,001) als bei der kontinuierlichen Fütterung (6,20 ± 1,45 g) (Tabelle 5). Die spezifische Wachstumsrate der Versuchsfische betrug bei kontinuierli-cher Fütterung im Mittel 0,89 ± 0,07 %, gegenüber 0,68 ± 0,05 % (T=6,095; df=10; p<0,001) bei einmaliger Fütterung. Folglich konnte auch ein signifikanter Unterschied im Futterquotienten festge-stellt werden. Dieser lag über den gesamten Zeitraum bei durchschnittlich 1,15 ± 0,09 bei der konti-nuierlichen Fütterung, im Vergleich zu 1,51 ± 0,11 bei einmal täglicher Futtergabe (T=6,152; df=10; p<0,001). Es zeigte sich zum Versuchsende ein stärkeres Auseinanderwachsen der Karpfen bei konti-nuierlicher Futtergabe. Zu Beginn war die relative Standardabweichung der Einzelgewichte in den Versuchsgruppen im Mittel mit 14 % in beiden Ansätzen noch gleich groß. Am Versuchsende ergab sich durch die kontinuierliche Fütterung jedoch eine signifikant höhere (T=9,325; df=10; p<0,001) Streuung der Einzelgewichte (17,23 ± 1,07 % respektive 23,29 ± 1,18 %).

Diskussion Bei der kontinuierlichen Fütterung kam es durch eine signifikant bessere Futterverwertung zu einem gesteigertem Wachstum gegenüber der einmaligen Fütterung. Dies spiegelte sich bei den eingangs genannten Studien bei Lachsen und Forellen nicht wieder. Anscheinend führte die größere, gleichzeitig aufgenommene Futtermenge bei der Einzelfütterung dazu, dass die magenlosen Karpfen nicht alle



Futterbestandteile optimal verdauen bzw. aufschließen konnten. Jedoch zeigte sich auch, dass durch die kontinuierliche Automatenfütterung die Karpfen etwas stärker auseinanderwuchsen. Eine Erklä-rung hierfür könnte sein, dass sich stärkere Fische vermehrt unter dem Automaten aufhielten und schwächere Tiere vom Futter wegdrängten. Wird alles Futter auf einmal verabreicht, ist aufgrund der dargebotenen Menge für alle Fische der Zugang gleich.

Schlussfolgerung Aus den vorliegenden Ergebnissen lassen sich verschiedene Empfehlungen ableiten. Wenn das Ziel ein möglichst homogener Fischbestand ist, z.B. für die Satzfischproduktion, empfiehlt sich eine einmalige Fütterung. Wenn dies zweitrangig ist, aber eine optimale Futterverwertung und ein maximierter Zu-wachs angestrebt werden, ist eine kontinuierliche Fütterung zu empfehlen. In Bezug auf Reinigungs-aufwand und nährstoffbedingte Wasserbelastung unter intensiven Mastbedingungen in Kreislaufanlagen sollte dies definitiv berücksichtigt werden. In wie weit sich die untersuchten Parame-ter durch mehrere Einzelgaben über den Tag verteilt oder eine kontinuierliche Fütterung über einen längeren Zeitraum beeinflussen lassen, kann Gegenstand zukünftiger Untersuchungen sein. Ebenso wäre zu ermitteln, in welchem Maße ein höheres Fütterungsniveau die untersuchten Leistungsparame-ter und die Wachstumshomogenität beeinflusst.

Literatur Almazán-Rueda, P., Schrama, J. W., & Verreth, J. A. (2004). Behavioural responses under different feeding methods and light regimes of the African catfish (Clarias gariepinus) juveniles. Aquaculture, 231(1), 347-359.

Dalsgaard, J., Verlhac, V., Hjermitslev, N. H., Ekmann, K. S., Fischer, M., Klausen, M., & Pedersen, P. B. (2012). Effects of exogenous enzymes on apparent nutrient digestibility in rainbow trout (Oncorhynchus mykiss) fed diets with high inclusion of plant-based protein. Animal feed science and technology, 171(2), 181-191.

Drew, M. D., Borgeson, T. L., & Thiessen, D. L. (2007). A review of processing of feed ingredients to enhance diet digestibility in finfish. Animal Feed Science and Technology, 138(2), 118-136.

Francis, G., Makkar, H. P., & Becker, K. (2001). Antinutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish. Aquaculture, 199(3), 197-227.

Gatlin, D. M., Barrows, F. T., Brown, P., Dabrowski, K., Gaylord, T. G., Hardy, R. W., & Overturf, K. (2007). Expanding the utilization of sustainable plant products in aquafeeds: a review. Aquaculture Research, 38(6), 551-579.

Jørgensen, E. H., & Jobling, M. (1992). Feeding behaviour and effect of feeding regime on growth of Atlantic salmon, Salmo salar. Aquaculture, 101(1), 135-146.

Naylor, R. L., Hardy, R. W., Bureau, D. P., Chiu, A., Elliott, M., Farrell, A. P., Forster, I., Gatlin, D. M., Goldburg, R. J., Hua, K., & Nichols, P. D. (2009). Feeding aquaculture in an era of finite resources. Proceedings of the National Academy of Sciences, 106(36), 15103-15110.

Schreckenbach, K., Steffens, W., & Zobel, H. (1987). Technologien, Normen und Richtwerte zur Fischproduktion. Institut für Binnenfischerei, Berlin.

Sveier, H., & Lied, E. (1998). The effect of feeding regime on growth, feed utilisation and weight dispersion in large Atlantic salmon (Salmo salar) reared in seawater. Aquaculture, 165(3), 333-345.

Wedekind, H. & Radandt, U. (2000). Fütterungshäufigkeit und Wachstum bei Regenbogenforellen. AUF AUF, Aquakultur und Fischereiinformation Baden-Württemberg, Heft 2: 6-8.

Yamamoto, T., Shima, T., Furuita, H., & Suzuki, N. (2002). Influence of feeding diets with and without fish meal by hand and by self-feeders on feed intake, growth and nutrient utilization of juvenile rainbow trout (Oncorhynchus mykiss). Aquaculture, 214(1), 289-305.

Autorenanschrift Alexander M. Greiling Bayerische Landesanstalt für Landwirtschaft - Institut für Fischerei Weilheimer Straße 8 82319 Starnberg, Germany E-Mail: alexander.greiling(at)lfl.bayern.de

Winkler et al.: Determination of polyphenol and crude nutrient content and nutrient digestibility of dried and ensiled white and red grape pomace cultivars


Determination of polyphenol and crude nutrient content and nu-trient digestibility of dried and ensiled white and red grape pomace cultivars

Anne Winkler1, Fabian Weber2, Robert Ringseis3, Klaus Eder3 and Georg Dusel1 1 University of Applied Science Bingen, DE 2 Institute Food Technology and Biotechnology, University Bonn, DE 3 Institute of Animal Nutrition and Nutrition Physiology, Justus-Liebig-University Gießen, DE

Abstract The present study aimed to determine the nutrient and energy content of fresh and ensiled grape pomace (GP) from different grape varieties (Germany), and to estimate the feed value of dried white, dried red and ensiled white GP by calculating nutrient digestibility and the content of metabolizable energy (ME) and net energy lactation (NEL) measured in sheep as a ruminant model. GP from red cultivars had higher contents of organic matter (OM), crude protein (CP), ether extract (EE), crude fibre (CF), total phenolic contents (TPC) and ME, whereas the concentrations sugar was lower than from white cultivars. Compared with untreated GP, ensiled GP had increased concentrations of CP, ether extract (EE), CF and a higher ME content and markedly decreased concentrations of sugar and TPC. The concentrations of dry matter and OM were not different between ensiled and fresh GP. Compared with dried GP, ensiled GP had a higher nutrient digestibility (OM; CP; CF; neutral detergent fibre [NDF]; acid detergent fibre [ADF]) and higher energy values (ME; NEL). The digestibility of OM, CP, EE and CF and the energy content were higher for dried red than for dried white GP, whereas the digestibility of NDFOM and ADFOM was lower for dried red than dried white GP. In conclusion, the re-sults show that both red and white GP are suitable dietary sources for enrichment with TPC. Com-pared with drying, ensiling of GP improves the feeding value and is a good possibility of preserving GP for ruminant feeding.

Introduction Germany has a total vineyard area of 102,000 ha on which a total volume of 8.4 million hectoliters wine was produced in 2013 (German Wine Institution 2014). During winemaking, considerable amounts of GP are produced as the most important by-product. GP consists mainly of skins (about 80% of wet weight, Jiang et al. 2011), but contains also varying amounts of seeds and stems. From a nutritional point of view, GP is interesting because it is an abundant and inexpensive source of poly-phenols (Toaldo et al. 2013), a class of secondary plant metabolites (especially flavonoids), whereby the effects of flavonoids are largely based on their anti-inflammatory activities through modulating the activities of key regulators of inflammation, and cell protection. In contrast to humans, in farm ani-mals the potential health-promoting effects of flavonoids have been investigated scarcely (Santos et al. 2014), despite the fact that inflammatory processes that promote metabolic problems and impair production are a frequent phenomenon in farm animals and thus dietary strategies to combat inflam-matory processes are of great demand. Although it has been already shown that GP, either in the fresh, dried or ensiled form, has a low energy content and can be included in feeding rations for rumi-nants, especially when fed near to maintenance (Baumgärtel et al. 2007), little is known about the feeding value and the polyphenol content of GP from different grape varieties. Thus, the present



study had two objectives: (1) to determine the content of crude nutrients, TPC and energy of GP from different grape varieties (Rhineland-Palatinate/Germany); and (2) to determine the feed value of dried white, dried red and ensiled white GP for sheep by calculating nutrient digestibility and the contents of ME and NEL.

Materials and methods a) Determination of the contents of crude nutrients and TPC of different GP cultivars From the vintage 2012, 5 randomly selected bunches of wine grape cultivars were obtained from 10 different wineries within the winegrowing area “Neustadt an der Weinstraße” (Rhineland-Palatinate, Germany). The selected white grape varieties were Pinot blanc (n = 4) and Riesling (n = 6); the red grape varieties were Dornfelder (n = 5), Pinot noir (n = 5) and Portugais bleu (n = 2). Sample preparation. Each fresh GP sample was divided into three portions and frozen at −24°C until processing and analysis. After thawing at ambient temperature, one portion of each GP sample was dried for 48 h at 60°C, milled and analyzed for their crude nutrients according to the official analytic methods (VDLUFA 2007). The second portion (~4 kg) of each GP sample was divided in two portions of approximately 2 kg each. One portion was mixed with 6.4 g of a chemical ensiling additive (47% sodium chloride, 30% sodium benzoate, 20% calcium formate, 3% sugar beet molasses), put into a plastic bag and sealed hermetically in 5 l buckets. The other portion was prepared similarly without using an ensiling agent. Finally, the closed plastic bags were weighted with stones, stored for 8 weeks and then partially dried at 60°C, ground and analyzed for their crude nutrients. The other part of en-siled samples and the third portion of each frozen GP were lyophilized and milled. Extraction of TPC from the obtained GP powder was carried out using an ASE 350 (Accelerated Solvent Extraction Sys-tem, Dionex Corporation, Thermo Scientific, Waltham, MA) as described by González-Centeno et al. (2013). The TPC of the extracted GP cultivars was measured spectrophotometrically in 96-well plates according to the Folin–Ciocalteu method (Singleton and Rossi 1965). The TPC was calculated using gallic acid (GA) as a standard for calibration and expressed as mg GA/g GP. The presented TPC con-tents are means of six measurements. Calculations and statistical analysis. Statistical analysis was carried out using analysis of variance and comparison of means between groups by Tukey’s HSD test with Statistica software (Version 8.0, StatSoft). Differences were considered as significant at p < 0.05. All experimental data represent means and standard deviations (mean ± SD). For each grape variety, three replicates were used for statistical evaluation (untreated, ensiled with an ensiling agent, ensiled without an ensiling agent). b) Determination of nutrient digestibility and energy content of dried and ensiled GP The digestibility trials were conducted in accordance with animal welfare legislation and were ap-proved by the Provincial Government of Coblenz, Germany. The digestibility trials (Trial 1: dried white GP; Trial 2: dried red GP; Trial 3: ensiled GP) were carried out according to the guidelines of Gesell-schaft für Ernährungsphysiologie (GfE) (1991) at the Ruminant Research Unit of the University of Applied Sciences Bingen. Origin and preparation of GP. The dried white GP (Riesling) and dried red GP (Pinot noir) for Trials 1 and 2, respectively, were obtained from a winery in Bingen (Rhineland-Palatinate). Because seeds and skins were separately supplied, they were blended at a ratio of 70:30 before starting the trials. The GP (Riesling) used for Trial 3 was obtained from a winery in Bingen (Rhineland-Palatinate) and imme-diately ensiled in a container without using an ensiling agent. Animal and housing. Eight male Shropshire sheep were used for the three trials in a 2 (animals) x 4 (treatments) cross-over design (n = 8) with four replicates per dietary treatment. Sheep were kept individually in metabolic cages with ad libitum access to water. They were randomly assigned to the treatment groups and received diets containing decreasing amounts of chopped hay (85%, 70%, 55%



and 40%) at the expense of increasing amounts of GP (15%, 30%, 45% and 60%). Thereby, the linearity of digestibility response of the test diets at different supplementation levels could be illustrat-ed by using linear regression analysis (Westreicher-Kristen et al. 2013). The sheep were fed twice a day at 08:00 and 17:00 h. The amount of feed was calculated to achieve a daily ME intake of the 1.2-fold of the mean maintenance requirement (0.9-1-1 kg/d; GfE 1991). According to GfE guidelines (GfE 1991), sheep were adapted to experimental diets, conditions and facilities for 14 d (week 1 in stable, week 2 adaptation to metabolic cages) prior to the sampling period. During the sampling period of 7 d, faeces were collected daily, weighted and sub-sampled (from each sheep on each treatment) by taking a 30% aliquot and frozen at −24°C for later analysis. Sub-samples of hay and GP were taken once a day and frozen at −24°C until analysis. At the end of each digestibility trial, all feed samples were pooled, homogenized and analyzed for the content of crude nutrients, gross energy and TPC. Analysis. The GP and hay batches (dry basis) of each trial were dried at 60°C for 48 h. The faeces samples (wet basis) were lyophilized and samples of feed and faeces were ground and analyzed for DM, crude nutrients, NDF, ADF and acid detergent lignin (ADL) according to the official analytical methods (VDLUFA 2007). Gross energy was determined using a bomb calorimeter. The TPC of the previously extracted GP cultivars was quantified as well. The analysed contents of crude nutrients, gross energy and TPC of dried white GP, dried red GP, ensiled GP and hay used in the three trials are listed below: trial 1 (dried white GP): DM=907 g/kg, OM=932 g/kg DM, CP=109 g/kg DM, EE=58 g/kg DM, CF=241 g/kg DM, Ash=68 g/kg DM, NDF=424 g/kg DM, ADF=437 g/kg DM, ADL=320 g/kg DM, TPC=56 mg GA/g GP, gross energy=21.3 MJ/kg DM; trial 2 (dried red GP): DM=901 g/kg, OM=967 g/kg DM, CP=112 g/kg DM, EE=50 g/kg DM, CF=214 g/kg DM, Ash=33 g/kg DM, NDF=341 g/kg DM, ADF=346 g/kg DM, ADL=236 g/kg DM, TPC=87 mg GA/g GP, gross energy=21.7 MJ/kg DM; trial 3 (ensiled white GP): DM=966 g/kg, OM=918 g/kg DM, CP=109 g/kg DM, EE=20 g/kg DM, CF=296 g/kg DM, Ash=82 g/kg DM, NDF=401 g/kg DM, ADF=448 g/kg DM, ADL=301 g/kg DM, TPC=46 mg GA/g GP, gross energy=18.9 MJ/kg DM; hay: DM=844 g/kg, OM=928 g/kg DM, CP=94 g/kg DM, EE=9 g/kg DM, CF=296 g/kg DM, Ash=69 g/kg DM, NDF=622 g/kg DM, ADF=363 g/kg DM, ADL=53 g/kg DM, TPC=not determined, gross energy=19.1 MJ/kg DM. Calculations. The energy values [MJ/kg DM] of GP and hay were calculated on the basis of digestible nutrients as suggested by the GfE (2001). The digestibility coefficient for nutrients and energy was calculated for each diet and animal based on the differences between quantities of nutrient intake and output (GfE 1991). The nutrient digestibilities were calculated by multiple linear regression based on a common intercept model and individual slopes for test sources according to Kluth et al. (2005) using the equation Y = a + bn * xn,where Y is the digestibility of GP (at an inclusion rate of 100%), a is the intercept, bn is the slope for test source n and xn is a given concentration originating from test source n [%]. To calculate the digestibility of GP at a dietary inclusion rate of 100%, the equation was ex-trapolated to x = 100.

Results and discussion a) Determination of the contents of crude nutrients and TPC of different GP cultivars Concerning cultivar, it was observed that red cultivars Pinot noir, Dornfelder and Portugais bleu had higher concentrations of OM, CP, CF and lower concentrations of sugar than the white cultivars Pinot blanc and Riesling, but these effects were significant only for specific cultivars (p < 0.05). No effect of cultivar was found with regard to the concentrations of DM and EE. Similar results were reported for GP from the Saale-Unstrut region in Germany (Baumgärtel et al. 2007) and from a Turkish winegrow-ing area (Basalan et al. 2011). Regarding colour, it was observed that fresh red GP had higher con-centrations of OM, CP, EE, CF and lower concentrations of sugar than the fresh white GP (p < 0.05). With regard to treatment, it was found that the GP ensiled with an ensiling agent had increased con-centrations of CP, EE, CF and markedly decreased concentrations of sugar compared to untreated GP (p < 0.05). The GP ensiled without additive had only increased concentrations of EE and markedly



decreased concentrations of sugar compared to untreated GP (p < 0.05). The concentrations of DM and OM were not different between ensiled (with and without additive) and fresh GP. The TPC of GP was influenced significantly by treatment and colour (Table 1). The ensiled GP had an approximately 50% lower TPC than the fresh GP (p < 0.05), without effect of the ensiling additive. A decrease in the content of TPC and/or polyphenol classes, like tannins, anthocyanins or saponins, during ensiling of GP has been also reported by Pirmohammadi et al. (2007) and Cardona et al. (2009) and is ascribed to polymerisation and oxidation of TPC, especially tannins (Ribereau-Gayon et al. 2006). Thus, our findings suggest that an enrichment of TPC in ruminant diets with the aim of inducing health-promoting effects associated with these compounds, e.g. anti-inflammatory effects, can be achieved at low inclusion levels (1%, DM basis) particularly with fresh or dried GP, rather than with ensiled GP. In our study, the TPC ranged between 44 mg GA (Riesling) and 65 mg GA (Portugais bleu) per g GP. Although the differences between cultivars were not significant, the average TPC of all red GP (58 mg GA/g GP) was significantly higher than of all white GP (48 mg GA/g GP). This finding is in agreement with investigations of Baumgärtel et al. (2007). In contrast to these results, De La Cerda-Carrasco et al. (2014) reported higher concentrations of TPC in white GP (Sauvignon blanc, Chardonnay) than in red GP (Cabernet sauvignon, Carmenere) obtained from a winegrowing area in Chile, being consistent with a more complete extraction of polyphenols from berries during red winemaking process. These variations in TPC content might be due to regional differences in the extraction process in winemaking and differences in the concentration of TPC between grape varieties.

Table 1: Analyzed crude nutrients, TPC and ME of selected GP

n DM OM CP EE CF sugar TPC

(g/kg) (g/kg DM)

(g/kg DM)

(g/kg DM)

(g/kg DM)

(g/kg DM)

(mg GA1)/ g

Effect of cultivars 4 Pinot blanc 365±36 879ab±31 89a±7 31±3 158a±24 318b±109 54±13 6 Riesling 363±33 860a±36 93a±9 27±5 175a±24 283b±60 44±11 5 Dornfelder 374±66 943b±23 138b±11 36±6 255b±49 69a±99 57±23 5 Pinot noir 421±59 912ab±60 102a±33 38±10 217ab±42 94a±146 56±16 2 Portugais bleu 395±104 953ab±19 99ab±2 32±9 244ab±14 213ab±1 65±18 Effect of colour

10 White 363±32 868a±34 91a±8 29a±5 168a±24 297b±80 48a±6 12 Red 397±66 932b±43 117b±28 36b±8 237b±43 103a±119 58b±13 Effect of treatment

22 Untreated 382±55 903±50 105a±25 33a±7 206a±50 191b±141 56b±11 22 Ensiled without EA2) 354±49 907±41 109ab±50 41b±6 231ab±24 0.7a±0.5 32a±11 22 Ensiled with EA 371±51 921±27 130b±15 43b±7 235b±24 4.8a±35 29a±12 1) GA= gallic acid; 2) EA= ensiling agent; ab Means not sharing the same superscript are significantly different (p < 0.05)

b) Determination of nutrient digestibility and energy content of dried and ensiled GP Digestibility of nutrients estimated by multiple regression analysis using a common intercept and indi-vidual slopes for test sources, and ME and NEL values calculated on the basis of digestible nutrients are shown in Table 2 and 3. The common intercept represents the digestibility of nutrients from a ration consisting of 100% hay. The digestibility of OM and the crude nutrients CP, CF, NDFOM and ADFOM was higher for ensiled GP (Trial 3) than for the dried GP (Trials 1 and 2), whereas the digesti-bility of EE did not differ between ensiled and dried GP. The estimated digestibility of OM, CP, EE and CF was slightly higher for dried red GP (Trial 2) than for dried white GP (Trial 1), except NDFOM and ADFOM. Both NDFOM and ADFOM were digested at lower rates from the test diets containing dried red GP (Trial 2) than dried white GP (Trial 1). According to the differences in nutrient digestibility between



ensiled GP and dried GP, the calculated ME and NEL values were highest for the ensiled GP. The ob-served improvement of OM, CP and fibre digestibility by ensiling can be largely explained by the deg-radation of cell structure in the course of microbial fermentation, thereby increasing the release of anti-nutritive tannins from GP (Ribereau-Gayon et al. 2006) and consequently increasing the OM di-gestibility of ensiled GP. Calculated ME and NEL values were approximately 0.8 MJ per kg DM higher for dried red GP than for dried white GP (Table 3).

Table 2: Nutrient digestibility of dried and ensiled Riesling as well as of Pinot noir GP based on common inter-cepts and slopes determined by multiple linear regression analysis

Slope Estimated digestibility (%) Common

intercept Trial 1-dried

white GP Trial 2-dried

red GP Trial 3- ensiled

white GP Trial 1- dried

white GP Trial 2-dried

red GP Trial 3- ensiled

white GP OM 52.2±1.48 -0.19±0.05 -0.14±0.01 0.00±0.04 34 39 52 CP 44.0±1.89 -0.30±0.05 -0.27±0.04 -0.17±0.12 14 17 27 EE 39.3±1.91 0.57±0.03 0.57±0.05 0.37±0.07 96 97 76 CF 54.6±1.34 -0.28±0.04 -0.28±0.03 -0.06±0.02 26 27 49 NDF 55.4±3.17 -0.32±0.10 -0.43±0.02 -0.18±0.08 23 12 38 ADF 44.8±3.70 -0.33±0.11 -0.35±0.00 -0.08±0.11 12 10 36

Table 3: Calculated content of ME and NEL (MJ/kg DM)1) of dried and ensiled Riesling as well as of Pinot noir GP

Hay Trial 1- dried white GP Trial 2- dried red GP Trial 3- ensiled white GP ME (MJ/kg DM) 7.22 5.76 6.54 7.36 NEL (MJ/kg DM) 4.20 3.13 3.64 4.16 1) Based on digestible (d) nutrients, equations according to GfE (2001); ME [MJ/kg DM] = 0.0312 · dEE [g/kg DM] + 0.0136 · dCF [g/kg DM] + 0.0147 · (dOM – dEE – dCF) [g/kg DM] + 0.00234 · CP [g/kg DM]; NEL [MJ/kg DM] = 0.6 {1 + 0.004 · (q – 57) · ME [MJ/kg DM]}; GE [MJ/kg DM] = 0.0239 · CP [g/kg DM] + 0.0398 · EE [g/kg DM] + 0.0201 · CF [g/kg DM] + 0.0175 · NfE [g/kg DM]; q = {ME [MJ/kg DM]/ GE [MJ/kg DM]} · 100.

Conclusion The present data suggest that both red and white GP are suitable dietary sources for the enrichment with TPC. Compared with drying, ensiling improves the feed value of GP and is a good possibility of preserving the seasonally produced by-product of winemaking. Despite the fact that ensiling causes a 50% loss of TPC content of GP, ensiled GP, which has a low energy content comparable with straw of wheat and oat, might replace other low-quality roughages for ruminants. In addition, our data clearly show that a distinction between white and red GP is required to calculate the ME content of GP cor-rectly.

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Corresponding author M. Sc. Anne Winkler Abteilung: Tierernährung Fachhochschule Bingen, Fachbereich 1- Life Science and Engineering Berlinstrasse 109, 55411 Bingen am Rhein E-mail: a.winkler(at)fh-bingen.de

Starkute et al.: The influence of solid state and submerged fermentation with Pediococcus pentosaceus KTU05-8, KTU05-9 and KTU05-10 strains on the free amino acids profile in lupine seeds


The influence of solid state and submerged fermentation with Pediococcus pentosaceus KTU05-8, KTU05-9, and KTU05-10 strains on the free amino acids profile in lupine seeds

Vytaute Starkute1, Elena Bartkiene1,Vadims Bartkevics2,3, Daiva Zadeike4 and Grazina Juodeikiene4 1 Department of the Food Safety and Quality, Veterinary Academy, Lithuanian University of Health Sciences, Kaunas, LT 2 Institute of Food Safety, Animal Health and Environment, Riga, LV 3 University of Latvia, Centre of Food Chemistry, LV 4 Department of Food Research and Technology, Kaunas University of Technology, Kaunas, LT

Abstract The aim of this study was to investigated the influence of submerged (SMF) and solid state (SSF) fermentation with bacteriocin-like inhibitory substances (BLIS) producing Pediococcus pentosaceus KTU05-8, KTU05-9, and KTU05-10 strains on free amino acids (FAA) profile in lupine seed varieties ‘Vilčiai’, ‘Vilniai’, and new bred hybrid lines No.1700 and No.1701. Results of the ANOVA test indicated that there is a significant effect of lupine varieties, fermentation method, type of microorganisms ap-plied for the fermentation on amino acids profile in lupine seeds and interaction of these factors (F(5.989)=1755.321, P<0.0001). The modeling of FAA concentration by using fermentation of lupine seeds could constitute a valuable contribution to the human and animal diet.

Introduction The amino acids profile is important because it gives information about the quality of the protein of fermented and non-fermented lupine and its nutritional value. Fermentation with lactobacilli increased concentrations of free amino acids in legumes (Curiel et al., 2015; Coda et al., 2015). The essential amino acids play important roles in the body, for example histidine helps in the removal of excess metals from the body, it stimulates the synthesis of collagen, which is the main structural protein in the extracellular space in the various connective tissues in animals, and can be used as a nutritional strategy to prevent loss of muscle after gastric bypass (Katsanos et al., 2016). Threonine is frequently found in the active centers of enzymes, like isoleucine. Methionine plays a special role in protein biosynthesis. The tyrosine by enzymatic oxidation is converted into melanin (Wu et al., 2016) The aim of this study was to investigated the influence of submerged (SMF) and solid state (SSF) fermentation with bacteriocin-like inhibitory substances (BLIS) producing Pediococcus pentosaceus KTU05-8, KTU05-9, and KTU05-10 strains on free amino acids (FAA) profile in lupine seed varieties ‘Vilčiai’, ‘Vilniai’, and new bred hybrid lines No.1700 and No.1701.

Material and methods Materials The lupine seed varieties ‘Vilciai’, ‘Vilniai’, and new bred hybrid lines No.1072 and No.1734 were obtained from the Voke Branch of Lithuanian Institute of Agriculture (Voke, Lithuania) in 2015. The



LAB strains Pediococcus pentosaceus KTU05-8, KTU05-9 and KTU05-10, previously isolated from spontaneous rye sourdough, were stored at -80 °C and cultured at 30°C temperature for 48 h in MRS medium (CM0359, Oxoid, Hampshire, United Kingdom) prior to be used. Fermentation of lupine seeds Water content was calculated with reference to moisture content of the raw materials and required humidity of the end product for solid state fermentation (SSF) (moisture content 45 %) and for submerged fermentation (SMF) (moisture content 65%). Fermentation was carried out for 48 hours at optimal temperature for Pediococcus pentosaceus KTU05-8, KTU05-9 and KTU05-10 cultivation (35 °C). Six different samples from each plant were made using different pediococci strains and different fermentation technologies (SSF and SMF). Control samples were not fermented lupine seeds. Determination of free amino acids (FAA) Free amino acids (FAA) were analyzed by gas chromatography with flame ionization detection after the ion-exchange solid phase extraction and chloroformate derivatization using EZ-Faast technology (Phenomenex). Standard solutions of the amino acids alanine (ALA), glycine (GLY), valine (VAL), leucine (LEU), isoleucine (ILE), threonine (THR), serine (SER), proline (PRO), asparagine (ASP), methionine (MET), glutamine (GLU), phenylalanine (PHE), lisine (LYS), histidine (HIS), and tyrosine (TYR), in addition of the internal standard (NVAL). Samples (1.00 g) were weighed in 15 mL polypropylene test tubes with screw caps and mixed with 7.5 mL of 0.1 M HCl, and subjected to ultrasonification in a water bath (t = 40 °C) for 15 minutes. The mixture was shaked and then centrifuged (3000 rpm, 15 min). An 2.5 mL aliquot of the mixture was transferred into another 15 mL polypropylene screw cap test tube and 7.5 mL of deionized water was added to 10 mL volume. Samples were then stored at -80 °C until analysis. The derivatized amino acids were analyzed using a GC-FID instrument (Agilent; 6890N) equipped with an auto-sampler (Agilent; 7683 Series). Aliquots of the derivatized amino acids (2 μL) were injected at 1:15 split ratio at 250 °C into a Zebron column (ZB-AAA, 10 m, 0,25 mm in diameter) programmed from 110-320 °C at 32 °C/min. Helium was used as a carrier gas at 1.5 mL/min in constant flow mode and nitrogen was used as a make-up gas. The detector temperature was 320 °C. Five different standard solutions with different concentrations (from 50 to 200 nmol/μL) of amino acid standards were used for the calibration of gas chromatograph.

Statistical analyses All analytical experiments were carried out in triplicate. Data were subjected to analysis of variance (ANOVA) using statistical package SPSS for Windows (Ver.15.0, SPSS, Chicago, Illinois, USA). The calculated mean values were compared using Duncan’s multiple range test with significance defined at P ≤ 0.05. In order to evaluate an influence of three different factors (fermentation method, different lupine seeds hybrids and application of different pediococci) and their interaction on free amino acids (FAA) in the lupine samples, the statistical analysis was performed using the three-way analysis of variance (ANOVA) and the Tukey HSD test as post-hoc test (statistical program R 3.2.1).

Results Essential and non-essential free amino acids (FAA) content (%) from extracted amino acids in submerged (SMF) and solid state (SSF) fermented lupine seeds is given in Table 1 and Table 2, respectively. In compare fermented and non fermented ‚Vilciai‘ variety lupine seeds, the content of certain essential amino acids (such as VAL, LEU, ILE, MET), were elevated in the SMF lupine. However, some essential amino acids (HIS), decreased in concentration appr. 50 percent in both (SSF and SMF) conditions. Also, it was found decreasing of some non-essential amino acids in SMF ‚Vilciai‘ variety lupine (GLY, ASP, GLU). In compare amino acids profile of SMF and SSF ‚Vilciai‘ variety lupine



samples, SMF was more effective, and higher increasing in amino acids concentration were found. Similar tendencies were found in ‚Vilniai‘ variety lupine seeds. The content of VAL, THR, and MET were increased in both (SMF and SSF) samples, however, fermentation in all cases decreased HIS concentration in `Vilniai‘ variety lupine samples. Different tendencies were established of non-essential amino acids changes in fermented ‚Vilniai‘ variety lupine seeds. In all cases, ALA and SER concentrations were increased, however, ASP, GLU, and TYR decreased in ‚Vilniai‘ variety lupine. In compare changes of amino acids concentration during SMF and SSF of new bred lupine hybrid lines No.1072 and No.1734, it was found, that in all cases concentration of essential amino acids THR and MET increased, and HIS decreased, however, non-essential amino acids ALA and SER increased and ASP and GLU decreased.

Table 1: Free amino acids (FAA) content (%) from extracted amino acids in submerged (SMF) and solid state (SSF) fermented lupine seeds

Samples Essential free amino acids VAL LEU ILE THR MET PHE LYS HIS

NF

Vilciai 4.35 8.34 4.99 3.37 0.16 5.52 6.42 5.17 Vilniai 4.32 7.05 4.83 3.45 0.15 4.75 7.16 4.34 No.1072 4.56 7.39 5.10 3.75 0.15 5.10 7.66 4.40 No.1734 4.69 8.43 5.12 4.26 0.17 5.58 5.83 4.30

SMF

Vilciai Pp8 7.66 11.90 5.40 5.29 2.09 6.79 4.50 2.43 Vilciai Pp9 6.39 11.05 4.11 5.06 1.73 5.03 3.81 2.30 Vilciai Pp10 6.48 10.61 4.19 5.11 1.64 4.06 4.18 2.39 Vilniai Pp8 6.75 10.93 4.95 5.92 2.09 6.22 7.64 2.70 Vilniai Pp9 6.68 12.62 4.51 4.98 2.18 5.84 6.15 2.15 Vilniai Pp10 5.98 11.00 4.09 5.42 1.95 3.72 7.03 3.02 No.1072 Pp8 7.32 11.98 4.90 6.37 1.66 6.90 5.10 2.92 No.1072 Pp9 6.43 11.68 3.89 5.50 1.77 5.90 3.82 2.31 No.1072 Pp10 8.22 13.62 5.11 6.64 1.90 5.29 4.71 3.34 No.1734 Pp8 6.80 11.32 4.71 5.78 1.81 6.24 4.22 2.13 No.1734 Pp9 7.29 12.67 4.54 6.78 2.04 5.65 4.09 2.15 No.1734 Pp10 6.37 11.07 4.05 6.22 1.95 3.95 4.93 2.51

SSF

Vilciai Pp8 4.20 8.99 4.71 3.67 0.80 5.31 7.13 3.87 Vilciai Pp9 4.81 8.39 5.53 4.23 0.78 5.43 6.25 3.94 Vilciai Pp10 4.52 9.10 5.09 3.91 0.85 5.64 7.52 4.39 Vilniai Pp8 5.73 5.37 3.63 6.86 0.93 3.28 6.86 2.92 Vilniai Pp9 4.86 4.90 2.59 6.31 0.87 2.43 3.02 3.23 Vilniai Pp10 5.03 5.14 3.43 6.74 1.09 0.98 10.81 4.24 No.1072 Pp8 5.21 4.79 3.17 6.96 0.81 2.95 1.63 4.04 No.1072 Pp9 4.10 3.91 2.26 5.92 0.87 1.61 5.68 2.54 No.1072 Pp10 4.03 2.89 2.10 5.62 0.54 0.64 5.98 3.76 No.1734 Pp8 6.94 7.98 4.85 7.23 1.30 4.44 6.42 2.27 No.1734 Pp9 4.51 3.31 1.97 5.83 0.70 1.49 5.86 3.14 No.1734 Pp10 4.03 3.14 2.25 4.45 0.63 0.82 23.35 2.45

NF – non fermented; SMF – submerged fermentation; SSF – solid state fermentation; Pp8 - Pediococcus pentosaceus KTU05-8; Pp9 - Pediococcus pentosaceus KTU05-9; Pp10 - Pediococcus pentosaceus KTU05-10; ALA – alanine; GLY – glycine; SER - serine; PRO - proline; ASP - asparagine; GLU - glutamine; TYR - tyrosine

Results of the ANOVA test indicated that there is a significant effect of lupine varieties (F(2.999)=13661.969, P<0.0001), fermentation method (F(1.00)=314814.116, P<0.0001), type of microorganisms applied for the fermentation (F(2.00)=86517.403, P<0.0001) and interaction of these factors (F(5.989)=1755.321, P<0.0001) on amino acids profile in lupine seeds. Since the interaction between factors has been determined as statistically significant no further statistical analysis of main factors were performed. Tukey’s HSD test was performed after ANOVA test with purpose to determine



which groups in the results set have notable differences. Results of this test clearly indicates signifi-cant (P<0.0001 in all tests) differences between all three-way interaction groups. Lupin may be a better alternative to soybeans as it is not genetically modified, has lower levels of phytoestrogens and is lower in cost (Villarino et al., 2015). Technological processes may modify the nutritional quality of plant proteins. One of the treatments used to increase health-promoting proper-ties of the products is the fermentation of raw materials. Fermentation of lupin significantly reduce the amount of anti-nutrients and improve the amino acid profile (Voa et al., 2015). Also, fermentation changes the organoleptic characteristics of foods through developing a wide diversity of flavors, aro-mas and textures. Moreover, fermentation may improve nutritional quality through enrichment of food substrates with vitamins, proteins, essential amino acids and essential fatty acids.The amounts of lysine, isoleucine, leucine, phenylalanine and tyrosine in lupin are comparable to the Food and Agricultural Organization (FAO) standards for amino acids of ideal reference protein appropriate for adults (FAO/WHO/UNU., 1991). The main limiting amino acids in lupin are the S-amino acids (methionine and cysteine), valine and tryptophan (Wu et al., 2016).

Table 2: Non-essential amino acids content (%) from extracted amino acids in submerged (SMF) and solid state (SSF) fermented lupine seeds

Samples Non-essential free amino acids ALA GLU GLY PRO SER TYR ASP

NF

Vilciai 2.93 25.88 3.79 4.56 5.11 8.78 10.64 Vilniai 3.14 26.58 3.83 4.35 5.02 10.61 10.43 No.1072 3.37 27.34 4.06 4.67 5.53 5.57 11.34 No.1734 3.99 26.20 4.59 4.70 6.11 5.64 10.39

SMF

Vilciai Pp8 5.01 17.00 3.24 4.69 8.42 8.18 7.40 Vilciai Pp9 4.72 19.25 2.75 4.13 11.44 12.32 5.91 Vilciai Pp10 5.36 18.38 2.99 4.07 11.14 13.04 6.36 Vilniai Pp8 6.09 18.88 3.27 3.56 9.24 3.21 8.57 Vilniai Pp9 6.78 17.46 3.53 3.93 8.56 4.86 9.74 Vilniai Pp10 7.23 15.80 3.38 3.41 10.80 7.52 9.64 No.1072 Pp8 4.21 18.17 3.68 4.24 12.89 4.00 5.66 No.1072 Pp9 4.14 19.34 3.06 4.20 13.52 8.74 5.72 No.1072 Pp10 5.61 14.37 4.32 5.17 11.63 3.64 6.45 No.1734 Pp8 5.71 19.00 3.66 4.51 11.68 5.71 6.73 No.1734 Pp9 6.78 15.85 4.43 5.24 11.96 4.36 6.14 No.1734 Pp10 7.68 17.03 4.33 4.66 13.83 4.58 6.82

SSF

Vilciai Pp8 3.31 28.05 4.11 4.76 6.52 3.68 10.89 Vilciai Pp9 3.48 24.69 4.55 5.33 6.84 3.45 12.31 Vilciai Pp10 3.43 24.03 4.32 5.15 7.15 3.34 11.56 Vilniai Pp8 6.58 13.18 5.23 6.85 21.22 4.38 7.00 Vilniai Pp9 7.59 11.41 5.53 7.03 26.15 7.40 6.67 Vilniai Pp10 9.31 16.87 5.25 3.46 18.57 1.79 7.29 No.1072 Pp8 6.94 20.01 5.54 6.73 18.80 4.64 7.79 No.1072 Pp9 7.19 21.07 4.90 5.25 25.36 3.82 5.52 No.1072 Pp10 7.79 15.73 5.28 6.38 31.67 1.20 6.40 No.1734 Pp8 7.46 16.72 4.12 5.32 13.81 3.24 7.90 No.1734 Pp9 6.90 11.57 5.44 7.05 29.35 7.19 5.70 No.1734 Pp10 6.48 7.76 4.32 5.56 22.17 7.45 5.15

NF – non fermented; SMF – submerged fermentation; SSF – solid state fermentation; Pp8 - Pediococcus pentosaceus KTU05-8; Pp9 - Pediococcus pentosaceus KTU05-9; Pp10 - Pediococcus pentosaceus KTU05-10; ALA – alanine; GLY – glycine; SER - serine; PRO - proline; ASP - asparagine; GLU - glutamine; TYR - tyrosine



Conclusion We conclude that lupine is suitable substrate for Pediococcus pentosaceus KTU05-8, KTU05-9 and KTU05-10 cultivation, and the modeling of FAA concentration by using fermentation of lupine seeds could constitute a valuable contribution to the human and animal diet.

References Curiel J. A., Coda R., Centomani I., Summo C. a, Gobbetti M., Rizzello C. G. (2015) Exploitation of the nutritional and functional characteristics of traditional Italian legumes: The potential of sourdough fermentation International Journal of Food Microbiology. Vol. 196, P. 51–61.

Coda R., Melama L.a, Rizzello C. G.b, Curielb J. A., Sibakov J. a, Holopainena U., Pulkkinen M. C, Sozera N. (2015) Effect of air classification and fermentation by Lactobacillus plantarum VTT E-133328 on faba bean (Vicia faba L.) flour nutritional properties. International Journal of Food Microbiology. Vol. 193, P. 34–42.

Katsanos C. S., , Madura J. A., Roust, L. R. (2015) Essential amino acid ingestion as an efficient nutritional strategy for the preservation of muscle mass following gastric bypass surgery. Nutrition. Vol. 32, P. 9–13.

Voa B. V., Buib D. P., Nguyen H. Q., Fotedara R. (2015) Optimized fermented lupin (Lupinus angustifolius) inclusion in juvenile barramundi (Lates calcarifer) diets. Aquaculture. Vol. 444, P. 62–69

Wu Q., Fang A, Li H, Zhang Y, , Yao S. (2015) Enzymatic-induced upconversion photoinduced electron transfer for sensing tyrosine in human serum. Biosensors and Bioelectronics. Vol. 77, P. 957–962.

Villarino C.B.J, Jayasena V., Coorey R., Bell S. & Johnson S.K. (2015) Nutritional, Health and Technological Functionality of Lupin Flour Addition to Bread and Other Baked Products: Benefits and Challenges, Critical Reviews in Food Science and Nutrition, DOI: 10.1080/10408398.2013.814044).

FAO/WHO (1991). Protein quality evaluation. Report of a jointv FAO-WHO expert consultation., FAO: Rome.


Vytaute Starkute Department of the Food Safety and Quality, Veterinary Academy, Lithuanian University of Health Sciences; Tilzes str. 18, LT-47181, Kaunas, Lithuania. E-mail: vytaute.starkute(at)gmail.com

Kesselring et al.: Silage fermentation quality in grass silages inoculated with Lactobacillus kefiri alone and a for-mulation containing L. kefiri


Silage fermentation quality in grass silages inoculated with Lac-tobacillus kefiri alone and a formulation containing L. kefiri

Jutta Kesselring1, Gudrun Boeck1, Karin Schoendorfer1, Theodora Hoeger2 and Gerd Schatzmayr2 1 Biomin Holding GmbH, AT 2 Biomin Research Center, AT

Abstract Lactobacillus kefiri DSM 19455 is a novel heterofermentative strain first registered in 2013 in the EU. To better characterize its effects as silage inoculant in grass silage alone and within a three-strain mixed silage inoculant, we ran two small-scale mini-silo laboratory trials from two consecutive mead-ow grass cuts on the same field. The forage in both trials was affected from drought stress typical for 2015 in Austria. L. kefiri DSM 19455 is demonstrated to be a typical heterofermentative silage bacte-rium, producing high levels of acetic acid. The typical disadvantages of the heterofermentative silage bacteria compared to homofermeters such as unfavourable effects of palatability, metabolic losses of dry matter and weaker efficacy in lowering the silage pH were not apparent when L. kefiri was part of a three-strain silage inoculant of homo- and heterofermenters: The novel formulation of biological silage inoculant containing L. kefiri DSM 19455, L. brevis DSM 23231 and L. plantarum DSM 19457 significantly improved the fermentation quality of grass silage, leading to fewer fermentation losses during fermentation and higher energy contents.

Introduction Multi-strain silage inoculants composed of homo- and heterofermentative Lactic Acid Bacteria (LAB) are the most prevalent biological silage inoculants today. Lactic acid, the main metabolite of homo-fermentative LAB, lowers the pH value of silage and improves feed palatability (Krizsan et al., 2007). Acetic acid is a heterofermentative metabolite which directly inhibits the growth of many spoilage bacteria, yeast and fungi, thereby prolonging the aerobic stability (or shelf life) of silage (McDonald, 1991). A good balance between lactic acid and acetic acid and homofermentative and heterofermenta-tive silage inoculant strains is essential to achieve both palatability and aerobic stability. Lactobacillus kefiri DSM 19455 is a novel heterofermentative strain proprietary to Biomin Holding GmbH, Austria. To better characterize its effects as silage inoculant in grass silage alone and within a three-strain mixed silage inoculant, we ran two small-scale mini-silo laboratory trials from two consecutive cuts on the same field.

Material and methods Meadow grass, harvested at two consecutive cuts in April and May 2015, was either treated with L. kefiri DSM 19455, Biomin® BioStabil Plus (L. plantarum DSM 19457, L. brevis DSM 23231 and L. kefiri DSM 19455) or left untreated (control). Each treatment was tested in 15 replicates (5 each for testing nutrient composition, fermentation characteristics and aerobic stability after 46 and 92 days, and 5 for testing the acidification and fermentation after 7 days) during a 92 day experimental period (at con-stant 20 °C). Mini-silos (plastic buckets with tightly closing lid) with a volume of 1.8 L and 5.8 L were fully filled and compacted with hydraulic pressure 6 bar (1.8 L buckets) and 8 bar (5.8 L buckets) to a density of 1 kg DM 5 L-1. The mini-silos were closed immediately after filling. In addition, three fresh



forage samples were analysed immediately at the time of filling the silos. The inoculant suspensions and water were checked for their actual LAB content on MRS medium (de Man, Rogosa and Sharpe agar), ISO 15214.

Experimental treatments T1: Negative control (no additives, but water) T2: 2x105 cfu g-1 forage L. kefiri DSM 19455. T3: 2x105 cfu g-1 forage L. plantarum DSM 19457, L. brevis DSM 23231 and L. kefiri DSM 19455 (Bio-min®BioStabil Plus, Biomin, Austria). Silages were analysed 46 and 92 days after ensiling for DM loss (corrected for volatiles), microbiology (LAB, clostridia, yeast and mould) and chemical composition (Weender). DM, DM (corrected for vola-tiles) crude protein (CP), neutral detergent fibre (NDF), pH, lactic acid, volatile fatty acids, alcohols and N-NH3 were measured as described elsewhere (Acosta Aragon et al., 2012).

Results Based on the WSC content of the herbage prior to ensiling (table 1), grass 1 (first cut) was easy to ensile and grass 2 (second cut) moderately difficult to ensile according to the EFSA opinion on silage additives guidelines (European Food Safety Authority 2012). The forage in trial 1 had low protein con-tent (drought stress) and low fibre content (early harvest) together with high sugar content (early harvest). In trial 2 the forage was typical for drought conditions with low protein content, high crude fibre and low energy content. High crude ash content indicates soil contamination. In the easy to en-sile first cut grass silage (grass 1) T2 and T3 led to a spike in lactic acid concentration already 7 days after ensiling at 84 g kg-1 DM (T2) and 123 g kg-1 DM (T3). In T2, this spike was followed by a steep decline of lactic acid in favour of acetic acid. T3 had stable lactic acid levels between day 7 and day 46 around 130 g kg-1 DM, whereas at day 90 the lactic acid concentrations were found to be roughly around 80 g kg-1 DM. The uncontrolled spontaneous fermentation in T1 produced a slow steady in-crease of lactic acid from day 7 (62 g kg-1 DM) to 90 (142 g kg-1 DM) together with elevated propionic acid levels (table 2).

Table 1: Chemical composition and microbial parameters of the forage prior to ensiling

Grass 1 sd Grass 2 sd Dry matter, g kg-1 359.0 6.24 345.3 6.43 Crude protein, g kg-1DM 121.0 4.58 88.34 1.53 Crude fat, g kg-1DM 20.3 0.58 20.34 0.58 Crude fibre, g kg-1DM 202.0 12.53 321.34 10.60 Crude ash, g kg-1DM 94.0 1.00 105.00 6.25 aNDFom, g kg-1 DM 420.33 12.42 579.00 15.59 ADFom, g kg-1 DM 233.33 4.04 359.34 12.34 WSC, g kg-1DM 185.00 12.17 44.67 4.93 pH 6.21 6.41 0.06 Buffer capacity, mequiv 100g-1 DM 3.82 0.04 4.01 0.32 Clostridia, log10 cfu g-1 3.87 3.78 Yeasts & molds, log10 cfu g-1 5.70 0.10 4.96 0.38 LAB, log10 cfug-1 3.17 0 3.43 0.71 ME, MJ kg-1 DM 10.88 0.15 8.80 0.07 NEL, MJ kg-1 DM 6.65 0.12 5.14 0.05 DM, dry matter; cfu, colony forming units; LAB, lactic acid bacteria; fibre fractions NDF and ADF are expressed on an organic matter basis; WSC, water soluble carbohydrates



Table 2: Effect of biological inoculants on chemical composition and fermentation characteristics of first cut grass silage (grass 1) after 92 days

T1 T2 T3 overall average X ± SD X ± SD X ± SD Dry matter, DM, g kg-1 307.00 ± 2.45 304.80 ± 5.36 328.20 ± 7.26 313.33 ± 12.01 Dry matter#, DMc, g kg-1 311.74 ± 3.33 333.32 ± 7.03 352.01 ± 7.27 332.35 ± 17.96 Crude protein, g kg-1 DM 141.00 ± 3.74 138.20 ± 1.79 133.60 ± 2.41 137.60 ± 4.07 Crude fat, g kg-1 DM 34.40 ± 1.34 32.00 ± 1.22 32.40 ± 0.55 32.73 ± 1.49 Crude fibre, g kg-1 DM 249.80 ± 12.79 240.40 ± 12.74 226.40 ± 6.80 238.87 ± 14.33 Crude ash, g kg-1 DM 108.40 ± 2.70 112.60 ± 5.50 100.60 ± 2.30 107.20 ± 6.22 WSC, g kg-1 DM 28.00 ± 13.96 15.40 ± 4.83 51.40 ± 7.23 31.60 ± 17.77 aNDFom, g kg-1 DM 448.60 ± 7.96 440.00 ± 6.04 423.40 ± 6.88 437.33 ± 12.62 ADFom, g kg-1 DM 300.20 ± 20.07 280.60 ± 27.54 258.60 ± 8.91 179.80 ± 25.76 Lactic acid, g kg-1 DM 142.10 ± 5.13 28.40 ± 6.40 82.77 ± 5.23 84.42 ± 48.34 Acetic acid, g kg-1 DM 21.25 ± 3.10 84.58 ± 8.69 22.96 ± 3.87 42.90 ± 30.98 Butyric acid, g kg-1 DM 0.00 ± 0.00 1.16 ± 2.59 1.78 ± 1.98 0.98 ± 1.90 Propionic acid, g kg-1 DM 9.34 ± 1.06 0.77 ± 1.06 3.58 ± 1.64 4.56 ± 3.88 Ethanol, g kg-1 DM 5.65 ± 5.33 5.91 ± 1.13 37.56 ± 2.84 16.38 ± 15.85 Ammonia N, g kg-1 total N 8.34 ± 0.98 6.76 ± 1.41 5.23 ± 0.43 6.78 ± 1.63 pH after 7 days 4.76 ± 0.05 4.19 ± 0.04 4.08 ± 0.24 4.34 ± 0.31 pH after 92 days 4.38 ± 0.35 4.40 ± 0.03 4.03 ± 0.03 4.29 ± 0.17 ME, MJ kg-1 DM 10.24 ± 0.14 10.26 ± 0.13 10.62 ± 0.11 10.37 ± 0.21 NEL, MJ kg-1 DM 6.15 ± 0.11 6.12 ± 0.13 6.43 ± 0.11 6.25 ± 0.16 DMc losses, g kg-1 DM 77.92 ± 8.53 59.85 ± 13.86 22.24 ± 20.41 53.32 ± 14.27 Dry matter#, DMc, DM corrected for volatiles

Table 3: Effect of biological inoculants on chemical composition and fermentation characteristics of ensiled second cut grass (grass 2) after 92 days

T1 X ± SD

T2 X ± SD

T3 X ± SD Overall average

Dry matter, DM, g kg-1 327.00 ± 3.77 321.00 ± 1.92 341.00 ± 2.51 329.80 ± 9.20 Dry matter#, DMc, g kg-1 380.81 ± 2.71 381.52 ± 1.92 388.95 ± 3.85 383.76 ± 4.68 Crude protein, g kg-1 DM 92.00 ± 1.87 95.00 ± 1.79 95.00 ± 2.17 93.87 ± 2.26 Crude fat, g kg-1 DM 32.00 ± 1.22 30.00 ± 1.00 32.00 ± 1.10 31.27 ± 1.39 Crude fibre, g kg-1 DM 362.00 ± 8.88 365.00 ± 6.35 349.00 ± 6.32 358.68 ± 9.90 Crude ash, g kg-1 DM 120.00 ± 4.28 123.00 ± 2.59 110.00 ± 1.30 117.53 ± 6.19 WSC, g kg-1 DM 3.00 ± 1.63 5.00 ± 2.63 4.00 ± 1.95 4.08 ± 2.06 aNDFom, g kg-1 DM 606.00 ± 10.46 592.00 ± 5.96 580.00 ± 5.76 592.60 ± 13.18 ADFom, g kg-1 DM 417.00 ± 10.08 406.00 ± 5.03 411.00 ± 8.71 411.40 ± 8.89 Lactic acid, g kg-1 DM 9.80 ± 6.26 11.95 ± 3.24 114.31 ± 1.97 45.35 ± 50.63 Acetic acid, g kg-1 DM 10.59 ± 0.85 55.52 ± 1.77 17.76 ± 1.92 27.96 ± 20.45 Butyric acid, g kg-1 DM 37.73 ± 1.87 3.30 ± 1.24 0.00 ± 0.00 13.67 ± 17.70 Propionic acid, g kg-1 DM 3.08 ± 0.46 13.51 ± 0.57 3.88 ± 0.18 6.82 ± 4.92 Ethanol, g kg-1 DM 8.38 ± 0.21 9.12 ± 1.18 4.40 ± 1.00 7.30 ± 2.30 Ammonia N, g kg-1 total N 12.27 ± 2.73 11.58 ± 0.97 8.64 ± 2.15 10.83 ± 2.52 pH after 7 days 5.16 ± 0.05 4.62 ± 0.05 4.28 ± 0.06 4.69 ± 0.38 pH after 92 days 5.11 ± 0.09 4.75 ± 0.02 4.19 ± 0.03 4.68 ± 0.39 ME, MJ kg-1 DM 8.31 ± 0.09 8.22 ± 0.05 8.56 ± 0.05 8.36 ± 0.16 NEL, MJ kg-1 DM 4.79 ± 0.07 4.73 ± 0.03 4.95 ± 0.04 4.82 ± 0.11 DM losses, g kg-1 FM 49.38 ± 6.63 33.20 ± 1.10 20.79 ± 2.02 34.45 ± 12.33 Dry matter#, DMc, DM corrected for volatiles; FM, fresh matter



Discussion The speed of acidification might explain how Biomin® BioStabil Plus helped avoid dry matter losses and preserve the feeding value of the forage (+0.28 MJ NEL in grass 1). In the moderately difficult to ensile second cut (grass 2), T1 was characterized by a butyric acid fermentation, T2 was dominated by acetic acid and propionic acid and T3 by a lactic acid fermentation (table 3). Accordingly, T3 was most effective in lowering the pH and preserving energy (+0.25 MJ NEL) and dry matter.

Conclusion L. kefiri DSM 19455 was demonstrated to be a typical heterofermentative silage bacterium, producing high levels of acetic acid. The typical disadvantages of the heterofermentative silage bacteria such as unfavourable effects of palatability, metabolic losses of dry matter and lower efficacy in lowering the silage pH were not apparent when L. kefiri was part of a three-strain silage inoculant of homo- and heterofermenters: The novel formulation of biological silage inoculant containing L. kefiri DSM 19455 significantly improved the fermentation quality of grass silage, leading to fewer fermentation losses during fermentation and higher energy contents.

References ACOSTA ARAGON, Y., JATKAUSKAS, J. & VROTNIAKIENE, V. 2012. The effect of a silage inoculant on silage quality, aerobic stability, and meat production on farm scale. ISRN veterinary science, 2012, 345927.

KRIZSAN, S. J., WESTAD, F., ADNOY, T., ODDEN, E., AAKRE, S. E. & RANDBY, A. T. 2007. Effect of volatile compounds in grass silage on voluntary intake by growing cattle. Animal, 1, 283-92.

MCDONALD, P. 1991. The Biochemistry of Silage.

EFSA Journal 2012. 10(1):2528


Dr. Jutta Kesselring Biomin Holding GmbH Erber Group Campus 1 3130 Getzersdorf E-mail: jutta.kesselring(at)biomin.net

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Ettle und Obermaier: Untersuchungen zum Einsatz von Lebendhefe in der Milchviehfütterung


Untersuchungen zum Einsatz von Lebendhefe in der Milchvieh-fütterung

Thomas Ettle und Anton Obermaier Bayerische Landesanstalt für Landwirtschaft, Institut für Tierernährung und Futter-wirtschaft, DE

Abstract A feeding trial using a total of 48 Simmental and Brown Swiss cows was conducted to evaluate the effects of live yeast supplementation on performance of dairy cows. Cows were equally distributed to two feeding groups and fed a Total Mixed Ration with or without live yeast supplementation over a period of 12 weeks. From a trial in rams there was no evidence for differences in digestibility of organ-ic matter or crude fibre. In the dairy cow feeding trial, daily feed intake was slightly higher in control than in yeast supplemented group. There were no effects on milk yield and milk composition. Live yeast supplementation of diets lead not to a higher reticulo-ruminal pH compared to control cows. It is concluded that live yeast addition has no effects on performance of dairy cows beyond the lactational peak.

Einleitung In der Praxis werden im Bereich der Milchviehfütterung bei steigenden Milchleistungen zunehmend Rationen mit vergleichsweise niedrigen Fasergehalten und andererseits hohen Gehalten an leicht lösli-chen Kohlenhydraten gefüttert. Es ist davon auszugehen, dass sich diese Art der Fütterung häufig in den Grenzbereichen einer wiederkäuergerechten Ernährung abspielt und den Pansen der Tiere belas-tet. Unter den Futterzusatzstoffen, die eingesetzt werden, um hier die Verhältnisse zu stabilisieren, finden sich auch Lebendhefen. Diese werden in der Praxis relativ häufig eingesetzt. Andererseits wird gerade aus der Praxis verstärkt nach aktuellen, unabhängigen Versuchsergebnissen zu den Effekten und zur Einsatzwürdigkeit von Lebendhefen in der Milchviehfütterung nachgefragt. Vor diesem Hinter-grund soll vorliegender Versuch klären, ob sich beim Einsatz einer am Markt verfügbaren Lebendhefe (Saccharomyces cerevisiae) Effekte auf Futteraufnahme und Milchleistungskriterien bei hochleistenden Fleckviehkühen ergeben.

Material und Methoden Für die Untersuchung wurden insgesamt 48 Milchkühe unter Berücksichtigung von Rasse (jeweils 16 Fleckvieh- und 8 Brown Swiss-Kühe), Laktationsstand, Zahl der Laktationen, Milchleistungskriterien und Futteraufnahme auf die Fütterungsgruppen „Kontrolle“ und „Versuch“ aufgeteilt. Zu Versuchsbe-ginn befanden sich die Tiere im Mittel am 109. Laktationstag der 3. Laktation. Die Kühe wurden in einem Offenfrontstall mit Liegeboxen gehalten, der mit 24 automatischen Wiegetrögen ausgestattet ist. Der Versuch wurde über eine Zeitdauer von 12 Wochen durchgeführt. Es wurde eine Totale Mischration (TMR) ad libitum angeboten, die in beiden Gruppen auf gleichen Anteilen Maissilage (31,3 % der TM), Grassilage (23,2 % der TM), Heu/Stroh (5,7 % der TM) Gras-cobs (3,9 % der TM) und Kraftfutter (35,8 % der TM) basierte. Rechnerisch war die TMR auf eine Milchleistung von 36 kg/Tier und Tag ausgelegt. Das Mineralfutter für die Versuchsgruppe, das in der Kraftfuttervormischung enthalten war, wurde so mit der Lebendhefe supplementiert, dass in der Ge-samtration eine Konzentration an Hefen von 4x108 KBE/kg TM erreicht wurde.



Die Futteraufnahme wurde tierindividuell über die Wiegetröge erfasst. Die Milchleistung wurde täglich gemessen, Milchproben wurden einmal je Woche vom Morgen- und Abendgemelk eines Tages gezo-gen. Das Lebendgewicht wurde nach dem Melken mit einer automatischen Durchlaufwaage erfasst. Zusätzlich wurde dreimal während der Versuchsperiode die Körperkondition mit dem BCS-System be-wertet und die Rückenfettdicke mit einem Ultraschallgerät gemessen. Der reticuloruminale-pH-Wert wurde mit einem pH-Wert-Bolus (smaxtec ®, Österreich) erfasst, wobei für die Auswertung ab Einga-be der Boli 50 Tage Messdauer (incl. 6 Tage Messdauer in Vorperiode mit einheitlicher Fütterung) berücksichtigt wurden. Je Gruppe waren 6 Tiere mit den Boli ausgestattet. Die Verdaulichkeit der bei-den TMR wurden nach den Leitlinien der GfE (1991) an Hammeln geprüft. Für beide Varianten kamen 5 Hammel zum Einsatz. Auf Basis der verdaulichen Rohnährstoffe wurden die Gehalte an ME und NEL nach den Vorgaben der GfE (2001) kalkuliert. Von den Kraft- und Grobfuttermitteln wurden monatli-che Mischproben erstellt, an denen die Rohnährstoffgehalte nach VDLUFA (1976) bestimmt wurden. Die Rohnährstoff- und Energiegehalte der TMR wurden aus den Analysenwerten der Einzelkomponen-ten und den über den Mischwagen erfassten tatsächlich täglich eingewogenen Mengen errechnet.

Ergebnisse und Diskussion Die Supplementierung von Rationen für Wiederkäuer mit Lebendhefen wird mit einem Potential zur Verbesserung der Faserverdaulichkeit in Verbindung gebracht (DeVries und Chevaux, 2014). In vorlie-gender Untersuchung waren jedoch weder die Verdaulichkeit der organischen Substanz noch die Ver-daulichkeit der Rohfaser durch die Zulage an Lebendhefe zur TMR beeinflusst (Tabelle 1). Vergleichbare Ergebnisse ergaben sich auch in einer Arbeit von Ettle und Schwarz (2002). Für beide TMR errechnete sich aus den Verdaulichkeiten und den Inhaltsstoffen ein Energiegehalt von 7,0 MJ NEL/kg TM, was sich mit der Rationsplanung deckt.

Tabelle 1: Verdaulichkeit der Rohnährstoffe beim Hammel

Versuchsgruppe Kontrolle Versuch

Verdaulichkeit, % OS 79,6 ± 1,7 79,2 ± 1,8 XL 78,9 ± 2,5 77,4 ± 3,1 XF 72,0 ± 1,8 72,0 ± 3,1 OR 81,3 ± 1,7 80,9 ± 1,7

Die Futteraufnahme lag in der Versuchsgruppe etwas niedriger, als in der Kontrollgruppe (Tabelle 2), was zu leicht erniedrigten täglichen Aufnahmen an Energie und nXP führte. Aus einer Metaanalyse wurden grundsätzlich positive Effekte einer Lebendhefezulage zu Rationen für Wiederkäuer abgeleitet (Desnoyers et al., 2009), wobei sich in dieser Untersuchung auch positive Effekte auf die Verdaulich-keit der organischen Substanz ergaben. Da nach Marden et al. (2008) mögliche positive Effekte der Hefezulage auf die Futteraufnahme durch eine Verbesserung der Verdaulichkeit der Zellwände erklärt werden können, ist auch erklärbar, warum in vorliegender Untersuchung keine Effekte auf die Fut-teraufnahme ersichtlich waren. Allerdings wurde in vorliegender Untersuchung lediglich die Verdau-lichkeit der Rohfaser geprüft, nicht aber die Verdaulichkeit der NDF und ADF.

Tabelle 2: Futteraufnahme, Energie- und Nährstoffaufnahme


TM-Aufnahme, kg/Tag 23,0 ± 4,8 22,4 ± 5,8 NEL-Aufnahme, MJ/Tag 161 ± 2,1 158 ± 2,5 XP-Aufnahme, g/Tag 3522 ± 15 3425 ± 18 nXP-Aufnahme, g/Tag 3542 ± 318 3453 ± 387



Zwischen den beiden Versuchsgruppen ergaben sich keine Unterschiede in der täglichen Milchleistung (Tabelle 3). Dies deckt sich mit den fehlenden Effekten auf die Verdaulichkeit der Rationen und mit den eher geringen Unterschieden in der täglichen Futteraufnahme. Wie der Übersicht von Robinson und Erasmus (2009) zu entnehmen ist, wird in der Literatur sowohl von positiven als auch von fehlen-den und negativen Effekten einer Lebendhefezulage auf die Milchleistung berichtet. Als Ursache für die Unterschiede können u. a. Rationsgestaltung, Höhe der Supplementierung, Leistungsniveau aber auch der Hefestamm bzw. das Produkt diskutiert werden. In der Tendenz ergibt sich in vorliegender Untersuchung bei etwas verringerter Futteraufnahme in der Versuchsgruppe und zwischen den Grup-pen vergleichbarer Milchleistung ein positiver Effekt der Hefesupplementierung auf die Futterverwer-tung. Bei den Milchinhaltsstoffen ergaben sich keine Unterschiede zwischen den Gruppen. Mögliche Effekte einer Hefesupplementierung könnten sich bei der Milchfettproduktion ergeben (van Straalen et al., 2013), was sich durch eine erhöhte ruminale Acetat-Produktion nach Hefesupplementierung (Marden et al., 2008) erklären ließe.

Tabelle 3: Milchleistung und Milchinhaltsstoffe


Milchleistung, kg/Tag 33,7 ± 5,4 33,8 ± 5,3 Milchfettgehalt, % 3,88 ± 0,43 3,82 ± 0,35 Milcheiweißgehalt, % 3,63 ± 0,21 3,63 ± 0,20 Milchlaktosegehalt, % 4,65 ± 0,26 4,70 ± 0,11 Milchharnstoffgehalt, mg/l 222 ± 33 211 ± 41 ECM, kg/Tag 33,6 ± 5,0 33,5 ± 4,9

Der Pansen-pH-Wert wurde vorliegend mit einem kabellosen Sensor, der im Netzmagen der Tiere zum Liegen kommt, erfasst. Im Mittel ergab sich sowohl in der Kontroll- und der Versuchsgruppe pH-Wert von 6,14±0,10 und 6,07±0,22. Von größerer Bedeutung als die mittleren täglichen pH-Werte sind die Schwankungen im Tagesverlauf bzw. die Zeitdauer der Unterschreitung von pH-Wert-Minima anzuse-hen (Steingass und Zebeli, 2008). Zur Abgrenzung einer subklinischen Pansenacidose zum „Normal-zustand“ wurde ein mittlerer täglicher Pansen-pH-Wert von unter 6,16 und oder ein Unterschreiten des Pansen-pH-Wertes von 5,8 für mehr als 5,2 h (Zebeli et al., 2008). Während der Versuchsphase ergab sich bei keinem Tier an keinem Tag ein Unterschreiten des pH-Wertes von 6,1 über 24 h hin-weg. Ein Unterschreiten des pH-Wertes von 5,8 über mehr als 5,2 h hinweg wurde in der Kontroll- und Versuchsgruppe an 22 und 75 Tiertagen von jeweils 264 möglichen Tiertagen gefunden. Da Unter-schiede im Niveau des pH-Wertes im Netzmagen und ventralen Pansensack auftreten könnten (GfE, 2014) bleibt unklar, wie diese Werte im Vergleich zur Literatur einzuordnen sind. Ein stabilisierender Effekt der Hefesupplementierung, wie wer z.B. bei Marden et al. (2008) gefunden wurde, ergibt sich in vorliegender Untersuchung bei der gewählten Messtechnik allerdings nicht.

Fazit In vorliegender Untersuchung wurde nach Hefesupplementierung eine etwas erniedrigte Futterauf-nahme bei mit der Kontrollgruppe vergleichbarer Milchleistung gefunden. In der Tendenz ergibt sich daraus eine verbesserte Futterverwertung in der Versuchsgruppe. Allerdings konnte durch die Zulage der Lebendhefe weder die Gesamtverdaulichkeit noch die Verdaulichkeit der Rohfaser beim Hammel gesteigert werden. Auch der Pansen-pH-Wert war durch die Zulage mit Lebendhefe kaum beeinflusst. Insgesamt lässt sich aus den Ergebnissen schließen, dass eine Zulage der Lebendhefe bei Milchkühen nach Überschreiten des Laktationsgipfels keine Effekte auf die Leistung zeigt.



Literatur DeVries, T.J., Chevaux, E. (2014): Modification of the feeding behavior of dairy cows trough live yeast supplementation. J. Dairy Sci., 97, 6499-6510

Ettle, T., Schwarz, F.J. (2002): Effect of yeast culture (Saccharomyces cerevisiae, Levucell) on performance of beef and dairy cattle. Proc. Soc. Nutr. Physiol. 11, 116

GfE (2001): Empfehlungen zur Energie- und Nährstoffversorgung der Milchkühe und Aufzuchtrinder. DLG-Verlag, Frankfurt a. Main, Germany.

GfE (2014): Evaluation of structural effectiveness of mixed rations for dairy cows – status and perspectives. Proc. Soc. Nutr. Physiol. 23, 166-179

GfE (Ausschuss für Bedarfsnormen der Gesellschaft für Ernährungsphysiologie) (1991): Leitlinien zur Bestimmung der Verdau-lichkeit von Rohnährstoffen an Wiederkäuern, J. Anim. Physiol. Anim. Nutr. 65, 229 - 234

Marden, J. P., Julien, C., Monteils, V., Auclair, E., Moncoulon, R. Bayourthe, C. (2008): How Does Live Yeast Differ from Sodium Bicarbonate to Stabilize Ruminal pH in High-Yielding Dairy Cows? J. Dairy Sci. 91, 3528–3535

Desnoyers, M., Giger-Reverdin, S., Bertin, G., Duvaux-Ponter, C., Sauvant, D. (2009): Meta-analysis of the influence of Saccharomyces cerevisiae supplementation on ruminal parameters and milk production of ruminants J. Dairy Sci. 92 , 1620–1632

Robinson, P.H., Erasmus, L.J. (2009): Effects of analyzable diet components on the responses of dairy cows to Saccharomyces cerevisiae based yeast products: A systematic review of the literature. Anim feed Sci. technol. 149, 185-198

Steingass, H., Zebeli, Q. (2008): Strukturbewertung von Rationen für Milchkühe. 35. Viehwirtschaftliche Fachtagung, LVFZ Raumberg- Gumpenstein, 19-25

Van Straalen, W.M., Kok, I., Walker, N., Phillips, P. (2013): Effekte einer Lebendhefezulage auf die Leistung sowie physiologi-sche Parameter hochleistender Milchkühe. Tagungsband Forum angewandte Forschung in der Rinder- und Schweinefütterung, 86-90

VDLUFA (1976): Methodenbuch Band III. Die chemische Untersuchung von Futtermitteln. 3. Auflage inklusive 1-7. Ergänzungs-lieferung. VDLUFA-Verlag, Darmstadt.

Zebeli, Q., Dijkstra, J., Tafaj, M., Steingass, H., Ametaj, B.N., Drochner, W. (2008): Modeling the adequacy of dietary fiber in dairy cows based on the responses of ruminal pH and milk fat production to composition of the diet. J. Dairy Sci. 91, 2046-2066

Autorenanschrift Thomas Ettle Institut für Tierernährung und Futterwirtschaft Grub Prof.-Dürrwaechter-Platz 3, 85586 Poing E-Mail: Thomas.Ettle(at)LfL.bayern.de

Deml et al.: Effekte einer Amylase und Protease Supplementierung auf den ruminalen Abbau von Stärke, Protein und Faser bei trockenstehenden Holstein Kühen


Effekte einer Amylase und Protease Supplementierung auf den ruminalen Abbau von Stärke, Protein und Faser bei trockenste-henden Holstein Kühen

Mirko Deml, Carmen Bolduan und Wilhelm Windisch Technische Universität München, DE

Abstract Eight non-lactating rumen-fistulated cows were used to study rumen starch, protein and fiber degra-dability according to the in situ-method with the aid of exogenous enzymes (amylase, protease) with following treatments: control, addition of amylase, addition of protease and addition of a combination of amylase and protease. The combination of both enzymes resulted in an increased ruminal degrada-tion of starch in maize grain. The protease treatment and the combination of amylase and protease led to an accelerated ruminal degradation of protein in soybean meal. NDF degradation in grass silage as the dominant source of dietary fiber was not affected by any treatment suggesting that enzyme addition did not affect this group of nutrients.

Einleitung Die Verdauung beim Wiederkäuer beruht in erster Linie auf der Fermentation des Futters im Pansen. Die Effizienz dieses Vorgangs wird unter anderem durch die chemischen Eigenschaften des Futters beschränkt. Futterzusatzstoffe können eine Möglichkeit sein, die Futterverwertung von Wiederkäuern zu verbessern. In dem nachfolgend beschriebenen Versuch wurden exogene Enzyme (Amylase, Pro-tease) als Futterzusatzstoffe eingesetzt. Die verwendeten Enzyme sollten den Zugang zu Stärke bei Mais im Pansen erleichtern, da dieser durch die chemische Zusammensetzung des Korns erschwert sein kann (Kotarski et al., 1992; Larson & Hoffman, 2008). Eine weitere Hypothese ist, dass durch den Einsatz einer Amylase Mikroorganismen im Pansen gefördert werden, welche selbst keine Amylase Aktivität aufweisen (Tricarico et al., 2008). Dies könnte wiederum zu einer gesteigerten Faserverdau-lichkeit führen. So konnte z. B. durch den Einsatz einer Amylase eine gesteigerte NDF-Verdaulichkeit in einigen Studien nachgewiesen werden (Tricarico et al., 2008; Klingerman et al., 2009; Gencoglu et al., 2010; Weiss et al., 2011; McCarthy et al., 2013). In dem vorliegenden Versuch sollte anhand der Nylon-Beutel-Technik überprüft werden, ob durch den Enzymeinsatz der ruminale Abbau einzelner Nährstoffe beeinflusst werden kann.

Material und Methoden Der Versuch wurde an acht trockenstehenden, pansenfistulierten Holstein Kühen durchgeführt. Alle Tiere wurden folgenden Behandlungen unterzogen: Kontrolle (Kon): keine Enzymzulage; Zulage von Amylase (Amy): 300 Amylase-Units (AU)/kg Trockenmasse (TM); Zulage von Protease (Prot): 15000 Protease-Units (PU)/kg TM; Kombination der beiden Enzympräparate (Amy+Prot): 150 AU + 7500 PU/kg TM. (1AU = Menge an Enzym, die 6 µmol p-Nitrophenol pro Minute von 1,86 mM Ethyliden-G7-p-Nitrophenyl-Maltoheptaosid bei pH 7,0 und 37 °C freisetzt; 1 PU = Menge an Serin Protease, die 1 µmol p-Nitroanilin (pNA) pro Minute von 1 mM Suc-Ala-Ala-Pro-Phe-pNA (C30H36N6O9) Substrat bei pH 9,0 und 37 °C freisetzt). Die Enzyme wurden direkt vor der Fütterung in die Ration eingemischt. Die Versuchstiere wurden als Lateinisches Quadrat (2×4×4) in vier Durchgängen eingesetzt. Während jedes Durchgangs erhielten



jeweils zwei Tiere dieselbe Behandlung. Anschließend wurden die Behandlungen getauscht, so dass jedes Tier jede der vier Behandlungen einmal erhielt. Jeder Messperiode ging eine 21-tägige Adaptati-onsphase voraus. Nach Abschluss jeder Messphase erfolgte eine 8-tägige Phase ohne jegliche En-zymsupplementierung. Die tägliche Futteraufnahme lag bei 7,0 kg T. Die Ration bestand aus 49 % Maissilage, 20 % Körnermais, 15 % Grassilage, 6 % Sojaextraktionsschrot und 10 % Heu. Die Ration wurde den Tieren täglich zu zwei gleichen Teilen um 7.00 Uhr und 16.00 Uhr vorgelegt. Der Zugang zu Wasser und Salzlecksteinen erfolgte ad libitum. Die Bestimmung der ruminalen Abbaubarkeit erfolgte anhand der Nylon-Beutel-Technik (Ørskov und McDonald, 1979). Dazu wurde von jedem Futtermittel und der TMR Probenmaterial (4 g TM) in Nylon-Beutel (Porengröße 50 µm) eingewogen und 4 Beutel pro Futtermittel und Inkubationszeit unmittelbar vor der Morgenfütterung für 1, 2, 3, 4, 5, 6, 9, 12, 24 und 48 Stunden im Pansen der Kühe inkubiert. Das Probenmaterial in den Beuteln wurde ebenfalls mit den Enzympräparaten versetzt, in dem glei-chen Verhältnis wie in der Ration. Nach Ablauf der Inkubationszeit wurden die Beutel in Eiswasser gelegt um eine weitere mikrobielle Aktivität zu unterbinden und anschließend gewaschen, getrocknet und zurückgewogen, um die Trockenmasseverluste der einzelnen Futterkomponenten zu ermitteln. Zur Bestimmung des ruminalen Nährstoffabbaus wurden die Rückstände der 4 Beutel pro Futtermittel und Kuh gepoolt, gemahlen und auf die jeweiligen Nährstoffe an den Zeitpunkten 1, 3, 6, 9, 12, und 24 h analysiert. Körnermais diente dazu den Stärkeabbau darzustellen, Sojaextraktionsschrot den Proteinabbau und bei Grassilage wurde der Abbau der neutralen Detergenzien-Faser (NDF) ermittelt. Die Analysen der NDF- und Rohproteingehalte erfolgte anhand der erweiterten Weender Futtermittel-analyse (VDLUFA, 2007), die Stärkegehalte wurden nach enzymatischer Vorbehandlung, unter Ver-wendung einer hitzestabilen Amylase, und anschließender photometrischer Glucosemessung bestimmt (Brandt et al., 1987). Die Ergebnisse wurden einer Varianzanalyse mit anschließendem Duncan-Test unterzogen. Gegen-stand der Varianzanalyse waren signifikante Mittelwertunterschiede zwischen den Behandlungen. Das Signifikanzniveau lag bei p<0,05.

Ergebnisse Die Ergebnisse des ruminalen Abbaus von Stärke bei Körnermais, Protein bei Sojaextraktionsschrot und NDF bei Grassilage sind in Tabelle 1 dargestellt. Die Untersuchungen zeigten einen numerisch erhöhten Stärkeverlust bei 1 und 3 h und einen signifikant erhöhten Stärkeverlust bei 6, 9, 12 und 24 h durch die Kombination der beiden Enzyme im Vergleich zur Kontrolle am Beispiel Körnermais. Die Supplementierung einer Amylase und einer Protease alleine zeigte keine Wirkung auf den Stärkeabbau von Körnermais. Der Proteinabbau von Sojaextraktionsschrot wurde durch die Protease-Behandlung am stärksten be-einflusst. Diese Variante erzielte die höchsten Abbauwerte, welche sich auch von 1 bis 9 h signifikant von der Kontrolle unterschieden. Die Kombination der beiden Enzyme führte auch hier zu signifikant erhöhten Abbauwerten im Vergleich zur Kontrolle von 1 bis 6 h. Die Amylase-Behandlung zeigte, au-ßer bei 1 h, keinen signifikant erhöhten Abbau im Bezug auf die Kontroll-Behandlung. Der Einsatz der verschiedenen Enzymsupplementierungen führte bei Grassilage bei keiner der einge-setzten Behandlungen zu einer Erhöhung des ruminalen NDF-Abbaus.



Tabelle 1: Ruminaler Abbau an Nährstoffen (%) von verschiedenen Futtermitteln bei unterschiedlicher En-zymsupplementierung

Stärkeverlust Körnermais Behandlung Inkubationszeit (h)

1 3 6 9 12 24 Kon 28,9 30,8 34,7bc 37,8b 43,5b 67,6b Amy 27,9 30,4 35,5ab 38,7b 44,0b 71,8ab Prot 27,8 30,2 33,1c 39,4b 44,9b 66,4b Amy+Prot 29,7 33,8 37,4a 42,1a 49,5a 73,9a SEM 1,6 1,2 0,8 1,0 1,4 2,1 Signifikanz n. s. n. s. ** * * * Proteinverlust Sojaextraktionsschrot Behandlung Inkubationszeit (h)

1 3 6 9 12 24 Kon 19,1d 25,1b 34,2b 51,5bc 61,9 93,8 Amy 20,8c 25,9b 36,9ab 49,5c 58,7 92,1 Prot 26,9a 34,0a 41,3a 63,2a 65,9 93,8 Amy+Prot 24,1b 31,4a 40,0a 58,4ab 70,0 93,9 SEM 0,6 1,0 1,8 2,7 3,5 1,8 Signifikanz *** *** * ** n. s. n. s. NDF-Verlust Grassilage Behandlung Inkubationszeit (h)

1 3 6 9 12 24 Kon 6,2 8,1 12,1 18,8 26,5 53,2 Amy 4,7 5,9 10,2 15,1 21,7 51,9 Prot 5,1 6,1 10,7 17,0 22,4 49,8 Amy+Prot 5,4 6,9 9,9 18,3 25,0 50,1 SEM 0,7 0,8 1,0 1,2 2,2 2,6 Signifikanz n. s. n. s. n. s. n. s. n. s. n. s. (Werte mit unterschiedlichen Hochbuchstaben unterscheiden sich signifikant; n. s.: nicht signifikant; *: p<0,05; **: p<0,01; ***: p<0,001)

Diskussion und Schlussfolgerung

In dem vorliegenden Versuch konnte Anhand der Nylon-Beutel-Technik ein signifikant erhöhter Tro-ckenmasseabbau bei Körnermais durch die Supplementierung der Kombination beider Enzympräparate festgestellt werden (Deml et al., 2015). Die jetzt vorliegenden Ergebnisse bestätigen die Vermutung, dass es sich hierbei um einen gesteigerten Stärkeabbau im Pansen handelt, da besonders bei Mais der Zugang der Stärke durch eine Matrix aus Proteinen und NSP (nicht-Stärke Polysaccharide) erschwert ist (Kotarski et al., 1992; Larson & Hoffman, 2008). Diesen negativen Eigenschaften des Maiskorns kann scheinbar durch die Verabreichung einer Kombination aus Amylase und Protease zum Teil ent-gegen gewirkt werden. Der Trockenmasseabbau bei Sojaextraktionsschrot lieferte nur vereinzelt signifikante Ergebnisse (an 2 von 10 Inkubationszeiten). Der Proteinabbau zeigt jedoch deutlich, dass in den Stunden 1 – 9 bzw. 1 – 6 h (für Prot bzw. Amy+Prot) ein erhöhter Proteinabbau von Sojaextraktionsschrot im Pansen stattfindet. Der NDF-Abbau war in dieser Untersuchung von besonderem Interesse, da doch einige wissenschaftli-che Veröffentlichungen der vergangenen Jahre von einer erhöhten NDF-Verdaulichkeit durch den Ein-satz einer Amylase berichtet hatten. Diese erhöhte NDF-Verdaulichkeit könnte laut Tricarico et al. (2008) auf einer gesteigerten Energiebereitstellung für faserspaltende Mikroorganismen durch die Amylase beruhen. In dem beschriebenen Versuch führte zwar nicht die Amylase-Behandlung, jedoch die Kombination der Amylase mit einer Protease zu einem gesteigerten Stärkeabbau im Pansen. Diese



gesteigerte Energiebereitstellung im Pansen hatte jedoch keinen Einfluss auf den ruminalen NDF-Abbau von Grassilage.

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VDLUFA (2007): Die chemische Untersuchung von Futtermitteln. Methodenbuch Band III. VDLUFA-Verlag, Darmstadt.

Weiss, W. P., Steinberg, W., Engstrom, M. A. (2011): Milk production and nutrient digestibility by dairy cows when fed exoge-nous amylase with coarsely ground dry corn. J. Dairy Sci. 94, 2492-2499.

Autorenanschrift Mirko Deml Lehrstuhl für Tierernährung Technische Universität München Liesel-Beckmann-Straße 2 85354 Freising E-Mail: mirko.deml(at)wzw.tum.de

Chaucheyras-Durandet et al.: Effect of a live yeast, Saccharomyces cerevisiae I-1077, used as a feed additive for ruminants, on in situ ruminal digestibility of alfalfa hay and fibre-associated microorganisms


Effect of a live yeast, Saccharomyces cerevisiae I-1077, used as a feed additive for ruminants, on in situ ruminal digestibility of alfalfa hay and fibre-associated microorganisms

Frédérique Chaucheyras-Durand1,2, Aurélie Ameilbonne1,2, Pascale Mosoni2

and Evelyne Forano2

1 Lallemand Animal Nutrition, Blagnac, FR 2 INRA, Saint-Genès Champanelle/Blagnac, FR

Introduction and Objective

The digestion of plant material and its subsequent conversion in energy to fulfill ruminant’s require-ments are performed through a complex symbiotic relationship of microbiota within the rumen. In this study, we investigated the effect of a live yeast, Saccharomyces cerevisiae I-1077, used as a feed additive for ruminants, on in situ ruminal degradation of alfalfa hay and on its colonisation by bacteria and fungi.

Material and methods

3 rumen cannulated cows were fed with grass silage/meadow hay. A first period of 4 weeks without yeast (-SC) was followed by a second 4 week-period (+SC) during which the cows received daily 1010 cfu of S.cerevisiae I-1077 (Levucell SC20, Lallemand SAS). Nylon bags containing 5g of chopped alfal-fa hay were incubated in the rumen during 2, 6, 12 and 24 hours. Bags were removed and residual DM and NDF were determined. Fibrobacter succinogenes, Butyrivibrio fibrisolvens and anaerobic fungi were quantified on fresh plant material incubated in an additional series of bags by real time PCR (Denman and Mc Sweeney, 2006; Stevenson and Weimer, 2007). Unincubated bags were used as controls.

Results

The live yeast supplementation induced a significant increase in dry matter disappearance and NDF degradation of alfalfa. The rate of degradation was particularly stimulated. The early colonisation of alfalfa particles by anaerobic fungi appeared to be improved; the populations of B.fibrisolvens were greatly promoted whatever the incubation time of the bags, whereas F.succinogenes populations, which were dominant, were not influenced by yeast supplementation.

Table 1: Effects of S.cerevisiae I-1077 on alfalfa hay degradation and particle associated microorganisms

2 6 12 24 -SC +SC -SC +SC -SC +SC -SC +SC DMD (%) 13.17 26.98* 17.45 32.6* 29.15 40.7* 41.53 50.84* NDFD (g/kg NDF) 83.74 211.21* 107.25 237.14* 183.12 261.41* 336.11 363.48 Anaerobic fungi (1) 3.52 3.56 2.29 3.29 3.73 5.82 11.61 10.87 B. fibrisolvens (2) 4.46 4.88* 5.61 6.66* 5.64 6.66* 6.33 7.45* F. succinogenes (2) 7.32 7.42 8.05 8.04 8.07 8.29 8.28 7.96* *P-Value <0.05 (Tukey- test); (1): μg of biomass/gDM; (2): log10 of 16S rDNA copy numbers/gDM

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Unbenannt-8 1 23.03.16 13:20

Chaucheyras-Durandet et al.: Effect of a live yeast, Saccharomyces cerevisiae I-1077, used as a feed additive for ruminants, on in situ ruminal digestibility of alfalfa hay and fibre-associated microorganisms


Conclusion The daily distribution of S.cerevisiae I-1077 can significantly improve degradation of alfalfa hay in the rumen and diversely affects feed particles associated microbial populations.

Corresponding author Frédérique Chaucheyras-Durand INRA Unit of Microbiology UR454 CR Clermont-Ferrand/Theix 63122 Saint- Genès Champanelle/France E-mail: frederique.chaucheyras(at)clermont.inra.fr

Hovenjürgen et al.: Einfluss mittelkettiger Fettsäuren auf die Entwicklung schweinespezifischer pathogener Keime in vitro


Einfluss mittelkettiger Fettsäuren auf die Entwicklung schwei-nespezifischer pathogener Keime in vitro

Dr. Michael Hovenjürgen, Dorothee Schulze Schwering und Jan-Bernd Reeken BEWITAL agri GmbH & Co. KG, DE

Abstract The reduction of antibiotic useinlivestock is still in the center of discussion. As an alternative to antibi-otic agents, organic acids play an important role in animal nutrition. Especially medium chain fatty acids are used because of their antibacterial effect mainly against gram-positive pathogens such as Streptococcus. However, the areas of application and the effects of the respective acids differ, so that synergistic effects can be caused by a precise selection and combination of acids. The objective of this study was to investigate the mechanisms of action of various fatty acids. Results should help to de-termine an efficient combination of active ingredients which inhibit particularly growth of pig specific pathogens.The active components lauric and myristic acid have a significant impact on bacterial growth and decrease it by increasing concentration. Low dosage of the combination product BEWI-FATRIX SynerG+ already reduces the growth of swine relevant pathogenic bacteria like Escherichia coli, Streptococcus suis, Salmonella spec. and Clostridium perfringens in vitro.

Einleitung Durch die gesetzlich geforderte Reduzierung des Antibiotikaeinsatzes in der Nutztierhaltung spielen organische Säuren in der Tierernährung eine immer größer werdende Rolle. Besonders mittelkettigen Fettsäuren kommt in der Tierernährung aufgrund ihrer energieliefernden Inhaltsstoffe und antibakte-riellen Wirkung eine besonders hohe Bedeutung zu (Tölle & Meyer, 2008). Diese Eigenschaften ma-chen sie sehr interessant für den Einsatz in der Schweinehaltung (Ferrara, 2012). Gerade hier ist der Infektionsdruck auf die Tiere, ausgelöst durch schweinespezifische pathogene Keime (Streptococcus suis, Escherichia coli, Salmonella spec. und Clostridium perfringens),ein Hauptgrund für den Einsatz von antibiotischen Arzneimitteln. Viele in vitro-Studien zeigten beim Einsatz von mittelkettigen Fettsäuren ausgeprägte antibakterielle Effekte sowohl gegen gramnegative als auch grampositive Keime (Kabara et al., 1972; Batovska, et al., 2009). Laurinsäure gilt als besonders aktiver antibakterieller Wirkstoff, wobei Glycerolmonolaurat noch wirksamer sein soll (Kabara, et al., 1972; Batovska, et al., 2009). Das in der Literatur beschrie-bene antimikrobielle Potenzial mittelkettiger Fettsäuren wurde bereits in der Praxis zur Anwendung gebracht. Eine große Anzahl an Zusatzstoffen findet in der Schweinefütterung ihre Anwendung. Es gilt hier, die Immunität der Tiere zu stärken und auch den Einsatz von Antibiotika zu reduzieren (Tölle & Meyer, 2008). Die Einsatzbereiche und die Wirkung der jeweiligen Säuren sind jedoch sehr verschie-den, sodass durch die Auswahl und die Kombination von Säuren synergistische Effekte hervorgerufen werden können. Vor diesem Hintergrund war die Zielsetzung die Wirkung verschiedener Fettsäuren zu untersuchen, um so eine effiziente Wirkstoffkombination zu ermitteln, die vor allem das Wachstum schweine-spezifischer pathogener Keime, wie Streptokokken effektiv hemmt.



Material und Methoden Mittels Bouillon-Mikrodilutionstest wurde zunächst der Einfluss der Fettsäuren Laurinsäure (C12), My-ristinsäure (C14) und des Fettsäure-Mixes Capryl/Caprinsäure (C8C10, im Verhältnis 60:40), sowie der Einfluss von Glycerolmonolaurat (GML) auf das Wachstum von Escherichia coli, Streptococcus suis, Salmonella poona und Clostridium perfringens untersucht. Zunächst wurden die verschiedenen Bakterienstämme mit Konzentrationen der genannten Wirkstoffe in Nährmedium inkubiert. Neben den reinen Fettsäuren wurde auch das Kombinationsprodukt BEWI-FATRIX SynerG+ sowie zwei Kombinationsprodukte (Kombinationsprodukt F und Kombinationsprodukt D) auf der Basis von Monolaurat getestet. Hierfür wurden von allen Wirkstoffen 10%-ige Stammlö-sungen hergestellt, indem jeweils 100mg C12, GML, BEWI-FATRIX SynerG+ sowie 100mg vom Kom-binationsprodukt F und D in 1ml 50%-igem Ethanol gelöst wurden. Die gleiche Menge C14 wurde in 1ml 99%-igem Ethanol gelöst. Der flüssige Fettsäuremix C8C10 wurde 1:10 mit Wasser verdünnt. Laurin- und Myristinsäure wurden in den Konzentrationen 0,2%, 0,4%, 0,6%, 0,8% und 1% einge-setzt. C8C10 und GML sowie die zugsammengesetzten Produkte (BEWI-FATRIX SynerG+, Kombinati-onsprodukte F und D) wurden in den Konzentrationen 0,1%, 0,2%, 0,3%, 0,4%, 0,5% und 0,6% getestet. In einem weiteren Schritt wurden von einer 18 bis 24 Stunden alten Reinkultur auf Blutagar einzelne Kolonien in 1ml Nährmedium resuspendiert. Kolonien von Escherichia coli und Salmonella poona wur-den in gepuffertem Peptonwasser resuspendiert, Streptococcus suis Typ 1 wurde in BHI-Bouillon (an-gereichert mit Blutserum, L-Cystein und NAD) resuspendiert. Für die Vorkultur von Clostridium perfringens wurden 11ml H-Medium mit einer Kolonie von Clostridium perfringens angeimpft und über Nacht anaerob bei 37°C bebrütet. Als Maß des Bakterienwachstums wurde bei einer Wellenlänge von 600nm die optische Dichte ermit-telt. Die Auswertung der Ergebnisse erfolgt über die Angaben der „minimal inhibitory concentration 50“ (MIC50). Die MIC50 beschreibt diejenige Wirkstoffkonzentration, bei welcher das Bakterienwachs-tum um 50% reduziert vorliegt. Das Bakterienwachstum wurde im Falle von Escherichia coli, Strep-tococcus suis und Salmonella poona über einen Zeitraum von 15 Stunden untersucht (stündliche Messung). Statistische Auswertung: Von allen Messwerten wurden im Anschluss an den Wachstumsversuch zunächst die Leerwerte sub-trahiert, um die Absorption des Mediums inklusive der Wirkstoffe herauszurechnen. Die neu berechne-ten Absorptionen bei der Wellenlänge von 600nm spiegeln somit das Bakterienwachstum unter den unterschiedlichen Wachstumsbedingungen wider.

Ergebnisse Die Wirkstoffe C12 und C14 zeigen einen deutlichen Einfluss auf das Bakterienwachstum. Mit zuneh-mender Konzentration dieser Wirkstoffe nimmt das Bakterienwachstum ab. Die Ergebnisstabelle zeigt, dass das Bakterienwachstum der vier Teststämme bereits bei einer Zugabe von 0,2% Laurinsäure um die Hälfte abnimmt. Des Weiteren hat der Zusatz von Myristinsäure, C8/C10 Mix und GML bereits in geringen Konzentrationen einen deutlichen Effekt auf das Wachstum von E-scherichia coli und den grampositiven Bakterien Streptococcus suis und Clostridium perfringens.



Tabelle 1: Minimal einzusetzende Konzentrationen der getesteten Wirkstoffe, bei denen das Bakterienwachstum in vitro um 50% reduziert wird

Auffällig ist, dass die MIC50 dieser Wirkstoffe in Bezug auf Salmonella poona im Bereich von 0,4%-1,0% liegen und somit in höheren Konzentrationen eingesetzt werden müssten um eine 50%-ige Re-duzierung des Salmonella poona Wachstums zu erzielen. Im Vergleich mit den beiden Kombinationsprodukten zeigt sich, dass die Zugabe von BEWI-FATRIX SynerG+ zum Nährmedium das Wachstum von grampositiven Keimen schon bei niedrigeren Konzent-rationen bereits um 50% hemmt. So reichen bereits 0,1% des Produktes BEWI-FATRIX SynerG+ aus, um das Keimwachstum von Streptococcus suis um 50% zu reduzieren. Weiterhin ist auch ein Einfluss von BEWI-FATRIX SynerG+ auf das Wachstum von Escherichia coli und Salmonella poona zu be-obachten. Schon 0,4% bzw. 0,6% des Produktes reichen aus, um die MIC50 zu erlangen. Es konnte ein Einfluss verschiedener Konzentrationen der antimikrobiellen Wirkstoffe Laurinsäure, Myristinsäure, Fettsäuremix C8/C10 und Glycerolmonolaurat auf das in vitro Wachstum schweinespezi-fischer pathogener Keime festgestellt werden. Des Weiteren zeigten auch die Kombinationsprodukte ab bestimmten Konzentrationen eine antimikrobielle Wirkung. Jedoch zeigten die Kombinationspro-dukte keine einheitliche Verbesserung gegenüber den einzelnen Wirkstoffen. Hier zeigte nur BEWI-FATRIX SynerG+ eine bessere Wirkung.

Diskussion Derzeit befinden sich einige Futterzusatzstoffe auf dem Markt, die den Infektionsdruck beim Schwein durch den Einsatz bestimmter antibakteriell wirkender mittelkettiger Fettsäuren im Futter senken kön-nen. In der vorliegenden in vitro Untersuchung konnte die Wirksamkeit verschiedener Wirkstoffe auf schweinespezifische pathogene Keime ermittelt werden. Die Ergebnisse und die Synergieeffekte der untersuchten Wirkstoffe wurden bei der Zusammensetzung des Ergänzungsfuttermittels BEWI-FATRIX SynerG+ berücksichtigt. Durch die Applikation von BEWI-FATRIX SynerG+ auf die verschiedenen Bak-terienkulturen konnte, im Gegensatz zu den Kombinationsprodukten D und F, auf Streptococcus suis bereits ab einer Applikationsmenge von 0,1% eine signifikante Wirkung nachgewiesen werden.

Schlussfolgerung Bei der Betrachtung der schweinespezifischen pathogenenBakterien Escherichia coli, Streptococcus suis, Salmonella poona und Clostridium perfringens ist festzustellen, dass sich das Bakterienwachstum in vitro bei einer geringen Einsatzmenge von BEWI-FATRIX SynerG+ bereits um 50% reduziert. Der in der Literatur beschriebene antimikrobielle Effekt von Laurinsäure wurde weiter belegt. Eine deutlich höhere Wirksamkeit von Glycerolmonolaurat, die in der Vergangenheit von einzelnen Autoren be-schrieben wurde, konnte in der vorliegenden Untersuchung hingegen nicht bestätigt werden. Vielmehr

MIC50 in % Escherichia coli

Streptococcus suis

Salmonella poona

Clostridium perfringens

C8C10 (60:40) 0,3% <0,2% >1,0% 0,1% Laurinsäure (C12) 0,2% 0,2% 0,2% 0,1% Myristinsäure (C14) 0,2% 0,2% 0,4-0,8% 0,3% Glycerolmonolaurat (GML) 0,4% 0,1% 0,6% 0,1% BEWI-FATRIX SynerG+ 0,4% 0,1% 0,6% 0,1% Kombinationsprodukt F >0,6% >0,6% >0,6% 0,2% Kombinationsprodukt D 0,5% 0,6% >0,6% 0,2%



bleibt festzuhalten, dass nur eine Kombination der Wirkstoffe und die daraus resultierenden Synergie-effekte die gewünschte Wirkung hervorrufen kann.

Literatur Batovska, D., Todorova, I., Tsvetkova, I. & Najdenski, H., 2009. Antibacterial Study of Medium Chain Fatty Acids and Their 1-Monoglycerides: Individual Effects and Synergistic Relationships , Sofia: Polish Journal of Microbiology .

Ferrara, F., 2012. Untersuchungen zum Einsatz von mittelkettigen Fettsäuren und kurzkettigen organischen Säuren in der Fütterung von Absatzferkeln, Berlin: Freie Universität Berlin.

Kabara, J., Swieczkowski, D., Conley, A. & Truant, J., 1972. Fatty Acids and Derivatives as Antimicrobial Agents, Michigan: Michigan State University.

Tölle, D. K.-H. & Meyer, C., 2008. Einsatz von mittelkettigen Fettsäuren (MCFA) in der Ferkelaufzucht. Landpost, 16 02, pp. 50-51.

Zentek, J. et al., 2011. Nutritional and physiological role of medium-chain triglycerides and medium-chain fatty acids in piglets, Berlin: Cambridge University Press.

Autorenanschrift

Dr. Michael Hovenjürgen BEWITAL agri GmbH & Co. KG Industriestraße 10 DE-46354 Südlohn-Oeding E-Mail: M.Hovenjuergen(at)bewital.de

FATRIX SynerG+®

Keimer et al.: Effects of a hydrolysed yeast (Kluyveromyces fragilis) on feed intake and zootechnical performance in weaned piglets


Effects of a hydrolysed yeast (Kluyveromyces fragilis) on feed intake and zootechnical performance in weaned piglets

Birgit Keimer1,2, Alexandra Schlagheck2, Rudolf Hartwigsen3 and Georg Thaller3

1 Humboldt Universität zu Berlin, Albrecht Daniel Thaer-Institut für Agrar- und Gartenbauwissenschaften, DE 2 Biochem Zusatzstoffe GmbH, Lohne, DE 3 Christian-Albrecht Universität zu Kiel, Institut für Tierzucht und Tierhaltung, DE

Abstract Early weaning of piglets is often accompanied by growth losses and diarrhea. Various nutritional ap-proaches for optimising the weaning transition and minimising enteric diseases have been tested in the past decade. Next to the diet composition (carbohydrates, protein source), the palatability of the diet seems to be promising to ensure a stable feed intake in the acute phase after weaning. Beside animal protein sources, some yeast products have been proposed to improve palatability. A feeding trial has been conducted to investigate the effect of hydrolysed yeast on feed intake and zootechnical performance in weaned piglets. The feeding trial was divided in two feeding phases (phase 1: d1-d25 after weaning, phase 2: d26-d40 after weaning). In the treatment group hydrolysed yeast was dosed with 1% in the first phase and 0.5% in the second phase. Feed intake was significantly improved by addition of hydrolysed yeast in the first phase (485 vs. 530 g per piglet/day, p<0.05) and overall peri-od (770 vs. 837g per piglet/day, p<0.05). Average weaning weight was about 9.0 kg per piglet and identical for both groups. After first feeding phase the yeast-supplemented piglets had significant higher body weights compared to control (17.7 vs. 18.7kg, p<0.05). No significant effect on final body weights could be observed, but treatment group was numerically improved (29.6 vs. 30.8kg, p=0.132). Average daily weight gain tended to be increased in treatment group compared to control (phase 1: 347 vs. 389g, p=0.061; phase 2: 799 vs. 806g, p=0.069). Feed conversion ratio was not affected.

Introduction Weaning is a stressful and critical period in piglet´s life. After weaning, piglets have to cope with ab-rupt separation from their mother and mixing with other litters in a new environment. As a result, a low and variable feed intake can be observed within the first days or weeks after weaning. This low and variable feed intake may result in negative impacts on intestinal physiology and performance (Pluske et al. 1997). The composition of the weaner diet plays an important role in controlling nega-tive impacts on intestinal physiology. Next to the amount and structure of carbohydrates, the protein source and concentration has a significant influence on intestinal microbiology, morphology and im-munology. High quality protein sources such as milk powder and porcine blood plasma are known to have posi-tive effects on intestinal morphology. Lalles et al. (2007) concluded that these effects often result from the stimulated feed intake. Therefore, it is extremely important to use tasty ingredients in diets for weaned piglets to ensure a high feed intake. Yeast extracts based on the yeast cytoplasm are known to improve palatability in diets for men and animal. Yeast extracts in combination or in re-placement to animal plasma proteins improve feed intake, weight gain and feed conversion ratio in weaned piglets (Carlson et al., 2005; Pereira et al., 2012; Rigueira et al., 2013). Due to economical



reason, yeast extracts are primarily used in human nutrition. Hydrolysed yeasts may create a good alternative to yeast extract for use in animal nutrition. Next to the flavor-enhancing properties of the yeast extract, hydrolysed yeast contains prebiotic-acting yeast cell wall components. To study the effects of a hydrolysed yeast on feed intake and zootechnical performance, a feeding trial was carried out at Hohenschulen research farm of the University Kiel.

Materials and methods Ninety post-weaning piglets ((DL x DE) x BHZP) were assigned equally into two homogeneous groups (9 animals per pen, n=5) according to body weight, litter, and gender. Piglets were weaned at ap-prox. 25 days of age. The experimental period lasted 40 days and was divided into two feeding phas-es; phase 1 (d1-d25 post weaning) and phase 2 (d26-d40 post weaning). Piglets were fed isoenergetic and isonitrogenous diets either without yeast (control) or with a hydrolysed yeast (Kluy-veromyces fragilis) dosed with 10 g per kg in phase 1 and 5 g per kg in phase 2 (table 1). Control diet was prepared according to composition of commercial diets for piglets. To balance the diets for ener-gy and protein level, wheat and soybean meal contents slightly vary between the diet-compositions. Feed and water were offered ad-libitum. Individual body weights (BW) were recorded at weaning and after each feeding phase. The feed in-take per piglet was estimated by dividing feed intake per pen by the number of piglets per pen. The feed conversion ratio (FCR) was calculated from feed intake per piglet and the recorded body weight gain per piglet for each period. Average daily weight gain (ADWG) was calculated by dividing weight gain by number of days for each period.

Table 1: Composition of control and treatment diets

Phase 1 Phase 2 Control Treatment Control Treatment Ingredients (g per kg) Wheat 46.1 44.6 47.2 46.9 Barley 18.0 18.0 15.0 15.0 Wheat bran 2.00 3.90 4.20 4.50 Soy protein concentrate 1.00 1.00 0.00 0.00 Soybean meal 18.9 17.6 18.2 17.7 Soybean oil 1.30 1.40 0.50 0.50 By-products from bakery 6.00 6.00 10.00 10.00 By-products from dairy 1.00 1.00 0.00 0.00 Premix 3.70 3.70 3.30 3.30 VitaLys Liquid 1.50 1.50 1.30 1.30 DL-Methionine 0.10 0.10 0.10 0.10 Threonine 0.20 0.20 0.20 0.20 Yeast cell wall 0.20 0.00 0.00 0.00 Hydrolysed yeast 0.00 1.00 0.00 0.50 Chemical composition (calculated) Energy, MJ ME/kg 13.6 13.6 13.4 13.4 Crude protein, % 17.5 17.5 17.0 17.0 Lysine, % 1.30 1.30 1.20 1.20 Crude fat, % 3.27 3.43 2.53 2.55 Crude fiber, % 2.88 3.00 2.95 2.96 Crude ash, % 4.95 4.98 5.16 5.16 Calcium, % 0.69 0.69 0.73 0.73 Phosphorus, % 0.50 0.50 0.55 0.55



All piglets were monitored regularly for any abnormalities and clinical signs of disease. Medical treat-ments and mortality were documented. All piglets were ear-tagged to ensure clear identification. Sta-tistical analysis was performed using SAS Mixed procedure.

Results

Overall, the piglets performed on a high level without any evident health problems in the present study. Figure 1 depicts the average feed intake per week for the first three weeks after weaning. Hy-drolysed yeast fed piglets started with a slightly higher feed intake in week 1 (239 vs 253g per day, p=0.290). Highest effects on feed intake could be observed in week 2 (318 vs.372g, p<0.05) and week 3 (621 vs. 694g, p=0.126).

Figure 2: Average daily feed intake per piglet in the first three weeks after weaning (Mean, SD, n=5)

The results of feed intake, weight gain and FCR per period are given in table 2. Significant effects on feed intake in the first feeding phase as well as for overall period could be observed. In the first feed-ing phase piglets in treatment group ate 9.3% more than the control ones. Overall feed intake was improved by 8.7%.

Table 2: Feed intake, ADWG (average daily weight gain) and FCR (feed conversion ratio) for each feeding phase (n=5)

Control Treatment p-value Mean SD Mean SD

Average daily feed intake (kg) Phase 1 (d1-d25) 0.485 ±0.05 0.530 ±0.04 0.016 Phase 2 (d26-d40) 1.269 ±0.05 1.347 ±0.13 0.494 Total (d1-d40) 0.770 ±0.05 0.837 ±0.07 0.037 ADWG (kg) Phase 1 (d1-d25) 0.347 ±0.04 0.389 ±0.02 0.061 Phase 2 (d26-d40) 0.799 ±0.03 0.806 ±0.04 0.069 Total (d1-d40) 0.512 ±0.02 0.545 ±0.02 0.138 FCR (kg feed per kg weight gain) Phase 1 (d1-d25) 1.40 ±0.06 1.36 ±0.08 0.338 Phase 2 (d26-d40) 1.59 ±0.07 1.67 ±0.16 0.673 Total (d1-d40) 1.50 ±0.04 1.53 ±0.11 0.454



Average weaning weight was about 9.0 kg per piglet and identical for both groups. After first feeding phase piglets had significant higher body weights compared to control (17.7 vs. 18.7kg, p<0.05). No significant effect on final body weights could be observed, but treatment group was numerically im-proved (29.6 vs. 30.8kg, p=0.132). In both feeding phases, piglets from treatment group tended to have higher daily weights gains compared to control. No significant effects on feed conversion ratio could be observed.

Discussion The hydrolysed yeast had a significant positive effect on feed intake, especially in the first three weeks after weaning. These results are in line with study of Carlson et al. (2005), who observed best effects of yeast extracts on feed intake in the first two weeks after weaning (+12%). They also com-pared the effect of yeast extract plus blood plasma as a positive control and found no significant dif-ferences in feed intake between both groups. Blood plasma increased feed intake by +10% in the first two weeks after weaning compared to negative control. As a result of the higher feed intake piglets gained more weight. In the first feeding period, FCR was numerically improved. Lalles et al (2007) reviewed that a high and stable feed intake after weaning can improve intestinal morphology (villi height and crypth depth), which can lead to a better feed utilisation. However, FCR in the second feeding period was numerically higher in treatment group. It can be assumed that the stimulation of feed intake during this period provides no adverse effects on FCR.

Conclusion The tested yeast product improved feed intake and zootechnical performance in piglets. It has to be considered that different yeast products that are available on the market vary origin and processing. Further research should investigate the effects of processing on the flavor-enhancing properties of yeast products.

References Brooks P.H., Moran, C.A., Beal J.D., Demeckova, V., Campbell, A. (2001) Liquid feeding for the young piglet. In The Weaner Pig: Nutrition and Management, 153-178, Wallingford, Oxon: CAB International

Carlson, M.S., Veum, T.L., Turk, J.R. (2005) Effects of yeast extract versus animal plasma in weanling pig diets on growth performance and intestinal morphology. J Swine Health Prod. 13(4):20-209

Lalles, J.-P., Bosi, P., Smidt, H., Stokes, C.R. (2007) Weaning – A challenge to gut physiologists. Livestock Science 108: 82-93

Pereira, C.M.C., Donzele, J.L., de Oliveira Silva, F.C, de Oliveira, R.F.M, Kiefer, C., Ferreira, A.S., Hannas, M.I., Brustolini, P.C. (2012) Yeast extract with blood plasma in diets for piglets from 21 to 35 days of age. R. Bras. Zootec. 41 (7): 1676-1682

Pluske, J.R., Hampsoon, D.J., Williams, I.H. (1997) Factors influencing the structure and function of the small intestine in the weaned pig: a review, Livestock Production Science 51: 215-236

Rigueira, L.C.M., Thomaz, M.C., Rigueira, D.C.M., Pascoal, L.A.F., Amorim, A.B., Budino, F.E.L. (2013) Effect of plasma and/or yeast extract on performance and intestinal morphology of piglets from 7 to 63 days of age. R. Bras. Zootec. 42 (7): 496-503

Corresponding author Birgit Keimer Biochem Zusatzstoffe GmbH Küstermeyerstrasse 16 49393 Lohne (Oldenburg) E-mail: keimer8(at)biochem.net

Baur et al.: Prüfung eines Cnicin-haltigen Extraktes aus Cnicus benedictus als Tränkwasserzusatz zur Stabilisie-rung der Darmgesundheit bei Absatzferkeln


Prüfung eines Cnicin-haltigen Extraktes aus Cnicus benedictus als Tränkwasserzusatz zur Stabilisierung der Darmgesundheit bei Absatzferkeln

Anja C. Baur1, Holger Kluge1, Gert Horn2, Jutta Kalbitz3, Antje Breitenstein3 und Gabriele I. Stangl1 1 Institut für Agrar- und Ernährungswissenschaften, Martin-Luther-Universität Halle- Wittenberg, DE 2 Exsemine GmbH, Salzatal, DE 3 BioSolutions Halle GmbH, Halle (Saale), DE

Abstract Cnicin is a sesquiterpene lactone which is characterized by a strong bitter taste. Cnicin exerts anti-bacterial, anti-inflammatory and cytotoxic properties. The current study aimed to investigate the im-pact of cnicin on health, food intake and growth of piglets. Therefore, sixty 28-day-old piglets were randomly allocated into three groups and received cnicin (extracted from Cnicus benedictus) in con-centrations of 0 mg/l (A, control), 20 mg/l (B) and 100 mg/l (C) with their drinking water for 3 weeks. Additionally, the antimicrobial capacity was elucidated in an in vitro model. Escherichia coli (E.coli)and Staphylococcus aureus (S.aureus)were incubated withthree concentrations of a cnicin solution (50 µg/ml, 100 µg/ml, 200 µg/ml) and an extract from Cnicus benedictus. Food intake, weight gain and the consistency of feces was not significantly differenent between the groups of piglets. However, cnicin was capable of reducing the growth of E.coli and S.aureus.

Einleitung Cnicin bzw. Sesquiterpenlactone zeichnen sich durch einen ausgeprägten Bittergeschmack aus, der bei Cnicus benedictus Schäden durch pflanzenfressende Insekten und Wildverbiss verhindert. Der verdau-ungsförderliche Bitterstoff könnte sich anregend auf die Futteraufnahme auswirken. Wie die Untersu-chungen von Bruno et al. (2003) und Bach et al. (2011) gezeigt haben, weist Cnicin eine erhebliche antibakterielle Wirkung gegenüber gramnegativen und grampositiven Bakterien aus und verfügt über eine hohe, mit Indometacin vergleichbare entzündungshemmende Aktivität (Schneider et al. 1987). Es zeigt weiterhin eine zytotoxische Aktivität gegenüber verschiedenen Tumorzelltypen (Bruno et al. 2005). In dieser Untersuchung wurde Cnicin über ein spezielles Extraktionsverfahren aus der Cnicus benedictus Krautdroge isoliert und dem Tränkwasser zugesetzt, um Effekte auf die Futteraufnahme, das Wachstum und die Tiergesundheit bei Absetzferkeln zu prüfen.

Material und Methoden Für die Untersuchungen wurden 60 Ferkel ((DExDL)xPietrain) im Alter von 28 Tagen mit einem Durch-schnittsgewicht von 7,6 kg randomisiert auf 3 Versuchsgruppen verteilt. Die Aufstallung erfolgte in Einzelbuchten, die mit 10 l Fülltränken zur Erfassung der Tränkwasseraufnahme ausgestattet waren. Das Tränkwasser der Kontrollgruppe A enthielt nur Wasser mit einem Zusatz an Ethanol, um eine vergleichbare Alkoholkonzentration wie in der Versuchsgruppe C einzustellen. In den Versuchsgruppen B und C wurde das Tränkwasser mit 20 bzw. 100 mg in Ethanol gelöstem Cnicin je Liter Wasser er-gänzt. Für die Supplementierung mit Cnicin wurde ein Dicksaft (Herstellung Bell Flavors & Fragrances



GmbH, Leipzig) mit einer Cnicinkonzentration von 1,48 % und einem Alkoholgehalt von 54 % herge-stellt. Cnicus benedictus zur Herstellung des Dicksaftes lieferte die Exsemine GmbH,Salzatal. Analy-tisch wurde die Cnicinkonzentration mittels HPLC von BioSolutions Halle GmbH kontrolliert. Über einen Zeitraum von 21 Tagen erfolgte täglich die Erfassung der Tränkwasseraufnahme und eine Bonitierung der Kotkonsistenz (1=fest, 2=breiig, 3=flüssig). An den Tagen 14 und 21 wurde den Tieren rektal Kotproben für eine Trockensubstanzbestimmung entnommen. Wöchentlich wurden die Tiere bis zum Versuchsende über 6 Wochen gewogen und die Futteraufnahme erfasst. Diese Daten dienten zur Be-stimmung der wöchentlichen Futterverwertung. Die Tiere erhielten in den ersten 14 Tagen ein Prestarterfutter mit 13,6 MJ/kg und 17,2 % Protein und von Tag 14-42 ein Starterfutter mit 13,6 MJ/kg und 16,6 % Protein. Ergänzt wurden die Futtermi-schungen mit Vitaminen, Spurenelementen und kristallinen Aminosäuren entsprechend der Empfeh-lungen der GfE (2006). Die Futtermischungen wurden pelletiert und ad libitum gefüttert. In einem in vitro Vorversuch wurde die antimikrobielle Aktivität der Reinsubstanz Cnicin in den Kon-zentrationen 50 µg/ml, 100 µg/ml und 200 µg/ml auf Escherichia coli und Staphylococcus aureus ge-prüft. Escherichia coli wurde in Luria Bertani und Staphylococcus aureus in Trypticase-Soy-Yeast-Extrakt-Medium bei 37°C und 170 rpm im Schüttelinkubator kultiviert. Als Kontrollen wurden jeweils Nährmedien ohne Cnicin sowie Nährmedium mit 2% Ethanol, welches als Lösungsmittel für Cnicin diente, mitgeführt. Diese Voruntersuchung diente zur Orientierung der geprüften Konzentration an Cnicin im Tränkwasser der Ferkel.

Ergebnisse und Diskussion Die in vitro Voruntersuchung zeigte einen deutlichen Einfluss der geprüften Cnicinkonzentrationen auf das Wachstum von Escherichia coli und Staphylococcus aureus (Abb.1a und b). Die Befunde zur anti-mikrobiellen Aktivität korrespondieren mit den Ergebnissen von Bruno et al. (2003). In dieser Untersu-chung lagen die minimalen Hemmkonzentrationen von Cnicin für Escherichia coli bei 12,5 µg/ml und für Staphylococcus aureus bei 25 µg/ml.

Abbildung 1: Wachstum von Escherichia coli in Luria Bertani (LB) Medium (A) und Staphylococcus aureus in

Trypticase-Soy-Yeast-Extract (TSY) Medium (B) unter Einfluss verschiedener Cnicin-Konzentrationen



Cnicin hatte keinen negativen Einfluss auf die Tränkwasseraufnahme. In der Gruppe, die die höchsten Cnicinkonzentrationen erhielt, war die Wasseraufnahme sogar am höchsten (Tab.1). Die Messung der Kottrockensubstanz am Tag 14 und 21 zeigte keine statistischen Unterschiede. In der zweiten Ver-suchswoche wurde eine signifikante Korrelation (r=0,32) zwischen der täglichen Aufnahme an Cnicin und der Kottrockensubstanz am Tag 14 ermittelt. Aus der täglichen Bonitierung der Kotkonsistenz über den gesamten Versuchszeitraum ergaben sich keine Tiere mit auffälligem Durchfall. Die ausschließliche Gabe der cnicinhaltigen Tränke in den 21 Tagen nach dem Absetzen hatte keinen Einfluss auf die Futteraufnahme, den Lebendmassezuwachs und die Futterverwertung (Tab.2). Eine Steigerung der Futteraufnahme durch Cnicin konnte nicht beobachtet werden. Auch nach dem Abset-zen der Cnicintränke wurden keine Leistungsunterschiede zwischen den Versuchsgruppen festgestellt (Tab.2).

Tabelle 1: Einfluss von Cnicin auf die Wasseraufnahme und die Fäzestrockensubstanz

Parameter Kontrolle Cnicin 20 mg/l

Cnicin 100 mg/l

SEM P-Wert

Tränke (Liter/Tag) Tag 1-7 0,9 1,0 1,1 0,06 0,132 Tag 7-14 1,4ab 1,2b 1,9a 0,09 0,015 Tag 14-21 1,6 1,7 2,1 0,09 0,086 Kot TS (%) Tag 14 30,4 28,3 31,5 0,61 0,092 Tag 21 28,0 28,9 29,6 0,53 0,446 SEM: standard error of the means Statistische Analyse mittels einfaktorieller Varianzanalyse (ANOVA). Mit unterschiedlichen Hochbuchstaben gekennzeichnete Werte unterscheiden sich signifikant voneinander (Tukey HSD-Test, p<0,05).

Tabelle 2: Einfluss von Cnicin auf die Futteraufnahme und die Gewichtszunahme während und nach der Tränkpe-riode

Parameter Kontrolle Cnicin 20 mg/l

Cnicin 100 mg/l

SEM P-Wert

Startgewicht (kg) 7,5 7,7 7,6 0,07 0,587 Gewicht Tag 21 11,9 11,9 11,9 0,16 0,982 Zunahme Tag 1-21 (g/Tag) 211 205 204 6,66 0,898 Futteraufnahme Tag 1-21 (g/Tag) 295 291 288 8,07 0,942 Futteraufwand Tag 1-21 (kg/kg) 1,41 1,45 1,43 0,02 0,671 Gewicht Tag 42 (kg) 22,5 22,4 22,4 0,30 0,971 Zunahme Tag 21-42 (g/Tag) 507 498 500 8,86 0,916 Futteraufnahme Tag 21-42 (g/Tag) 809 807 797 13,58 0,936 Futteraufwand Tag 21-42 1,60 1,62 1,59 0,01 0,476 SEM: standard error of means Statistische Analyse mittels einfaktorieller Varianzanalyse (ANOVA).

Schlussfolgerungen Cnicin zeigt in vitro eine starke antimikrobielle Wirkung. Obgleich Cnicin eine mögliche appetitfördern-de Eigenschaft zugesprochen wird, unterschied sich die Futteraufnahme der cnicinbehandelten Ferkel nicht von denen der Kontrollgruppe. Ein Einfluss von Cnicin auf das Wachstum und die Kotkonsistenz



war bei den offensichtlich gesunden Tieren ebenfalls nicht zu beobachten. Es ist jedoch nicht auszu-schließen, dass sich bei Ferkeln mit Durchfallerkrankungen Cnicin als gesundheitsförderlich erweist.

Danksagung Für die freundliche Unterstützung des Projektes sei der Bundesstiftung Umwelt (DBU) herzlich gedankt.

Literatur BACH, S. M.; FORTUNA, M. A.; ATTARIAN, R.; DE TRIMARCO; J. T.; CATALAN, C. A. N.; AV-GAY, Y; BACH, H., 2011: Antibacterial and Cytotoxic Activities of the Sesquiterpene Lactones Cnicin and Onopordopicrin. Natural Product Communications 6 (2), 163-166

BRUNO, M.; ROSSELLI, S.; MAGGIO, A.; RACCUGLIA, R. A.; NAPOLITANO, F.; SENATORE, F., 2003: Antibacterial Evaluation of Cnicin and Some Natural and Semisynthetic Analogues. Planta Medica 69 (3), 277-281

BRUNO, M.; ROSSELLI, S.; MAGGIO, A.; RACCUGLIA, R. A.; BASTOW, K. F.; WU, CH.-CH.; LEE, K.-H., 2005: Cytotoxic Activity of Some Natural and Synthetic Sesquiterpene Lactones. Planta Medica 71 (12), 1176-1178

Gesellschaft für Ernährungsphysiologie (GfE), 2006: Empfehlungen zur Energie- und Nährstoffversorgung von Schweinen. DLG-verlag, Frankfurt (Main)

SCHNEIDER, G.; LACHNER, I., 1987: Beitrag zur Analytik und Wirkung von Cnicin. Planta Medica 53, 247-251

Autorenanschrift Anja Christina Baur Institut für Agrar- und Ernährungswissenschaften Von-Danckelmann-Platz 2 06120 Halle-Saale E-Mail: Anja-Christina.Baur(at)landw.uni-halle.de

Suarez and Gallissot: Effect of an algae-clay mix on the use by broiler chickens of a diet containing corn DDGS


Effect of an algae-clay mix on the use by broiler chickens of a diet containing corn DDGS

Maria Garcia Suarez and Marie Gallissot Olmix SA, Brehan, FR

Abstract This study was set up to evaluate the effect of supplementing an algae-clay mix on zootechnical per-formance of broiler chickens fed with corn DDGS. 414 day-old chicks were randomly distributed to eighteen pens, allocated to one of three groups re-ceiving different diets: the standard diet (C-), the test diet (T-), containing corn DDGS at the level of 10%, and the test diet supplemented with 0.1% of algae-clay mix (T+). Three different feeds were distributed from D0-D9, D9-D18 and D18-D31. Group weighing of the animals (D0, D9, D18 and D31) and litter quality scoring (D18) were performed. Results were submitted to analysis of variance. Results show a significant decrease of ADWG in the finishing period (-8.61%, P = 0.04) and the total period (-7%, P = 0.02) in the T- group compared to the control. In finishing and total periods, the ADWG of the T+ group is significantly higher than in the T- group (respectively +11.72%, P = 0.02 and +7.13%, P = 0.03) and is similar to the control. On the other hand, in the starter period, C- and T- groups show a significantly higher ADWG than the T+ group. Mortality was non-significantly lower in the T+ group than in C- and T- groups. No visible impact was observed on litter quality. In the end, this study shows a positive effect of the algae-clay mix on growth performance of broiler chickens fed with the test diet, raising the interest of its use in the utilization of such diets.

Introduction With an increasing competition worldwide, the poultry industry is more than ever pushed to improve its productivity. Meanwhile, the supply of raw materials is changing, both from an economic point of view (high prices volatility) and a quality point of view (emergence of new raw materials and by-products, which use is not always optimized). In this context, feed supplementation with specific addi-tives or premixes can contribute to improve the use of diets containing low digestible ingredients (Shalash et al, 2009; Ouhida et al, 2000), while maintaining high levels of performance and produc-tivity. In the last years, several studies highlighted the ability of clays to improve feed digestibility (Tauquir and Nawaz, 2001; Reichardt, 2008; Habold et al, 2009). This way, Olmix developed a prod-uct associating algae extracts (Ulva sp and Solieria chordalis) and clay (bentonite), which aims at the better use of diets containing by-products. The objective of the present study was to evaluate the effect of this algae-clay mix (MFeed+) on the growth performance of broiler chickens fed a diet con-taining by-products and raised under field conditions.

Materials and methods The study was conducted in a commercial broiler farm in Brittany, France. A total of 414 day-old chicks (ROSS PM3) were selected and randomly distributed to 18 pens of 1.03 m² lined up along the building. Randomization was done per block of 3 pens taking into account broilers initial weight. Pens were randomly allocated to one of the three groups, differing by their diet composition (Table 1): the



standard diet (C-), the test diet (T-), containing corn DDGS at the level of 10%, and the test diet sup-plemented with 0.1% of algae-clay mix (T+). All broilers received three successive feeds: a starter feed (D0-D9), a grower feed (D9-D18) and a finisher feed (18-D31), manufactured specifically for the trial. The standard diet and the test diet were formulated to be isoenergetic and iso-digestible amino acids for each stage. Feed was distributed ad libitum manually by the farmer (one feeder per pen) and water was distributed via the usual drinking system. Housing system was a Colodaro type with con-crete floor and dynamic ventilation. Litter was composed of straw during the trial. Chicks were vac-cinated with a coccidiostat at one day-old, by spraying on the feed, prior to their allocation to the different groups. The rest of the building was used to raise chickens of the same age, following the standard management of the farm.

Table 1: Composition of the diets

Starter D0-D9 Grower D9-D18 Finisher D18-D31

Standard Test Standard Test Standard Test Composition (%) Corn 32.00 33.52 36.70 51.21 40.20 59.82 Wheat 25.00 18.60 26.50 5.60 26.50 - Corn DDGS - 10.00 - 10.00 - 10.00 Sodium bicarbonate 0.20 - 0.20 - 0.10 - Soyabean meal 36.00 30.70 30.00 25.10 26.50 21.90 Dibasic calcium phosphate 1.65 1.58 1.20 1.25 1.15 1.20 Sepiolite - - 0.50 0.50 1.00 0.99 Sodium chloride 0.20 0.27 0.20 0.26 0.25 0.26 Crude soy oil 2.00 2.83 2.20 3.50 2.00 3.41 DL-Methionine 0.25 0.22 0.20 0.20 0.15 0.20 Lysine HCl 0.10 0.18 0.20 0.28 0.10 0.17 Choline 0.30 0.30 0.30 0.30 0.30 0.30 Mineral premix 1.00 1.00 1.00 1.00 1.00 1.00 Vitamins premix 0.80 0.80 0.80 0.80 0.75 0.80 Theoretical nutritional value (%) Crude fat 4.50 4.50 6.00 6.00 6.00 6.00 Crude fiber 3.50 3.50 3.50 3.50 3.00 3.00 Crude protein 21.50 21.50 19.00 19.00 18.00 18.00 Methionine 0.56 0.56 0.50 0.50 0.45 0.45 Lysine 1.24 1.24 1.15 1.15 1.00 1.00 Ash 6.50 6.50 6.00 6.00 6.50 6.50 Calcium 1.00 1.00 0.90 0.90 0.90 0.90 Phosphorus 0.70 0.70 0.60 0.60 0.50 0.50 Sodium 0.14 0.14 0.14 0.14 0.14 0.14 Metabolizable energy (kcal/kg) 2849 2849 2981 2981 3049 3049

Animals were group weighed (per pen) at day 0 and at the end of each feeding period (D9, D18 and D31). Refused feed was weighed at the end of each feeding period to measure the feed intake (FI) and calculate the feed conversion ratio (FCR) for each period (D0-D9, D9-D18, D18-D31 and D0-D31). Calculation of the Average Daily Weight Gain (ADWG) was also made for each period for the different groups. Mortality was taken into account: date of deaths was used to assess an average number of present birds per period and weight of dead birds was used to adjust the ADWG per pen and per peri-od. A qualitative scoring was given to the litter at day 18 for each pen, using the scoring system de-scribed by Aubert et al., (2011):

- score 1: dry and crumbly, - score 2: crumbly but slightly damp, - score 3: crumbly but partly caking, - score 4: caking with crumbly litter when digging, - score 5: completely caking or damp.



FI, FCR and ADWG were submitted to an analysis of variance. Mortality data were compared with Pearson Chi-2 test. Systat® software was used to conduct all statistical analyses with a significance level of 5%.

Results Average initial weight of the animals did not differ among the groups (P=0.771) and so the groups are comparable at the start of the trial. Feed intake, feed efficiency and growth rate performance are displayed in table 2. Results show a significant decrease of FI and ADWG in the finishing and total periods in the T- group compared to the control. In finishing and total periods, FI of the T+ group is significantly higher than in the T- group (respectively +8.1%, P< 0.01 and +5.5%, P< 0.01) and is similar to the control. ADWG follows the same evolution: +11.6% (P = 0.03) in finisher and +7.1% (P = 0.04) in total period in favor of the T+ group compared to T-, and similar values between C- and T+ groups. On the other hand, in the starter period, C- and T- groups show a significantly higher ADWG than the T+ group. The FCR is higher in the starter period in T+ group compared to C- and T- groups (P < 0.01). However, it tends to be lower in the other phases. In the total period, the lowest FCR is obtained with the supplemented test diet (T+). Cumulated mortality over 31 days varies between 1.5 and 5.1% among the three groups, with the lowest value for the supplemented group (T+). Main causes of mortality were associated with sudden death and regular sorting by the farmer of broilers with late growth or locomotive troubles. No visible impact on litter quality was observed.

Table 2: Zootechnical performance of the animals

C- T- T+ Effect1 Average feed intake (g/chicken) Starter 301.6 289.1 289.3 Grower 726.7 693.2 704.3 Finisher 1821.4b 1661.1a 1796b D**, S** D0-D31 2849.5b 2645.1a 2790.5b D**, S* Average Daily Weight Gain (g/chicken) Starter 27.8b 26.8b 25.6a S* Grower 51.9 49.0 51.4 Finisher 74.3b 67.9a 75.8b D*, S* D0-D31 54.3b 50.5a 54.1b D*, S* Feed conversion ratio Starter 1.21a 1.20a 1.26b S** Grower 1.56 1.58 1.52 Finisher 1.89 1.88 1.82 D0-D31 1.69 1.69 1.66 1 From the analysis of variance, taking into account the effect of the diet (D) and the effect of the supplementation (S). *P≤0.05; **P≤0.01. a, b On a same line, different letters indicate a significant difference

Discussion The study shows a negative effect of the incorporation of corn DDGS in the diet on zootechnical per-formance of the birds. The lower feed intake and growth rate observed with this test diet are com-pensated by the supplementation with the algae-clay mix. This positive effect could be explained by the incorporation of the algae-clay mix in the diet. Indeed, Reichardt (2008) and Habold et al. (2009) mention the ability of clays to enhance the contact between enzymes and nutrients and highlight the



presence of enzymatic cofactors in clays, through the presence of metallic ions, allowing an increased activity of enzymes. Metallic ions such as zinc and copper actually have the capacity to activate some enzymes (Niederhoffer, 2000; Jondreville et al, 2002; Williams, 1960). This way, the presence of Montmorillonite and Ulva sp and Solieria chordalis macroalgae in the tested mix, significant sources of metallic ions (Kim, 2012), may have favored the activity of some digestive enzymes and thus contrib-uted to the increased performance of the broilers fed the supplemented diet.

Conclusion This study shows the zootechnical efficacy of the algae-clay mix in diets of broilers raised up to 31 days, at 0.1% in a corn-wheat diet containing 10% of corn DDGS, and highlights its interest for the valorization of such diets. In a context where the high volatility of cereals prices pushes towards the use of alternative ingredients, this algae-clay mix may be used to reduce the feed cost while maintain-ing a standard level of performance.

References Aubert C., Rousset N., Allain E., Ponchant A., 2011. TeMA., (18), 12-16.

Habold C., Reichardt F., Le Maho Y., Angel F., Liewig N., Lignot J.H., Oudar H., 2009. Brit J Nutr, (102), 249-257.

Jondreville C., Revy P.S., Jaffrezic A., Dourmad J.Y., 2002. INRA Prod. Anim., 15 (4), 247-265.

Kim S.K., 2012. In : Handbook of marine macroalgae. (Wiley-Blackwell) John Wiley and Sons.

Niederhoffer E.C., 2000. Southern Illinois Univ.

Ouhida I., Perez J.F., Piedrafita J., Gasa J., 2000. Anim Feed Sci Tech, (85), 183-194.

Reichardt F., 2008. In : thesis (Université Louis Pasteur Strasbourg).

Shalash S.M.M., Ali M.N., Sayed M.A.M., El-Gharby H.E., Shabaan M. 2009. Int. J. Poult. Sci., (8), 545-552.

Tauquir N.A., Nawaz H, 2001. Int J Agri Biol, (3), 149-150.

Williams R.J.P., 1960. Nature, (188), 322.

Corresponding author Maria Garcia Suarez Olmix SA ZA du Haut du Bois 56580 Brehan, France E-mail: animalcare.ts(at)olmix.com

Ion et al.: The energy effect of guanidino acetic acid in phytase-supplemented broiler diets


The energy effect of guanidino acetic acid in phytase-supplemented broiler diets

Anca Ion1, Mario Müller2 and Lavinia Stef3 1 Evonik Nutrition & Care GmbH Essen Sucursala Bucuresti, Bucharest, RO 2 Evonik Nutrition & Care GmbH, Hanau, DE 3 University of Agricultural Studies and Veterinary Medicine of Banat in Timisoara, RO

Abstract CreAMINO®, containing 96 % of guanidino acetic acid, has repeatedly been proven to enable broilers to overcome a certain energy deficiency in their rations. So far, an energy reduction of 50 kcal AMEN/kg compared to the standard feed can be compensated by adding CreAMINO® to the feed. The use of feed enzymes, in particular phytase, is considered standard in broiler industry. These enzymes also exert a nutritional matrix with a claimed energy saving potential being similar or even higher than that of CreAMINO®. Theoretically, the use of feed enzymes could let the energy effect of CreAMINO® in such diets disappear. Therefore, the CreAMINO® supplementation to a phytase-containing ration was investigated. The addition of CreAMINO® (600 g/t) to an energy-reduced (-50 kcal AMEN/kg), phytase-containing diet enabled broilers to perform, at least, at the level of control group for all parameters investigated. The animals in the negative control were not able to cope with the energy deficiency of 50 kcal AMEN/kg compared to standard in the absence of CreAMINO®. Earnings after feed cost increased 4.73 EUR/100 birds in the energy-reduced, CreAMINO®-supplemented treatment compared to the control group.

Introduction and Objective Creatine is a naturally occurring component in the animal's body tissue and plays a central role in energy metabolism. CreAMINO® contains 96 % of guanidino acetic acid being the natural precursor of creatine in energy metabolism, and impacts positively on energy metabolism. Common effects of CreAMINO® supplementation are: decreased feed conversion ratio, and improved weight gain. Besides this, CreAMINO® has repeatedly been proven to enable broilers to overcome a certain energy defi-ciency in their rations. So far, it is suggested that an energy reduction of 50 kcal AMEN/kg compared to the standard feed can be compensated by adding CreAMINO® to the feed. The use of feed enzymes, in particular phytase, is considered standard in broiler industry. Producers of feed enzymes also put nutritional matrices to their products. Quite often, their claimed energy sav-ing potential is similar to or even higher than that of CreAMINO®. Therefore, it is of huge interest whether the supplementation of CreAMINO® to an energy-reduced, phytase-containing diet is still as effective as described above. The objective of this study was to check the energy sparing effect of CreAMINO® in a broiler diet al-ready being supplemented with phytase by considering live performance. The trial was conducted in the research facility of University of Agricultural Studies and Veterinary Medicine of Banat in Timisoara, Romania.



Experimental Design A total of 240 Ross 308 day-old male chicks (feather sexed) were randomly distributed to three die-tary treatments (T I to T III, see table 1) consisting of ten replicates with eight birds each. Broilers were fed in a three-phase feeding regime with starter, grower and finisher diet from 1 to 10, 11 to 24, and 25 to 36 days of age, respectively.

Table 1: Nutritional treatments

Treatment Energy, kcal AMEN/kg Phytase* CreAMINO®

I control Starter Grower Finisher

3,025 3,100 3,150

1,500 FYT/kg 1,500 FYT/kg 250 FTU/kg

- - -

II energy-reduced (- 50 kcal AMEN/kg) plus CreAMINO®

Starter Grower Finisher

2,975 3,050 3,100


600 g/t 600 g/t 600 g/t

III energy-reduced (- 50 AMEN kcal/kg)

Starter Grower Finisher

2,975 3,050 3,100


- - -

*…due to different premix composition two different phytase products were used

All raw materials were analyzed prior to feed calculation. Latest recommendations of Evonik (Evonik 2012) were used to formulate feed composition of treatment I (control). Energy, and all nutrients including amino acids on an SID basis were in line with these specifications. In treatment II (energy-reduced (- 50 kcal AMEN/kg) plus CreAMINO®), the energy content of the feed was reduced by 50 kcal AMEN/kg across all phases, and CreAMINO® was added to compensate for this energy deficiency. The feed of treatment III (energy-reduced (- 50 kcal AMEN/kg)) was calculated as a negative control still containing the phytase, but no CreAMINO®. Nutritional parameters in the feed actually fed to the animals are crucial for the outcome of this kind of investigations. Regarding energy reduction it can clearly be stated that the intended pattern, as well as the degree of energy decrease (- 50 kcal AMEN/kg) has been achieved within the respective nutritional treatments. Levels of amino acids across the treatments matched feed calculation, and were found to be on an absolutely comparable level. Thus, the intended trial design has been con-firmed by analysis! Feed intake (FI), and body weight (BW) were recorded at the end of each phase. Feed conversion ratio (FCR), as well as average daily gain (ADG) were calculated according to the feeding phases. Also, mortality was recorded during the entire trial. Statistics were run using ANOVA procedure of R software.

Results and discussion Parameters of live performance obtained in the trial are displayed in table 2. Mortality was on a very low level of 0.75 % with no differences between the treatments observed. There was a treatment effect on FI. It amounted to about 3,200 g in control, whereas the birds fed the energy-reduced, CreAMINO®-supplemented diet ate about 130 g more (p<0.01). FI in the energy-reduced group was comparable to control. Generally, FI was slightly below ROSS performance objec-tives (Aviagen 2014).



Table 2: Live performance

Parameter control energy-reduced plus CreAMINO® energy-reduced p

CreAMINO® - 0.06 % - Energy level standard - 50 kcalAMEN/kg - 50 kcalAMEN/kg

Mean SEM Mean SEM Mean SEM FI, g 3,192a 38.0 3,331b 42.1 3,159a 28.3 <0.01

Initial BW, g 41.1 0.27 41.3 0.19 40.7 0.33 ns

BW, day 36, g 2,201b 32.6 2,301c 27.0 2,097a 14.4 <0.001

ADG, g 61.1b 0.90 63.9c 0.76 58.3a 0.40 <0.001

FCR, kg/kg 1.45a 0.01 1.45a 0.01 1.51b 0.01 <0.001

FCRcorr*, kg/kg 1.45a 0.01 1.43a 0.01 1.53b 0.01 <0.001

*…FCR corrected to average BW of 2,200 g

Initial BW was the same across the treatments and did not impact on final results. Control group achieved a final BW of 2,201 g. Birds of treatment II (energy-reduced plus CreAMINO®) performed significantly better reaching a BW of 2,301 (p<0.001). Usually, CreAMINO® supplementation to an energy-reduced diet compensates the energy deficiency. The finding, that CreAMINO® addition ena-bles birds to perform even better than the standard-fed control, has been observed in previous trials every now and then. Most probably, a perfect environment as indicated by that low mortality could be the proper explanation for such an outcome. Reducing energy by 50 kcal AMEN/kg only (Treatment III) resulted in a significant drop of BW in comparison to the other treatments (p<0.001). These birds weighed 100 g less than the control, and even 200 g less compared to energy-reduced plus CreAM-INO®. According to the respective ROSS objective the broilers of energy-reduced plus CreAMINO® matched, whereas the other groups were below the expectations. Results for ADG showed the same statistical pattern as final BW (p<0.001). FCR, as measured, was similar in control and treatment II. Those birds achieved a FCR of 1.45 kg/kg. It is significantly less than the FCR results for the energy-reduced treatment with 1.52 kg/kg (p<0.001). Due to the huge differences in final BW, FCR was cor-rected to the overall average BW of 2,200 g. A change in FCR of about 2.2 points per 100 g of live weight gain in the range of 2,100 to 2,300 g of body weight was assumed for this adjustment. This correction made the treatment-related differences even more obvious. The energy-reduced, CreAM-INO®-supplemented group ended up with a FCR of about 1.43 kg/kg being ten points below energy-reduced group. The economy behind the different treatments was assessed according to the trial conditions, its re-sults, the price situation as of summer 2015, and a cost of 7 EUR/kg CreAMINO® (Table 3). Actual feed cost reflected the respective adjustments of feed specifications as well as the differences in feed intake. They were in a range of 100.31 (energy-reduced) to 108.09 EUR/100 birds for energy-reduced plus CreAMINO®. For comparison, in control feed cost amounted to 104.19 EUR/100 birds. Due to the strong difference in final BW the revenues differed considerably. With 180.97 EUR/100 birds fed the energy-reduced treatment performed worst, whereas energy-reduced plus CreAMINO® achieved the highest revenue (198.58 EUR/100 birds). Balancing feed cost and revenue the treatment energy-reduced plus CreAMINO® ended up with earnings of 90.49 EUR/100 birds and exceeded the other nutritional treatments by far. Difference to control amounted to 4.73 EUR/100 birds, that to energy-reduced reached almost 10 EUR/100 birds. Thus, higher feed cost have been more than compensated by increased live performance.

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Table 3: Economical assessment of the nutritional treatments

Parameter control energy-reduced plus CreAMINO® energy-reduced Feed cost Starter feed, EUR/t 356.46 354.54 348.63 Grower feed, EUR/t 330.25 328.34 319.05 Finisher feed, EUR/t 319.48 317.57 311.9 Feed intake, g/bird 3,192 3,331 3,159 Feed cost, EUR/100 birds 104.19 108.09 100.31 Revenue, EUR/kg LW* 0.863 0.863 0.863 LW, kg 2,201 2,301 2,097 Revenue, EUR/100 birds 189.95 198.58 180.97 Earnings after feed cost, EUR/100 birds 85.76 90.49 80.66 Difference to control, EUR/100 birds - 4.73 - 5.10 Earnings after feed cost**, % 100.0 105.5 94.1 *…according to DGS Magazin 45/2015; **…Treatment I (control) = 100 %

Conclusions The supplementation of CreAMINO® (600 g/t) to an energy-reduced (-50 kcal AMEN/kg), phytase-containing diet enabled broilers to perform, at least, at the level of control group for all parameters investigated. The animals in the negative control were not able to cope with the energy deficiency of 50 kcal AMEN/kg compared to standard in the absence of CreAMINO®. Earnings after feed cost increased 4.73 EUR/100 birds in the energy-reduced, CreAMINO®-supplemented treatment compared to the control group.

References Aviagen (2014): ROSS 308 Broiler, Nutrition Specifications

Evonik (2012): Recommendations for Broilers

Corresponding author Dr. Mario Müller Evonik Nutrition & Care GmbH Rodenbacher Chaussee 4 D-63457 Hanau, Germany E-Mail: mario.mueller(at)evonik.com

Alijosius et al.: Single and combined effects of mannanoligosaccharides and dietary endo-1,4-β-xylanase and endo-1,3/1,4-β-glucanase on the performance and some intestinal function of laying hens


Single and combined effects of mannanoligosaccharides and di-etary endo-1,4-β-xylanase and endo-1,3/1,4-β-glucanase on the performance and some intestinal function of laying hens

S. Alijosius, R. Gruzauskas, V. Kliseviciute, V. Sasyte, A. Raceviciute-Stupeliene and A. Dauksiene Institute of Animal Rearing Technologies, Lithuanian University of Health Sciences, Kaunas, LT

Abstract The goals of the study were to assess the effects of prebiotic mannanoligosaccharides (MOS) alone or in combination with dietary enzymes, composed mainly of endo-1.4-β-xylanase and endo-1.3/1.4-β-glucanaseon the productivity and some intestinal function of laying hens during the laying period (38-46 weeks of age). Fourty 38 wk of age Lohman Brown laying hens were assigned to 4 dietary treat-ments for 8 wk. The dietary treatments were: 1) control, 2) compound feed supplemented with endo-1.4-β-xylanase and endo-1.3/1.4-β-glucanase (E group, enzyme dosage 0.05 g/kg, activity 1400 AXC/g and 2000 AGL/g), 3) feed supplemented with MOS (P group, prebiotic dosage 1 g/kg), 4) feed supplemented with endo-1.4-β-xylanase and endo-1.3/1.4-β-glucanase (EP group, enzyme dosage 0.05 g/kg, activity 1400 AXC/g and 2000 AGL/g) plus MOS (dosage 1 g/kg). The results suggested that inclusion of combination of enzymes and MOS hadn't effect on the egg production parameters, except for the feed conversion ratio to produce 1 kg of egg mass: it was decreased by 11%. The en-zymes and MOS lowered the quantity of SCFA, expect propionic acid – it was increased in addition of enzymes. Enzymes and MOS did not influence the pH in the GIT of laying hens. In group with en-zymes and MOS increased dry matter content of the Ileum, but decreased in Cecum.

Introduction It is well known the benefits of xylanase and β-glucanase supplementation for poultry production. The cereal NSP (arabinoxylans and β-glucans) present in wheat or triticale are known to contribute to the anti-nutritive effects of compound feed due to their property of increasing intestinal viscosity where absorption of nutrients takes place, resulting in an impaired growth and productivity of animals. Feed enzymes are typically added to animal feed to improve nutrient digestibility and animal performance (Smeets et al., 2014) within the gastrointestinal tract (Pariza and Cook, 2010). However, the enzymes take their highest activity in small intestine of laying hens, and some of undegraded feed had reachedthe Cecum. The highest activity of microfora takes place at Cecum and it is important to sup-port the colonization and proliferation of microflora. For a substrate to be classified as a prebiotic the substrate cannot be hydrolyzed by the stomach or small intestine, it must select for beneficial bacteria in the large intestine, and fermentation of the substrate should have beneficial effects for the host. Most prebiotics are carbohydrates and oligosaccharides. There are wide range of prebiotics, such as fructooligosaccharides, galactooligosaccharides and etc. (Gaggia et al., 2010).MOS is a prebiotic, component of the yeast Saccharomyces cervisiae. MOS is lowering gut pH through lactic acid produc-tion, inhibiting or preventing colonization of pathogens, modifying metabolic activity of commensal bacteria and stimulation of the immune system Prebiotics have also been shown to increase short chain fatty acids, lowering the pH of the cecum and stimulating growth of Bifidobacterium and Lacto-bacillus (Hajati and Rezaei, 2010).



There are wide range of studies (results of them are contradictory) representing effects of prebiotics and multi-enzymes on productivity and physiology of laying hens. We had hyphothesed, that prebiotics MOS (level of 1 g/kg) and enzymes composed of endo-1.4-β-xylanase and endo-1.3/1.4-β-glucanase (dosage 0.05 g/kg, activity 1400 AXC/g and 2000 AGL/g) have effect on productivity and gastrointestinal physiology of the laying hens.

Material and methods The feeding trial was conducted on laying hens of the cross Lohman Brown of 38 weeks of age. Fourty laying hens were divided into 4 groups: C – control; E, P and EP – experimental; 10 laying hens in each group. The birds were fed with 125 g per day of compound feed (with or without sup-plementation) for 8 weeks. The Control group (C) received a feed based on corn, wheat, triticale and soybean meal; E group received basal diet supplemented with enzymes (endo-1.4-β-xylanase and endo-1.3/1.4-β-glucanase, dosage 0.05 g/kg, activity 1400 AXC/g and 2000 AGL/g), P group received basal diet supplemented with prebiotic MOS (dosage 1 g/kg), EP group received basal diet supple-mented with enzymes and prebiotic (endo-1.4-β-xylanase and endo-1.3/1.4-β-glucanase, dosage 0.05 g/kg, activity 1400 AXC/g and 2000 AGL/g and MOS, dosage - 1 g/kg). Rovabio® Excel AP enzyme mixture (containing mainly endo-1.4-β-xylanase and endo-1.3/1.4-β-glucanase) used in this study was provided by Adisseo (France) and produced from Penicillium funiculosum. Agrimos® (Lalleman Inc., France) is a specific combination of mannanoligosaccharides and glucose (β-glucans) extracted from the yeast cell walls of Saccharomyces cerevisiae. A compound feed was formulated to meet the nutri-ent and energy requirements for laying hens. During the feeding trial, the laying hens were held in the individual cages with stationary drinking–bowl and feeding boxes under the same feeding and keeping conditions. At the end of the trial from each group 5 laying hens (5 birds × 4 groups of birds = total of 20 birds) were selected and killed according to the recommendations for euthanasia of experi-mental animals (Close et al., 1997). The intestinal content of Duodenum, Ileum, Cecum and Rectum was collected for the determination of pH and dry matter. In the remaining Cecum content, short-chain fattyacids (SCFA) were determined. The value of pH of different segments of the GI tract was measured immediately by using a digital pH meter “Inolab pH 730” (WTW, Germany); dry matter content was determined by drying the chyme at 105°C until total desiccation and calculating the difference between the dried and non-dried contents of the intestinal sections. The concentration of SCFA was measured by HPLC system (Varian Inc., USA) (Kliseviciute et al., 2014). Statistical analyses were done using SPSS 22 software. Results were expressed as mean ± standard deviation (SD). Significance differences between treated samples were evaluated by Levene’s t–test. Differences were considered significant at P<0.05.

Results and discussion The results of the present study showed lower feed conversion ratio for the production of 1 kg egg mass in the group fed diets supplemented with enzymes and MOS compared to the C group. For the other productivity characteristics, i. e. egg weight, egg production and feed intake did not differ signif-icantly among treatment groups (P>0.05) (Table 1). Some earlier studies had reported about beneficial effects on the egg production of hens when they were fed diets supplemented with MOS (Bozkurt et al., 2012; Bozkurt et al. 2012a) and enzymes (Mir-zaie at al., 2012). Enzyme supplementation has been reported to improve egg production (Scheideler et al., 2005), egg weight (Mirzaie et al., 2012) and feed conversion ratio (Yörük et al., 2006).



Table 1: Productivity parameters of laying hens (38-46 weeks of age) fed diets supplemented with enzymes and MOS

Age of the laying hens (weeks)

Groups C E P EP

38-46

Feed intake (g) 1714.05±64.99 1694.61±68.34 1702.03±55.71 1698.40±75.11

Egg weight (g) 63.68±5.22 67.71±4.60 64.78±4.26 65.58±3.88

Egg production (%) 84.8±10.76 88.9±12.17 88.2±11.39 89.1±9.85

Feed conversion ratio (kg of feed/kg of eggs mass) 2.32a±0.36 2.07ab±0.39 2.17ab±0.29 2.10b±0.21

a, b Means within a row with different superscripts differ significantly (P<0.05)

The SCFA concentrations in the cecum of laying hens are shown in Table 2.The lowest level of volatile fatty acid in hens' cecum content was noted for the butyric acid in experimental EP group (4.76 μmol/g). Distinct differences between control and experimental groups were observed for the content of acetic acid in the cecum of laying hens: in the P groups it compound by 10.65 μmol/g and in the EP group - 17.19 μmol/g (P<0.05).

Table 2: Effect of enzymes and MOS on short-chain fatty acids concentration in the cecum of laying hens (μmol/g)

Parameters Groups C E P EP

Acetic 67.77±2.01a 62.17±7.90ab 57.12±5.54b 50.58±6.82b

Propionic 14.57±5.35 14.50±3.57 14.67±1.48 13.35±4.01 Butyric 16.21±4.35a 11.49±2.12ab 11.76±4.32ab 4.76±1.69b

a, b Means within a row with different superscripts differ significantly (P<0.05)

The effects of enzymes and MOS on the dry matter content and pH of the chymus of different gastro-intestinal tract segments are shown in Table 3.

Table 3: Effect of enzymes and MOS on pH value and dry matter content in the digesta of different parts of laying hens gastrointestinal tract

Segments of gastro-intestinal tract

Groups C E P EP

Dry matter, % Duodenum 14.95±2.35 18.25±2.69 16.35±1.57 15.69±2.14 Ileum 18.41±1.79a 18.09±2.03ab 16.96±1.76ab 26.70±16.61b Cecum 24.45±2.67a 22.74±2.22ab 21.12±3.13ab 19.86±8.55b

Rectum 21.73±0.50 19.12±0.00 21.31±1.43 21.52±1.73 pH Duodenum 5.89±0.12 5.88±0.19 5.60±0.12 5.95±0.21 Ileum 5.93±0.66 5.57±0.40 5.21±0.40 5.70±0.56 Cecum 6.13±0.48 6.79±0.32 5.86±0.31 5.73±0.29 Rectum 5.75±0.23 7.14±0.00 5.22±0.23 5.45±0.70 a, b Means within a row with different superscripts differ significantly (P<0.05)

The results indicated that in EP group (diets supplemented with enzymes and MOS) the dry matter level of Ileum chymus were significantly increased (P<0.05), but the dry matter of Cecum were signif-



icantly decreased compared to the C group (P<0.05).There was no significant difference in duodenum and rectum among the experimental groups.Gastrointestinal pH was not influenced by enzymes and MOS interaction (Table 3).

Conclusion During the experimental 8–week feeding of laying hens the addition of enzymes and MOS hadn’t ef-fect on productivity parameters (except of feed conversion ratio for the production of 1 kg egg mass) of laying hens in comparison to the control group. In experimental groups, which diets were supple-mented with enzymes and MOS were get the lower acetic acid concentration. Enzymes and MOS did not influence the pH in the gastrointestinal tract segments of laying hens. In group with enzymes and MOS increased dry matter content of the Ileum, but decreased in Cecum.

References Bozkurt M., Tokuşog˘lu Ö., Küçükyılmaz Ö., Akşit H., Çabuk M., Çatlı A., Seyrek K., Çınar M. (2012) Effects of dietary mannan oligosaccharide and herbal essential oil blend supplementation on performance and oxidative stability of eggs and liver in laying hens. Italian Journal of Animal Science 11:e41

Bozkurt M., Kucukyilmaz K., Catli , A.U., Cınar M., Bintaş E., Coven F. (2012a) Performance, egg quality, and immune response of laying hens fed diets supplemented with mannan-oligosaccharide or an essential oil mixture under moderate and hot environmental conditions. Poultry Science 91, 1379–1386

Close B., Banister K., Baumans V., Bernoth E.M., Bromage N., Bunyan J., Erhardt W., Flacknell P., Gregory N., Hackbarth H., Morton D., Warwick C. (1997) Recommendations for euthanasia of experimental animals: Part 2. DGXT of the European Com-mission. Laboratory Animals 31,1-32. Gaggia F., Mattarelli P., Biavati B. (2010) Probiotics and prebiotics in animal feeding for safe food production. International Journal of Food Microbiology 141, 15–28

Hajati H., Rezaei M. (2010) The Application of Prebiotics in Poultry Production. International Journal of Poultry Science 9, 298–304

Kliseviciute V., Gruzauskas R., Grashorn M.A., Raceviciute-Stupeliene A., Sasyte V., Svirmickas G.J., Bliznikas S. (2014) Effect of different supplementation levels of whole Triticale grown in Lithuania to broiler diets on performance and parameters of functioning of the digestive tract. European Poultry Science 78, 1–13

Mirzaie S., Zaghari M., Aminzadeh S., Shivazad M., Mateos G.G. (2012) Effects of wheat inclusion and xylanase supplementation of the diet on productive performance, nutrient retention, and endogenous intestinal enzyme activity of laying hens. Poultry Science 91, 413–425

Pariza M.W., Cook M. (2010) Determining the safety of enzymes used in animal feed. Regulatory Toxicology and Pharmacology,56, 332–342

Scheideler S.E., Beck M.M., Abudabos A., Wyatt C.L. (2005) Multiple-enzyme (Avizyme) supplementation of corn–soy-based layer diets. The Journal of Applied Poultry Research 14, 77–86

Smeets N., Nuyens F., Campenhout L.V. , Niewold T. (2014) Enhancement of xylanase efficacy in the precence of other polysaccharidases and protease. Proceedings of the XIVth European Poultry Conference, Stavanger, p.430

Yörük M.A., Gül M., Hayirli A., Karaoglu M. (2006) Multi-Enzyme Supplementation to Peak Producing Hens Fed Corn-Soybean Meal Based Diets. International Journal of Poultry Science 5, 374–380.

Corresponding author Saulius Alijosius Department of Animal Sciences Lithuanian University of Health Sciences Veterinary Academy Tilzes str. 18, LT– 47181 Kaunas E-mail: Saulius.Alijosius(at)lsmuni.lt

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Benarbia et al.: Effect of citrus extract and saponins plant extract on broiler zootechnical performances: comparative study to synthetic growth promoters


Effect of citrus extract and saponins plant extract on broiler zo-otechnical performances: comparative study to synthetic growth promoters

Mohammed el Amine Benarbia1, Valantino Arnaiz2, Alssendra Caussilas2, Juan Manuel Garcia1 and Pierre Chicoteau1 1 NORFEED SAS, Beaucouzé, FR 2 Montana, La Molina Lima, PE

Introduction Modern livestock management has to meet sustainability standards. One of the most important of these standards is the respect of the one health concept. Indeed this emerging concept considers the link between environmental, animal and human health. On the other hand, modern livestock have to meet the growing demand for animal protein source. This is a challenging situation knowing that the use of antibiotic growth promoter and coccidiostat has been banned and/or is being banned in several countries. This ban is justified by the fact this kind of use of antibiotic and/or coccidiostat is not with-out having several side effects not only on animal health but also on human and environment health. As a consequence of this ban many alternatives to AGP appears in the market. Citrus extract (CE) and saponins rich plant extract (SRPE) based feed additive belong to the most promising alternatives. Indeed, many trials showed that feed supplemented either with citrus and/or saponins riche plant enhances zootechnical parameters in various species (Juin et al., 2003a; Nordi et al., 2014). Thanks to the prebiotic effect of citrus extract, this last is considered as an efficient growth promoter due to the enhancement of intestinal health. This is achieved by modulation gut flora in favor of beneficial bacte-ria by several mechanism (Coelho et al., 2013; Unno et al., 2015). Saponin rich plant is also efficient growth promoter. Their main mechanism of action is also in modulating gut flora and enhancing de-fense again coccidiosis. Indeed several studies showed their efficacy in controlling this disease and improving animal health. The aim of this study was to study the effect of feed supplementation with well-characterized citrus extract CE (Nor-Spice AB®) and saponins rich plant extract SRPE (NorponinXO2) on zootechnical pa-rameters of broiler and to compare it to AGP supplementation.

Material and methods The study has been performed in an experimental farm in Lima (Montana, Peru). 264 birds (Cobbs 500) were randomly divided in 3 groups (8 replicate of 11 birds) depending on diet supplementation as showed in the following table.

Group 1: negative control 2: positive control 4: CE (Nor-Spice AB®) + SRPE (Norponin XO™)

Feed Standard growing diet baczin®1000 ppm+ neomycin 150

ppm+ Decoquinate 500 ppm

Nor-Spice AB® 250 ppm Norponin XO™ 500 ppm

Animals had continuous access to water and feed. Birds were raised until day 42. Feed intake and mortality were recorded daily and live weight weekly. Zootechnical parameters were calculated (FCR, ADG). ANOVA was performed for statistical analysis.



Results Live weight: Group of animal raised with feed supplemented with growth promoters tends to have higher live weight compared with group of animals raised with feed without any supplementation. No significant difference has been observed between group of animals raised with synthetic growth pro-moters and CE, SRPE group. This last have a tendency to have highest live weight.

Feed Conversion Ratio (FCR): Group of animals raised without any supplementation had tendency to have the highest FCR. Feed supplementation either with synthetic or natural feed additives had a ten-dency to reduce FCR. This tendency was more marked in the group of animal raised with feed sup-plemented with the combination of CE and SRPE.

Livability: Group of animal raised with feed supplemented with CE and SRPE had the highest livability rate (100%) compared to the other group. Where group of animals raised without any feed supple-mentation had the lowest viability rate among all the groups of animals.

1.52

1.54

1.56

1.58

1.6

1.62

1.64

1.66

1.68

Negative control Positive control CE+SRPE

Figure 2: Feed Conversion ratio (FCR)

Figure 1: Final live weight of animal after 42 days expressed in gr



Discussion This trial shows that phytogenic feed additive is as efficient as synthetic growth promoters are. In-deed, there were no significant difference between animal raised with feed supplemented with natural phytogenic additive and animals raised with feed supplemented with synthetic growth promoters in terms of zootechnical parameters. This may be explained by the complementary action of both phyto-genic feed additives. Citrus extract are well described for their positive effects on broiler zootechnical parameters (Goliomytis et al., 2015; Juin et al., 2003b). This is due the action of several molecules like citroflavonoids, pectic oligosaccharide, and limonene among others. The main target of these molecules is gut microbiota. Some of them have a well described prebiotic effect like POS and citrofla-vonoids that enhance the growth of beneficial bacteria like Bifidobacterium and lactobacillus (Abuelsaad et al., 2014; Parhiz et al., 2015; Unno et al., 2015). Other molecule like limonene have action in inhibiting several pathogenic bacteria (Subramenium et al., 2015; Vuuren and Viljoen, 2007). Saponin rich plants have also the gut and its microbiota as a target for their beneficial effect on ani-mal health and productivity. Hassan et al. (2008) demonstrated in their study that diet supplementa-tion with guar (saponins rich plant) ameliorate coccidiosis symptoms and reduced oocyst excretion after broiler challenge with Eimeria tenella (Alfaro et al., 2007; Hassan et al., 2008). This is mainly explained by the ability of saponins to make stable link to cell membrane sterols, which is behind cell lysis. In addition to their effect on coccidiosis, saponins have been described having other positive effect that may explain thein positive effect on animal performance. Several studies showed that sap-onins rich plant enhance palatability, nutrient digestion and also intestinal epithelial cell renewal (Ab-bas, 2010; Zentek et al., 2013). Intestinal health plays a key role in animal health and productivity. Inside the gut, microbiota is a cornerstone of intestinal health. Indeed, a well-balanced microbiota is crucial for a functional and healthy intestine. Microbiota interacts with many physiological systems. The well-described interac-tions are those linked to digestion, immunity and the nervous system. Data remain scarce but there is no doubt that a good management of intestinal microbiota is crucial in the management of animal health and productivity. Citrus extract and saponins rich plant extract are a blend of molecules that affect intestinal microbiota. This may be the key mechanism of action that explains their positive ef-fect on zootechnical performances. Thanks to the diversity of their molecules and complementary action, phytogenic feed additive are an efficient alternative to synthetic growth promoters. However, good characterization and understanding of their mechanism of action is mandatory for their good use. This study demonstrated that when the characterization of molecules is mastered, phytogenic

Figure 3: Livabilty expressed in %



feed additive are as efficient as synthetic ones. These data open doors for new era in livestock man-agement in a more sustainable way that respects animal, human and environmental health.

References Abbas, R.J. (2010). Effect of Using Fenugreek, Parsley and Sweet Basil Seeds as Feed Additives on the Performance of Broiler Chickens. Int. J. Poult. Sci. 9, 278–282.

Abuelsaad, A.S.A., Allam, G., and Al-Solumani, A.A.A. (2014). Hesperidin inhibits inflammatory response induced by Aeromonas hydrophila infection and alters CD4+/CD8+ T cell ratio. Mediators Inflamm. 2014, 393217.

Alfaro DM, Silva AVF, Borges SA, Maiorka FA, Vargas S, Santin E (2007). Use of Yucca schidigera extract in Broiler diets and its effects on performance results obtained with different Coccidiosis control methods. J Appl Poult Res 16:248–254.

Coelho, R.C.L.A., Hermsdorff, H.H.M., and Bressan, J. (2013). Anti-inflammatory properties of orange juice: possible favorable molecular and metabolic effects. Plant Foods Hum. Nutr. Dordr. Neth. 68, 1–10.

Goliomytis, M., Kartsonas, N., Charismiadou, M.A., Symeon, G.K., Simitzis, P.E., and Deligeorgis, S.G. (2015). The Influence of Naringin or Hesperidin Dietary Supplementation on Broiler Meat Quality and Oxidative Stability. PloS One 10, e0141652.

Hassan, S.M., El-Gayar, A.K., Cadwell, D.J., Bailey, C.A., and Cartwright, A.L. (2008). Guar meal ameliorates Eimeria tenella infection in broiler chicks. Vet. Parasitol. 157, 133–138.

Juin, H., Elgaard, T., and Chicoteau, P. (2003a). Effect of a citrus extract (NOR-SPICE AB) on broiler performances. Br. Poult. Sci. 44, 810–811.

Nordi, E.C.P., Costa, R.L.D., David, C.M.G., Parren, G. a. E., Freitas, A.C.B., Lameirinha, L.P., Katiki, L.M., Bueno, M.S., Quirino, C.R., Gama, P.E., et al. (2014). Supplementation of moist and dehydrated citrus pulp in the diets of sheep artificially and naturally infected with gastrointestinal nematodes on the parasitological parameters and performance. Vet. Parasitol. 205, 532–539.

Parhiz, H., Roohbakhsh, A., Soltani, F., Rezaee, R., and Iranshahi, M. (2015). Antioxidant and anti-inflammatory properties of the citrus flavonoids hesperidin and hesperetin: an updated review of their molecular mechanisms and experimental models. Phytother. Res. PTR 29, 323–331.

Subramenium, G.A., Vijayakumar, K., and Pandian, S.K. (2015). Limonene inhibits streptococcal biofilm formation by targeting surface-associated virulence factors. J. Med. Microbiol. 64, 879–890.

Unno, T., Hisada, T., and Takahashi, S. (2015). Hesperetin Modifies the Composition of Fecal Microbiota and Increases Cecal Levels of Short-Chain Fatty Acids in Rats. J. Agric. Food Chem. 63, 7952–7957.

Vuuren, S.F. van, and Viljoen, A.M. (2007). Antimicrobial activity of limonene enantiomers and 1,8-cineole alone and in combination. Flavour Fragr. J. 22, 540–544.

Zentek, J., Gärtner, S., Tedin, L., Männer, K., Mader, A., and Vahjen, W. (2013). Fenugreek seed affects intestinal microbiota and immunological variables in piglets after weaning. Br. J. Nutr. 109, 859–866.

Corresponding author Mohammed el Amine BENARBIA NORFEED SAS 3 rue Amedeo Avogadro 49070, Beaucouzé, France E-mail: amine.benarbia(at)nor-feedsud.fr

Buckiuniene et al.: Effect of lycopene on egg quality parameters and fatty acids composition in the egg yolk, using rapeseed and linseed oil in laying hens nutrition


Effect of lycopene on egg quality parameters and fatty acids composition in the egg yolk, using rapeseed and linseed oil in laying hens nutrition

V. Buckiuniene¹, R. Gruzauskas¹, A. Bajorinaite¹, A. Raceviciute-Stupeliene¹, V. Kliseviciute¹, V. Sasyte¹,A. Dauksiene¹, S. Alijosius¹ and G. Svirmickas² 1 Lithuanian University of Health Sciences, Veterinary Academy, Institute of Animal Rearing Technologies Kaunas, LT 2 Lithuanian University of Health Sciences Institute of Animal Science of Veterinary Academy, Baisiogala, Radviliskis district, LT

Abstract The aim of this study was to investigate the effects of dietary supplemented rapeseed oil and linseed oil with lycopene on laying hens eggs qualitative parameters, fatty acid concentration on egg of 30-37 weeks of age of laying hens. A total of 40 Lohman Brownn laying hens which were 30 weeks old were assigned to four treatment groups (10 hens per each treatment group) and fed with the experimental diets for 8 weeks. The I control group compound feed was supplemented with rapeseed oil (4.5 %), the II control group - was supplemented with linseed oil (4.5 %), I experimental group – rapeseed oil (4.0 %) + lycopene (25 g/kg) and II experimental group – linseed oil (4.0 %) + lycopene (25g/kg) and keeping in the same conditions. On egg quality parameters in experimental groups statistically significant results get on yolk color intensity and eggshell hardness (P<0.05). Linseed oil with lycopene statistically significant increase total of monounsaturated fatty acids concentration (MUFA), it increased 1.97 percent (P<0.05) com-pared with I control group, PUFA in I experimental group (T3) increased 2.37% (P<0.05), but in II experimental group (T4) it decreased - 3.495(P<0.05) compared with control groups. Omega 3 in-creased in all experimental groups from 1.75 to 3.75 (P<0.05), copmpared with control groups. The results of this study clearly demonstrate that supplemention of lycopene and different oils had effect on eggshell strength, MUFA, PUFA and omega 3 concentration on egg yolk.

Introduction Egg is an important source of nutrients for human and can be used effectively in a wide range of food products due to its excellent functional properties. The colour of egg yolk is one of the determinant factors of consumer choice. Eggs with bright reddish-yellow yolk were perceived as excellent. It was well documented that the colour and composition of egg yolk can be modified through the alterations of laying hen diets (Parpinella et al., 2006). In recent years, consumers have become interested infunctional eggs (those containing high selenium, highpolyunsaturated fatty acids (PUFAs) and herbal antioxidants etc.; Sahin et al.2008; Jung et al. 2011; Ghasemi et al. 2014; Zhang & Kim, 2014). Egg is considered one of the most complete foods in human nutrition, as its composition includes protein of excellent biological value, in addition to essential amino acids, vitamins, fatty acids and minerals (Alleoni & Antunes, 2001). Yolk colour is one of the important criteria that affect the consumer’s choices and acceptance. In most countries, deep coloured egg yolk receive popular



acceptance (Guyonnet, 2013), because it is believed to be of better quality and healthier (Hernandez et al., 2000). Eggs provide a completely packaged, highly nutritious food including essential nutritional components (Laudadio et al., 2014). As previously mentioned, the egg is often referred to as one of the original and natural functional foods. Numerous studies have demonstrated that variations in rations fed laying hens can greatly impact the level of nutrients such as vitamins, minerals, omega-3 polyunsaturated fatty acids (PUFA) and lutein (Schiavone and Barroeta, 2011). Recent studies have shown that diets enriched with antioxidant substances can be used to attenuate the negative effects of environmental stress, as the detrimental effects of environmental stress could be partly due to induction of oxidative stress (Bollengier-Lee et al., 1998; Seven et al., 2010; Chauhan et al., 2014). Lycopene is a strong antioxidant among dietary carotenoids and can prevent the produc-tion of ROS (reactive oxygen species) and associated undesirable effects (Palozza et al. 2011). The aim of this study was to investigate lycopene and different oils on egg qualitative parameters and fatty acids concentration in the egg yolk.

Material and methods Feeding trial was conducted laying hens of Lohman Brownn strain at the age of 30 weeks. The laying hens were divided into 4 groups, 10 laying hens in each group. I group is the control group which was added rapeseed oil 4.5% (T1) and II group was also control group, added linseed oil 4.5% E (T2) in, III (I experimental) group – rapeseed oil 4.0% + lycopene 25g/kg (T3) and IV (II experimental) group - linseed oil + lycopene 25g/mg (T4). During the feeding trial, the laying hens were held in the individual cages with stationary drinking-bowl and feed box under the same feeding and holding con-ditions. The laying hens were fed with compound feed 125 g per day (NRC, 1994). Egg weight, albumen high, Haugh unit, intensity of egg yolk color are established by multifunctional automatic egg characteristics analyzer „Egg Multi-Tester EMT-5200“, hardness of eggshell – by „Egg Shell Force Gauge MODEL–II“ device, and thickness of eggshell – by electronic micrometer „MI-TUTOYO“. Extraction of lipids for fatty acid analysis was performed with chloroform/methanol (2:1 v/v) as de-scribed by Folch et al. (1957). Fatty acid methyl esters (FAME) were prepared using the procedure of Christopherson and Glass (1969). Lipid quality indices, i.e., atherogenic index (AI) and thrombogenenicity index (TI), were calculated according to Ulbricht and Southgate (1991). AI =[C12:0+(4×C14:0) + C16:0]/[n-6 PUFA + n-3 PUFA + MUFA]; TI =[C14:0 + C16:0 + C18:0]/[(0.5 × MUFA) + (0.5 × n-6 PUFA) + (3 × n-3 PUFA) + n-3/n-6PUFA]. Peroxidizability index was determined according to Witting L. A. ir Horwitt M. K. (1964) methods and calculated by formula: IP= (0.025*monoenoic acid)+(1*dienoic acid)+(2*trienoic acid)+(4*tetraenoic acid)+(6*pentaenoic acid)+(8*hexaenoic acid) hypocholesterolemic/Hypercholesterolemic ratio (h/H) was determined by Fernández et al. (2007). Statistical Analysis.The results of the experiment were analysed using the 1-way ANOVA test, and significant differences between groups were determined by Duncan’s multiple range test. Statistica 8.0. for WindowsTM software was used. Differences were considered significant at P<0.05.



Results Qualitative research results of eggs of laying hens of 30-37 weeks of age are presented in Table 1. The eggshell hardness in experimental groups (T3 and T4) increased from 12 to 47%, compared with control groups (T1 and T2) (P<0.05). When analyzed Haugh unit in T3 and T4 groups it decreased from 7 to 9% (P>0.05), compared with control groups. The compound feed supplement with different oils and lycopene (T3 and T4) had effect on the intensity of yolk color, it increased from 2.50 to 4.05 times (P<0.05) compared with control groups. To the rest egg qualitative parameters, the different oils and lycopene did not had statistically significant effect.

Table 1: Effect rapeseed, linseed oils and lycopene on the egg quality parameters (per all period)

Parameters T1 T2 T3 T4 Egg weight, g 60.95±2.40 63.01±3.93 60.32±2.12 63.56±2.14 Eggshell hardness, kg/m² 28.91±2.51 27.90±3.95 32.43±3.37* 41.06±2.71* Albumen height, mm 6.36±2.19 6.64±1.92 5.83±0.79 6.76±1.24 Haugh unit 76.78±3.99 77.60±2.58 71.54±7.33 70.90±5.71 Yolk color intensity 2.02±0.31 3.39±1.19 8.19±0.16* 8.49±0.38* Eggshell weight, g 5.432±0.37 5.374±0.85 5.527±0.35 5.389±1.31 Eggshell thickness, mm 0.31±0.06 0.30±0.11 0.33±0.02 0.33±0.06 *- data statistically significant at P<0.05

The fatty acid concentration of the eggs are presented in Table 2. Monounsaturated fatty acids (MUFA) in II experimental group (T4) increased statistically – 1.97% (P<0.05), but in I experimental group (T3) it decreased by 3.47% (P<0.05) compared with control groups. Polyunsaturated fatty ac-ids (PUFA) in T3 increased 2.37% (P<0.05), in T4 it decreased 3.49% (P<0.05) compared with con-trol groups. Omega 6 in T4 group decreased 3.7 times (P<0.05) compared with T1 control group. Omega 3 in all experimental groups increased from 1.75 to 3.7 percent (P<0.05) compared with con-trol groups. Omega6 and omega 3 ratio also in all experimental groups increased from 8.35 to 16.55% (P<0.05) compared with control groups. To the rest parameters, lycopene did not have sta-tistically significant results.

Table 2: Effect of rapeseed, linseed oil and lycopene on the fatty acids concentration of content of egg yolk (%)

Fatty acid T1 T2 T3 T4 Total of saturated fatty acid (SFA) 31.00±1.03 29.34±0.27 31.97±0.97 30.76±1.56 Total of mono unsaturated fatty acid (MUFA)

43.67±2.50 43.55±1.23 40.20±0.83* 45.52±1.00*

Total of polyunsaturated fatty acid (PUFA) 24.72±1.47 26.77±1.19 27.09±0.69* 23.28±1.37* PUFA/SFA 0.80±0.04 0.91±0.04 0.85±0.04 0.76±0.08 Trans-isomers 0.13±0.01 0.13±0.02 0.15±0.01 0.16±0.05 Total of omega-6 fatty acid 1.65±0.15 7.18±0.50 0.87±0.10 1.94±0.20* Total of omega-3 fatty acid 23.07±1.37 19.59±0.93 26.23±0.60* 21.34±1.27* Total of omega-6/total omega-3 fatty acid ratio

14.00±1.01 2.74±0.20 30.55±2.91 11.09±1.09

IA 0.34±0.02 0.29±0.01 0.35±0.02 0.34±0.03 IT 0.80±0.03 0.54±0.01 0.88±0.04 0.78±0.06 h/H 2.95±0.16 3.35±0.07 2.79±0.17 2.95±0.27 IP 36.95±1.07 47.40±2.73 37.52±1.58 37.04±1.88* *- data statistical significantly (P<0.05)



Discussion Our results is agree with Olson et al (2008), they determined that carotenoids contribute to egg yolk color, and therefore, changes indietary carotenoids should modify the color of egg yolk. As previously mentioned, the egg is often referred to as one of the original and natural functional foods. Numerous studies have demonstrated that variations in rations fed laying hens can greatly impact the level of nutrients such as vitamins, minerals, omega-3 polyunsaturated fatty acids (PUFA) and lutein (Schiavone and Barroeta, 2011). Studies have showed that lycopene consumption has a cardioprotec-tion effect in humans and animals (Arab and Steck, 2000; Rissanen, 2006) by up- regulating the redox status such as improving antioxidant enzyme activities and antioxidant vitamin contents (Luo and Wu, 2011), and optimizing the plasma lipid profile (Sahin et. al., 2006a; Upaganlawar and Balaraman, 2012). There is limited data on impact of rapeseed, linseed oils and lycopene which were used in laying hens nutrition and it‘s effect on egg qualitative parameters and fatty acids composition. So, in our trial, statistical signifficant results were get in eggshell hardness (it increased from 12 to 47% (P<0.05)) and yolk color intensity (it increased from 2.50 to 4.05 times (P<0.05)). Supplemented compound feed with rapeseed, linseed oil and lycopene statistically increased MUFA, PUFA and omega 3 composition.

Conclusion The results clearly demonstrate that different oils and lycopene had statistically significant effect on eggshell strenght and MUFA, PUFA and omega 3 concentration on the egg yolk.

Acknowledgement Financed by the ministry of Agriculture of the Republic of Lithuania; project MT 11/30.

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Arab, L. and S. Steck. 2000. Lycopene and cardiovascular disease. Am. J. Clin. Nutr. 71:1691S-1695S.

Bollengier-Lee, S., M. A. Mitchell, D. B. Utomo, P. E. V. Williams and C. C. Whitehead. 1998. Influence of high dietary vitamin E supplementation on egg production and plasma characteristics in hens subjected to heat stress. Br. Poult. Sci. 39:106-112.

Chauhan, S. S., P. Celi, B. J. Leury, I. J. Clarke and F. R. Dunshea. 2014. Dietary antioxidants at supranutritional doses improve oxidative status and reduce the negative effects of heat stress in sheep. J. Anim. Sci. 92:3364-3374.

Christopherson S.W. and Glass R.L. 1969. Preparation of milk fat methylesters by alcoholysis in an essentially nonalcoholic solution. J. Dairy Sci. 52: 1289–1290.

Fernández M., Ordóñez J.A., Cambero I., Santos C., Pin C. and De la Hoz L. 2007. Fatty acid compositions of selected varieties of Spanish dry ham related to their nutritional implications. Food Chemistry. 101: 107–112.

Folch J., Less M. and Sloane-Stanley G.H. 1957. A simple method for isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry. 226: 497–509.

Ghasemi R, Torki M, Ghasemi HA, Zarei M. 2014. Single or combined effects of date pits and olive pulps on productive traits, egg quality, serum lipids and leucocytes profiles of laying hens. J Appl Anim Res. 42:103–109.

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J. B. Olson, N. E. Ward, and E. A. Koutsos. 2008. Lycopene Incorporation into Egg Yolk and Effects on Laying Hen Immune Function. Poultry Science 87:2573–2580.



Jung S, Hee-Han B, Nam K,Ahn DU, Lee JH, Jo C. 2011. Effect of dietary supplementation of gallic acid and linoleic acid mixture or their synthetic salt on egg quality. Food Chemist. 129:822-829.

Laudadio, V., E. Ceci, N.M.B. Lastella, M. Introna and V. Tufarelli. 2014. Low-fiber alfalfa (Medicago sativa L.) meal in the laying hen diet: Effects on productive traits and egg quality. Poult. Sci., 93: 1868-1874.

Luo, C. and X. G. Wu. 2011. Lycopene enhances antioxidant enzyme activities and immunity function in N-Methyl-N′-nitro-N-nitrosoguanidine-induced gastric cancer rats. Int.J. Mol. Sci. 12:3340-3351.

NRC – National Research Council. 1994. Nutrient requirements of poultry. 9th ed. National Academy Press Washington, D.C., USA.

Palozza P, Parrone N, Simone R, Catalano A. 2011. Role of lycopene in the control of ROS-mediated cell growth: implications in cancer prevention. Curr Med Chem 18:1846–1860

Parpinella GP, Meluzzi A, Sirri F, Tallarico N, Versari A (2006) Sensory evaluation of egg products and eggs laid from hens fed diets with different fatty acid composition and supplemented with antioxidants. Food Res Int 39:47–52.

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Corresponding author Buckiuniene V. Lithuanian University of Health Sciences, Veterinary Academy, Institute of Animal Rearing Technologies Kaunas, LT E-mail: vilija.buckiuniene(at)lsmuni.lt

Sobczak et al.: The effect of dietary supplementation with butyric acid or sodium butyrate on egg production and physiological parameters in laying hens


The effect of dietary supplementation with butyric acid or sodi-um butyrate on egg production and physiological parameters in laying hens

Alicja Sobczak, Aleksandra Drażbo, Maria Hanuszewska and Krzysztof Kozłowski University of Warmia and Mazury in Olsztyn, PL

Abstract A total of 432 Lohmann Brown laying hens at 48 weeks of age were randomly assigned to three die-tary treatments (9 replicates of 16 birds each). The hens were housed in three-tier battery cages for 24 weeks. All birds were fed iso-nitrogenous and iso-caloric diets in mash form, and had free access to water. Control group (T1) hens were fed a basal diet, group T2 received a basal diet supplemented with an encapsulated source of butyric acid (at 500 g/t feed) and group T3 received a basal diet sup-plemented with 70% of protected sodium butyrate (at 700 g/t feed). The number and weight of eggs laid, feed intake, feed conversion, egg quality, bone mineralization and the gastrointestinal tract pa-rameters of hens were determined throughout the experiment. Dietary supplementation with butyric acid and sodium butyrate contributed to a significant increase (P<0.05) in eggshell thickness, eggshell weight as a percentage of total egg weight, higher activity of bacterial enzymes in the cecum, and higher concentrations of butyric acid in the cecal contents. Dietary supplementation with butyric acid and sodium butyrate improved eggshell quality and increased the concentrations of butyric acid in the cecal digesta in laying hens. Hens fed diets supplemented with butyric acid were characterized by a higher calcium content of bones and lower ammonia concentrations in the cecum. It can be concluded that both butyric acid sources had a beneficial influence on eggshell quality, tibia parameters and selected gastrointestinal tract parameters.

Introduction Gut health, which is a key prerequisite for good bird performance and economically viable poultry production (Samik et al., 2007), is influenced by the gut microbiota (Dhawale, 2005). Butyric acid is the primary energy source for metabolism in intestinal epithelial cells in poultry, it is necessary for proper development of gut-associated lymphoid tissue (GALT), and is considered to be an important growth modulator of intestinal microflora (Leeson et al., 2005; Antongiovanni et al., 2007). Organic acids such as fumaric, propionic and butyric acids and their salts have been found to exert varied ef-fects on egg production and egg quality parameters depending on their source, the amount of organic acids used, location, environmental conditions and the composition of the diets (Gama et al., 2000). In several experiments, dietary supplementation with organic acids (including butyric acid and sodium butyrate) positively affected growth performance, feed conversion ratio, carcass quality, serum lipid profile and histomorphometrical parameters of the small intestine in broilers (Mansoub, 2011; Chamba et al., 2014; Gasemi et al., 2014; Kamal and Ragaa, 2014). Soltan (2008) demonstrated that a mix-ture of organic acids increased egg production and improved eggshell quality in laying hens. The objective of this study was to determine the effect of dietary supplements containing butyric acid or sodium butyrate on selected parameters of egg production, egg quality, and intestine development and function in laying hens.



Materials and methods The experiment was conducted in the experimental facilities of the Department of Poultry Science, University of Warmia and Mazury in Olsztyn, Poland. The experimental materials comprised of 432 Lohmann Brown laying hens aged 48 weeks, raised in accordance with the breeder’s recommenda-tions (LTZ, 2014). The hens were reared for 24 weeks. At 48 weeks of age, the hens were randomly assigned to three dietary treatments (9 replicates of 16 birds each) and were kept in pairs in Big Dutchman double-sided three-tier battery cages. The birds were housed in an environmentally con-trolled house with a day length of 15 h light:9 h dark. The diets, offered ad libitum in mash form, were formulated to meet the nutrient and energy requirements of laying hens (Smulikowska and Rutkowski, 2005). T1 (control group) hens were fed a basal diet based on wheat, triticale, soybean meal and corn, with no feed additives, T2 hens received a basal diet supplemented with 500 g of en-capsulated butyric acid/t of feed, and T3 hens were fed a basal diet supplemented with 700 g of pro-tected sodium butyrate/t of feed. Both products are coated. Feed intake and feed conversion were recorded at four-week intervals. Eggs were collected from each cage six times per week. Egg quality was analyzed at four-week intervals (at 52, 56, 60, 64, 68 and 72 weeks of age). Each time, egg quality was evaluated on 15 eggs per group selected randomly based on the average egg weight for a given experimental period. Eggshell thickness and breaking strength, yolk color, albumen quality (Haugh unit score) and the percentage composition of egg com-ponents were determined. On the last day of the experiment, at 72 weeks of age, 9 birds from each group were selected randomly to determine gastrointestinal tract parameters. Viscosity, dry matter content and digesta pH were determined in the small intestine. Samples of fresh cecal digesta were used for immediate analyses to determine: digesta pH, the hydration rate of the cecal contents, am-monia and short-chain fatty acids (SCFA) concentrations, while the remainder was transferred to mi-crofuge tubes and stored at -70°C until analyses of bacterial enzymatic activity. On the last day of the experiment, at 72 weeks of age, 12 birds from each group were selected randomly to determine bone mineralization parameters. Tibia samples collected from laying hens were separated from the muscles and cartilage, and were assayed for the mean content of DM, ash, Ca and P.

Results Table 1: Production parameters of laying hens and egg quality

Performance parameter Groups P T1 T2 T3 Egg weight, g 66.10 ± 1.75 65.91 ± 1.18 65.88 ± 1.61 0.346 Egg mass, g/hen 62.85 ± 1.70 62.67 ± 1.41 62.46 ± 2.09 0.894 Laying rate, % 95.14 ± 1.37 95.17 ± 1.93 94.93 ± 1.73 0.948 Feed intake, g/hen 116.6 ± 3.1 116.4 ± 3.0 116.1 ± 3.8 0.932 FCR, g feed/g egg mass 1.862 ± 0.038 1.861 ± 0.038 1.862 ± 0.041 0.998 Egg quality Egg Shell thickness, mm 0.345b ± 0.034 0.358a ± 0.033 0.359a ± 0.028 0.003 Shell strength, N 36.78 ± 9.30 38.69 ± 7.76 38.93 ± 7.28 0.156 Yolk color, points 11.70 ± 0.83 11.66 ± 0.80 11.88 ± 0.61 0.113 Haugh units 81.39 ± 7.43 83.05 ± 6.14 82.71 ± 7.07 0.235 Egg composition, % Yolk 25.78 ± 0.79 25.70 ± 0.58 26.00 ± 0.60 0.431 Albumen 64.38 ± 2.25 64.17 ± 1.90 63.76 ± 2.06 0.125 Shell 9.83b ± 0.98 10.13a ± 0.87 10.23a ± 0.86 0.009 Values in same rows with no common superscript are significantly different (P0.05)



Table 2: Selected parameters of gastrointestinal tract and bone mineralization

Item Groups P T1 T2 T3 Butyric acid in cecal digesta, µmol/g 14.5b ± 1.2 18.6ab ± 1.2 21.9a ± 2.3 0.005 Ammonia in the cecum, mg/g 0.53ab ± 0.03 0.48b ± 0.02 0.57a ± 0.03 0.038 Tibia calcium content1, % 22.68 ± 1.25 23.49 ± 0.49 23.25 ± 0.68 0.081 Values in same rows with no common superscript are significantly different (P0.05) 1Data related to fresh matter

Conclusions Dietary supplementation with butyric acid and sodium butyrate improved eggshell quality and in-creased the concentrations of butyric acid in the cecal digesta in laying hens. Hens fed diets supple-mented with butyric acid were characterized by a higher calcium content of bones and lower ammonia concentrations in the cecum. It can be concluded that both butyric acid sources had a beneficial influ-ence on eggshell quality, tibia parameters and selected gastrointestinal tract parameters.

References Antongiovanni M, Boccioni A, Petacchi F, Leeson S, Minieri S, Martini A and Cecchi R (2007) Butyric acid glycerides in the diet of broiler chickens: effects on gut histology and carcass composition. Ital. J. Anim. Sci. 6, 19-25.

Chamba F, Puyalto M, Ortiz A, Torrealba H, Mallo JJ and Riboty R (2014) Effect of partially protected sodium butyrate on performance, digestive organs, intestinal villi and E. coli development in broiler chickens. Int. J. Poult. Sci. 13, 390-396.

Dhawale A (2005) Better eggshell quality with a gut acidifier. Poultry Int. 44, 18-21.

Gama NMSQ, Olivera MBC, Santin E and Berchieri J (2000) Supplementation with organic acids in diets of laying hens. Ciencia Rur., Santa Maria 30, 499-502.

Gasemi HA, Akhavan-Salamat H, Hajkhodadadi I, Khaltabadi-Farahani AH (2014) Effects of dietary organic acid blend supplementation on performance, intestinal morphology and antibody-mediated immunity in broiler chickens. Acta Adv. Agric. Sci. 2, 64-74.

Kamal AM and Ragaa NM (2014) Effect of dietary supplementation of organic acids on performance and serum biochemistry of broiler chicken. Nat. Sci. 12, 38-45.

Leeson S, Namkung H, Antongiovanni M and Lee EH (2005) Effect of butyric acid on the performance and carcass yield of broiler chickens. Poult. Sci. 84, 1418-1422.

LTZ (2014) Lohmann Tierzucht GmbH. Manual of Lohmann Brown Hens. www.ltz.de.

Mansoub NH (2011) Comparitive effect of butyric acid and probiotic on performance and serum composition of broiler chickens. Adv. Environ. Biol. 5, 1188-1191.

Samik KP, Gobinda H, Manas KM and Gautam S (2007) Effect of organic acid salt on the performance and gut health of broiler chickens. J. Poult. Sci. 44, 389-395.

Smulikowska S and A Rutkowski (2005) Nutrient requirements of poultry. Feeding recommendations and nutritive value of feed. 4th ed. The Kielanowski Institute of Animal Physiology and Nutrition of the Polish Academy of Sciences, Jabłonna by Warszawa (in Polish).

Soltan MA (2008) Effect of dietary organic acid supplementation on egg production, egg quality and some blood serum parameters in laying hens. Int. J. Poult. Sci. 7, 613-321.

Corresponding author Dr. habil. Krzysztof Kozłowski Faculty of Animal Bioengineering Department of Poultry Science University of Warmia and Mazury in Olsztyn Oczapowskiego 5, 10-719 Olsztyn, Poland E-mail: kristof(at)uwm.edu.pl

Paleckaitis et al.: Effect of dietary mannan oligosaccharides and organic acids on common carp health and productivity


Effect of dietary mannan oligosaccharides and organic acids on common carp health and productivity

Mindaugas Paleckaitis , Ieva Kudlinskiene , Alius Pockevicius and Romas Gruzauskas Lithuanian University of Health Sciences, Veterinary Academy, Kaunas, LT

Abstract Thesis aim: to evaluate the effect of Mannan Oligosaccharide and Organic acids mix influence on health, productivity, intestinal villous height. The control group was fed compound feed without addi-tives; 1 research group – was fed with compound feed enriched with 0.2 percent prebiotics (Mannan Oligosaccharides); II research group – was fed compound feed enriched with prebiotics (0.2) and organic acids mix (0.2 percent). For further researches 10 carps from each group were randomly se-lected. Fish were weighed, measured the body cranial, middle and tail parts length, and body height. The samples were taken from the front and back sides of the intestinal tract for microbiological and histological (were fixed in 10 % formalin phosphate buffer) examination. This research has shown that MOS and organic acids have improved health, weight gain, feed conversion ratio, specific growth rate and intestinal villus size of common carp. After summarizing the results it was concluded that in order to improve health and productivity of common carp it is appropriate to supplement its feed by adding 2kg/t of MOS and 2kg/t organic acids (butter, propionic, sorbic, caprylic, lauric).

Introduction In order to improve global fish demand, aquaculture is not only expanding its volumes but also is intensifying fish growing rates. In order to obtain as many fish from the smallest reservior as possible, health and productivity of these fish decreases inevitably. While safe and healthy food demand is growing scientists are looking for natural means of fish health stimulation. Supplementing Mannan oligosaccharides and organic acids to fish feed is a great alternative for solving these problems. Thesis aim:to evaluate the effect of Mannan Oligosaccharide and Organic acid mix influence on health, productivity, intestinal villous height.

Materials and methods Feeding trial. Feeding experiment began on March in 2014 with one year old carps, average weight of these carps was 104 g, experiment lasted for a 7 months. There were used 3 reservoirs near each other, all equal in size (90 areas), 2700 units of carps was weighted, divided in to three groups, 900 units per each and let in those three reservoirs. The control group was fed compound feed without additives; 1 research group – was fed with compound feed enriched with 0.2 percent prebiotics (Man-nan Oligosaccharide abstracted from yeast Saccharomyces cerevisiae); II research group – was fed compound feed enriched with prebiotics (0.2 percent Mannan Oligosaccharide abstracted from yeast Saccharomyces cerevisiae) and organic acid (0.2 percent) mix. For further researches 10 carps from each group were randomly selected. Fish were weighed, meas-ured the body cranial, middle and tail parts length, and body height. Were fixed a common internal organs and intestines weight. The samples were taken from the front and back sides of the intestinal tract for microbiological and histological (were fixed in 10 % formalin phosphate buffer) examination.



Calculation of total makeweight was performed by A. G. J. Tacon (1990), weight of captured fish is subtracted from the weight from primary quantity of fish. Feed conversion efficiency (how many kg of feed is used to get one kilogram of production) was calculated by dividing quantity of used feed from makeweight (Shalaby et al., 2006). The percent of mortality was calculated by M. A. Genc et al. (2006).

Statistical data analysis. Statistical data analysis was performed with Microsoft Office Excel 2010 and (IBMSPSS) programs.

Results The 1st research group was fed compound feed with MOS additives weight gain was 30.36 percent bigger then that of the control group, also it was observed 2.77 percent better efficiency of feed con-version and 18 percent higher specific coefficient of growing comparing data with control group. These parameters of efficiency also was performed by Y. Staykov and others (2005) research. They also used 0,2 percent MOS additives, the trial results showed that experimental groups weight gain was 11,6 percent higher, and feed conversion was better in 17,6 percent. V. Culjak and others (2006), in their research used 0.6 percent MOS, in final result carps weight was 24 percent bigger, and feed conversion was better from 2.06till 1.60 percent. 2nd research group which was fed compound feed with organic acids showed significant increase in weight gain (31.84 percent) compared with control group, better efficient in feed conversion (1,6 per-cent), also bigger average carp weigh (10,40 percent), better specific coefficient of growing too (20 percent). Because of very limited information about organic acids use in carp nutrition and it effect on productivity, we compared our research results with researches which was made with broilers. These results matches with data of M. F. Denli et al. (2003) who used organic acid in birds feed and have noticed a better feed conversion parameters in growth. Also S. Adil and others (2011) research with broilers determined significant increase in weight gain and bigger efficient in feed conversion com-pared data with control group.

Table 1: The results of carps growth speed and feed conversion

Pond

Nr..

Feed

add

itive

s

Parameter

Date

Fish

Quan

tity,

un

its.

Tota

l wei

ght,

kg

Aver

age

wei

ght,

g

Yiel

d, p

ct.

Fish

wei

ght

kg/h

a

Used

feed

, kg

.

Bree

d

Age,

ye

ars.

1. Control group (without additives)

10.06 Carps 1+ 275 443 1610 30,6 482 1260

2. I research (MOS)

10.06 Carps 1+ 485 636 1310 53,9 699 1900

3. II research (MOS+OA)

10.06 Carps 1+ 361 650

1797 40,1 657 1990



1 Schedule: The results of mortality on control and experimental groups, %

The research results showed that 2nd research group which was given feed with MOS and organic acids additives, mortality was 9.56 percent less, and the 1st research group which was given feed with only MOS 27 percent less than that of the control group. V. Culjak and others (2006) established MOS influence on carps health and productivity, they had determined 16.7 percent mortality decrease in the research group, while Y. Staykov and others (2005) find even – 46.51 percent decrease of mortality. Those results confirmed results of current research results, which determined MOS improve fish health and condition of immune system (Zhou, Li, 2004; Staykov et al., 2005; 2007; Torrecillas et al., 2007). The decrease of carp mortality may associate with the MOS property directly (by stimulating non-specific immunity) and indirectly (by improving the good microflora growth) stimulate the immune system functions (Song et al., 2014).

Table 3: The results of the histological research (the front side)

Group Intestinal villus length, (µ m) Crypt depth, (µ m) Intestinal villus/Crypt ratio Control 2829±105* 333.7±36.7 8.47±1.26*

I research (MOS) 3159±243 343.2±25.7 9.20±0.55

II research (MOS +OR) 3179.4±71* 242.2±13.8 13.13±0.78*

*p<0,05

After measuring carp intestinal villus and crypts depth with CellSens Dimension program it can be seen that 1st research group villus was 10.44 percent (p<0.05) , 2nd group 11.02 percent ( p<0,05) longer comparing with control group. Depth crypt in 1st research group are 2.77 % less in deep and in 2nd group – 27.4 percent less in deep comparing it with control group. Villous of the first part of intes-tinal and ratio with crypt is better comparing with control group in 7.8 percent from 1st group and 35.4 percent from 2nd group (Table 3). The results of back intestinal side: the villus in 1st reseach group – 1.25 % longer, crypt depth 2.07 % less, ratio of villus and crypt – 7.9 %, better then control croup. Villus long of second part of guts from 2nd group – 11.16 percent (p<0.05) longer, depth of crypt 4.77 percent (p<0.05) less in depth, and proportion of villous and crypt – 15.4 percent (p<0.05) better then control group (Table 4).



Table 4: The results of histological research (the back side)

Group Intestinal villus length, (µ m) Crypt depth (µ m) Intestinal villus/Crypt ratio

Control 1958±120* 222.4±15.7* 8.8±0.9* I research

(MOS) 1982.9±62.5 217.8±25.4 9.1±1.15

II research (MOS +OR) 2204±118* 211.8±20.0* 10.4±0.8*

*p<0,05

Summarizing data, we can affirm that MOS and organic acid mix additives to feed improves better development of fish intestinal villus and that enlarges intestinal surface.

Conclusion This research has shown that MOS and organic acids have improved health, weight gain, feed conver-sion ratio, specific growth rate and intestinal villus size of common carp. After summarizing the results it was concluded that in order to improve health and productivity of common carp it is appropriate to supplement its feed by adding 2kg/t of MOS and 2kg/t organic acids (butter, propionic, sorbic, caprylic, lauric).

References Adil S., Banday T., Ahmad Bhat G., Salahhudin M., Raquib M., Shanaz S. Response of broiler chicken to dietary supplementation of organic acids. Journal of Central European Agriculture. 2011. 12(3). P.4 98-508.

Culjak V., Bogut G., Has-Schon E., Milakovic Z., Canecki K. Effect of Bio-Mos® on performance and health of juvenile carp. In: Nutritional Biotechnology in the Feed and Food Industries: Proceedings of Alltech’s 22nd Annual Symposium (Suppl., Abstracts of Posters presented). April 23-26, Lexington, KY, USA. 2006. P. 90.

Denli M. F., Okan K., celik. Effect of dietary probiotic, organic acid and antibiotic supplementation to diets on broiler performance and carcass yield. 2003. P. 89-91.

GENC M.A., AKTAS M., GENC E., YILMAZ E. Effects of dietary mannanoligosaccharide on growth, body composition and hepatopancreas histology of Penaeus semisulcatus (de Haan 1844). Aquaculture Nutrition. 2007. P. 156–161.

Tacon A. G. J. Standard method for nutritional and feeding of farmed fish and shrimp. Argent librations press, Vol 1. 1990. P. 117.

Torrecillas S., Caballero M. J, Sweetman J., Makol A., Izquierdo M. S. Effects of feeding Bio- Mos on European sea bass (Dicentrarchus labrax) uvenile culture. Presented at Alltech’s Technical Seminar Series held in Dublin, 21 st November, Ireland. 2007. P. 13.

Staykov Y., Denev S. A, Spring P. The Influence of Dietary (Bio-Mos®) on the Growth Rate and Immune Function of Common Carp (Cyprinus Carpio L.). In: Lessons From The Past To Optimise The Future – Aquaculture Europe 2005 (Eds. B. Howell and R. Flos), August 5-9th, Tronheim, Norway, European Aquaculture Society, Special Pub No 35. 2005. P. 431-432.

Zhou X. Q, Li Y. L. The effects of Bio-Mos on intestinal microflora and immune function of juvenile Jian carp (Cyprinus carpio Var. Jian). Nutritional biotechnology in the feed and food industries: Proceedings of Alltech’s 20th annual symposium; Lexington, KY, USA. 2005.

Corresponding author Mindaugas Paleckaitis Lithuanian University of Health Sciences, Veterinary Academy Kaunas, Lithuania E-mail: mi7jazz(at)gmail.com

Korzekwa et al.: Positive effects of a flavonoid-rich phytogenic product on growth, survival, and bacterial infection of Pacific White Shrimp


Positive effects of a flavonoid-rich phytogenic product on growth, survival, and bacterial infection of Pacific White Shrimp

Monika Korzekwa, Tilman Wilke and Sven Brenner Dr. Eckel GmbH, Niederzissen, DE

Abstract The effects of Anta®Ox FlavoSyn (Dr. Eckel GmbH, Niederzissen, Germany) a polyphenol-rich product, on body weight, total production, survival rate, average daily growth (ADG), feed conversion ratio (FCR), and bacteria counts in pacific white shrimp were studied under farm conditions. Postlarvae 12 were stocked in 6 x 5 ponds (0.8 ha each) at a density of 750,000 PL12/ha with seawater of 25-30 ppt salinity. Shrimp were distributed into two groups (with three replicates/treatment). They were fed five times per day with commercial pelleted feed (control group), or feed supplemented with 800 ppm Anta®Ox FlavoSyn. After 60 days of feeding the highest total production and survival rate were ob-served in the Anta®Ox FlavoSyn group, being 5399.7 ±952.0 kg and 91.0 ±13.1 %, respectively, while those of control group were 4697.3 ±1616.7 kg and 63.0 ±22.1 %, respectively. However, this difference within the experimental groups was not statistically significant (p>0.05). The same could be observed for body weight, ADG and FCR which were in the range of 11.8-12.4 g, 0.16-0.18 g/day, and 1.35-1.55, respectively. Total bacteria number (NA count), Vibrio spp. (TCBS count), and V. parahae-molyticus (CHROMagar Vibrio count) in the hepatopancreas of Anta®Ox FlavoSyn 800 ppm-fed shrimp were significantly lower than those in the control group. In addition, histopathological study of hepatopancreas revealed sign of bacterial infection in control shrimps but not Anta®Ox FlavoSyn-fed shrimp. The present study therefore strongly suggests that Anta®Ox FlavoSyn had a positive effect on shrimp production.

Introduction Shrimp farmers around the world have the same goal to keep their production system in a balance. Endemic pathogens and epidemic diseases (like EMS/AHPN caused by Vibrio parahaemolyticus) con-stantly put this balance at risk. In the past it was commonplace to keep farming system in balance by the preventive use of antibiotics. Customers and retailers in Europe, the US and other markets have become more and more sensitive to the abundance of antibiotic usage in animal production. Hence, shrimp farmers need alternative tools to keep their animals healthy. Functional feed additives are an alternative way to strengthen the natural defence mechanism of the animals and therefore to keep the production system at an equilibrium. The farmer gains benefits from via high growth rates, good feed conversion and overall low mortality.

Materials and methods Experimental animals. The on-farm trial was conducted in a shrimp farm located in Chantaburi prov-ince, Eastern Thailand. Six earthen ponds with an area of around 0.8 ha each were used. Pacific white shrimp (Litopenaeus vannamei) post-larvae 12 (PL12) were stocked at a density of 750,000 PL12/ha with seawater of 25-30 ppt salinity. The two experimental groups consisted of a control group, in which shrimp were fed commercial pelleted feed (containing 36% crude protein and 6% lipid from Charoen Pokphand Foods Public Company Limited (CPF), Thailand), and a treatment group, in which shrimp were fed pelleted feed supplemented with 800 ppm of Anta®Ox FlavoSyn (Dr. Eckel GmbH,



Niederzissen, Germany). Each group was tested with three replicates. The shrimp were fed five times a day for 60 days. The feeding rate was adjusted according to the shrimp weight throughout the 60-day experimental period (Limsuwan and Chanratchakool, 2004). The production, growth and survival rate were recorded at the end of the trial. Bacterial study. Sixty shrimps were sampled from each group at the 60th day. The hepatopancreas of each shrimp were aseptically removed, homogenized, and spread on NA (general media for bacterial culture), TCBS (selective media for Vibrio spp. culture), and CHROMagar Vibrio (selective media for detection of V.parahaemolyticus) using the spread plate method and then incubated at 37°C for 24 hours. Finally, all of the bacterial colonies were counted to calculate the CFU/g unit. Histological study. At the end of the trial, 60 shrimps from each group were sampled, fixed with Da-vidson’s fixative solution for histological examination of the hepatopancreas, and then embedded in paraffin and stained with hematoxylin and eosin (H&E) as described by Bell and Lightner (1988). Statistical analysis. At the end of the experiment, the data from all of the experimental groups were statistically compared using a one-way analysis of variance (ANOVA) and Duncan’s New Multiple Range testing. Differences were considered significant if p<0.05.

Results After 60 days of dietary administration, body weight, total production, survival rate, average daily growth (ADG), and feed conversion ratio (FCR) were not significantly different among all experimental groups, despite the fact that survival rate and total production of shrimps fed on Anta®Ox FlavoSyn 800 ppm appeared superior to the control group, being 91.0 ±13.1, and 63.0 ±22.1 %, respectively for survival rate, and 5,399.7 ±952.0, and 4,697.3 ±1616.7 kg, respectively for total production (Ta-ble 1).

Table 1: Body weight, total production, survival, average daily growth (ADG), and feed conversion ratio (FCR) of Pacific white shrimp after 60 days of feeding with three different diets

Experimental groups Body weight (g)

Total production (kg)

Survival rate (%)

ADG (g/day) FCR

Control 12.3 ±2.8a 4697.3 ±1616.7a 63.0 ±22.1a 0.18 ±0.04a 1.55 ±0.11a Anta®Ox FlavoSyn 11.8 ±1.8a 5399.7 ±952.0a 91.0 ±13.1a 0.16 ±0.02a 1.35 ±0.14a The data are presented as the mean ± standard deviation. Means in the same column with different superscripts are significant-ly different from each other (p<0.05).

For the bacterial study, total number of bacteria (NA count), Vibrio spp. (TCBS count), and V. para-haemolyticus (CHROMagar Vibrio count) in hepatopancreas of Anta®Ox FlavoSyn 800 shrimp were 13.7 ±87.1, 1.5 ±1.4, and 0.7 ±1.6 x 105 CFU/g, respectively, which were significantly lower than the control group about one order of magnitude (Table 2).

Table 2: The number of bacteria (105 CFU/g) in the hepatopancreas of Pacific white shrimp after feeding four different diets for 60 days

Experimental groups NA count TCBS count CHROMagar Vibrio count Control 122.3 ±71.3b 11.6 ±11.2b 6.8 ±6.8b Anta®Ox FlavoSyn 13.7 ±87.1a 1.5 ±1.4a 0.7 ±1.6a The data are presented as the mean ± standard deviation. Means in the same column with different superscripts are significant-ly different from each other (p<0.05).

For the histological study, the hepatopancreas of control-fed shrimps showed sign of atrophy and bacterial infection. About 20-80% of hepatopancreatic cells were damaged. In contrast, hepatopan-



creas of shrimp fed Anta®Ox FlavoSyn appeared more normal, the necrotic cells were only in the range of 0-10% (Figure 1).

Figure 1: Hepatopancreas of control shrimps (A: 50x, B: 100x) showed sign of atrophy and bacterial infection, while those of Anta®Ox FlavoSyn-fed shrimp (C: 50x, D: 100x) appeared normal and healthy

Discussion Polyphenols are a major group of plant secondary metabolites that have one or several phenolic hy-droxyl groups. They are widespread in many commonplace foods and beverages such as fruits, vege-tables, cereals, legumes, tea, wine, and beer (Bravo, 1998; D’Archivio et al., 2007; Wink and Schimmer, 2010). Their wide ranging biological effects include antioxidant, anti-inflammatory and antimicrobial activities and make polyphenols useful in promotion good health (Neyestani, 2008; Xia et al., 2010; Landete, 2012). There are many reports about the positive effects of the polyphenol-rich feed additive extracted from grape pomace (Vitis vinifera) and, to a lesser extent, spent hops (Humu-lus lupulus) on the health of pigs, chickens and dairy cows (Brenes et al., 2010; Viveros et al., 2011; Gessner et al., 2012, 2013; Fiesel et al., 2014; Koch et al. 2015). Our previous laboratory study on shrimp health strongly suggested similar effects (Niyamosathaet al., 2015). However, the effects of these compounds on the health of shrimp have never been evaluated in farm condition. The present study showed that Anta®Ox FlavoSyn could increase survival rate and total production of pacific white shrimp reared in earthen ponds. However, these benefits were not significant statistical-ly, probably as a result of great variation among experimental groups, which in turn is not unusual for farming conditions. Nevertheless, all bacteria counts (NA, TCBS, and CHROMagar Vibrio) in hepato-pancreas of Anta®Ox FlavoSyn-fed shrimps were significantly lower than those in the control group and this finding is consistent with the histopathological study which looked normal, indicating that

A B

C D



shrimp fed on Anta®Ox FlavoSyn were indeed healthier. The lower bacterial infection may account for better survival rate, which in turn, lead to higher total production of the Anta®Ox FlavoSyn-fed group. The lower bacterial number in Anta®Ox FlavoSyn-fed shrimps observed in the present study was in-consistent with the previous study under laboratory conditions which found no significantly difference in number of bacteria among the experimental groups (Niyamosathaet al., 2015). The reason of this incongruity is not totally understood, but lies most likely in the difference of environmental factors between controlled laboratory condition and farm conditions, i.e. shrimp in one pond might be ex-posed to different set of conditions compared to the other ponds. Shrimp fed on Anta®Ox FlavoSyn might be expose to more suitable pond condition so that grape pomace’s polyphenols in Anta®Ox FlavoSyn could exert their health promoting activity more effectively. Grape skin is rich in flavonols (e.g., quercetin, myricetin, and kaempferol), flavanols (e.g., catechin, epicatechin, and procyanidin), hydroxycinnamic acid, anthocyanins, and resveratrol, while flavanols (e.g., catechin, epicatechin, and procyanidin) and hydroxybenzoic acid can be found in abundant amounts in grape seeds (Montealegre et al., 2006; Xia et al., 2010). One or several of these polyphenols of grapes may account for many biological effects, e.g. antioxidant, antimicrobial, and anti-inflammatory activities, even if the identity of the active substances and their precise mechanisms of action have yet to be investigated.

Conclusion In conclusion, this study revealed the beneficial results of using Anta®Ox FlavoSyn in shrimp farming. Shrimp fed on Anta®Ox FlavoSyn tend to have numerically higher survival rate and total production compared to control groups. In addition, Anta®Ox FlavoSyn-fed shrimp also had lower number of bacteria and a more normal appearance of their hepatopancreas while those of the control group showed obvious signs of bacterial infection. In summary, Anta®Ox FlavoSyn is a promising feed addi-tive to be use in shrimp farming for increasing shrimp production.

References Broussard C. T., C. L. Hofacre, R. K. Page and O. J. Fletcher. 1986. Necrotic enteritis in cage-reared commercial layer pullets. Avian Dis. 30: 617-619.

Bell, T.A. & D.V. Lightner. 1988. A handbook of normal penaeid shrimp histology. World Aquaculture Society, Baton Rouge, Louisiana, USA, pp. 114.

Bravo, L. 1998. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr. Rev., 56(11): 317-333.

Brenes. A., A. Viveros, I. Goñi, C. Centeno, F. Saura-Calixto & I. Arija. 2010. Effect of grape seed extract on growth perfor-mance, protein and polyphenol digestibilities, and antioxidant activity in chicken. Span. J. Agric. Res., 8(2): 326-333.

D’Archivio, M., C. Filesi, R.D. Benedetto, R. Gargiulo, C. Giovannini & R. Masella. 2007. Polyphenols, dietary sources and bioa-vailability. Ann. Ist. Super. Sanita., 43(4): 348-361.

Fiesel, A., D.K. Gessner, E. Most & K. Eder. 2014. Effects of dietary polyphenol-rich plant products from grape or hop on pro-inflammatory gene expression in the intestine, nutrient digestibility and faecal microbiota of weaned pigs. BMC Vet. Res., 10: 1-11.

Gessner D.K., R. Ringseis, M. Siebers, J. Keller, J. Kloster, G. Wen & K. Eder. 2012. Inhibition of the pro-inflammatory NF-κB pathway by a grape seed and grape marc meal extract in intestinal epithelial cells. J. Anim. Physiol. Anim. Nutr., 96(6): 1074-1083.

Gessner, D.K., A. Fiesel, E. Most, J. Dinges, G. Wen, R. Ringseis, & K. Eder. 2013. Supplementation of a grape seed and grape marc meal extract decreases activities of the oxidative stress-responsive transcription factors NF-κB and Nrf2 in the duodenal mucosa of pigs. Acta. Vet. Scand., 55(1): 1-10.

Koch C.; D. K. Gessner; F. J. Romberg; A. Winkler; G. Dusel; E. Herzog; E. Most; K. Eder. 2015. The effect of a polyphenol-rich plant extract on milk performance and the expression of genes of stress of the endoplasmic reticulum and inflammation in the liver of dairy cows in early lactation. Boku-Symposium Tierernährung (14), Wien, pp. 118-122.

Landete, J.M. 2012. Updated knowledge about polyphenols: functions, bioavailability, metabolism, and health. Crit. Rev. Food. Sci. Nutr., 52(10): 936-948.



Limsuwan, C. & P. Chanratchakool. 2004. Shrimp aquaculture industries of Thailand. National Research Council of Thailand, Bangkok, Thailand, 206 pp.

Montealegre, R.R., R.R. Peces, J.L.C. Vozmediano, J.M. Gascuena, & E.G. Romero. 2006. Phenolic compounds in skins and seeds of ten grape Vitis vinifera varieties grown in a warm climate. J. Food. Comp. Anal., 19: 687-693.

Neyestani, T.R. 2008. Polyphenols and immunity. In: F.D. Meester & R.R. Watson (ed.). Wild-type food in health promotion and disease prevention; The Columbus concept. Humana Press, Totowa, New Jersey, USA, pp. 413-434.

Niyamosatha, H., N. Chuchird and T. Rairat. 2015. Effect of Dietary Polyphenol-Rich Feed Additive from Grape Pomace on Growth, Survival, and Tolerance to Vibrio Infection in Pacific White Shrimp (Litopenaeus vannamei). KU Fish Res Bull 39 (2): 1-9.

Viveros, A., S. Chamorro, M. Pizarro, I. Arija, C. Centeno & A. Brenes. 2011. Effects of dietary polyphenol-rich grape products on intestinal microflora and gut morphology in broiler chicks. Poult. Sci., 90(3): 566-578.

Wink, M & O. Schimmer. 2010.Molecular modes of action of defensive secondary metabolites. In: M. Wink (ed.). Annual plant reviews volume 39: Functions and biotechnology of plant secondary metabolites. 2nd ed. Blackwell Publishing Ltd, Singapore, pp. 21-161.

Xia, E.Q., G.F. Deng, Y.J. Guo & H.B. Li. 2010. Biological activities of polyphenols from grapes. Int. J. Mol. Sci., 11(2): 622-646.

Corresponding author Monika Korzekwa Dr. Eckel GmbH Technical Sales Im Stiefelfeld 10 56651 NiederzissenGermany Phone +49 (0) 2636 9749 28 E-Mail: m.korzekwa(at)dr-eckel.de www.dr-eckel.de

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Anta®Ox FlavoSyn ist eine einzigartige pflanzliche Formulierung. Die sorgfältige Kombination beinhaltet eine große Bandbreite wertvoller Polyphenole, abgerundet durch weitere sekundäre Pflanzeninhaltsstoffe. Umfang-reiche Forschungen in Zusammenarbeit mit verschiedenen Universitäten und Versuchsgütern haben die Wirksamkeit eindrucksvoll dokumentiert: in vivo − für alle Tierarten!

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Raceviciute-Stupeliene et al.: Carcass traits and meat quality analysis of pigs for fattening in their rations used different forms of selenium


Carcass traits and meat quality analysis of pigs for fattening in their rations used different forms of selenium

Asta Raceviciute-Stupeliene, Romas Gruzauskas, Vilma Kliseviciute, Vilma Sasyte and Saulius Bliznikas Institute of Animal Rearing Technologies, Lithuanian University of Health Sciences, Veterinary Academy, Kaunas, LT

Abstract The aim of this study was to determine the effect of different forms of selenium on productivity, car-cass traits and meat quality of pigs for fattening.Sixty 62-d-old pigs (Landrace x Yorkshire (mother) and Pietrain x Duroc (father)) for fattening which were individually weighed and were randomly as-signed to 2 dietary treatments with 4 replicate stalls of 30 pigs each. The pigs were fed for 13 weeks ad libitum with a standard wheat-barley-soybean meal compound diet supplemented with 0.3 mg/kg Na2SeO3 and 75 mg/kg vit. E (Control group) and with 0.3 mg/kg Na2SeO3 + 0.2 mg/kg Se-methionine and 75 mg/kg vit. E (Experimental group). Meat traits in live pigs were measured by ultra-sonic equipment Piglog 105. The samples for the analysis of α, γ tocopherols (vit. E) and selenium were taken from the M. longissimus dorsi between 12 and last rib. The results of conducted trial showed, that the mixture of inorganic and organic Se source (0.3 mg/kg Na2SeO3 + 0.2 mg/kg Se-methionine and 75 mg/kg vitamin E) hadn’t effect on pigs for fattening productivity parameters and carcass traits, but resulted in a greater tissue accumulation of Se than only inorganic forms of Se.

Introduction Selenium has been established as an essential trace element that is one of the most important factors in human’s and animal’s nutrition (Xia et al., 2004). In pigs, as well as in other domestic animal spe-cies, Se metabolism depends closely of Se forms and sources. Generally, organic Se has greater bioa-vailability and rates of tissue retention than inorganic Se (Juniper et al., 2011; Liao et al., 2012; Speight et al., 2012). Many studies have established that selenomethionine and selenized yeast (SY) are the most appropriate source of Se for use in animal nutritional supplements because of their greater bioavailability and lower toxicity (Wang, Xu, 2008; Skřivan et al., 2012). Ševčíková et al. (2006) recommends maintaining selenium concentration in feed between 0.1 mg/kg to 0.15 mg/kg of body weight. On the other hand Ryu et al. (2005) states that selenium concentration, including native selenium, cannot exceed 0.5 mg/kg of feed. Supplementation of the swine diet with vitamin E during the growing and finishing periods may im-prove pork quality. Vitamin E is a potent antioxidant and has been demonstrated to decrease lipid oxidation, improve average daily gain and feed efficiency of pigs (Cannon et al., 1996). Vitamin E and selenium act closely and synergistically. These two elements are essential components of anti-oxidative system and suppress oxidative processes of polyunsaturated fatty acids in cellular mem-branes (Skřivan et al., 2008). So the aim of this study was to determine the effect of different forms of selenium on productivity, carcass traits and meat quality of pigs for fattening.



Materials and methods The pigs for fattening were kept in the stalls and its keeping condition was accorded with the Council Directive 2008/120/EC of 18 December 2008 laying down minimumstandards for the protection of pigs. The feeding trial was performed with sixty 62-d-old Landrace x Yorkshire (mother) and Pietrain x Du-roc (father) pigs for fattening which were individually weighed and were randomly assigned to 2 die-tary treatments with 4 replicate stalls of 30 pigs each. The pigs were fed for 13 weeks ad libitum with a standard wheat-barley-soybean meal compound diet supplemented with 0.3 mg/kg Na2SeO3 and 75 mg/kg vit. E (Control group) and with 0.3 mg/kg Na2SeO3 + 0.2 mg/kg Se-methionine and 75 mg/kg vit. E (Experimental group). The diet was formulated to meet the nutrient and energy requirements for pigs for fattening (NRC, 1998). The data recorded during the feeding phase were live weight (LW) at 62, 90, 118 and 150 day from the start of the study, average daily gains (ADG) and feed: gain ratio (F:G) during the periods 62-90 days, 91-118 days, 119-150 days and from the start of the study (62–150 days). Before pigs slaughtering, fat thickness and muscularity (two measurements of fat thickness and thick-ness of the M. longissimus dorsi) was measured by ultrasound equipment „Piglog-105“(SFK Technolo-gy, 1991). At the end of the trial (150 days) from each group 8 pigs for fattening (8 pigs x 2 groups = total of 16 pigs) were selected and slaughtered according to standard procedures. The samples for the analysis of α, γ tocopherols (vit. E) and selenium were taken from the M. longissimus dorsi between 12 and last rib. The accumulation of Se was determined with the atomic absorption spectrometric method (Neugebauer et al., 2000) and vitamin E with the AOAC Official Method 975.43 by HPLC. Statistical analysis.Data were analyzed using one-way analysis of variance (ANOVA) with Statistica software package version 8.0 (StatSoft Inc., 2007). Means were compared with a PLSD Fisher‘s test. The differences between the control and experimental groups were considered to be statistically sig-nificant for P < 0.05.

Results and discussion The effects of dietary treatments (Se source and vitamin E addition) on pig growth performance are presented in Table 1. Final body weight (BW) and average daily gain (ADG) did not differ among treatments. Likewise, no difference in feed: gain ratio (F:G) were observed among the treatments groups during the study.

Table 1: Growth performance of pigs for fattening fed diets with different sources of Se and vitamin E (kg)

Items Control group Experimental group BW (initial) 26.67 ± 0.57 26.55 ± 0.47 BW (final) 116.30 ± 1.29 118.07 ± 1.69 ADG F:G ADG F:G I feeding period (62 – 90 days) 1.015 2.08 1.058 2.06 II feeding period (91 – 118 days) 0.968 2.75 1.005 2.58 III feeding period (119 – 150 days) 1.034 3.13 1.023 3.13 All periods (62 – 150 days) 1.006 2.65 1.029 2.59

Mateo with colleagues (2007) in the experiment with growing-finishing pigs fed diets with organic and inorganic selenium addition also didn’t find influence on the performance parameters. Contrarily Payne and Southern (2005) and Upton et al. (2009) determined, that the use of two type of selenium source (inorganic and organic) in poultry diet increased the weight of broiler chickens in comparison with control group.



In our study, the lack of difference among dietary treatments on growth performance indicates that pigs in the control and experimental diets different Se form utilized the same. The rather high dietary vitamin E level (75 mg/kg) might also explain the absence of response to supplementary Se on growth performance (Li et al., 2011).

Table 2: The influence of different sources of Se and vitamin E on pigs for fattening muscularity (independent by pigs’ sex)1

Group Age in days Weight, kg Fat thickness, mm Mouscles

thickness, mm Muscularity, % 1 point 2 point Control 150 110.4±1.38 14.7±0.94 14.6±0.95 53.9±2.85 56.79±0.91 Experimental 150 109.1±1.57 14.5±0.76 14.5±0.84 57.4±1.79 57.34±0.92 1 From each group selected 8 pigs for fattening (for the measurement with „Piglog-105“ is selected pigs from 85-110 kg)

By measuring the fattening pigs’ thickness of muscle (M. longissimus dorsi) and fat and by calculating the percentage of muscularity (Table 2), we can conclude that no addition of inorganic selenium, no combination of Na2SeO3 and Se-methionine and vitamin E hadn’t significant effect on the fat thickness and muscularity.

Table 3: The influence of different sources of Se and vitamine E on the accumulation of α, γ tocopherol and sele-nium in the meat of pigs for fattening

Group α-tocopherol, mg/kg γ- tocopherol, mg/kg Selenium μg/g DM (95%) Control 4.290.89 1.370.09 0.678±0.129a Experimental 4.880.83 1.430.04 1.142±0.091b a, b – means within each rows with different superscripts are significantly different at P < 0.05; PLSD Fisher‘s test, mean ± SEM

The α- and γ- tocopherols and Se concentrations in M. longissimus dorsi (expressed per DM) are summarized in Table 3. No effect of dietary treatments on vitamin E accumulation in muscle were observed. By analysing the accumulation of different Se form in tissues there were observed, that in muscle, only organic Se addition in the experimental ration has shown the ability to improve total Se content in the muscle. Indeed, inorganic selenium additions did not increase muscle Se contents, indi-cating a specific effect of organic source in this tissue. Our results confirmed previous research, which indicated that a lower proportion of Se was retained in muscle when inorganic Se source was fed to pigs (Mahan et al., 1999). It is probably that selenomethionine, was nonspecifically incorporated into muscle protein during growing period, which is characterized by a high rate of protein synthesis. Therefore, selenomethionine from organic Se sources can be readily incorporated into structural pro-teins because of no distinction between Met and selenomethionine by tRNA during protein synthesis in muscle (Schrauzer, 2003).

Conclusions 1. Inorganic and organic selenium mixtures (experimental group) hadn’t effect on pigs for fattening productivity parameters and carcass traits. 2. Feeding diets supplemented with mixture of inorganic and organic Se source (0.3 mg/kg Na2SeO3 + 0.2 mg/kg Se-methionine; 75 mg/kg vitamin E) resulted in a greater tissue accumulation of Se than only inorganic forms of Se.

Acknowledgments Part-financed by the ministry of Agriculture of the Republic of Lithuania project MT 11/30.



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Upton J. R., Edens F. W., Ferket P. P. (2009): The effects of dietary oxidized fat and selenium source on performance, glutathione peroxidase, and glutathione reductase activity in broiler chickens. Journal of Applied Poultry Research 18, 193–202.

Wang Y. B., Xu B. H. (2008): Effect of different selenium source (sodium selenite and selenium yeast) on broiler chickens. Anim. Feed Sci. Technol. 144:306–314.

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Corresponding author Asta Racevičiūtė-Stupelienė Lithuanian University oh Health Sciences Veterinary Academy. Kaunas, Lithuania. E-mail: asta.raceviciutestupeliene(at)lsmuni.lt

Václavková et al.: Fatty acid profile of pork from a local Prestice Black-Pied pigs and commercial hybrid pigs


Fatty acid profile of pork from a local Prestice Black-Pied pigs and commercial hybrid pigs

Eva Václavková, Jaroslava Bělková, Miroslav Rozkot, Stanislava Kuchařová and Jan Lipenský Institute of Animal Science Prague, Department of Pig Breeding, Kostelec nad Orlicí, CZ

Abstract The aim of the study was to compare fatty acid profile in meat from local Czech pig breed Prestice Black-Pied and commercial fatteners (Czech Large White x Czech Landrace) x (Hampshire x Pietrain). Pigs were divided in two groups (10 animals in each) and three months before slaughter fed ad libi-tum with the feed mixture with ground linseed addition (70 g · kg-1). The fatty acid content was eval-uated by gas chromatography in the samples of M. longissimus dorsi. There were found significant differences in most monitored fatty acids between Prestice Black-Pied pigs and hybrid pigs. Samples of M. longissimus dorsi from Prestice pigs were characterised by significantly higher (P<0.05-0.01) content of linoleic, alpha-linolenic, arachidonic acid and EPA. On the contrary, the content of myristic, palmitic, stearic and DHA were significantly (P<0.01) lower compared with hybrid pigs. The content of total SFA was significantly (P<0.01) lower and content of PUFA was significantly (P<0.01) higher in Prestice pigs. The omega-6/omega-3 PUFA ratio was lower in hybrid pigs and corresponds with the recommendation for human nutrition.

Introduction Interest in polyunsaturated fatty acids (PUFA) arises from their potential in therapeutic applications, and food and nutritional applications. They occur throughout animal, plant, algae, fungi and bacteria. They are found widely in many lipid compounds such as membranes, storage oils, glycolipids, phos-pholipids, sphingolipids and lipoproteins. Fatty acids have a diverse function in the body. They serve as a source of metabolic energy; provide structural integrity to cell membranes, are substrates for hormone production, and help to regulate cellular function, immune function, and inflammation. Ome-ga-3 fatty acids are promoted as conferring broad health benefits by preventing and treating a wide variety of inflammatory diseases. In cell culture and animal studies these essential fatty acids have potent immunomodulatory effects (Kirk, 2007). Meat fatty acid composition can be changed via the diet, linoleic, alpha-linolenic and long-chain PUFA content responds quickly to feeding higher levels of alpha-linolenic acid to pigs. The relative propor-tion of fatty acid composition is influenced by numerous factors including diet, fatness, age (body weight), gender, breed, environmental temperature, depot site, maintenance and hormones. The potential for dietary variation of lipid composition in monogastric animals is much greater than in ru-minants (Nurnberg et al., 1998). In pigs, the PUFA linoleic and alpha-linolenic cannot be synthesized and tissue concentrations respond rapidly to dietary changes. Saturated fatty acids (SFA) and mono-unsaturated fatty acids (MUFA) on the other hand are synthesized and their concentrations are less readily influenced by diet (Wood et al, 2004). Meat fatty acid composition is also influenced by genetic factors, although to a lower extent than dietary factors. The level of fatness also has an effect on the meat fatty acid composition. The contents of SFA and MUFA increase faster with increasing fatness than content of PUFA, resulting in a decrease in the relative proportion of PUFA and consequently in the PUFA/SFA ratio. Differences in fatty acid composition between breeds and genotypes can be large-



ly explained by differences in fatness. However, these effects are much smaller than the effects that can be achieved by dietary means (DeSmet et al., 2004). Přeštice Black-Pied pig is Czech national breed. The importance of this breed lies in its good reproduc-tion performance, adaptability, good vitality and resistance to diseases (Lustykova et al., 2008). The breed is characterised by higher backfat thickness and very good meat quality. The aim of the study was to evaluate the effect of linseed in diet of fattening Prestice Black-Pied pigs on their carcass value and meat quality.

Material and methods The aim of the study was to compare fatty acid profile in M. longissimus dorsi from local Czech pig breed Prestice Black-Pied and commercial fatteners (Czech Large White x Czech Landrace) x (Hamp-shire x Pietrain). Pigs were divided into two groups (10 animals in each) and three months before slaughter fed ad libitum with the feed mixture with ground linseed addition (70 g · kg-1). The samples of M. longissimus dorsi were collected 24 h after slaughter, packed in PE bags and stored at -20°C. The feed mixture was determined 9.13 rel. % of omega 3 PUFA and omega6/omega 3 PUFA ratio 2.91. Fatty acid composition of meat and backfat was determined after chloroform-methanol extrac-tion of total lipids (Folch et al., 1957). Alkaline trans-methylation of fatty acids was performed as de-scribed by Raes et al. (2003). Gas chromatography of methyl esters was carried out using an Agilent 6890M chromatograph (Agilent Technologies, Inc. local distributor HPST, Ltd., Prague 4, Czech Re-public). The statistical evaluation was performed using the computer program QCExpert (TriloByte Statistical Software Ltd.). Data were presented as the mean, standard deviation (SD) of each group and the significance levels.

Results The content of selected fatty acids (in rel. %) is illustrated in Table 1. There were found significant differences in most monitored fatty acids between Prestice Black-Pied pigs and hybrid pigs.

Table 1: The content of selected fatty acids (rel. %) in M.longissimus dorsi of Prestice Black-Pied pigs and commercial hybrid pigs

Fatty acid PC H myristic C 14:0 1.066 ± 0.089** 2.090 ± 0.153** palmitic C 16:0 21.637 ± 1.558** 25.426 ± 0.448** stearic C 18:0 10.695 ± 1.138** 12.770 ± 0.532** oleic C 18:1-n9 40.008 ± 4.231 40.360 ± 0.662 linoleic C 18:2-n6 11.885 ± 4.554** 6.155 ± 0.564** α-linolenic C 18:3-n3 2.016 ± 1.524* 1.118 ± 0.065* arachidonic C 20:4-n6 1.699 ± 0.451** 1.209 ± 0.325** EPA C 20:5-n3 0.185 ± 0.030** 0.041 ± 0.007** DHA C 22:6-n3 0.037 ± 0.008** 0.064 ± 0.015** SFA 34.025 ± 2.702** 41.443 ± 0.781** MUFA 47.429 ± 4.932 48.485 ± 0.843 PUFA 18.545 ± 6.862** 10.073 ± 1.054** Omega-6 PUFA 14.446 ± 4.535** 8.071 ± 0.979** Omega-3 PUFA 2.751 ± 1.552* 1.801 ± 0.109* omega-6/omega-3PUFA 6.189 ± 2.248* 4.473 ± 0.388* *P<0.05 **P<0.01 PC – Prestice Black-Pied pigs H – Hybrid pigs (CLW x CL) x (H x Pn) SFA – saturated fatty acids MUFA – monounsaturated fatty acids PUFA - polyunsaturated fatty acids



Samples of M. longissimus dorsi from Prestice pigs were characterised by significantly higher (P<0.05-0.01) content of linoleic acid, alpha-linolenic acid, arachidonic acid and EPA. On the contrary, the con-tent of myristic, palmitic, stearic and DHA were significantly (P<0.01) lower compared with hybrid pigs. The content of total SFA was significantly (P<0.01) lower (34.03 vs. 41.44 rel. %) and content of PUFA was significantly (P<0.01) higher (18.55 vs. 10.07 rel. %) in Prestice pigs. The content of MUFA was at the same level in both groups (P˃0.05). The M. longissimus dorsi samples of Prestice pigs were also characterised by higher content of omega-6 and omega-3 PUFA. But the ratio of these two groups of PUFA was lower in hybrid pigs (P<0.05).

Discussion Differences in fatty acid profile among different pig breeds can be explained by the differences in lean meat content and fat (DeSmet et al., 2004). Zhang et al. (2007) found differences in fatty acid con-tent in Duroc pigs, Chester White, Berkshire, Yorkshire, Landrace and Hampshire pigs. The highest concentration of saturated fatty acids and the lowest concentration of PUFA were found in Duroc pigs. Prestice Black-Pied breed is characterised by higher content of intramuscular fat (Dostálová et. al., 2012). The concentration of SFA depends on intramuscular fat content. There can be found higher concentration of SFA and lower proportion of unsaturated fatty acids in pigs with higher intramuscular fat content (Altmann et al., 1992). According Wood et al. (2004), traditional pig breeds have higher proportion of SFA and lower proportion of PUFA. Teixeira and Rodrigues (2013) evaluated the physi-cal-chemical characteristics of pork from a local breed Preto Alentejano and a commercial breed. Preto Alentejano breed presented the higher percentages of SFA (P<0.01) and MUFA (P<0.001) fatty acids. Renaudeau et al. (2005) found differences between fatty acid composition in Large White pigs and Creole pigs. There were higher percentage of intramuscular fat and higher saturated backfat fatty acid concentration in Creole pigs (40,0% vs. 37,9%). The concentration of linoleic and linolenic acid was lower compared with Large White pigs. On the contrary, there was found lower concentration of SFA and higher concentration of PUFA in our experiment. The differences in growth intensity, fat deposi-tion and meat quality in Basque pigs and Large White pigs were monitored by Alfonso et al. (2005). Basque pigs had a lower growth intensity and higher fat deposition. There were found differences in meat quality, especially in fatty acid content, between the breeds. But these differences were not significant. Csapó et al. (1999) did not find significant differences in fatty acid content among Manga-litsa breed, Large White x Mangalitsa and Mangalitsa x Duroc pigs. Kasprzyk et al. (2015) investigated the effects of breed on the fatty acid compositions of the Longissimus thoracis et lumborum of gilts and barrows of local Polish breed Pulawska and commercial breed Polish Landrace breed. Higher con-tents of intramuscular fat, C16 : 1, C18 : 1 and MUFA were found in the longissimus muscle of Pu-lawska pigs compared with Polish Landrace pigs. Furman et al. (2010) compared fatty acid composition of meat and adipose tissue from Krskopolje pigs (Slovenian local breed) and commercial fatteners. The M. longissimus dorsi of Krskopolje pigs contained less stearic acid than that of commer-cial fatteners. The Krskopolje pigs had the lowest proportion of SFA. The same result was obtained in our experiment. Yu et al. (2013) compared meat quality traits between Lantang (Chinese indigenous breed) and Landrace pigs. Fatty acid analysis demonstrated the lower MUFA and higher PUFA per-centage in Lantang longissimus dorsi than that in Landrace. In our trial, the MUFA content was not significantly different (P˃0.05).

Conclusion There were found differences for fatty acid concentration in M. longissimus dorsi between local Czech pig breed Prestice Black-Pied pig and commercial hybrid pigs. Prestice pigs were characterised by higher content of total PUFA, omega-6 and omega-3 PUFA and lower content of SFA. The omega-6/omega-3 PUFA ratio was lower in hybrid pigs and corresponds with nutritional recommendation.



Acknowledgement This study was supported by the Ministry of Agriculture of the Czech Republic – research project NAZV QJ1210253.

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Yu K., Shu G., Yuan F.F., Zhu X.T., Gao P., Wang S.B., Wang L.N., Xi Q.Y., Zhang S.Q., 2013: Fatty Acid and Transcriptome Profiling of Longissimus Dorsi Muscles between Pig Breeds Differing in Meat Quality. International Journal of Biological Science, 9:108-118.

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Corresponding author Ing. Eva Vaclavkova, Ph.D. Institute of Animal Science Prague Uhrineves Department of Pig Breeding in Kostelec nad Orlici Komenskoho 1239 51741 Kostelec nad Orlici, Czech Republic E-mail: vaclavkova.eva(at)vuzv.cz

Loibl et al.: Zum Einfluss von Störfaktoren auf zootechnische Parameter bei Mastschweinen – ein Beitrag zur Tierwohlforschung


Zum Einfluss von Störfaktoren auf zootechnische Parameter bei Mastschweinen – ein Beitrag zur Tierwohlforschung

Peter Loibl1, Wolfgang Preißinger1, Günther Propstmeier1, Simone Scherb1 und Wilhelm Windisch2 1 Bayerische Landesanstalt für Landwirtschaft, Institut für Tierernährung und Futter wirtschaft, DE 2 Technische Universität München, Lehrstuhl für Tierernährung, DE

Abstract A feeding trial with 96 fattening pigs was conducted to assess the influence of disturbing situations in typical stable routine on performance parameters and feed-intake-behaviour. Three different treat-ments were designed to picture different disturbances. Treatments included e. g. changes of several animals between pens, feed deprivation (max. 24 h) or feeding of different feed compositions (for max. 48 h). Disturbances induced short-term depressions of performance but had no overall impact. To analyse the effects on feed-intake-behaviour adequate statistical methodology has further to be established. Individual feed-intake-patterns and short-term deviations thereof should be a candidate for a novel animal associated indicator of animal welfare.

Einleitung Für die Tierwohlforschung ist es unerlässlich, objektiv messbare, tierbezogene Indikatoren zu finden und zu etablieren (Veissier und Forkman, 2008). Die momentan gängige Beurteilungspraxis des Wohl-befindens des Tieres, bzw. der Qualität des Tierwohls in einer bestimmten Haltungsumgebung zielt jedoch in hohem Maße auf Umgebungsfaktoren, wie die verfügbare Fläche pro Tier, die Zahl der ge-haltenen Tiere pro Gruppe oder das Vorhandensein von organischem Beschäftigungsmaterial ab (Wel-fare Quality®, 2009). Eine direkte Messung des Wohlbefindens am Tier findet dabei meist nicht statt. Hierzu fehlen v. a. tierbezogene Indikatoren, bzw. diese sind, wie z. B. Blutproben oder Speichelkor-tisolbestimmung nur schwer im laufenden Betrieb erfassen. Darum wird im Moment auf die Verhalten-serforschung und -beobachtung gesetzt um Tierwohl zu „messen“ (von-Borell et al., 2001; Conte et al., 2014). Im hier vorgestellten Projekt wird untersucht, ob zootechnische Leistungen oder das Futteraufnahme-verhalten in ad-libitum-Fütterung als ein Tierwohlindikator unter praxisnahen Bedingungen dienen können. Hierfür wurden Mastschweine und Aufzuchtferkel kurzfristigen Störungen ausgesetzt. Anhand dieser Situationen sollte herausgefunden werden, ob kurzfristiger Stress die Leistungen des Tieres beeinträchtigt und das Futteraufnahmeverhalten messbar verändern.

Material und Methoden Der Fütterungsversuch wurde am Lehr-, Versuchs- und Fachzentrum Schwarzenau der bayerischen Landesanstalt für Landwirtschaft (LfL) mit 96 Mastschweinen durchgeführt. Hierzu stand ein Mastab-teil mit acht Buchten zur Verfügung (Vollspaltenboden ohne Einstreu, jeweils 13 m2), die von jeweils einem Futterautomaten (Schauer Agrotronic GmbH, Prambachkirchen, Österreich) mit automatischer Tiererkennung und Messung der verzehrten Futtermenge pro Besuchszeitpunkt bedient wurden. Die Buchten wurden mit jeweils 12 Tieren der Rasse Pi x (DL x DE) unter Berücksichtigung von Wurf,



Geschlecht und Anfangsgewicht gleichmäßig belegt. Zu Versuchsbeginn waren die Tiere im Mittel 74 Tage alt und wogen 32 kg. Als Mastendgewicht wurden 120 kg LM angestrebt. Mit dem Erreichen des Mastendgewichtes wurden die Tiere im Versuchsschlachthaus Schwarzenau geschlachtet. Alle Tiere wurden zur Futtereiweißminimierung dreiphasig gefüttert (Anfangsmast (AM): 30-60 kg, Mittelmast (MM) 60-90 kg, Endmast (EM) 90 kg bis Schlachtung). Jede Behandlungsgruppe wurde, außer bei experimentellen Abänderungen, mit der gleichen Futtermischung gefüttert. Die Mischung bestand aus Gersten- (40 %), Weizen- (39-46 %) und Sojaschrot (18-12 %) und wurde mit einem handelsüblichen Mineralfutter ergänzt (3-2 %). Mit diesen Komponenten konnte ein Energiegehalt von 13,3 MJ ME bis 13,5 MJ ME (Anfangs- bis Endmastration) erreicht werden. Der Rohproteingehalt wur-de im Versuchsverlauf von 17,6 % auf 15,8 % gesenkt. Jeweils zwei der acht Buchten des Mastabteils wurden einer von vier Behandlungen zugeordnet, die praxisrelevante Störsituationen repräsentieren sollten (Tabelle 6). Neben einer Kontrollgruppe ohne Störungen (Gruppe A) wurde bei Gruppe B die Futterzufuhr variiert (Futterentzug für 12 h, bzw. 24 h und restriktive Fütterung für 48 h). Bei Gruppe C wurden jeweils 3 Tiere zwischen den beiden Buchten der Behandlungsvariante getauscht, die verfügbare Wassermenge verringert, bzw. nach Ausfällen die Gruppengrößen wiederausgeglichen. In der vierten Gruppe wurde die Futterzusammensetzung kurz-fristig verändert (nur Getreide für 48 h, die doppelte Menge Mineralfutter für 48 h und 48 h AM-Futter in der EM). Wegen technischer Probleme konnte erst zwei Wochen nach Mittelmastbeginn mit dem Einbringen der Störungen begonnen werden.

Tabelle 6: Zeitplan des Versuches mit Futterumstellung und Störungen

Behandlung Mastwoche A B C D 1 Versuchsbeginn (Anfangsmastration) 6 Futterumstellung von der Anfangsmast- auf die Mittelmastration

8 Kontrolle Futterentzug für 24 h Tausch von jeweils 3 Tieren zwischen den Buchten Nur Getreide für 48 h

10 Kontrolle Futterentzug für 12 h Tränkewasserdurchfluss auf 0,6 l/min reduziert

100 % mehr Mineralfutter für 48 h

11 Futterumstellung von der Mittelmast- auf die Endmastration

12 Kontrolle 60 % der durchschnittlichen Tagesmenge, 48 h

Ausgleich der Gruppengröße nach Ausfällen

Anfangsmastfutter für 48 h

14-16 Beginn des sukzessiven Schlachtens der schlachtreifen Tiere

Es wurden tierindividuell die gefressene Futtermenge pro Besuch und pro Tag und die Besuchszeit erfasst. Die Tiere wurden wöchentlich gewogen. Für die statistische Auswertung und Berechnung der Leistungszahlen wurde SAS 9.3/9.4 (Copyright© 2015, SAS Institute Inc.) verwendet. Signifikante Mittelwertunterschiede der Behandlungen wurden mit der Option LSMEANS der PROC GLM des SAS-Systems identifiziert.

Ergebnisse und Diskussion Tabelle 7 zeigt zusammengefasst die Mittelwerte der zootechnischen Leistungen der vier Behand-lungsgruppen. Sechs Tiere fielen krankheitsbedingt aus. In der Anfangs- und Mittelmast kam es bei den täglichen Zunahmen zu signifikanten Unterschieden. Ab der achten Mastwoche wurden die Tiere gezielt mit Störungen konfrontiert (s. o.). Gruppe B zeigte aber bereits in der Anfangsmast mit 706 g Tageszunahmen signifikant niedrigere Leistungen gegen-über den anderen Gruppen (durchschnittlich 766 g). Hier kam es in der Anfangsmast vorübergehend zu Schwanzbeißen, wodurch das niedrigere Niveau bei den Zunahmen erklärt wird. Dies kann in der



Tendenz bei der verringerten Futteraufnahme wiedergefunden werden. In der Mittelmast kam es zu folgender Entwicklung der Zunahmen: Die Kontrollgruppe (Gruppe A) nahm mit 932 g um durch-schnittlich 101 g pro Tag mehr zu als die anderen Behandlungen (831 g). Die Tiere schienen also in der Mittelmast auf die Störungen zu reagieren. Die täglichen Futteraufnahmen folgten nämlich dem gleichen Trend. Es kam zu einer Verringerung um 0,24 kg/Tag im Vergleich zur Kontrolle (2,24 kg/Tag (Gruppe A) zu 2,10 kg/Tag (Gruppen B bis C)). Gruppe D hatte in der Mittelmast mit 2,72 kg/kg ge-genüber den anderen Gruppen (2,45 kg/kg) eine niedrigere Futtereffizienz. Auch dies spricht für einen Behandlungseffekt, da hier in der Mittelmast v. a. mit dem Weglassen des Soja- und Mineralfutteran-teils in der achten Mastwoche die Verwertbarkeit des vorgelegten Futters reduziert wurde. Über alle Parameter zeigte sich aber, dass der Einfluss der Störungen ab der Endmast statistisch nicht mehr signifikant war. Dies spricht dafür, dass die Tiere mit zunehmendem Alter weniger empfindlich reagierten. Auch bei den Gesamtleistungen und den Schlachtparametern konnten keine signifikanten Unterschiede festgestellt werden.

Tabelle 7: Zoologische Leistungen der unterschiedlichen Behandlungsgruppen

Behandlung A B C D Signifikanz (p<0,05) Tierzahl n 23 24 20 23 --- Masttage d 103 104 104 105 0,96 Lebendmasse Anfang kg 32 32 32 33 0,82 Beginn MM kg 59 57 58 60 0,36 Beginn EM kg 85 80 82 82 0,07 Ende kg 114 111 113 114 0,48 Zunahmen/Tag AM g 774a 706b 754ab 769a 0,05 MM g 932a 821b 866ab 807b 0,01 EM g 845 887 867 774 0,59 Gesamt g 828 789 789 785 0,29 Futteraufnahme/Tag AM kg 1,69 1,59 1,68 1,67 0,24 MM kg 2,24a 2,02b 2,14ab 2,15ab 0,03 EM kg 2,47 2,40 2,30 2,40 0,33 Gesamt kg 2,19 2,07 2,09 2,13 0,23 Futtereffizienz (kg Futter pro kg Zunahme) AM kg/kg 2,19 2,27 2,23 2,17 0,13 MM kg/kg 2,41a 2,46a 2,49a 2,72b 0,03 EM kg/kg 3,17 3,02 3,11 3,12 0,81 Gesamt kg/kg 2,67 2,65 2,68 2,74 0,41 Schlachtparameter Fleischfläche cm2 61,0 57,8 60,7 59,5 0,09 Fettfläche cm2 15,3 14,3 15,0 14,2 0,16 Muskelfleisch % 60,5 60,7 59,5 61,0 0,63 AM = Anfangsmast (30-60 kg); MM = Mittelmast (60-90 kg); EM = Endmast (90 kg bis Schlachtung)

Wie die vorliegende Fütterungsstudie zeigte, war die unmittelbare Wirkung kurzfristiger Störungen im jeweiligen Mastabschnitt erkennbar, insbesondere in der Mittelmast. Für eine genauere Betrachtung der Effekte v. a. in der Anfangsmast wurde bereits ein Anschlussversuch durchgeführt. Kompensatori-sche Effekte verwischen jedoch die Folgewirkungen auf die Gesamtleistung der Tiere. Die Detektion von Störungen des Tierwohls auf der Basis zootechnischer Leistungsdaten ist somit auf wenige Wo-chen begrenzt.Deswegen entschloss man sich das Futteraufnahmeverhalten als kurzfristig reagie-renden Faktor genauer zu betrachten. In Abbildung 1 ist beispielhaft die tierindividuelle Verteilung der gefressenen Futtermenge im Tagesverlauf des Durchschnitts der Wochen sieben bis neun des Tieres 33621 (Gruppe A) dargestellt.



Abbildung 1: Durchschnittliche Futteraufnahme im Tagesverlauf des Tieres 33621 (Gruppe A) in den Wochen 7, 8

und 9

Die Futteraufnahme schien einem Rhythmus zu folgen. So lagen die wöchentlichen Verzehrsma-xima konstant bei etwa 07:00 und 13:00 Uhr und nahmen mit fortschreitender Mastdauer men-genmäßig zu. Für zukünftige Analysen soll die statistische Methodik entsprechend weiterentwickelt werden, dass die Konstanz der Aufnahme untersucht werden kann und kurzfris-tige Änderungen erkannt werden können. Wird dies möglich, so könnte man den individuellen Futteraufnahmeverlauf als tierbezogenen Tierwohlindikator etablieren.

Schlussfolgerungen Im vorgestellten Versuch wurde untersucht, welchen Einfluss in der praktischen Stallroutine vor-kommende Störungen auf das Tier haben. Da sich nur kurzfristige (auf den einzelnen Mastab-schnitt begrenzte Einflüsse) auf zootechnische Leistungen zeigten, wird zukünftig analysiert, ob und wie die Futteraufnahme beeinflusst wird. Hierfür müssen statistische Methoden gefunden und etabliert werden, um einen Normalzustand definieren zu können und kurzfristige Abwei-chungen desselben absichern zu können. So könnte man durch detaillierte Analyse des Futter-verzehrsverhaltens einen neuen, objektiven und tierbezogenen Tierwohlindikator erarbeiten.

Literatur Conte, S., Bergeron, R., Grégoire, J., Gète, M., D’Allaire, S., Meunier-Salaün, M.-C., Devillers, N. (2014): On-farm evaluation of methods to assess welfare of gestating sows. In: Animal 8 (7), S. 1153-1161.

Veissier, I. und Forkman, B. (2008): The Nature of Animal Welfare Science. In: Annual Review of Biomedical Sciences 10, S. T15-T26.

von-Borell, E., Bockisch, F.-J., Büscher, W., Hoy, S., Krieter, J., Müller, C., Parvizi, N., Richter, T., Rudovsky, A., Sundrum, A., Can den Weghe, H. (2001): Critical control points for on-farm assessment of pig housing. In: Livestock Production Science 1-2 (72), S. 177-184.

Welfare Quality® (2009): Welfare Quality® assessment protocol for pigs (sows and piglets, growing and finishing pigs). Welfare Quality® consortium, Lelystad, Netherlands.



Autorenanschrift Peter Loibl Lehrstuhl für Tierernährung Liesel-Beckmann-Straße 2 85354 Freising Tel.: 0049 172/3639054 E-Mail: peter.loibl(at)wzw.tum.de

Rozkot et al.: Minischweine und eine spezielle Diät für die Erforschung des metabolischen Syndroms


Minischweine und eine spezielle Diät für die Erforschung des metabolischen Syndroms

Miroslav Rozkot1, Pavel Klein2, Petr Daněk1, Eva Václavková1, Jaroslava Bělková1 und Jan Lipenský1 1 Das Forschungsinstitut für Tierproduktion, v.v.i., Abt. für Schweinezucht Kostelec nad Orlici, Prag (VÚŽV), CZ 2 Contipro Pharma, a.s., Dolní Dobrouč, CZ

Abstract Type 2 diabetes and metabolic syndrome represents major health problem in human population. Both these nosological units are complex and polyfactorial. Similarly like in humans, also in pigs DM II and MS is associated with obesity, glucose intolerance, insulin resistance and high level of plasmatic tri-glycerides. Pigs are therefore valuable preclinical models. Crucial factor for development of diet-induced DM II and MS is inherited predisposition. Feeding diabetogenic diet to Kostelec minipigs led to morbid obesity, but not to development of DM II or MS. These results suggest that Kostelec minipigs are DM II – resistant and can not be used as a model of this disease.

Einleitung Diabetes Typ II (DM II) und das metabolische Syndrom (MS) stellen eines der wichtigsten gesundheit-lichen Probleme der heutigen menschlichen Population dar. Diese beiden nosologischen Einheiten sind kompliziert und polyfaktoriell. Ähnlich wie bei Menschen sind DM II und MS auch bei Schweinen mit Adipositas, Glukose-Unverträglichkeit, Insulinresistenz und einem hohen Spiegel von plasmatischen Triglyzeriden verbun-den. Schweine stellen deshalb ein wertvolles präklinisches Modell dar (Christoffersen et al 2013, Jo-hansen et al 2001). Der entscheidende Faktor für die Entstehung einer diätbedingten DM II und MS ist die genetische Prädisposition.

Material und Methoden Im Experiment wurden 21 Minischweine vom Typ Minnesota eingesetzt (Jungsauen und Kastraten), die in 3 Gruppen eingeteilt wurden (experimentell I, experimentell II, Kontrollgruppe). Diät und Fütte-rungsplan für die Gruppen I und II: KPB (Komplette Mischfutter) 30%, Rinderfett 35%, 35% Saccha-rose. Erste Gruppe erhalten Futtermittel regelmäßig, zweimal am Tag, die zweite Gruppe wurde zeitweise einmal am Tag zur Simulation eines ungesunden Lebensstils, zugeführt. Verwendete Tests: Glukosetoleranztest: Der orale GTT wurde jeden Monat bei allen Tieren durchgeführt, und zwar laut demselben Protokoll wie bei Menschen. Es wurde Blut aus den Ohrengefäßen entnommen (Klein und Sojka, 2012) und der Glukosespiegel wurde mithilfe eines handelsüblichen Glukometers be-stimmt.Fettmessung: Das Rückenfett wurde jeden Monat mit einem Ultraschallgerät gemessen (So-nomark SM100M) Blutuntersuchung: Blut für die Analyse ausgewählter Marker wurde im 2., 5. und 8. Monat des Expe-riments aus den Ohrengefäßen entnommen.



Ergebnisse

0

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100

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140

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0 2 4 6 8 10 12

time (months)

live

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Group II (irregularlyfed animals)

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Group I Group II Control Group I Group II Control Group I Group II Control

Glucose (mmol/l) 2,45 2,08 1,82 2,9 3,16 2,57 2,47 2,34 1,55 GHbA1c (mmol/mol) 4,71 4,57 5,6 1,94 2,06 2,03 9 10,43 8,25 Cholesterol (mmol/l) 2,55 2,16 2,2 4,41 4,32 2 3,03 2,96 2,43

HDL (mmol/l) 0,93 0,82 1,73 2,49 2,32 1,65 1,21 1,16 0,86 LDL (mmol/l) 1,13 0,98 0,77 2,11 1,87 0,7 1,35 1,38 1,19 TAG (mmol/l) 0,74 0,52 0,25 0,57 0,92 0,25 0,47 0,65 0,38

atherogenity index 0,77 0,85 0,25 1,81 1,59 1,83

Abbildung 1: Kontrollgruppe Abbildung 2: Studiengruppe



Diskussion Die Fütterung dieses Typs von Minischweinen mit einer diabetogenen Diät führte zu einer morbiden Adipositas, aber nicht zur Entstehung von DM II oder MS.

Schlussfolgerungen Diese Ergebnisse deuten im Gegensatz zu Publikationen anderer Autoren an, dass zumindest dieser Typ von Minischweinen DMII-resistent ist und nicht auf diese Weise als Modell dieser Erkrankung ein-gesetzt werden kann.

Acknowledgement Unterstützt durch Grant MZE RO 0714 und NAZV QJ 1210253

Literatur Christoffersen B., Golozoubova V., Pacini G., Svendsen O., Raun K., 2013: The young Göttingen minipig as a model of childhood and adolescent obesity: influence of diet and gender.Obesity (Silver Spring). Jan;21(1):149-58. doi: 10.1002/oby.20249.

Johansen T., Hansen H.S., Richelsen B., Malmlöf R., 2001:The obese Göttingen minipig as a model of the metabolic syndrome: dietary effects on obesity, insulin sensitivity, and growth hormone profile. Comp Med. Apr;51(2):150-5.

Klein P., Sojka M., 2012: A simple method for aseptic collection of blood from minipig metatarsal veins. Research in pig bree-ding, 6, (2) s33-36.

Autorenanschrift Ing. Miroslav Rozkot, CSc. Das Forschungsinstitut für Tierproduktion, v.v.i., Prag (VÚŽV) Abt. für Schweinezucht Kostelec nad Orlici Komenskeho 1239 51741 Kostelec nad Orlici, Czech Republic E-Mail: rozkot.miroslav(at)vuzv.cz

Preißinger et al.: Pellets oder schrotförmiges Futter an Abrufstationen für Mastschweine - Auswirkungen auf Mast- und Schlachtleistung


Pellets oder schrotförmiges Futter an Abrufstationen für Mast-schweine - Auswirkungen auf Mast- und Schlachtleistung

Wolfgang Preißinger, Günther Propstmeier und Simone Scherb Bayerische Landesanstalt für Landwirtschaft, Institut für Tierernährung und Futter-wirtschaft Grub/Schwarzenau, DE

Abstract Growth and carcass traits of growing and finishing pigs fed diets with different physical form (coarse meals or pelleted feed) were examined. In the experiment 96 pigs with an average bodyweight (BW) of 33 kg were randomly allocated into 4 groups. The trial was divided in a grower (30-70 kg BW) and finisher (70-120 kg BW) phase. The experiment included the following treatments: A, coarse meal in grower and finisher phase; B, coarse meal in grower and pelleted feed in finisher phase; C, pelleted feed in grower and coarse meal the finisher phase; D, pelleted feed in grower and finisher phase. All diets based on wheat, barley and soybean meal. There were no differences in the composition of the diets for each phase, but crude protein and amino acid contents were lower in the finisher diets. The diets were fed individually by an automatic feeder (Schauer Agrotronic – GmbH, Austria). The average daily feed intake was about 2.1 kg in all treatments. The feed intake was not affected by the physical form of the feed. Coarse meal fed in the grower and finisher phase or only fed in the finisher phase resulted in lower average daily gains (805 respectively 792 g vs. 867 respectively 852 g) and in adverse feed conversion ratios (2.63 respectively 2.68 kg/kg vs. 2.53 respectively 2.50 kg/kg). The majority of carcass traits were not affected by the physical form of the feed, but back fat thickness and area of back fat were lower when coarse meal was fed in the finisher period.

Einleitung Abrufstationen für Mastschweine werden seit längerem in der Mastleistungsprüfung eingesetzt. Auch für Fütterungsversuche werden sie am Lehr-, Versuchs- und Fachzentrum (LVFZ) für Schweinehaltung der Bayerischen Landesanstalt für Landwirtschaft (LfL) in Schwarzenau genutzt. Gegenüber der Flüs-sigfütterung (Langtrog mit Sensor) zeigten sich aber bei nahezu identischen Futtermischungen deut-lich niedrigere Mastleistungen an den Abrufstationen. Die Tiere erhielten dort das Futter als trockenen Schrot. Insbesondere in der Endmast ab 100 kg Lebendmasse (LM) ließen Futteraufnahme und Zu-wachs der Tiere an den Abrufstationen nach. In der Mastleistungsprüfung mit gleicher Technik, aber pelletiertem Futter, werden auch im höheren LM-Bereich ansprechende Leistungen erzielt. Obwohl in der Literatur von positiven Effekten auf Tiergesundheit und Immunantwort bei grob geschrotetem gegenüber pelletiertem Futter berichtet wird (Betscher et al., 2010; Kamphues, 2010; Wintermann et al., 2010; Capai et al., 2015) wurde in vorliegender Untersuchung an den Abrufstationen schrotförmi-ges mit pelletiertem Futter unter dem Aspekt der Funktionssicherheit dieser Stationen verglichen.

Material und Methoden Der Fütterungsversuch mit 96 Mastschweinen wurde am LVFZ Schwarzenau bis zu einer angestrebten LM von ca. 120 kg durchgeführt. Für den Versuch wurden die Tiere der Rasse Pi x (DL x DE) nach LM, Abstammung und Geschlecht ausgewählt und gleichmäßig auf folgende vier Gruppen aufgeteilt: Behandlung A: Anfangsmast Schrot, Endmast Schrot



Behandlung B: Anfangsmast Schrot, Endmast Pellets Behandlung C: Anfangsmast Pellets, Endmast Schrot Behandlung D: Anfangsmast Pellets, Endmast Pellets Die Schweine wurden in 8 Buchten zu je 12 Tieren auf Betonspalten ohne Einstreu gehalten. Sie wa-ren zu Versuchsbeginn im Durchschnitt 74 Tage alt und wogen ca. 31 kg. Pro Behandlungsgruppe wurden 2 Buchten gemischtgeschlechtlich aufgestallt. Die Futterzuteilung erfolgte über Abrufstationen mit integrierter Futterverwiegung für das Einzeltier (Compident MLP, Schauer Agrotronic, GmbH). Die LM wurden alle 2 Wochen am Einzeltier erfasst. Bei Erreichen von ca. 120 kg LM wurden die Tiere nach den Vorgaben der Mastleistungsprüfung (D) an 3 Terminen im Versuchsschlachthaus Schwar-zenau geschlachtet. Die pelletierten Mastfutter wurden in der Versuchsmahl- und Mischanlage der Stoffwechselanlage Grub des Institutes für Tierernährung und Futterwirtschaft hergestellt und pelletiert. Die schrotförmi-gen Futter wurden in der Mahl- und Mischanlage des LVFZ Schwarzenau erstellt. Die Futteruntersu-chen wurden im Labor der Abteilung Qualitätssicherung und Untersuchungswesen der LfL in Grub (AQU 3) nach VDLUFA-Richtlinien durchgeführt (VDLUFA, 2012).

Ergebnisse und Diskussion Die Versuchsrationen (Tabelle 1) basierten auf Weizen, Gerste, und Sojaextraktionsschrot sowie einem handelsüblichen Mineralfutter. Die Versuchsfutter unterschieden sich nur hinsichtlich ihrer Konfektio-nierung und waren bei den wesentlichen Inhaltsstoffen vergleichbar.

Tabelle 1: Versuchsfutter, Zusammensetzung und analysierte Inhaltsstoffe (88 % T)

Anfangsmast (30-70 kg LM) Endmast (70-120 kg LM Konfektionierung Schrot Pellets Schrot Pellets Weizen % 40 40 45 45 Gerste % 39 39 39 39 Sojaextraktionsschrot % 18 18 13,5 13,5 Mineralfutter 1) % 3 3 2,5 2,5 ME MJ 13,33 13,37 13,56 13,44 Rohfett g 22 23 23 23 Rohfaser g 34 34 33 33 Rohprotein g 172 172 169 164 Lysin g 10,7 10,6 9,0 8,6 Calcium g 7,3 7,4 6,2 6,7 Phosphor g 4,8 4,7 4,3 4,5 1) 8 % Lysin; 1,5 % Methionin; 1,5 % Threonin

Die Gehalte an Lysin waren bei den pelletierten Futtermischungen etwas niedriger. Eine Ursache dafür könnte die Hitzeeinwirkung durch das Pelletieren sein. Die Konfektionierung des Futters zeigte keinen Einfluss auf den täglichen Futterabruf aus den Statio-nen. In der Anfangsmast wurden im Mittel zwischen 1,69 (Behandlung C) und 1,80 kg (Behandlung B) Futter abgerufen. In der Endmast waren es im Mittel zwischen 2,37 (Behandlung A) und 2,50 kg (Be-handlung B). Mit Werten zwischen 2,12 und 2,19 kg Futter pro Tier und Tag lag der Futterabruf in den einzelnen Behandlungen im Mittel der Mast eng beieinander. Die durchgängige Fütterung von Pellets führte gegenüber der durchgängigen Fütterung von Schrot zu knapp 50 g höheren täglichen Zunahmen. Bei Einsatz von Pellets in der Endmast (Gruppen B und D) wurden über den gesamten Versuchszeitraum mit 852 und 867 gegenüber 805 und 792 g bei den Gruppen A und C (Schrot in der Endmast) signifikant höhere tägliche Zunahmen festgestellt. Die Er-



gebnisse in der Anfangsmast waren zum Teil widersprüchlich. So ergaben sich in diesem Mastab-schnitt bei Schrot (Gruppe A und B) mit 765 und 835 g sowie bei Pellets (Gruppe C und D) mit 770 und 820 g täglichen Zunahmen signifikante Unterschiede bei gleicher Futterkonfektionierung. In der Endmast gab es zwischen Schrot (Gruppe A und C) mit 819 und 792 g sowie Pellets (Gruppe B und D) mit 878 und 865 g täglichen Zunahmen keine statistisch abzusichernden Unterschiede bei gleichem Futtertyp. Aufgrund der vergleichbaren Futteraufnahmen und der niedrigeren täglichen Zunahmen war im Mittel der Mast bei schrotförmigem Endmastfutter (Gruppen B und D) der Futteraufwand je kg Zuwachs mit 2,64 bzw. 2,68 kg Futter signifikant höher als bei pelletiertem Endmastfutter und Werten von 2,52 bzw. 2,49 kg. In der Anfangsmast zeigten sich mit einem Futteraufwand von 2,19 und 2,13 kg Futter je kg Zuwachs bei Fütterung von Pellets (Gruppe C und D) keine signifikanten Unterschiede. Mit 2,34 gegenüber 2,15 kg war der Futteraufwand bei Schrotfütterung (Gruppe A und B) jedoch statistisch abzusichern. Nach dem Futterwechsel in den Gruppen B und C war im 2. Mastabschnitt nur in Grup-pe C (Wechsel von Pellets auf Schrot) mit 3,09 kg der Futteraufwand gegenüber den anderen Ver-suchsgruppen signifikant höher.

Tabelle 2: Futterabruf und Aufzuchtleistungen (LSQ-Werte)

Gruppen A Schrot-Schrot

B Schrot-Pellets

C Pellets-Schrot

D Pellets-Pellets

Sign. p1)

Tiere/Ausfälle n 23/1 23/1 20/3 23/1 Masttage n 112 109 114 109 Lebendmasse Beginn Versuch kg 32,1 30,3 30,7 31,3 0,1603 Ende Anfangsmast kg 69,6 71,2 68,5 71,4 0,1973 Ende Versuch kg 122,0 124,8 121,5 123,9 0,1936 Tägliche Zunahmen Anfangsmast g 765a 835b 770a 820b 0,0047 Endmast g 819a 878b 792a 865b 0,0013 Gesamt g 805a 867b 792a 852b <0,0001 Futterabruf/Tier, Tag Anfangsmast kg 1,79 1,80 1,69 1,75 0,1646 Endmast kg 2,37 2,50 2,44 2,43 0,2671 Gesamt kg 2,12 2,19 2,12 2,13 0,4909 Futteraufwand (Futter/kg Zuwachs) Anfangsmast kg 2,34a 2,16b 2,19b 2,14b <0,0001 Endmast kg 2,89a 2,85a 3,08b 2,82a 0,0010 Gesamt kg 2,63a 2,53b 2,68a 2,50b 0,0002 1) Irrtumswahrscheinlichkeit

Die Schlachtleistungen waren gut und lagen im üblichen Rahmen für Mastschweine bayerischer Gene-tik. Das bezahlungsrelevante Kriterium Muskelfleischanteil (MFA) lag im Geschlechtermix zwischen 59,7 (Behandlung B) und 61,5 % (Behandlung A). Mit Ausnahme der Parameter Speckmaß und Fett-fläche sowie des Fleischanteils im Bauch ließen sich zwischen den Versuchsgruppen keine Unterschie-de statistisch absichern. Schrotförmiges Futter in Vor- und Endmast bzw. nur in der Endmast führte zu geringerer Fettausprägung (Speckmaß, Fettfläche) sowie einem höheren Fleischanteil bzw. weniger Fett im Bauch. Die Unterschiede ließen sich bei diesen Parametern zum Teil statistisch absichern.



Tabelle 3: Schlachtparameter nach LPA-Richtlinien (D) (LSQ-Werte)

Gruppen A Schrot-Schrot

B Schrot-Pellets

C Pellets-Schrot

D Pellets-Pellets

Sign. p1)

Schlachtgewicht kg 100,0 98,6 102,1 102,6 0,6645 Ausschlachtung % 82,2 79,1 83,9 82,6 0,4060 Fleischfläche cm² 62,7 62,8 60,4 62,2 0,5918 Fettfläche cm² 16,4a 19,5b 17,2a 18,2ab 0,0318 Fleisch-Fett-Verhältnis 1: 0,27 0,31 0,28 0,29 0,1219 Speckmaß mm 12,9a 15,3b 14,0ab 14,4b 0,0073 Fleischmaß mm 70,4 72,0 70,1 70,3 0,5790 Muskelfleischanteil % 61,5 59,7 60,5 60,2 0,0630 Fleischanteil im Bauch % 59,8a 56,1b 58,5ab 57,1b 0,0280 1) Irrtumswahrscheinlichkeit

Zusammenfassung und Schlussfolgerung Die Futterkonfektionierung (Pellets oder Schrot) hatte keinen Einfluss auf den täglichen Futterabruf bei Abrufstationen für Mastschweine (Schauer Compident MLP) mit Trogverwiegung. Die Funktionsfä-higkeit dieser Stationen ist somit auch bei Einsatz von geschrotetem Futter gewährleistet. Unter dem Aspekt der Schlachtkörperqualität (Verfettung) und der Tiergesundheit (Wintermann et al., 2010) ist Schrot als Futter von Vorteil. Stehen jedoch maximale Mastleistungen im Fokus, so ist bei dieser Technik dem pelletierten Futter der Vorzug zu geben.

Literatur Betscher, S.; Beinecke, A.; Kamphues, J. (2010): Effekte der Futterstruktur (Vermahlungsgrad/Konfektionierung ) auf immunologische Parameter – Sekretionsmuster der Muzine, Anzahl IgA-sezernierender Plasmazellen - in der Schleimhaut des Magen-Darm-Trakts junger Schweine. In: M. Gierus, H. Kluth, M. Bulang und H. Kluge (Hrsg.): 11. Tagung Schweine- und Geflügelernährung, 23.-25. November 2010 Lutherstadt Wittenberg, 118-123.

Cappai, M.G.; Koop, F.; Pistis, L.; Sander, S.J.; Dimauro, C.; Pinna, W.; Kamphues, J. (2015): Morphometric traits oft he Pey-er`s patch (as a aign of local immune response) in relation to different physical forms of one complete diet fed to growing pigs. Proc. Soc. Nutr. Physiol. (24) 28.

Kamphues, J. (2010): Zur Bedeutung der Struktur für Schweine: Nicht nur für die Gesundheit des Magen-Darm-Traktes von Interesse! In: M. Gierus, H. Kluth, M. Bulang und H. Kluge (Hrsg.): 11. Tagung Schweine- und Geflügelernährung, 23.-25. November 2010 Lutherstadt Wittenberg, 124-126.

VDLUFA-Methodenbuch Band III: Die Untersuchung von Futtermitteln 3. Aufl. 1976, 8. Ergänz.lief. 2012, VDLUFA-Verlag Darmstadt.

Wintermann, M.; Betscher, S.; Mößeler, A.; Schulze Langenhorst, C.; Stalljohann, G.; Beinecke, A.; Kamphues, J. (2010): Einfluss von Vermahlungsgrad (grob/fein), botanischer Zusammensetzung (Gerste/Weizen) und Angebotsform (trocken/flüssig) verschiedener Futtermittel auf die Magengesundheit von Schweinen. In: M. Gierus, H. Kluth, M. Bulang und H. Kluge (Hrsg.): 11. Tagung Schweine- und Geflügelernährung, 23.-25. November 2010 Lutherstadt Wittenberg, 127-129.

Autorenanschrift Dr. Wolfgang Preißinger Bayerische Landesanstalt für Landwirtschaft Institut für Tierernährung und Futterwirtschaft Dienstort Schwarzenau Stadtschwarzacher Str. 18 D-97359 Schwarzach a. Main E-Mail: Wolfgang.Preissinger(at)LfL.bayern.de

Bichl et al.: Zearalenone Adsorption to Lignocellulose Binders – Differences in vitro and in vivo


Zearalenone Adsorption to Lignocellulose Binders – Differences in vitro and in vivo

Gerlinde Bichl1, Heidi Schwartz-Zimmermann2, Sebastian Fruhauf1, Karl Schedle3, Sabine Masching4, Dian Schatzmayr1 and Franz Berthiller2 1 BIOMIN Research Center, Tulln, AT 2 BOKU Vienna, Dept. for Agrobiotechnology (IFA-Tulln), CD-Laboratory for Mycotoin Metabolism, Tulln, AT 3 BOKU Vienna, Dept. for Agrobiotechnology (IFA-Tulln), Institute for Animal Nutri- tion, Livestock Products, and Nutrition Physiology (TTE), Vienna, AT 4 BIOMIN Holding GmbH, Getzersdorf, AT

Abstract The mycotoxin zearalenone (ZEN) causes high losses in pig breeding and fattening due to its oestro-genic effects. Binding substances are often added to feed in order to reduce the bioavailability of ZEN during digestion. In the present work, lignocellulose and lignosulfonate binders were tested for their binding efficiency in vitro and in vivo. The in vitro binding assay showed adsorption rates over 85% at pH 3.0 and pH 6.5 (0.2% binder (w/v), 1 µg/ml ZEN). The substances were further tested in a feeding trial with nine pigs divided in three groups using a Latin square design. Animals received feed contam-inated with 0.15 mg/kg ZEN during the relevant periods. The feed of the treatment groups contained also 1 kg/t lignosulfonate or 2.5 kg/t lignocellulose. Urine samples were collected per pig and period. The creatinine content was determined for each sample. ZEN and its metabolites were measured after a methanol/acetonitrile precipitation of the urine using LC-MS/MS system. The measured concentra-tions were related to the creatinine content for better comparison. The measured ZEN biomarker in pigs was ZEN-glucuronide (ZEN-GlcAc) with 73.4 ± 22.5 ng/mg creatinine in the ZEN group. 83.2 ± 25.6 [ng/mg creat.] ZEN-GlcAc were measured in the treatment group with lignosulfonate and 79.7 ± 28.3 [ng/mg creat.] ZEN-GlcAc with lignocellulose. These results show no significant differences between the treatment groups by in vivo testing. Hence, lignocellulose binders cannot be used for effective adsorption of dietary ZEN during digestion in pigs.

Introduction Zearalenone (ZEN) is one of the most important mycotoxins. ZEN (Figure 4) is produced by several Fusarium species and is mostly found in cereals like maize and wheat. ZEN is known for its oestrogen-ic effects to mammals because it is able to bind to oestrogen receptors. The oestrogenic activity also

depends on the species specific binding affinity. The most vulnera-ble animal species are pigs. ZEN causes a significantly higher fre-quency of stillborn piglets, reduced litter size and fetal weight. ZEN is rapidly absorbed from the gastrointestinal tract in mammals after oral intake. The bioavailability reaches up to 85% of the in-gested dose (Fink-Gremmels and Malekinejad, 2007). ZEN has several closely related metabolites. Some metabolites, like alpha-, beta-zearalenol (α-, β-ZEL) are formed during metabolism in plants, animals and humans through a reduction of the keto-

Figure 4: Chemical structure of zearalenone



group at position 7. Conjugation with glucuronic acid (ZEN-GlcAc) in animals is also possible during metabolism. New detoxification strategies and binders are created regularly. The literature for in-stance describes in vitro mycotoxin binding to lignocellulose based products like peach stones or grape pomace (Lopičić et al., 2013; Avantaggiato et al., 2014). In the present work, two lignocellulose bind-ers were tested in vitro and in vivo to evaluate the efficiency of the binding of dietary ZEN during the digestion in pigs. The measurement of ZEN and its formed metabolites in pig urine can determine the amount of bioavailable ZEN in feed during the in vivo trial.

Material and methods In vitro adsorption tests at different pH values The two binders lignosulfonate and lignocellulose were tested in vitro with 0.2% binder (w/v) and 1 µg/ml ZEN at pH 3.0 and 6.5. The tests were carried out as described in Fruhauf et al., 2012. Feeding trial This feeding trial was carried out as a modified triplicate 3 x 3 Latin square design on the University of Natural Resources and Life Sciences, Vienna. Animals were allotted randomly according to body weight (BW) and litter (pigs from 3 different litters were used each time) to individual metabolic cages (Ehret, Tulln, Austria). In the feeding trial, nine male castrated pigs (30-70 kg body weight) were kept in metabolic cages and received feed contaminated with 0.15 mg/kg ZEN. The pigs were divided in three groups of three pigs each and two groups received feed additives mixed into the feed. Product one contained 1 kg lignosulfonate per ton of feed and product two contained 2.5 kg lignocellulose per ton. The three groups were rotated over the duration of the trial with a break between the periods, as shown in Table 4. The feed was composed of maize, barley, soy beans, amino acids, vitamins and micro nutrients to reach the nutrient requirements of the GfE (2006).

Table 4: Application plan for feeding trial of ZEN and feed additives lignosulfonate and lignocellulose

Time Applied feed pig 1 pig 2 pig 3 pig 4 pig 5 pig 6 pig 7 pig 8 pig 9 day ≤ 1 Feed without added products day 2-7 ZEN ZEN + lignosulfonate ZEN + lignocellulose day 8-14 Feed without added products day 15-21 ZEN + lignocellulose ZEN ZEN + lignosulfonate day 22-28 Feed without added products day 29-35 ZEN + lignosulfonate ZEN + lignocellulose ZEN

The urine was collected separately from each pig, but the samples from within each week were pooled. Urine sample preparation In order to normalize the excreted volume of urine, the concentration of creatinine in urine samples was measured. Urine was diluted with water by a factor of 5000 in two steps. The diluted solution was measured by LC-MS/MS and quantification was performed using external calibration between 6 and 600 ng/mL creatinine. Samples collected in the treatment weeks were worked up in duplicate. Urine samples containing more than 1100 mg/L creatinine were diluted to 1000 mg/L creatinine before measurement of ZEN metabolites. 200 μL of the prepared urine (about or below 1000 mg/L creatinine) was transferred into 1.5 mL Ep-pendorf reaction tubes. Then, 600 μL of methanol (MeOH)/acetonitrile (50/50; v/v) was added and the solution was shaken for 30 min. After centrifugation, the supernatants were transferred into a new



Eppendorf reaction tube and the residues were re-extracted with 200 μL MeOH/water (80/20; v/v). The extract was vortexed and centrifuged before it was added to the supernatants. The combined supernatants were evaporated to dryness using a gentle stream of pressurized air and taken up in 200 μL of MeOH/water (30/70; v/v). After vortexing and centrifugation the solution was transferred into an HPLC microvial. For quantification of ZEN and ZEN-GlcAc, external calibration from 1 – 300 µg/L was used. The calcu-lated concentrations in urine (µg/L) were divided by the concentration of creatinine (mg/L) and multi-plied by 1000 in order to obtain concentrations in ng/mg creatinine. Measurement methods for urine samples The samples were measured on a 1290 Agilent HPLC system coupled to a 4000 QTrap mass spec-trometer from Sciex. For the determination of creatinine in urine a Gemini® C18 column (150 x 4.6 mm, 5 μm, Phenomenex) was used. The mobile phases were MeOH/H2O/acetic acid (HAc) (40/59.9/0.1; v/v) and MeOH/HAc (99.9/0.1; v/v) with a flow rate of 800 μL/min and gradient elution. The injection volume was 2 μL, the column oven temperature 30 °C and the total run time was 4.8 min. The MS was operated in MRM mode after positive electrospray ionisation. The LC eluent was transferred into the ion source between 1.2 – 1.7 min. The monitored MRM transitions are shown in the bottom of Table 5. For the determination of ZEN and its metabolites, a Kinetex® C18 column (150 x 2.1 mm, 2.6 μm, Phenomenex) was used. The gradient elution was performed with mobile phase MeOH/H2O (20/80; v/v) and MeOH/H2O (98/2; v/v), both containing 5 mM ammonium acetate, at a flow rate of 250 μL/min. The injection volume was 5 μL, the column oven was set to 30 °C and the total run time was 14 min. The mass spectrometer was operated in MRM mode with negative polarity and the col-umn effluent was directed to the MS between 2 – 9.5 min. A selection of monitored mass transitions is shown in Table 5.

Table 5: Selection of MRM transition data for measurement of ZEN metabolites and creatinine

Q1 Mass (Da)

Q3 Mass (Da)

Time (msec)

ID Declustering potential (V)

Collision energy (V)

495.1 319.0 20 ZEL-GlcAc quant -80 -38 495.1 112.8 20 ZEL-GlcAc qual -80 -28 493.1 317.0 20 ZEN-GlcAc quant -85 -36 493.1 175.0 20 ZEN-GlcAc qual -85 -26 319.1 275.1 20 ZEL quant -95 -30 319.1 160.0 20 ZEL qual -95 -42 317.1 131.0 20 ZEN quant -90 -42 317.1 175.0 20 ZEN qual -90 -34 114.0 86.0 30 creatinine quant 20 15 114.0 44.2 30 creatinine qual 20 15

Results and discussion The in vitro adsorption showed more than 85 % binding for ZEN to lignosulfonate or lignocellu-lose (0.2% binder (w/v), 1 µg/ml ZEN) at pH 3.0 and pH 6.5. Because of these encouraging data, the substances were tested in a feeding trial. The in vivo testing showed different results. In general, the most important ZEN biomarkers in the analysis of pig urine are ZEN-GlcAc, ZEN, α-ZEL-GlcAc and α-ZEL. The results from the feeding trial are shown in Table 6. ZEN-GlcAc was the major metabolite. ZEN and α-ZEL were not detected. Traces of α-ZEL-GlcAc were detected, but quantification was hampered by co-eluting matrix. The measured concentrations were summarized for the individual sample treatment groups. The averages of the



treatment groups show no significant differences to each other with concentrations between 73.4 – 83.2 ng ZEN-GlcAc/mg creatinine. The differences between the pigs of one group were more pro-nounced than those originating from the addition of lignocellulose binders. Therefore, the tested lig-nosulfonate and lignocellulose didnot cause any changes in ZEN adsorption and metabolism in pigs.

Table 6: Measured concentrations of ZEN metabolites in pig urine [ng/mg creatinine]

ZEN-GlcAc [ng/mg creatinine] Time pig 1 pig 2 pig 3 pig 4 pig 5 pig 6 pig 7 pig 8 pig 9 day 1 0 0 0 0 0 0 0 0 0 day 2-7 71.7 113 99.1 59.1 68.3 82.2 45.7 117 88.3 day 14 0 0 0 0 0 0 0 0 0 day 15-21 63.4 108 118 57.9 77.2 65.5 70 111 67 day 28 0 0 0 0 0 0 0 0 0 day 29-35 55.5 122 113 57.8 60.6 59.2 51.7 42.3 82.3 group average STDEV ZEN 73.4 22.5 ZEN +lignosulfonate 83.2 25.6 ZEN +lignocellulose 79.7 28.3

Conclusion The efficiency of lignocellulose in vitro binding has been published several times. Also in the present work, the in vitro adsorption tests showed high binding of ZEN to lignocellulose and lignosulfonate materials. However, the analysis of ZEN biomarkers in pig urine indicates no differences of ZEN me-tabolism with and without lignocellulose binders. The addition of lignosulfonate or lignocellulose to pig feed with ZEN showed no reduced amount of bioavailable ZEN. Because of this reason lignosulfonate and lignocellulose cannot be used effectively for adsorption of dietary ZEN during digestion in pigs. Furthermore, this study highlights the necessity of proper in vivo studies before application of feed additives.

References Avantaggiato, G., Greco, D., Damascelli, A., Solfrizzo, M., Visconti, A. 2014. Assessment of Multi-mycotoxin Adsorption Efficacy of Grape Pomace. Journal of Agricultural and Food Chemistry 62: 497-507.

Bichl, G. 2014. Zearalenone – Development of an HPLC-MS/MS Method for the Determination of Zearalenone Biomarkers. FH Wiener Neustadt Campus Tulln.

Fink-Gremmels, J., Malekinejad, H. 2007. Clinical effects and biochemical mechanisms associated with exposure to the mycoestrogen zearalenone. Animal Feed Science and Technology 137: 326-341.

Fruhauf, S., Schwartz, H., Ottner, F., Krska, R., Vekiru, E. 2012. Yeast cell based feed additives: studies on aflatoxin B1 and zearalenone. Food Additives and Contaminants 29: 217-231.

Lopičić, Z. R., Bočarov-Stančić, A. S., Stojanović, M. D., Milojković, J. V., Pantić, V. R., Adamović, M. J. 2013. In vitro evaluation of the efficacy of peach stones as mycotoxin binders. Zbornik Matice Srpske za Prirodne Nauke 124: 287-296.

Corresponding author Gerlinde Bichl MSc BIOMIN Resarch Center Technopark 1 3430 Tulln, Austria E-mail: gerlinde.bichl(at)biomin.net

Morton et al.: Analysis of the effectiveness of fibre analysis techniques at the determination of the properties of lignocellulose sources and their corresponding effects on broiler production characteristics


Analysis of the effectiveness of fibre analysis techniques at the determination of the properties of lignocellulose sources and their corresponding effects on broiler production characteristics

Jeffrey Morton1, Arthur Kroismayr1 and Nikola Puvača2

1 Agromed Austria GmbH, AT 2 Patent co., RS

Abstract The effectiveness of current chemical analysis techniques were evaluated by comparing the effect of two Lignocellulose sources, which according to chemical analysis have similar composition, on produc-tion characteristics in Ross 308 strain Broilers. A total of 888 birds were equally divided into three treatments, treatments T1, T2, and T3, were all fed a standard broiler diet with an addition of 0.8% LC product A, 0.8% LC product B, and 0.4% LC product B respectively. Weight gain was shown to be higher in T2 and T3 when compared to T1 (p<0.05). Treatment T3 had a higher feed consumption during week 3 followed by a lower intake in week 4 (p<0.05). Treatment T2 showed a lower feed conversion ratio then T3, T1 had no difference to treatments T2 and T3 during week 3 (p<0.05). No significant differences were noted in litter moisture content across treatments (p>0.05). These find-ings indicate that the chemical analysis used to determine their content was not adequate at deter-mining the properties of the two products, and thus ineffective as a basis for dietary recommendation.

Introduction Dietary fibre content in poultry nutrition has become an important topic for nutritionists and produc-ers. With many studies showing the benefits of including fibre sources in both layer and broiler diets (Roberts et al., 2007; Jimeénez-Moreno et al., 2015). As this idea, that fibre is essential in poultry diets gets more established, the question has become “what is the right amount of fibre?” While cur-rent chemical analysis (TDF, Van Stoet) are better than in the past (Weender), they still do not accu-rately evaluate the qualities of a fibre source. The present study was conducted to evaluate such inaccuracies, by evaluating differences in perfor-mance in broilers fed one of two fibre sources that are known to have a positive effect on production characteristics in broilers (Liu et al., 2009). Both products are lignocellulose based, and are according to chemical analysis, are very similar. A chemical analysis with regards to fibre content can be seen in table 1.

Materials and methods For the trial, 888 day-old Ross 308 strain broilers were equally divided into 3 treatments with 8 repli-cates each. The groups were evenly distributed in the barn, and microclimate was carefully monitored to ensure that barn position did not create false results. The dietary treatments consisted of a com-mercially available broiler diet with the fibre treatment added “on top” for the starter and grower phases, but left out during the finisher phase. The treatments were as follows: T1= base diet + Lig-nocellulose (LC) A 0.8%, T2= base diet + LC B 0.8%, T3 base diet + LC B 0.4%. The composition of the three phase base diet can be seen in Table 2. During the duration of the trial, feed and water were provided ad libitum.



Table 1: Chemical analysis of the fibre products

LC A* LC B+ % Difference XF (%) 67.6 59.6 11.8 ADF (%) 70.8 69.3 2.1 ADL (%) 23.9 26.9 11.1 NDF (%) 84.8 82.5 2.7 * (Sancho, 2013), +(Agrolab group, 2016)

Table 2: Composition of the three phase base diet

Nutrients, % of as-fed basis Diet mix+

Starter Grower Finisher Dry matter 89.6 89.2 88.9

Moisture 10.4 10.8 11.1 Crude protein 22.0 19.0 17.0 Crude fat 5.1 5.1 5.6 Crude fibre 3.5 3.2 3.6 Crude ash 6.3 6.0 5.5 Ca 1.0 1.0 0.9 P 0.8 0.8 0.7 Metabolizeable Energy, MJ/kg 12.5 12.8 13.0

Feed consumption, body weight and litter quality for each treatment, were measured at the end of each week for the duration of the trial. Liter quality was determined by analyzing moisture content, determined by drying at 105oC for 24 hours until a constant weight was achieved. Statistical analysis of results was conducted using the statistical software Statistica 12 for Windows (StatSoft, Inc., Tulsa, OK, U.S.A.). Fisher’s l.s.d. post-hoc multiple range test was used to determine any differences between treatments. For all analyses a p value of .05 was used.

Results and discussion Body weight was measured at the end of each week and the average from each group was deter-mined this data is listed in table 3. It can be noted that there are significant differences between all three trial treatments from the end of the first week on, except at the final weighing, where T2 and T3 were not significantly different (p>0.05). It can be noted that the heaviest chickens were in T2 in every weighing after the start of the trial, achieving a final weight of (2203.90g). From the table, av-erage daily gain (ADG) can be calculated as 48.86, 51.63, and 50.68 for T1, T2, and T3 respectively. It is particularly interesting to note in the context of this study, that T3 generally out performed T1 in weight gain, despite the lower inclusion rate, and thus lower levels of all fibre types. It was however, apparently not the lower levels of fibre that caused this effect as T2 out performed T3 in all but the final weighing. Feed consumption was carefully monitored throughout the trial, these results are shown in table 4. It is interesting to note that the only significant differences (p<0.05) were measured at the end of weeks three and four, showing that T3 had a different feed consumption than T1 and T2. It can be noted however, that during week three T3 ate much more than the other groups but during the fol-lowing week, T3 ate significantly less. The lack of difference between T1 and T2, shows that the birds in T1 and T2 ingested roughly the same amount of feed, and thus ingested roughly the same amount of fibre according to the chemical analysis, and yet they still ended up with very different growth rates.



Table 3: Average chicken body weight measured weekly, g

Age T1: LC A 0.8% T2: LC B 0.8% T3: LC B 0.4% Day 0 35.52a 2.82 35.38a 2.66 34.97a± 2.77 Day 7 133.66c 6.18 140.40a 9.61 135.35b± 8.19 Day 14 335.93c 22.42 352.68a 18.44 341.85b± 23.30 Day 21 703.12c 61.99 754.28a 46.60 728.14b± 54.90 Day 28 1169.00c 117.79 1221.80a 107.89 1195.62b± 82.87 Day 35 1558.26c 282.82 1659.34a 274.92 1608.63b± 273.70 Day 42 2088.05b 340.75 2203.90a 333.42 2163.79a± 331.78 * Values with different superscripts in the same row, have significant difference (p<0.05)

Table 4: Average feed consumption per bird, g

Age T1: LC A 0.8% T2: LC B 0.8% T3: LC B 0.4% Day 7 162.71a ±29.50 163.57a ± 30.51 172.21a ± 27.56 Day 12 284.11a ±17.03 292.33a ± 10.16 288.85a ± 13.55 Day 21 535.79b ± 20.51 537.76b ± 62.77 596.26a ± 59.39 Day 28 740.61a ± 45.43 791.13a ± 48.59 736.97b ± 49.63 Day 35 932.75a ± 55.77 995.96a ± 74.75 927.71a ± 71.94 Day 42 702.99a ± 285.45 734.01a ± 259.54 749.01a ± 300.86 * Values with different superscripts in the same row, have significant difference (p<0.05)

Table 5: Feed conversion ratio

Age T1: LC A 0.8% T2: LC B 0.8% T3: LC B 0.4% Day 7 1.21a ± 0.22 1.16a ± 0.21 1.27a ± 0.22 Day 12 1.33a ± 0.08 1.29a ± 0.09 1.34a ± 0.09 Day 21 1.40ab ± 0.03 1.31b ± 0.10 1.46a ± 0.11 Day 28 1.50a ± 0.12 1.46a ± 0.11 1.49a ± 0.06 Day 35 1.70a ± 0.11 1.66a ± 0.09 1.68a ± 0.04 Day 42 1.62a ± 0.17 1.58a ± 0.16 1.62a ± 0.14 * Values with different superscripts in the same row, have significant difference (p<0.05)

Despite the significantly large differences in growth, a significant difference in feed conversion ratio (FCR) only occurred at the end of the third week, and only showed that T3 had a higher FCR than T2 1.46 and 1.31 respectively (Table 5). Numerically speaking however, T2 was shown to have the low-est FCR at each measuring throughout the trial. The lack of significant results in FCR can likely be explained by the trend towards higher feed consumption in correlation with the average growth rate of each group. The highest mortality rate in the trial occurred in T3 (3.7%), significantly higher than T2 (1%) (p<0.05), but not significantly different from T1 (1.7%). However, none of the groups fell outside of the normal range for mortality (National Chicken Council, 2015). The European broiler index (EBI) for each group were 304.6, 330.6, and 307.4 for treatments T1, T2, and T3 respectively. No significant difference was seen between the treatments in EBI; though, as might be expected based on the re-sults presented above, there was a noticeable trend towards a higher EBI in T2. Litter moisture was measured weekly this data is presented in Figure 1. The results for moisture con-tent are very similar with each trial treatment showing a spike in litter moisture at some point in the trial. The spikes are likely associated with a diet change, as they were noted 1 week after a dietary change in all treatments. The similarities are not surprising as both products are advertised as having a positive effect on litter quality.

Unbenannt-1 1 21.03.16 11:28

Morton et al.: AGROMED


0

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Day 0 Day7 Day 14 Day21 Day28 Day35 Day 42

T1: LC A 0.8% T2: LC B 0.8% T3: LC B 0.4%

Figure 1: Litter Moisture Content

Conclusion Based on the obtained results, it can be determined that both of these LC fibre concentrates, despite the similar values from the chemical analysis, had very different effects on effects on chicken perfor-mance. This is particularly interesting when considering their small inclusion rate and the overall effect their differences had on diet composition. This trial showed that current techniques could not accu-rately determine the differences between two different LC sources. It can by extension be determined that the chemical analysis used to evaluate fibre types is not an accurate way of determining fibre quality in general. The authors thus, propose that research should be conducted towards the devel-opment of a more accurate method of analysis so that appropriate inclusion rates may be recom-mended to producers.

References Agrolab Group (2016) Report 1778129 – 697003, 697004

Jiménez-Moreno, E., de Coca-Sinova, A., González-Alvarado, J. M., Mateos G. G. (2016): Inclusion of insoluble fiber sources in mash or pellet diets for young broilers. 1. Effects on growth performance and water intake. Poultry Science 95, 41-52

Liu, J., Trautwein, J., Pietsch, M., Dusel, G. (2009) Einfluss von Lignocellulose in der Broilerfütterung. 8th BOKU-Symposium Tierenährung

National Chicken Council (2015): U.S. Broiler Performance. http://www.nationalchickencouncil.org/about-the-industry/statistics/u-s-broiler-performance/

Roberts, S.A., Xin, H., Kerr, B.J., Russell, J.R., Bregendahl, K. (2007) Effect of Dietary Fiber and Reduced Crude Protein on Nitrogen Balance and Egg Production in Laying Hens. Poultry Science 86: 1716-1725

Sancho R. C. (2013): Estudio Para La Mejora Del Rendimiento De La Canal En Conejos De Engorde En El Momento Del Sacrificio. Masters Thesis: Universidad Zaragova

Corresponding author Jeffrey Morton

Agromed Austria GmbH E-mail: morton(at)agromed.at

Chrastinová et al.: The effects of inorganic and organic zinc dietary supplementation on performance of rabbits and meat mineral elements content


The effects of inorganic and organic zinc dietary supplementa-tion on performance of rabbits and meat mineral elements con-tent

Ľubica Chrastinová1, Klaudia Čobanová2, Mária Chrenková1, Mária Polačiková1, Zuzana Formelová1, Andrea Lauková2, Ondrej Bučko3, Ľubomíra Grešáková2, Matúš Rajský1 and Ľubomír Ondruška1

1 National Agricultural and Food Centre, Research Institute of Animal Production, Ni- tra, RS 2 Institute of Animal Physiology Slovak Academy of Sciences, Košice, RS 3 Slovak University of Agriculture in Nitra, RS

Abstract The effects of the feed administered zinc from inorganic or organic sources on selected performance parameters of rabbits and mineral elements content in meat were the priority of this study. A total of 96 broiler rabbits 49th day of age (both male and female) were divided into 4 groups (control C and 3 experimental groups – 1EG, 2EG and 3EG) with 24 animals in each group (replicated 6 x 2 x 2). The rabbits were fed with complete pelleted diet adlibitum and had free access to drinking water during the experiment. The feed mixture was additionally administered: the dose 27.47 g ZnSO4.H2O in 1st experimental group, the dose 38.46 g of Glycinoplex-Znin 2nd group (2EG), and the dose 66.67 g Bioplex Zinc in 3rd group (3 EG); each per 100 kg. The fattening experiment lasted 48 days. The ex-perimental results show that: dietary supplementation of rabbit with zinc was carried out to determine its effect on growth of live weight and consumption of feed per unit of live weight growth.The in-creased bioavailability of microelements, when presented in inorganic (ZnSO4.H2O) form, may be due to improved absorption of trace elements in muscle tissues. On the other hand, feed mixtures with high zinc supplementation in organic form, resulted in decreased levels of zinc in muscle tissues from all monitored elements (P, Na, K, Fe, Zn).Our results suggest a bioequivalence of zinc chelates and Zn sulphate as efficacious source of zinc in nutrition of rabbits.

Introduction Zinc is recognized as an essential trace element in human and animal nutrition. The role of micro-minerals in health cannot be over emphasized; zinc has been a modifier of wide spectrum of biological

activities. Its deficiency has been related to various dysfunctions and alterations of normal cell metabolism. Brandt (2009) says that zinc ions are often coordinated to the amino acid side chains of aspartic acid, glutamic acid, cysteine and histidine in proteins. The theoretical and computational description of this zinc binding in proteins (as well as that of other transition metals) is difficult. Relations between min-eral elements in the organisms are shown in the following figure. In this study, supplementation of rabbits with zinc salt was conducted to determine its effects on reproductive performance and growth rate following improvement in the quality and quantity of non-traditional meat as a source of protein for the consumers (Under-wood, 1977; Alikwe et al. 2011). The role of zinc in the animal or-



ganism has begun to gain special attention. Zinc participates actively in protein synthesis and carbo-hydrate metabolism. The aim of this study was to reveal the effects of orally administered zinc from inorganic or organic sources on growth performance in rabbits and selected parameters of meat quali-ty.

Material and method Experiment was realized on 96 broiler rabbits, line M91 (49 days old, both male and female) divided into 4 groups (control group - CG and 3 experimental groups - 1EG, 2EG, 3EG) with 24 animals in each group (replicated 6 x 2 x 2). Rabbits were kept in standard cages, two animals per cage. They were fed with complete pelleted diet (pellets of 3.5 mm in diameter) adlibitum and had free access to drinking water from nipple drinkers during the experiment. The fattening experiment lasted 48 days. The basal diets were composed of: dehydrated lucerne meal 36 %, extracted sunflower meal 5.5 %, extracted rapeseed meal 5.5 %, wheat bran 9 %, oats 13 %, malt sprouts 15 %, DDGS (dried distillers grains with soluble) 5 %, barley grains 8 %, sodium chloride, mineral and vitamin mixture, limestone 1 %. The diet did not contain any anticoccidial drug. The rabbits in group C were fed with the unsupplemented basal diet with no zinc additive. The feed mixture was additionally administered: in 1stexperimental group by dose 27.47 g ZnSO4.H2O (Zinc sulphate monohydrate),in 2ndgroup (2EG) by dose 38.46 g of Glycinoplex-Znand in 3rd group (3 EG) dose 66.67 g Bioplex -Zn, each per 100 kg. The chemical composition of all feeds was determined by Weende (AOAC, 1995). Dietary supplemen-tation of rabbit with zinc was carried out to determine its effect on growth of live weight and con-sumption of feed per unit of live weight growth. Rabbit’s body weight and feed consumption were measured every week of the experiment. Mortality and morbidity were also daily recorded in groups, over the entire period of the experiment. In the morning on 91st day of age (6 weeks after all experi-mental procedures) 6 animals from each group were electrically stunned and killed by cutting the carotid artery and jugulars then the carcasses were refrigerated for 24 h at 4° C. The samples of Mus-culus longissimus dorsi (MLD) (50 g) were used to study parameters characterizing the content of nutrients (content of water, proteins, fat). They were estimated using an INFRATEC 1265 (Germany) spectroscope and expressed in g.100 g-1 original matter. The amino acids composition after acid hy-drolysis by 6M HCl and sulfuric amino acids after oxidation hydrolysis were analysed by ion-exchange chromatography on the AAA400 (Ingos Prague, Czech Republic). For macro and micro element analy-sis were samples (2 g ) ashed at 550 °C, the ash was dissolved in 10 ml of HCl (1:3) and minerals were determined with the atomic absorption spectrometry (AAS) method, phosphorus content was determined with molybdovanadate reagent on Camspec M501 (Spectronic Ltd, UK). Mineralized sam-ples were analysed for Ca, Mg, Na, K, Fe, Zn, Cu and Mn content. The spectrometer AAS iCE 3000 (Thermo, UK) was used for mineral content determination. Mineral nutrients content in feeds and meat were estimated in graphite corvette through electro thermal atomization. Ca content was esti-mated at the wave length of 422.7 nm, Mg at 285.2 nm, Na at 589.0 nm, K at 766.5 nm, Fe at 248.3 nm, Zn at 213.9 nm, Cu at 324.8 nm, Mn at 279.5 nm and content of P at 410.0 nm as phosphomo-lybdenic yellow (Official Journal L 206, 29/07/1978, p.0043-0055). All these analyses were performed in triplicates. Results were evaluated by statistical method such as: significance of differences, analysis of variance, one-way ANOVA and t-test which were performed at P level (less than 0.05). The aim of this study was to reveal the effects of orally administered zinc from inorganic or organic sources on selected parameters of meat quality.

Results and discussion The trial was carried out from July to August 2014 in the experimental house of the National Agricul-tural and Food Centre - Research Institute for Animal Production Nitra, Slovak Republic. Each experi-mental procedure, which involves animals, was approved by the State Veterinary and Food Institute of



the Slovak Republic. For the subsequent 6 weeks, the rabbits were fed the identical basal diet (BD) supplemented with equivalent dose of Zn (100 mg.kg-1) from various sources. Diets for experimental group 1, 2 and 3 consisted of same BD supplemented with Zn in the form of Zn sulphate, Zn chelate of glycine hydrate (Glycinoplex-Zn) and Zn chelate of protein hydrolysate (Bioplex- Zn), respectively. The complete mixture contained: crude protein 173 –176 g, fat 36 – 37 g, fibre 145 – 158 g, N-Free Extract 461 – 472 g, organic matter 831 – 832 g, Ca 7.3 – 7.6 g, P 6.9 – 7 g, Mg 2.6 – 2.7 g, Na 1.4 – 1.6 g, K 10.8 – 11.7 g, Fe 343 – 457 mg, Zn 126.4 mg, Cu 20.3 – 22.7 mg, Mn 165 mg and 10.9 – 11.4 MJ.kg-1per kg original mater.

Table 1: Performance of rabbits in response to dietary supplementation with zinc from inorganic or organic sources (mean ± SD)

Characteristics (n = 24) Control-C 1EG - with ZnSO4.H2O

2EG - with Glycino-plex- Zn

3EG -with Bioplex- Zn

Initial weight g 1637 ± 119 1633 ± 33 1663 ± 183 1638 ± 93 Final weight g 2971 ± 160 3004 ± 229 3049 ± 207 2954 ± 189 Feed intake g.day-1 133.93 132.66 130 132.83 Feed conversion ratio g.g-1 4.23 4.08 4.20 4.26 Carcass yield % 59.24 ± 0.78 59.41 ± 1.59 60.12 ± 0.45 58.37 ± 3.37

Total process of rabbit’s growth rate, averaged daily feed intake, feed intake and feed efficiency is shown in table 1. Among the experimental groups no significant difference was noted in feed intake, feed conversion ratio and carcass value in the fattening experiment. During the experiment, the ani-mals did not show any health problems. Results of selected meat quality parameters (content of wa-ter, content of proteins, fat and amino acids and mineral composition) are presented in table 2.

Table 2: The effect of dietary zinc supplementation on selected chemical characteristics of MLD muscles 24 h post mortem (mean ± SD)

Characteristics (n = 6) Control-C 1EG - with ZnSO4.H2O

2EG - with Glycinoplex- Zn

3EG -with Bioplex- Zn

Water g.100.g-1 74.61 ± 0.44 74.89 ± 0.19 74.74 ± 0.55 74.64 ± 0.44 Protein g.100.g-1 23.49 ± 0.43 23.31 ± 0.19 23.51 ± 0.27 23.67±0.57 Proportional es-sential AA

% 52.37 52.41 52.37 52.38 Proportional non essential AA

% 47.63 47.59 47.63 47.62

Ash 0.9± 0.01 1± 0.01 1± 0.01 1± 0.02 Fat g.100.g-1 0.92 ± 0.23 1.04 ± 0.13 0.89 ± 0.12 0.91± 0.21 Energetic value kJ.100g-1 428.15 ± 5.68 429.65 ± 4.55 427.30 ± 8.82 430.79 ± 8.69

Mineral composition of the raw muscles MLD (mean± SD) Calcium g.1000.g-1 0.072 ± 0.01 0.111± 0.03a 0.131 ± 0.05a 0.151 ± 0.04A Phosphorus g.1000.g-1 2.270 ± 0.06 2.228 ± 0.21 1.901 ± 0.04a 1.895 ± 0.16a Magnesium g.1000.g-1 0.216 ± 0.01 0.246± 0.04 0.255 ± 0.01 0.273 ± 0.01 Sodium g.1000.g-1 0.330 ± 0.02 0.354± 0.05 0.320 ± 0.05 0.338 ± 0.03 Potassium g.1000.g-1 3.671 ± 0.24 3.786± 0.14 3.547 ± 0.05 3.571 ± 0.19 Iron mg.1000.g-1 7.4062 ± 1.12 9.333± 2.53a 4.674 ± 0.91 4.765 ± 0.84 Zinc mg.1000.g-1 30.712 ± 3.00 38.375± 6.45a 26.367 ± 3.83 26.282 ± 3.45 Copper mg.1000.g-1 1.325 ± 1.13 2.154 ± 0.84a 3.979 ± 0.11A 4.219 ± 0.26A Manganese mg.1000.g-1 0.395 ± 0.08 0.639 ± 0.32A 0.670 ± 0.12A 0.433 ± 0.05a Within a row, means with the same superscript letters are different: a (p ≤ 0.05); A (p ≤ 0.01);

No differences in zinc bioavailability between inorganicand organic sources have been reported in rabbits (Underwood, 1977; Guimaraes and Motta, 2000; De Blas and Wiseman, 2010). No significant difference in Zn levels was observed in kidney cortex and muscle tissue between the control and the



group of animals fed supplemented diets with Glycinoplex-Zn and with Bioplex-Zn. The Zn level in-creased (P ˃ 0.05) in animals fed with supplemented diets of zinc sulphate monohydrate. A statistically significant difference (P ≤ 0.01) was obtained in the case of copper content, where the experimental group ranged between 3.979 mg.kg-1 and 4.219 mg.kg-1 and the control group was on the level 1.325 mg.kg-1 muscle tissues. A statistically significant difference was found out in the case of higher manganese content in experimental groups, which ranged from 0.639 mg.kg-1 to 0.670 mg.kg-1 muscle tissues (P ˃ 0.01) compared with the control group (0.395 mg.kg-1). Because of zinc’s environmental impact, the maximum level allowed in the EU for rabbit feeds is 150 mg.kg-1. In addi-tion, the adverse effect of high zinc intake on copper availability has to be considered, results of re-search of Maret and Sandstead on Zinc requirements and the risks and benefits of zinc supplementation (2006) supports our findings. It is necessary to continue the observations and to test also distinct levels of supplements.

Conclusion The experimental results show that:

Dietary supplementation of rabbits with zinc was carried out to determine its effects on growth of live weight and consumption of feed per unit of live weight growth.

Average values of Ca, Mg in MLD muscle of experimental groups were higher than in control group.

On the other hand, feed mixtures with high zinc supplementation in organic form, resulted decreasing level of zinc in muscle tissue from all monitored elements (P, Na, K, Fe, Zn).

Our results suggest bioequivalence of zinc chelates and Zn sulphate as efficacious source of zinc in nutrition of rabbits.

Acknowledgement The work was supported by the project of APVV – 0667-12 “Zinc in animal nutrition and consumer safety“.

References ALIKWE, P. C. N. - OJIEZEH, T. I. - OLAGBOYE, S. A. 2011. Effects of zinc supplement on rabbits performance and growth rate In: Journal of Agriculture and Social Research (JASR) Vol. 11, No. 2, p. 46-50.

AOAC 1995. Official Methods of Analysis. Washington, D.C.: Association of Official Analytical Chemists,1018 p.

BRANDT, E.G.-HELLGREN,N.- BRINCK, T.- BERGMAN, T.- EDHOLM, O. 2009. Molecular dynamics study of zinc binding to cysteines in a peptide mimic of the alcohol dehydrogenase structural zinc site. Phys. Chem. (PCCP), 2009, 11 (6): 975-983.

De BLAS, C. - WISEMAN, J. 2010. Nutrition of the Rabbit. 2nd Ed. Cabi, www. CABI org., 2010,325 p. ISBN 978-1-84593-669-3.

GUIMARAES, C. S. - MOTTA, F. W. 2000. Bioavailability of dietary zinc sources for fatteningrabbits. In: Blasco, A. (ed.) Proceedings of 7th World Rabbit Congress, Valencia University Publications, Valencia, Spain, 2000, p. 255–261.

MARET, W. - SANDSTEAD, H. H. 2006. Zinc requirements and the risks and benefits of zinc supplementation. Journal of Trace Elements in Medicine and Biology, vol. 20, 2006, p. 3–18.

UNDERWOOD. E. I. 1977. Trace Elements in Animaland Human Nutrition (4th Ed.) AcademicPress. London. p.196-242.

Corresponding author Mária Chrenková NAFC - Research Institute for Animal Production Nitra, Hlohovecká 2, 951 41 Lužianky, Slovak Republic Tel.: +421 37 6546 217 E-mail: chrenkova(at)vuzv.sk

Sager: Gesamtelementgehalte und deren Querbeziehungen in handelsüblichem Mischfutter für Rinder, Kälber, Schafe, Pferde, Schweine, Ferkel, Geflügel und Wild


Gesamtelementgehalte und deren Querbeziehungen in handels-üblichem Mischfutter für Rinder, Kälber, Schafe, Pferde, Schweine, Ferkel, Geflügel und Wild

Manfred Sager AGES Wien, AT

Abstract Quasi-total element concentration data of commercially available mixed feed has been compiled and sorted according to the target animals deer, cattle, calves, sheep, horses, pigs, piglets, and poultry. Within concentration ranges and concentration ratios, some differences were found due to the use intended. In particular, higher Ca levels for poultry, higher Cu for piglets, higher Co for calves, and higher Mo for pigs were detected. Within boxplots as well as within plots of element ratios against one another, some discriminations between target animals were found, which might be used to classify feeds of unknown use.

Schriftsatz Um tierartenspezifische Unterschiede und Querbeziehungen von Gesamtelementgehalten in kommer-ziell erhältlichen Mischfuttermitteln herauszuarbeiten, wurden in Erweiterung der amtlichen Kontrolle 21 Elemente in verschiedenen Ergänzungsfuttermitteln nach üblichem geeignetem Aufschluss mit ICP-OES Multi-Elementanalyse bestimmt. Zur vorliegenden Auswertung steht ein Datensatz von 555 Pro-ben zur Verfügung, sortiert nach dem Verwendungszweck für Wiederkäuer (Wild, Rinder, Schafe), Pferde, Schweine und Geflügel. Die Unterscheidung Rind – Kalb und Schwein - Ferkel erwies sich da-bei sowohl im Hinblick auf die zum Teil unterschiedlichen Konzentrationsbereiche, als auch Konzentra-tions-verhältnisse, als gerechtfertigt. Hinsichtlich der Hauptelemente sticht besonders das hohe Ca beim Geflügel hervor, welches für Eier-schalen benötigt wird, und auch zu höherem Ca/P und Ca/Mg Verhältnissen führt. Im Gegensatz dazu ist die Konzentration an P beim Kalb am höchsten und bei Schaf und Pferd am niedrigsten, beim Ver-hältnis K/P ist es genau umgekehrt. Na- und Fe-reicheres Futter bekommen Schweine und Ferkel, der Bereich an Na-Konzentrationen in Futtermittel für Kälber und Schafe schwankt aber sehr, und kann den Bereich für Schweine übersteigen. Im Zusammenhang dazu hat das Na/Mg Verhältnis bei Wild und Rind ein Minimum, und bei Ferkeln ein Maximum, und umgekehrt Ba/Na im Wildfutter ein Maxi-mum. Ausgenommen bei Schafen und Geflügel enthalten die Ergänzungsfuttermittel mehr Zn als Mn, das Schweinefutter enthielt mehr Mo als für andere Zielgruppen. Obwohl Fe-Salze im Gegensatz zu Al bewusst zugesetzt werden, bleibt das Verhältnis Fe/Al für sämtliche Tierarten überraschend konstant, was möglicherweise an der Aufschlussmethode liegt, bei welcher Alumosilikate grossteils nicht gelöst werden. Hohes Cu in Ferkelfutter und tiefstes in Schaffutter bildet sich auch in den Verhältnisse Cu/K und Cu/Mn ab. Höhere Co- Gehalte findet man in vielen, aber nicht in allen, Futtermittel für Kälber, was deutlich auch an den Verhältnisse Co/Ni, Co/Mo und Co/Fe sichtbar wird. Überraschenderweise sind Co/Ca und Co/Mg nicht nur bei Kälbern, sondern auch bei Ferkeln erhöht. Das Verhältnis Co/Ni be-wegt sich für andere Zielgruppen als Kälber im geochemisch erwartbaren Verhältnis von etwa 0,5.

Sager: Gesamtelementgehalte und deren Querbeziehungen in handelsüblichem Mischfutter für Rinder, Kälber, Schafe, Pferde, Schweine, Ferkel, Geflügel und Wild


Beim Verhältnis Ba/Cr zeigt die Gruppe Rind-Kalb-Schaf-Pferd eine höhere Tendenz als die Gruppe Wild-Schwein-Ferkel-Geflügel, das Verhältnis Ba/Ni hingegen ist nur bei Schaf und Wild etwa höher als beim Rest. Wird Faktorenanalyse mit Daten aller Proben in mehreren Varianten mit unterschiedlichen Variab-lenkombinationen durchgeführt, so ergeben sich keine deutlich unterscheidbaren Hauptkomponenten, da die meisten Elemente ihre höchste Gewichtung im 1. Faktor haben. Dagegen treten nur K-Mo bzw. Al-Ba mit starken Gewichtungen in eigenständigen Hauptkomponenten auf. Die Zuordnung von Fe und Mg ist oft mehrdeutig, sie stammen vermutlich aus verschiedenen Quellen. Mit Clusteranalyse werden die vorhandenen Datensätze hauptsächlich in 2 Gruppen getrennt, und der Rest verteilt sich auf die vorgegebene Zahl an Clustern. Trends sind erkennbar, aber eine eindeutige Zuordnung zu einer Tierart gelang nicht. Trägt man ausgewählte Elementverhältnisse doppeltloga-rithmisch gegeneinander auf, so lassen sich abgrenzbare Bereiche für verschiedene Tiere unterschei-den. Im Diagramm K/P gegen Na/Mg findet man getrennte Bereiche für Pferd und Wild, und für Rind und Kalb. In der Darstellung Ca/P gegen Cu/K unterscheidet sich Geflügelfutter von Futter für Pferde, Schweine, Schafe und Ferkel, aber auch Ferkelfutter von jenem für Schweine, Pferde und Schafe. Mit Ca/Mg gegen Co/Mn lassen sich Futter für Kuh und Schaf, Wild und Geflügel, sowie Pferd und Geflügel unterscheiden. Ba/Cr gegen Co/Ni ergibt getrennte Bereiche für Pferd-Geflügel und Schaf- Geflügel.

Zusammenfassung Dies zeigt, dass bei gezielter Fragestellung im Vergleich mit vorhandenen Datensätzen eine Zuordnung zu einer Zieltierart auf Grund der Elementzusammensetzung möglich erscheint.

Literatur M. Sager: Micro- and macro-element composition of animal feedstuffs sold in Austria. Ernährung/Nutrition 30 (11), 455 – 473 (2006)

M. Sager: Analysis of less-bioactive elements in green plants, food and feed samples (Sc-Y-La-Ce-Rb-Cs-Ti), Ecol. Chem. Eng. Vol. 17, No. 3, 289-295 (2010)

Corresponding author Doz. Dr. Manfred Sager Sonderuntersuchungen Elementanalytik AGES Wien Spargelfeldstrasse 191 1220 Wien E-Mail: manfred.sager(at)ages.at

Unbenannt-9 1 23.03.16 13:29

Ghareeb et al.: Impacts of the fusarium mycotoxin deoxynivalenol on animal health and production


Impacts of the fusarium mycotoxin deoxynivalenol on animal health and production

Khaled Ghareeb1,2, Wageha A. Awad3,4 and Josef Böhm1 1 Institute of Animal Nutrition and Functional plant compounds, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, AT 2 Department of Animal Behaviour and Management, Faculty of Veterinary Medicine, South Valley University Qena, EG 3 Department of Animal Hygiene, Poultry and Environment, Faculty of Veterinary Medicine, South Valley University Qena, EG 4 Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, AT

Abstract The mycotoxin deoxynivalenol (DON) worldwide cereal contaminants raise concerns for animal health following contaminated feed ingestion. DON is a type B trichothecene produced by Fusarium gramine-arum and Fusarium culmorum and mainly produced in the field conditions or during storage. The cur-rent study was conducted to investigate the impacts of DON feeding on serum clinical chemical parameters. One day boiler chicks were weighed and divided to a control (10 birds fed with basal diet) and DON group (10 birds fed with basal diet artificially contaminated with 10 mg DON/kg feed). At 35 d old, all birds were slaughtered and blood was collected from all birds for investigating the clinical chemical parameters. The results show that DON in broiler feeds decreased (P = 0.005) the level of alanine transaminase (ALT) compared with control, in contrast, increased (P = 0.002) the serum cho-lesterol concentration, the amount of circulating triglycerides compared with controls. These results indicate that DON in chicken feed has the ability to affect the serum clinical biochemistry which could suggest, at least in a part, that DON impairs the health of chicken when present in their feeds. Taken together, DON in the feed of broilers produced alteration in the clinical chemical parametersand high-light the need of a specific hazard characterization for DON risk assessment.

Introduction The worldwide occurrence of DON in food and feed together with its high toxic potentials in humans and animals concludes its significance as a risk factor for health. Toxicity of DON relies on its ability to cross the biological barriers and to affect the functions and viability of the cells forming such organ systems (Awad et al., 2012a, Awad and Zentek, 2015).DON mycotoxicosis in humans and animals may lead to gastrointestinal irritation or necrosis, haematological disorders, diarrhoea, vomiting, de-creased gain in body weight and injury to the haematopoietic systems in bone marrow, spleen, thy-mus and lymph nodes and may cause immunological alterations (Pestka and Smolinski, 2005). It is known that chickens have a higher tolerance to DON than other animals and are frequently ex-posed to DON through their cereal-rich diet (Eriksen and Pettersson, 2004; Awad et al., 2008a, b, 2013, 2014). However, low and moderate levels of contamination are responsible for various immuno-logical modulations and effects on the gut health of chickens (Ghareeb et al., 2013, 2014, 2015). DON produces its toxic effects by inhibiting protein synthesis: it binds to the 60S subunit of eukaryotic ribo-somes, impairs the function of the peptidyl transferase (Feinberg and Mclaughlin, 1989) and inhibits



the elongation of protein synthesis (Ehrlich and Daigle, 1987). Tissues with a high protein turnover and rapidly proliferating cells, such as the liver, immune cells and the gastrointestinal tract, are espe-cially sensitive to DON (Awad et al., 2012a). Many literatures have shown that DON alters the intestinal structure (Awad et al., 2006a, b, 2011a), interferes with the intestinal epithelial barrier function (Awad et al., 2004, 2005; Pinton et al., 2009, Awad and Zentek, 2015), affects nutrient absorption (Awad et al., 2007a, b, 2011a, b) and decreases the expression of the tight-junction claudin proteins in pigs (Pinton et al., 2009). In addition, DON was shown to suppress the antibody response to infectious bronchitis vaccine (IBV) in broiler chickens (Ghareeb et al., 2012). Actively dividing cells are the main targets of DON and therefore feeding birds with DON-contaminated diets can increase their susceptibility to enteric infections. The systematic responses such as change in the profile of liver and kidney enzymes, and blood clinical chemical parameters may give a significant conclusion on how DON intoxication affects the haemosta-sis and the biology of animal body. Investigating the changes in blood of challenged animals can facili-tate the diagnosis of DON and other mycotoxin exposure (Andretta et al. 2012).Therefore, the current experiment was conducted to address the toxic impacts of DON oral exposure on clinical biochemical serum parameters.

Materials and methods Experimental Study Design Twenty 1 d old broilers of a commercial strain were procured from a commercial hatchery. The birds were weighed at the beginning of the experiment, randomly divided into two dietary treatments (10 birds for each treatment). The birds were housed in temperature-controlled batteries during the 5 weeks experimental period. Photoperiod and heating followed standard recommendations. The tem-perature started at 35°C (from day 0 to day 3) and was gradually reduced (2°C/week) to 25°C for the remainder of the experiment according to normal management practice. During the first 2 weeks, chicks were provided with 24 h of light, after which lighting was decreased gradually (2 h daily) to 20 h by the third week and remained at this level until the end of the trial. The control group was fed starter and grower diets based on wheat, soya HP, fat and a premix with vitamins, minerals, amino acids, salt, and monocalcium phosphate. The DON group was fed the start-er and grower diets supplemented with 10 mg feedgrade DON per kg diet. Chicks were fed the starter diets from d 1 to 13 and the grower diets from d 14 to 35. Feed and water were offered ad libitum. Representative feed samples were taken at the beginning of the starter and grower periods and were analyzed for nutrient content and Fusarium mycotoxins.

Performance of Chicks The weight of each individual bird was taken at 1d of the experiment for all dietary groups. Feed in-take and body weight gain were measured weekly and along the experimental trial for all groups. Consequently daily feed intake per bird and the average daily body weight gain were calculated.

Blood Biochemical Parameter Blood was collected in tubes with anticoagulant (Li-Heparine) from all birds at slaughtering on d 35 of the experiment for blood biochemical parameter assays. After centrifugation at 3000 × g for 10 min, the plasma were collected for determination of total protein, albumin, Uric acids, creatinine kinase (CK), cholesterol, triglycerides, lactate dehydrogenase, γ-glutamyltransferase (GGT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) by automatic clinical chemistry analyzer at the central Laboratory of Veterinary Medicine University (Vienna, Austria).



Statistical Analysis Statistical program SPSS (version 20; SPSS GmbH, SPSS Inc., Munich, Germany) was used for data analysis. The Kolmogorov Smirnov test was used to test the normal distribution of the data. Performance, biochemical, and hematological variables were analyzed by independent samples t-test was used between the two groups. The probability values of 0.05 (p ≤ 0.05) were considered significant.

Results and discussion Fusarium species produce a vast array of mycotoxins, many of which are economically important in regard to animal production. The current study demonstrated clear effects of DON on physiological functions of broilers.The chronic DON intoxication with low or moderate concentrations is expected in poultry species and can produce harmful consequences on the health and welfare of birds. In the current study, it was found that the daily body weight gain was numerically lower for birds fed DON compared with their control counterparts. Furthermore, the present results show that serum enzymes indicating liver cell damage such as LDH, ALT, AST and GGT were not all affected by dietary DON. The decreased level of ALT and increased amount of cholesterol in the current study (Figure 1, 2) may suggest liver problem and/ or kidney affection. The liver weight was not affected in this study; however, absolute and relative weight of kidney was reduced, suggesting that the reduced level of ALT is due to the reduced weight of kidney due to DON intoxication. Moreover, increased the amount of cholesterol may indicate that the birds are stressed due to DON feeding. Interestingly, a higher level of triglycerides may suggest that DON in chicken feeds affect lipid metabolism.

Figure 1: Effects of DON on clinical chemical serum parameters



Figure 2: Effects of DON on lipid metabolism

In conclusion, the present study showed that feeding of wheat contaminated with Fusarium mycotoxin negatively affects the growth performance of chicken and altered the blood chemical components.

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Autorenanschrift Prof. Josef Böhm University of Veterinary Medicine Vienna (Vetmeduni), Department for Farm Animals and Veterinary Public Health, Institute of Animal Nutrition and Functional plant compounds Veterinärplatz 1, A-1210 Vienna, Austria E-Mai: Josef.Boehm(at)vetmeduni.ac.at

Autorenverzeichnis


Autorenverzeichnis

Abdallah, F. 94 Alencikiene, G. 49 Ali, H. 94 Alijosius, S. 176 Alijosius, S. 167 Ameilbonne, A. 142 Arnaiz, V. 172 Awad, W.A. 230 Bajorinaite, A. 176 Bartkevics, V. 124 Bartkiene, E. 38, 124 Baur, A.C. 154 Becker, C. 55 Bělková, J. 198, 207 Benarbia, M.A. 172 Berthiller, F. 214 Bichl, G. 214 Bliznikas, S. 194 Boeck, G. 129 Bohlmann, J.T. 10 Böhm, J. 230 Bolduan, C. 138 Breitenstein, A. 154 Brenner, S. 188 Brestenský, B. 88 Buckiuniene, V. 176 Bučko, O. 223 Buffler, M. 55 Caussilas, A. 172 Cernauskiene, J. 79 Chaucheyras-Durand, F. 142 Chicoteau, P. 172 Chizzola, R. 74

Chrastinová, L. 108 Chrastinová, Ľ. 223 Chrenková, M. 108, 223 Čobanová, K. 223 Daněk, P. 207 Dauksiene, A. 167, 176 Deml, M. 138 Detvanová, L. 99 Doležal, P. 99 Dovidaitiene, G. 79 Drażbo, A. 181 Dusel, G. 117 Eder, K. 117 Eipper, J. 69 Ettle, T. 134 Faix, S. 74 Farahat, M. 94 Forano, E. 142 Formelová, Z. 108, 223 Fruhauf, S. 214 Gallissot, M. 59, 158 Garcia, J.M. 172 Ghareeb, K. 230 Gierus, M. 69, 84 Goodarzi Boorojeni, F. 15 Greiling, A. 113 Grešáková, Ľ. 223 Gruzauskas, R. 49, 79, 103, 167, 176, 184, 194 Gübitz, G.M. 6 Hanuszewska, M. 181 Hartwigsen, R. 64, 150 Hechenberger, P. 69 Heger, J. 88

Autorenverzeichnis


Hernandez-Santana, A. 94 Hoeger, T. 129 Horn, G. 154 Hovenjürgen, M. 145 Humer, E. 44 Ion,A. 162 Ivanišinová, O. 74 Drouillard 1 Juodeikiene, G. 38, 124 Kalbitz, J. 154 Kalhotka, L. 99 Kampheus, J. 25 Kantautaite, J. 38 Karásek, F. 99 Keimer, B. 150 Kesselring, J. 129 Khidr, R. 92 Khol-Parisini, A. 44 Klein, P. 207 Klementaviciute, J. 79 Klevenhusen, F. 44 Kliseviciute, V. 103, 167, 176, 194 Kluge, H. 154 Korzekwa, M. 188 Kozłowski, K. 181 Kraugerud, O.F. 21 Kroismayr, A. 218 Krungleviciute, V. 38 Kuchařová, S. 198 Kudlinskiene, I. 49, 79, 184 Lauková, A. 223 Leithner, M. 84 Lipenský, J. 198, 207 Loibl, P. 202 Lübke, K. 113

Mader, A. 15 Masching, S. 214 Miezeliene, A. 49 Miladinovic, D. 21 Monkeviciene, I. 38 Morton, J. 218 Mosoni, P. 142 Mrkvicová, E. 99 Müller, M. 162 Münnich, M. 44 Nitrayová, S. 88 Obermaier, A. 134 Oceľová, V. 74 Ondruška, Ľ. 108, 223 Ots, M. 49 Paleckaitis, M. 184 Patráš, P. 88 Pavlata, L. 99 Pisarčíková, J. 74 Plachá, I. 74 Pockevicius, A. 184 Polačiková, M. 108, 223 Preißinger, W. 202, 210 Propstmeier, G. 202, 210 Puntigam, R. 69 Puvača, N. 218 Raceviciute-Stupeliene, A. 103, 167, 176, 194 Rajský, M. 223 Reckmann, K. 64 Reeken, J.-B. 145 Ribitsch, D. 6 Rimbach, M. 64 Ringseis, R. 117 Rozkot, M. 198, 207 Sager, M. 227

Autorenverzeichnis


Sasyte, V. 103, 167, 176, 194 Schatzmayr, D. 214 Schatzmayr, G. 129 Schedle, K. 69, 84, 214 Scherb, S. 202, 210 Schlagheck, A. 150 Schneeberger, E. 84 Schoendorfer, K. 129 Schulze Schwering, D. 145 Schwartz-Zimmermann, H. 214 Schwarz, C. 69 Sederevičius, A. 38 Sobczak, A. 181 Stangl, G.I. 154 Stankevicius, R. 38, 49, 79 Stanyte, G. 79 Starevicius, D. 38 Starkute, V. 124

Šťastník, O. 99 Stef, L. 162 Štenclová, H. 99 Suarez, M.G. 59, 158 Svirmickas, G. 103, 176 Thaller, G. 64, 150 Václavková, E. 198, 207 Weber, F. 117 Wedekind, H. 113 Wieß, S. 6 Wilke, T. 188 Windisch, W. 55, 138, 202 Winkler, A. 117 Zadeike, D. 124 Zaujec, K. 108 Zebeli, Q. 44, 49 Zelvyte, R. 38 Zentek, J. 15

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