Tierärztliche Hochschule Hannover Density gradient centrifugation ...

Tierärztliche Hochschule Hannover

Density gradient centrifugation of stallion semen

INAUGURAL – DISSERTATION

zur Erlangung des Grades einer

Doktorin der Veterinärmedizin

- Doctor medicinae veterinariae -

( Dr. med. vet. )

vorgelegt von

Gesa Stuhtmann

Uelzen

Hannover 2011

Wissenschaftliche Betreuung: Prof. Dr. H. Sieme

Klinik für Pferde

Reproduktionsmedizinische Einheit der Kliniken

1.Gutachter: Prof. Dr. H. Sieme

2. Gutachter: Prof. Dr. H. Bollwein

Tag der mündlichen Prüfung: 15.11.2011

To Heinrich and my family

Contents

1 Introduction _____________________________________________________- 1 -

2 Review of Literature_______________________________________________- 3 -

2.1 Sperm selection preparation methods _____________________________________ - 3 -

2.1.1 Migration procedure ___________________________________________________________ - 3 -

2.1.1.1 Swim- up _______________________________________________________________ - 3 -

2.1.1.2 Migration- sedimentation __________________________________________________ - 4 -

2.1.2 Filtration ____________________________________________________________________ - 4 -

2.1.2.1 Glass wool filtration ______________________________________________________ - 4 -

2.1.2.2 Molecular glass wool filtration ______________________________________________ - 5 -

2.1.2.3 Glass beads filtration ______________________________________________________ - 5 -

2.1.2.4 Sephadex columns________________________________________________________ - 5 -

2.1.2.5 Upstream method_________________________________________________________ - 6 -

2.1.3 Electrophoretic system _________________________________________________________ - 6 -

2.1.4 Sperm- specific antibodies ______________________________________________________ - 6 -

2.1.5 Density gradient centrifugation __________________________________________________ - 6 -

2.1.5.1 Principles of the selection __________________________________________________ - 6 -

2.1.5.2 Solutions for density gradient centrifugation ___________________________________ - 7 -

2.1.5.3 EquiPureTM and EquiPureTM Pro_____________________________________________ - 8 -

2.1.5.4 Iodixanol ______________________________________________________________ - 10 -

2.1.6 Review of studies comparing different selection methods_____________________________ - 11 -

3 Own experimental studies _________________________________________- 13 -

3.1 Materials and Methods ________________________________________________ - 13 -

3.1.1 Animals and semen collection for thesis 1 _________________________________________ - 13 -

3.1.2 Animals and semen collection for thesis 2 _________________________________________ - 13 -

3.1.3 Experimental design for thesis 1_________________________________________________ - 14 -

3.1.4 Experimental design for thesis 2_________________________________________________ - 16 -

3.2 Sperm analysis for thesis 1 and 2 ________________________________________ - 18 -

3.3 Statistical analysis for thesis 1 __________________________________________ - 18 -

3.4 Statistical analysis for thesis 2 __________________________________________ - 19 -

3.5 EquiPureTM Pro centrifugation, a method for sperm clean-up and its effects on sperm

quality of cold- stored and frozen- thawed stallion sperm____________________ - 20 -

3.5.1 Abstract ____________________________________________________________________ - 20 -

3.5.2 Introduction _________________________________________________________________ - 21 -

3.5.3 Materials and Methods ________________________________________________________ - 22 -

3.5.3.1 Animals and semen collection _____________________________________________ - 22 -

3.5.3.2 General semen processing_________________________________________________ - 23 -

3.5.3.3 First experiment_________________________________________________________ - 23 -

3.5.3.4 Second experiment ______________________________________________________ - 24 -

3.5.3.5 Sperm analysis__________________________________________________________ - 24 -

3.5.3.6 Statistical analysis _______________________________________________________ - 25 -

3.5.4 Results _____________________________________________________________________ - 26 -

3.5.5 Discussion __________________________________________________________________ - 33 -

3.6 Sperm quality parameters of stallion spermatozoa prepared by iodixanol

sedimentation density gradient centrifugation of fresh, cooled-stored and frozen-

thawed semen________________________________________________________ - 38 -

3.6.1 Abstract ____________________________________________________________________ - 38 -

3.6.2 Introduction _________________________________________________________________ - 39 -

3.6.3 Materials and Methods ________________________________________________________ - 40 -

3.6.3.1 Animals and semen collections_____________________________________________ - 40 -

3.6.3.2 General semen processing before centrifugation _______________________________ - 40 -

3.6.3.3 Centrifugation methods ___________________________________________________ - 41 -

3.6.3.4 General semen processing after centrifugation_________________________________ - 41 -

3.6.3.5 Sperm analysis__________________________________________________________ - 42 -

3.6.3.6 Sperm selective medium __________________________________________________ - 42 -

3.6.3.7 Statistical analysis _______________________________________________________ - 43 -

3.6.4 Results _____________________________________________________________________ - 43 -

3.6.5 Discussion __________________________________________________________________ - 49 -

4 Summarizing, Discussion and Conclusions ___________________________- 54 -

5 Summary ______________________________________________________- 58 -

6 Zusammenfassung_______________________________________________- 60 -

7 References _____________________________________________________- 63 -

8 Appendix_______________________________________________________- 81 -

8.1 EquiPure EquiPureTM Pro centrifugation, a method for sperm clean-up and its effects

on sperm quality of cold- stored and frozen- thawed stallion sperm ___________ - 81 -

List of abbreviations

AI artificial insemination

ALH amplitude of lateral head displacement

ATP adenosintriphoshate

CASA computer assisted sperm analysis

C Celsius

CC cushion centrifugation

CENTR routine centrifugation

DFI denaturation fragmentation index of sperm DNA

DNA deoxyribonucleic acid

DS diluted semen

EPP EquiPureTM Pro centrifugation

EPS externalization of phosphatidylserine

Et al. et alii

EXTEN extended semen

FITC/PNA-live percentage of Syto stained spermatozoa

FITC/PNA-AR percentage of FITC/PNA stained acrosomes

GAPDH glyceraldehyde 3-phosphate dehydrogenase-S

g/ml gram/milliliter

h hour

HIV human immunodeficiency virus

ICSI intra-cytoplasmatic sperm injection

INRA National Institute for Agricultural Research

IODIX iodixanol-density gradient centrifugation

IVF in vitro fertilization

min minute

mOsm/kg water milliosmoles per kilogram of water

µm/sec micrometer/second

PI propidiumiodid

PMS progressively motile sperm

% percentage

Rp correlation index

ROS reactive oxygen species

SCSA sperm chromatin structure analysis

SD standard deviation

VAP average path velocity (µm/sec)

VCL curvilinear velocity (µm/sec)

WSSM centrifugation without sperm selective medium

x-ray x-radiation

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1 Introduction

In horse breeding, artificial insemination (AI) has replaced natural mating almost completely.

This development has been associated with decreased fertility rates, both when using cooled-

stored or frozen-thawed semen (Aurich and Aurich, 2006, Johannisson et al. 2009).

Sperm exhibits great male-to-male variability in composition and quality. This is especially

true for stallions, because they are not selected for fertility parameters, as has been the case

for boars and bulls (Colenbrander et al. 2003, Loomis 2006). In addition, stallions are gener-

ally retained for breeding for a longer time than other farm animals. To achieve high preg-

nancy rates, spermatozoa must be of good quality, i.e. they must show good motility, normal

morphology, sufficient metabolism for energy production, high membrane integrity, chroma-

tin structure and acrosome integrity (Graham, 1996, Love and Kenney, 1998). Apart from

viable and morphologically normal spermatozoa, an ejaculate contains dead spermatozoa,

morphologically abnormal or immature spermatozoa and seminal plasma. These might have

detrimental effects on the fertile spermatozoa (Mortimer, 2000, Morrell, 2006a).

In order to select spermatozoa of superior quality for assisted reproduction techniques such as

in-vitro fertilization (IVF) or intra-cytoplasmatic sperm injection (ICSI), sperm separation

techniques are used (Mortimer 1994, Mortimer 2000, Henkel and Schill, 2003). Different

sperm separation and clean-up techniques are available, such as sperm migration (swim-up),

adherence methods for elimination of impaired spermatozoa (glass wool filtration, glass wool-

sephadex-filtration, Leucosorb®) and selection of subpopulations on the basis of their size

and density (density gradient centrifugation) (Mortimer, 2000, Sieme, 2004a).

In our study we tested two different density gradient solutions and evaluated the quality of the

spermatozoa that were selected using these methods. Sperm morphology, motility and veloc-

ity, were determined, as well as membrane integrity, acrosome membrane integrity, and

chromatin integrity. Furthermore, survival during cold-storage and after freezing and thawing

were examined. The first results section on this thesis describes the use of the novel formula-

tion EquiPureTM Pro (Nidacon International AB, Sweden). This solution contains colloidal

silica particles stabilized with covalently bound hydrophilic silane. It uses a top layer that also

contains a recombinant serine protease and a bottom layer that contains a protease inhibitor.

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The second results section describes the use of a 60 % solution of iodixanol (OptiPrepTM,

Progen, Heidelberg, Germany). Solutions with different densities were tested, that were pre-

pared by dilution of a iodixanol stock solution with Hank´s buffered salt solution.

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2 Review of Literature

2.1 Sperm selection preparation methods

The first sperm separation method to be applied was simple washing. Mahadevan and Baker

(1984) described one of the first washing procedures followed by swim-up (for review see

Henkel and Schill, 2003). Subsequent separation techniques were based on different princi-

ples including migration, filtration and density gradient centrifugation.

Migration techniques include: swim-up, under-lay and migration-sedimentation methods (Tea

et al. 1984, Zavos et al. 2000). Filtration methods use glass wool filtration, sephadex beads or

membranes (Paulson et al. 1977, Van der Ven et al. 1988, Drobnis et al. 1991, Agarwal et al.

1991). For density gradient centrifugation, several solutions are available. The most fre-

quently used of these in human fertility treatment is Percoll, consisting of polyvinylpyrroli-

done (PVP)-coated silica particles (Sato et al. 1990, Ord et al. 1990). After Percoll was with-

drawn from clinical use in 1996, different solutions were tested as potential replacements.

These included silanecoated silica particles such as Puresperm, Isolate, SpermGrad-100, Sil-

Select Plus (Claassens et al. 1998, Mousset- Simeon et al. 2004), iohexol (Nycodenz) (Gel-

lert- Mortimer et al. 1988), the dimer of iohexol iodixanol (OptiPrep), and the synthetic poly-

saccharide Ficoll (Bongso et al. 1989).

Henkel and Schill (2003) stated, that a sperm separation technique should be quick, easy to

handle, low in cost and should produce a high yield of motile spermatozoa. It should avoid

damaging the cells and eliminate dead spermatozoa, bacteria and reactive oxygen species

(ROS). Finally, it should be able to clean a large volume of samples. None of the techniques

that are currently available meet all of these requirements. Consequently, in human reproduc-

tion, the method chosen depends on the quality of the ejaculate. The swim-up method, for

example, can only be used when an ejaculate contains a high number of motile sperm.

2.1.1 Migration procedure

2.1.1.1 Swim- up

The classical swim-up procedure is the oldest and, until recently the most commonly used

technique. With this method, a solution is placed on top of the sperm pellet that is obtained by

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centrifugation and the motile sperm are allowed to move into this solution during a 60 min

incubation at 37°C. This technique results in a high percentage of motile, morphologically

normal spermatozoa in the solution layer, while other cells remain in the pellet. The yield of

motile spermatozoa, however, is low. This technique is thought to negatively affect sperm

quality because of the centrifugation step applied prior to swim-up. In the resulting pellet, the

sperm cells are closely packed together with cell debris which can release reactive oxygen

species (ROS) (Henkel and Schill, 2003). In order to avoid this, the swim-up technique can be

performed directly on extended semen. This is done by pipetting extended semen below a

medium layer and incubation for 15-60 min. Mortimer (2000) reviewed this method and

found detrimental effects on the chromatin structure. A further modification of the swim-up

method is the “Sperm Select System” (Select Medical Systems, Williston, Vt). The medium

that is used as an upper layer for motile sperm to move into consists of hyaluronate.

Zimmermann et al. (1994) found this method to be a useful technique for clinical human

sperm preparation (for review see Mortimer, 2000).

2.1.1.2 Migration- sedimentation

The migration-sedimentation technique is a combination of swim-up and sedimentation. This

method was first developed by Tea et al. (1984) and starts with a swim-up procedure using

extended semen without a centrifugation step. This method uses tubes with an inner cone.

First the spermatozoa swim up into the supernatant medium and after that they sediment into

the inner cone. Even though the selection of motile sperm is good when using this methods, it

results in a low yield. Zavos et al. (2000) modified the technique and used a multi-chamber

tube. Neither method has been used widely (for review see Henkel and Schill, 2003).

2.1.2 Filtration

2.1.2.1 Glass wool filtration

The glass wool filtration method has been first described by Paulson and Polakoski (1977).

Semen was added to a column containing densely-packed glass wool fibres. The mechanism

of selection is based on the motility of the spermatozoa and the filtration effect of the glass

wool. The outcome is influenced by the chemical structure, the surface characteristics and the

thickness of the glass wool fibres. The advantages of this procedure are that it can be used for

processing whole ejaculates, for oligo-and asthenozoospermatic ejaculates, and involves di-

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rect processing of the semen without a centrifugation step. After selection, a centrifugation

step can be included to remove the seminal plasma (for review Henkel and Schill, 2003). The

glass wool filtration method selects a high percentage of chromatin intact cells (Henkel et al.,

1994).

2.1.2.2 Molecular glass wool filtration

A modification of the glass wool filtration method was described by Grunewald et al. (2007).

They combined magnetic-activated cell sorting using annexin V-conjugated microbeads with

glass wool filtration. The microbeads bind externalized phosphatidylserine (EPS) which is

present on apoptotic cells. Phosphatidylserine is normally present on the inner side of the

plasma membrane, but is externalised during apoptosis. This method was demonstrated to be

efficient in removing apoptosis-activated spermatozoa (Grunewald et al., 2001, Paasch et al.,

2004). Magnetic-activated cell sorting was also applied after a density gradient centrifugation,

which was shown to improve semen quality as measured by motility, viability and apoptosis

indices (Said et al. 2005, Said et al. 2008).

2.1.2.3 Glass beads filtration

Description on what is done using this method showed good results in terms of selection of

motile sperms (Daya et al. 1987). However, it did not find a wide use in human reproduction,

because of concerns of contamination of the insemination medium with glass beads.

2.1.2.4 Sephadex columns

Sephadex columns produce a higher yield of motile, morphologically normal spermatozoa, as

compared to swim-up procedures. In addition, use of this method resulted in higher pregnancy

rates (for review see Henkel and Schill, 2003). Sephadex was introduced in the preparation of

frozen boar semen, where this resulted in a significant improvement in motility and acrosome

integrity of post-thaw spermatozoa. Similar results were found for human, water buffalo and

bovine semen (for review see Rutz et al. 1991). When the “Spermprep column” which con-

tains sephadex was used for stallion semen, this also resulted in a significant improvement in

percentages of live cells (Rutz et al. 1991).

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2.1.2.5 Upstream method

The upstream method was introduced by Farhang (2009). For this method, a tube is divided

into two parts by a nylon mesh, and semen and medium are placed on the mesh in the upper

part of the tube on top of the mesh. Whereas immotile sperm, cell debris, and seminal plasma

sink into the lower part of the tube, the motile spermatozoa will move upstream and stay in

the medium above the mesh. Use of this method results in selection of sperm with good motil-

ity and normal morphology (Farhang, 2009).

2.1.3 Electrophoretic system

Ainsworth et al. (2005) describe the use of an electrophoretic system for selection of motile,

viable, morphologically normal and chromatin intact spermatozoa on the basis of their size

and electronegative charge. This method was further developed by Microflow® technology

and allows for selection of a large number of spermatozoa in a short time and is easy to per-

form without a skilled technician (Ainsworth et al., 2005).

2.1.4 Sperm- specific antibodies

In the study of Anslinger et al. (2007) they used monoclonal antibodies directed against the

testicular angiotensin-converting enzyme that were coupled with biomagnetic beads. They

showed that use of such antibodies increase in the relative number of sperm selected from an

epithelial cell mixture. Furthermore, they determined that this technique is non-toxic.

2.1.5 Density gradient centrifugation

2.1.5.1 Principles of the selection

The most popular method to separate suspension of mixed populations of biological particles

is density gradient centrifugation. Gradients are either continuous, i.e. the density of the gra-

dient gradually increases from the top to the bottom, or discontinuous, consisting of layers of

different density with clear boundaries between the layers.

The different components of an ejaculate, such as spermatozoa, cellular debris, bacteria, leu-

cocytes, reactive oxygen species and seminal plasma, all have different densities. In addition,

sperm that have different properties have different densities. Such properties include differ-

ence in viability, motility, morphology, chromatin integrity, plasma and acrosomal membrane

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integrity. Mature cells, for example, have a density of more than 1.12 g/ml and they are more

dense than immature cells. Leucocytes have a density of less than 1.12 g/ml.

During density centrifugation, particles will remain in the part of the gradient corresponding

to their own density (the isopycnic point).

Selection efficiency is dependent the volume and the density of the gradient layers used, and

that of the extended semen, as well as the g-force and time of centrifugation (Mortimer,

2000).

Density gradient centrifugation is applied to semen to select spermatozoa of superior quality

for AI in human reproduction (Mortimer, 2000) and animal reproduction (Morrell, 2006a).

Furthermore, the method is performed to clean-up an ejaculate of bacterial and viruses such as

HIV, hepatitis C or B in human reproduction (Englert et al. 2004) or the equine arteritis virus

(Morrell and Geraghty, 2006b) and porcine reproductive and respiratory syndrome virus in

animal reproduction.

2.1.5.2 Solutions for density gradient centrifugation

Several types of density centrifugation solutions have been used to select spermatozoa, in-

cluding solutions composed of: albumin (human: Ericsson, 1977, Goodeaux and Kreider,

1978, Pruitt et al. 1987, stallion: Turner et al. 1998), Ficoll (Bongso et al. 1989), Accudenz

(Sbacia et al. 1996), polysaccharide beads (Ohashi et al. 1992), the plant polysaccharide ara-

binogalactan (Turner and Arns, 1997) and Percoll (Gorus et al. 1981, Buzby et al. 1993).

Density solutions should not cause osmotic stress to cells and have a high density. Sucrose,

cesium chloride, Ficoll and Metrizamide could not be used for this purpose because dense

solutions of those media are hypertonic and highly viscous (Mortimer, 2000).

The most commonly used density centrifugation solution was Percoll, which consists of poly-

vinylpyrrolidone (PVP)-coated silica particles. Percoll improves semen quality (Claassens et

a., 1998) and is able to separate spermatozoa from bacteria and reactive oxygen species (Ait-

ken et al. 1988). Selection of sperm using Percoll was found to result in a higher pregnancy

rate (Hyne et al. 1986, Ord et al. 1990). For stallion semen, the use of Percoll was optimized

by different researchers (Sato et al. 1990, Parsch et al. 1989). Sieme et al. (2003) found a bet-

ter recovery rate for Percoll as compared to swim-up, or glass wool centrifugation methods,

although there were fewer progressively motile sperm and detrimental effects were seen when

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using Percoll (Sieme et al. 2003, Mortimer, 1994). Avery and Greve (1995) suggested that

detrimental effects on bull spermatozoa after centrifugation with Percoll are due to interaction

of the spermatozoa with polyvinylpyrrolidone.

Nycodenz, used as x-ray contrast medium, was also tested for density gradient centrifugation.

This medium includes iohexol, which is an iodinated cyclic hydrocarbon. Gellert-Mortimer et

al. (1988) found better survival rates for oligozoospermic and asthenozoospermic semen

processed by Nycodenz as compared to Percoll gradients (for review see Henkel and Schill,

2003).

Several products containing silanecoated silica particles were introduced for density gradient

centrifugation including: IxaPrep (MediCult, Copenhagen), PureSperm (Nidacon Laoratories

AB, Gothenburg, Sweden), Isolate (Irvine Scientific, Santa Ana, CA, USA). All these prod-

ucts have no detrimental effects on the sperm and are low in toxicity. Many authors tested

their selection efficiency and compared with selection using Percoll. Several authors found no

differences between Isolate and PureSperm compared to Percoll (Centola et al. 1998, Claas-

sens et al. 1998, Mousset-Simeon et al. 2004. In contrast, Chen and Bongso et al. (1999)

showed higher values (recovery rate, percentage of progressively motile sperm) for PureS-

perm-treated spermatozoa (reviewed by Henkel and Schill, 2003).

Goodeaux and Kreider (1978) used bovine serum albumin for clean-up of stallion semen, this

resulted in increased percentage of motile and progressively motile sperm. In an insemination

trial, a foaling rate of 60% was obtained for bovine serum albumin selected spermatozoa

compared to 40 % for Tyrode-treated (control) semen.

2.1.5.3 EquiPureTM and EquiPure

TM Pro

Silane-coated silica particles stabilized with covalently bound hydrophilic silane are the com-

ponents of EquiPureTM and EquiPureTM Pro (Nidacon International, Sweden). Gradient cen-

trifugation with EquiPureTM selects the motile, viable, morphological normal spermatozoa

from immotile, dead sperm and sperm with damaged chromatin, as well as the seminal plasma

and microorganisms. The pH-value is between 7.4 and 7.8, the osmolality between 300 - 310

mOsm/kg H2O and endotoxin levels are < 1.0 EU/ml. EquiPureTM was used by MacPherson

et al. (2002) to improve sperm quality and by Morrell and Geraghty (2006b) and Bandte

(2007) to remove equine arteritis virus from stallion semen.

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After the withdrawal of Percoll in 1996, PureSperm (Mousset-Simeon et al. 2004) became a

often used medium for density gradient centrifugation in human assisted reproduction. Equi-

Pure that is used for clean-up of stallion semen has similar composition as PureSperm. Mous-

set-Simeon et al. (2004) compared selection of viable motile sperm for use for cryopreserva-

tion, using PureSperm and Percoll, iodixanol and three other silanecoated silica particle solu-

tions. Similar results were found for Percoll and PureSperm and the author concluded that

PureSperm is a good alternative for Percoll. These findings were confirmed by others (Cen-

tola et al. 1998, Claassens et al. 1998). Söderlund and Lundin (2000) found higher percentage

of hyperactivated sperm with higher velocity parameters after PureSperm treatment. Varner et

al. (2008) demonstrated an increase in pregnancy rate from 59 % to 91 % for EquiPureTM -

treated semen.

The EquiPureTM products were further developed, which resulted in the EquiPureTM Pro prod-

uct line. In this product, the top layer contains a recombinant serine protease and the bottom

layer contains a protease inhibitor. The solutions are optimized for stallion semen and are

ready-to-use. The enzyme in the top layer is thought to have a positive influence on the en-

zyme system of the spermatozoa and this might improve motility and the fertility. Pattinson et

al. (1990a, b), for example, evaluated the effects of different enzymes on antibody-mediated

spermagglutination. They found that trypsin supported in-vitro oocyte penetration. Also for

zona-free hamster ova, it was found that sperm treatment with trypsin improved motility and

in-vitro fertilization (Cohen and Aafjes, 1982). Rios and Barros (1997) evaluated a trypsin-

like enzyme, necessary for the spermatozoa of a shrimp (Rhynchocinetes typus) to penetrate

the egg-coat. Trypsin-like enzymes seems to play a role in the acrosome reaction (Meizel and

Lui, 1976) and are involved in the binding of mouse spermatozoa to the zona pellucida

(Saling, 1981). The enzyme glyceraldehyde 3-phosphate dehydrogenase-S (GAPDH) is

bound to the fibrous sheath of mammalian spermatozoa (Westhoff and Kamp, 1997). This

enzyme plays an important role in generating energy by glycolysis. Miki et al. (2004) demon-

strated infertility and the loss of progressive motility in knock-out mice for this enzyme.

Figenschau and Bertheussen (1999) suggest that trypsin stimulates GAPDH, and because of

this trypsin-treated semen thus should have higher ATP concentrations and show better motil-

ity than non-treated semen. Blevins et al. (2008) added trypsin to BoviPure and found an in-

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crease in fertilization rate (88.4% versus 63.1%) and a significantly higher number of em-

bryos that could be used for transfer (70.3 versus 51.8%) as compared to the standard method.

Figure 1: Density gradient centrifugation with EquiPureTM Pro from Nidacon International AB, Sweden

2.1.5.4 Iodixanol

OptiPrep is a 60 % solution of iodixanol in water (OptiPrepTM, Progen, Heidelberg, Ger-

many). The chemical formula for iodixanol is: (5,5´- ((2-hydroxy-1-3propanediyl)-bis (ace-

tylamino)) bis (N,N´-bis (2,3dihydroxypropyl-2,4,6-triiodo-1,3-benzenecarboxamide)) and is

a dimer of Nycodenz (iohexol) (AxisShield, Norway). Iodixanol was originally developed as

an x-ray contrast medium. It has a density of 1.32 g/ml and can be diluted with common phy-

siological saline solutions to any lower density. It is suitable for purification of cells because

it is metabolically inert, non-ionic and non-toxic and it does not expose cells to osmotic stress.

Iodixanol is used for isolation of organelles and a variety of different cell types (Claassens et

al. 1998). The first use of iodixanol for stallion semen was the cushion centrifugation method

(Revell et al. 1997, Delhomme et al. 2004, Escot, 2005, Sieme et al. 2006, Loomis, 2006). For

human semen, several authors demonstrated the selection efficiency of iodixanol (Claassens

et al. 1998, Pickering et al. 1989, Smith et al. 1997, Anderson and Grinsted, 1997 Tucker,

2002). Also for bull semen a protocol is available (Axis Sheets Application Sheet C16 4th edi-

tion). Patel et al. (1998) found that bovine semen could be improved using iodixanol (Opti-

Prep) and Nycodenz (for review see Fisher et al., 1998). Up to now, iodixanol has not been

used for the selection of stallion semen.

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Figure 2: Purification of motile sperm cells in a discontinuous iodixanol gradient application Sheet C 16,

Axis Shield PoC AS, Norway

2.1.6 Review of studies comparing different selection methods

Sieme et al. (2003) evaluated the use of different selection methods on recovery rates and

stallion sperm quality parameters. Methods included: Percoll gradient centrifugation, glass

wool filtration and Sephadex column and swim-up methods. The highest losses of spermato-

zoa were seen using the swim-up method. Percoll centrifugation resulted in a significantly

higher recovery rate compared to glass wool, Sephadex and swim- up. The filtration methods

and swim-up treatment, however, resulted in significantly better progressive motility than the

control group and after Percoll centrifugation. The decrease in motility of Percoll-treated se-

men was attributed to detrimental effect of Percoll on spermatozoa ( Mortimer, 1994, Avery

and Greve, 1995). Other studies that compared the effect of Percoll centrifugation and swim-

up methods on the yield and quality of the selected spermatozoa, showed conflicting results.

Some authors found no difference in the yield of motile spermatozoa (Smith et al. 1997),

whereas others demonstrated a higher total recovery rate and percentage of motile sperm after

Percoll centrifugation (Sharma et al. 1997) (for review Tucker and Jansen, 2002). Johnson et

al. (1996) tested glass wool filtration and the mini-Percoll column. They found a higher yield

and higher percentage of motile sperm for the filtration method. For Sephadex and glass wool,

Lopez et al. (1993) obtained higher percentage of morphologically normal sperm after Per-

coll centrifugation (for review Tucker and Jansen, 2002). For the preparation of stallion se-

men, Buzby et al. (1993) evaluated the differences between Percoll, Sephadex and the swim-

up method. They found no significant differences between the methods in terms of recovery

of motile and viable spermatozoa. Use of a Percoll centrifugation resulted in a higher percent-

age of motile spermatozoa directly after centrifugation. After 240 minutes, however, a signifi-

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cant decrease in the percentage of viable cells was seen for sperm that were selected using

Percoll centrifugation or the swim-up method, whereas no differences motility were found.

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3 Own experimental studies

3.1 Materials and Methods

3.1.1 Animals and semen collection for thesis 1

In the first experiment, three ejaculates were collected from four warmblood horses (4-7 years

old) and two riding ponies (9 years old) that participated in the AI-program of the State Stud

of Lower Saxony in Celle, Germany. Semen collections were done every other day during the

2008 breeding season. In the second experiment, two ejaculates were collected from each of

two warmblood stallions.

Semen collections were performed using an artificial vagina (Hanover model, Klug 1993)

(Minitüb, Landshut, Germany), a phantom (Model “Celle” Klug 1993) and a teaser mare. Ste-

rile gauze filter was performed to remove the gel-portion.

3.1.2 Animals and semen collection for thesis 2

Three ejaculates were collected from each of six warmblood stallions (5 to 11 years old), that

participated in the AI-program of the National Stud of the State of Lower Saxony in Celle,

Germany. Prior to the onset of the study, semen was collected over a period of one week to

stabilize the extragonadal sperm reserves. The study was performed at the beginning of the

breeding season 2009.

Semen collections were done every other day, using an artificial vagina (Hanoverian model;

Minitübe, Landshut, Germany). A phantom was used, and a teaser mare was fixed in front of

the phantom. The collection device was equipped with a sterile gauze filter to remove gel.

For the second experiment, in which semen processed as described above was used for cryo-

preservation, two groups of stallions were used that differed in freezeability. Stallions were

divided into “good” and “poor” freezers (n=3 per group) according to post-thaw motility prior

to the current experiment: stallions with progressive post-thaw sperm motility ≤ 35 % in more

than 3 out of 10 ejaculates were classified as “poor freezers” and stallions above these criteria

were assigned to the “good freezers” group.

- 14 -

3.1.3 Experimental design for thesis 1

In the first experiment, the effect of selective centrifugation methods on sperm quality pa-

rameters was tested. Sperm quality was assessed after cooled-storage as well as after freezing

and thawing. In the second experiment, the effects of varying freezing extender versus Equi-

PureTM Pro ratios on survival after freezing and thawing were analyzed.

General semen processing

The volume of gel-free semen was measured using a graduated cylinder, and the sperm con-

centration was determined using a photometer (Sperma Cue; Minitüb, Tiefenbach, Germany).

Aliquots of raw semen were immediately snap-frozen in liquid nitrogen for later analysis of

the chromatin integrity via the sperm chromatin structure assay (SCSA, Evenson and Jost,

2000).

An aliquot of gel-free raw semen was diluted with INRA 82 to a sperm concentration of 25 x

106 cells/ml for direct analysis of sperm motility and velocity parameters, and plasma mem-

brane integrity, and acrosomae membrane integrity. For centrifugation processing, three 30 ml

samples were prepared of 30 x 106 cells/ml in INRA 82. Centrifugation protocols are de-

scribed in detail below.

Centrifugation methods

EquiPureTM Pro centrifugation: (EPP)

The protocol described by Nidacon International AB was used in this study, with modifica-

tions. The volumes of the top and the bottom layers and extended semen were adjusted, as

well as the force and time for centrifugation, this was done based on preliminary studies. Ten

ml of bottom layer was added in a 50 ml conical plastic tube, after which 10 ml of top layer

was carefully added on top. Finally, 30 ml diluted semen was placed on top. Centrifugation

was done for 15 min at 330 x g.

Centrifugation without sperm selective medium: (WSSM)

Thirty ml semen diluted at 30 x 106 cells/ml in INRA82 was added in a 50 ml centrifugation

tube without sperm-selective medium. Centrifugation was performed for 15 min at 330 x g.

- 15 -

Cushion centrifugation: (CC)

OptiprepTM (Progen, Heidelberg, Germany) was used as a cushion fluid. Thirty ml semen di-

luted at 30 x 106 cells/ml in INRA82 was added to a 50 ml plastic tube, and 2 ml of cushion

fluid was layered underneath using a syringe with a sterile needle (2.00 X 80 mm). Centrifu-

gation was done for 20 min at 1000 x g according to Sieme et al. (2006).

First experiment: Further semen processing after centrifugation:

After centrifugation with either EquiPureTM Pro or WSSM, the supernatant was aspirated,

leaving a pellet of approximately 2 ml. Following CC, the supernatant was aspirated and the

cushion fluid was removed separately with a syringe. In all cases, the pellet was transferred

into a clean tube, using a syringe with a sterile needle.

For all three centrifugation preparations, the sperm number in the pellet was determined using

a Thoma Neu counting chamber. The recovery rate was calculated by dividing the cell num-

ber recovered after centrifugation with the original cell number before centrifugation. The

concentrated sperm solution obtained after each of the centrifugation preparation, was divided

into two parts.

One part was diluted with INRA 82 to a final concentration of 25 x 106 sperms/ml and stored

at + 5°C up to 72h. The other part was diluted with INRA 82 containing 5.0 % egg yolk and

glycerol, resulting in a final concentration of 2.5 % glycerol and 100 x 106 sperms/ml. After

dilution in freezing extender, samples were equilibrated for two hours at +5°C. After which it

was packaged in 0.5 ml straws that were cooled from + 5°C down to –140°C at 60°C/min in a

automated cell freezer (IMV, L´Aigle, France) and stored in liquid nitrogen until further anal-

ysis. Thawing was done in a water bath at 37°C for 30 seconds.

Second experiment: Further semen preparation after centrifugation:

The EquiPureTM Pro centrifugation protocol and recovery of the sperm pellet was similar as

described for the first experiment.

After estimation of the sperm concentration, the concentrated sperm solution obtained after

EquiPureTM Pro centrifugation was split into four equal parts. One part was diluted with

INRA 82 containing 5.0 % egg yolk and 2.5 % glycerol (freezing extender) to 100 x 106

- 16 -

sperms/ml, similar as it was done for the first experiment. The other parts were diluted with

freezing extender at a 1:1, 1:2 or 1:3 ratio (extended semen : freezing extender)

Sperm selective medium

Iodixanol was used for density centrifugation; its chemical formula is: 5,5´-((2-hydroxy-1-

3propanediyl)-bis(acetylamino))bis(N,N´-bis(2,3dihydroxypropyl-2,4,6-triiodo-1,3-

benzenecarboxamide)). A 60% iodixanol solution in water with a density of 1.32 ± 0.001 g/ml

(20° C) is commercially available as OptiPrepTM (Progen, Heidelberg, Germany). Before den-

sity centrifugation, isoosmotic solutions with different densities were prepared fresh by dilu-

tion of the 60% solution of iodixanol with Hanks Buffered Salt Solution (Invitrogen, France),

according the recommendation of AxisShield OptiPrepTM application sheet C16. The top layer

of 1.09 g/ml is a 16% iodixanol solution and the bottom layer of 1.165 g/ml is a 30% solution.

The densities of the media were checked by measuring using bench-top refractometer (Zeiss).

3.1.4 Experimental design for thesis 2

General semen processing before centrifugation

Standard spermatological characterization included determination of the volume of the gel-

free portion in a measuring cylinder and estimation of the sperm concentration via photomet-

ric measurement (Sperma Cue, Minitübe, Tiefenbach, Germany). An aliquot of the raw semen

was snap frozen in liquid nitrogen and stored for chromatin integrity analysis using the sperm

chromatin structure assay (SCSA; Evenson and Jost, 2000). The ejaculates were diluted

EquiProTM (Minitübe, Landshut), and split into three portions. For both centrifugation meth-

ods, the concentration was adjusted to 1 x 109 sperm/ml. A volume of 20 ml was used for io-

dixanol density gradient centrifugation and 40 ml for routine centrifugation. Control samples

were further diluted with EquiProTM to a final concentration of 50 x 106 sperm/ml.


Iodixanol density gradient centrifugation (IODIX):

The protocol for iodixanol density centrifugation of extended stallion semen, was optimized

in preliminary studies (data not shown). Iodixanol of different densities was prepared as de-

scribed in detail in section 3.2.3.6. A 10 ml top layer with a density of 1.09 g/ml was added in

a 50 ml conical plastic tube, after which 10 ml bottom layer of 1.165 g/ml was pipetted un-

- 17 -

derneath the top layer. The extended semen was carefully layered on top of the iodixanol so-

lutions. After centrifugation at 1000 x g for 20 minutes, spermatozoa show up as a band be-

tween the two layers. The supernatant was removed by aspiration with a syringe connected to

a sterile disposable needle. When the band that contained the sperm was reached this was

transferred into a clean tube using a new syringe.

Routine centrifugation (CENTR):

Routine centrifugation was performed by centrifuging extended semen in a 50 ml tube at 600

x g for 10 minutes, after which the supernatant was removed by aspiration with a syringe

connected to a sterile disposable needle. The resuspended sperm pellet was transferred into a

clean tube.

General semen processing after centrifugation

After centrifugation, for the resuspended pellet the number of spermatozoa was determined

using a haemocytometer (Thoma Neu, Hecht, Sontheim, Germany) and the volume was

measured, in order to determine the total sperm number and calculate the recovery rate. The

resuspended sperm pellet was divided into two parts, and one part was used for storage at 5°C

up to 72 h, whereas the other part was used for cryopreservation.

For cooled-storage, the resuspended pellet was diluted with EquiProTM to a final concentra-

tion of 50 x 10 6 sperm/ml. Sperm morphology, motility parameters, plasma membrane integ-

rity, acrosome membrane integrity and chromatin integrity were assessed immediately, as

well as after 24 h, 48 h and 72 h of storage at 5°C.

For cryopreservation, the centrifuged samples were diltued with EquiProTM to a concentration

of 100 x 106 sperm/ml, after which they were diluted with an equal amount of freezing ex-

tender (EquiProTM containing 5 % glycerol). After two hours of equilibration at 5°C, the se-

men was packed in 0.5 ml straws, cooled from + 5°C to –15°C at 10°C/min and -15°C to –

140°C at 25°C/min in controlled rate freezer (IMV, L´Aigle, France). Straws were transferred

in liquid nitrogen, and stored there until further analysis. Thawing was done in a water bath at

37°C for 30 seconds, after which samples were analyzed for sperm motility and velocity,

morphology, plasma membrane integrity, acrosome membrane integrity and chromatin integ-

rity.

- 18 -

3.2 Sperm analysis for thesis 1 and 2

Sperm motility parameters, plasma membrane integrity (viability), acrosome membrane integ-

rity, and chromatin integrity were assessed prior to and directly after centrifugation protocols,

as well as during storage at 5°C (24h, 48h, 72h) and after dilution with freezing extender and

after freezing and thawing.

Sperm motility parameters were assessed via computer-assisted sperm analysis (CASA;

SpermVisionTM, Minitüb, Tiefenbach, Germany) using 20 µm analyzing chambers (20 mi-

cron, SC 20-01-04-B, Leja, GN Nieuw-Vennep, NL) as described by Nicolae (2006). Motility

parameters included: percentage of progressively motile sperm (PMS), curvilinear velocity

(VCL in µm/sec), velocity average- path (VAP in µm/sec) and the amplitude of lateral head

deviation (ALH in µm).

Morphology was evaluated for 200 eosin-nigrosin stained cells, according to Weitze (2001).

Sperm VitalStainTM was used for the eosin-nigrosin staining (Björndahl et al. 2004).

Plasma membrane integrity (PMI) and the positive acrosome membrane integrity (PAS) of

spermatozoa were evaluated using a flow cytometer (Cell Lab QuantaTM SC, Beckman Coul-

ter, Krefeld). Therefore, 485 µl HBS-solution was mixed with 5 µl sperm. This mixture was

stained with 2,5 µl propidium iodide (PI) and 7,5 µl fluorescein labeled peanut agglutinin

(FITC-PNA), and incubated for 15 min in the dark at +37°C, according to Ashworth et al.

(1995) and Thomas et al. (1997).

Chromatin integrity was assessed using SCSA-assay according to the protocols of Evenson

and Jost (2000, 2001). Using this method, a DNA fragmentation index (% DFI) can be deter-

mined that is indicative for chromatin integrity. For these measurements, a FACScan flow

cytometer was used (Becton-Dickson, Heidelberg).

3.3 Statistical analysis for thesis 1

The analysis of fixed and random effects and their interactions were tested with the GLM

procedure using statistical software SAS (SAS, 2008). Residuals were tested for normality

using UNIVARIATE procedure. The centrifugation methods were used as a fixed effect (four

classes) whereas the stallion (six classes) and the ejaculate number (three classes) were set as

- 19 -

random effects. Differences in the means of the four sperm treatment methods were separated

using Tukey test comparisons (Tukey-Kramer multiple range test, Zhou, 2001). Data were

taken to be statistically significant when (p≤0.05). Correlations were analyzed with Pearson

correlation coefficient.

3.4 Statistical analysis for thesis 2

Statistical analysis was done using SAS software (SAS, 2008), using the GLM procedure. The

residuals were tested for normality using the UNIVARIATE procedure. Fixed effects were

the three different semen preparation methods (IODIX, CENTR and EXTEN), whereas ran-

dom effects were stallion (six classes) and ejaculate (three classes). For cryopreserved sam-

ples, the fixed effects were the two groups “poor freezers” and “good freezers”. Differences

were considered significant at a probability level of (p≤0.05). Correlations were analyzed with

Pearson correlation coefficient. Differences in means were detected using Tukey test com-

parisons, according to Zhou (2001).

- 20 -

3.5 EquiPureTM Pro centrifugation, a method for sperm clean-up and its

effects on sperm quality of cold- stored and frozen- thawed stallion

sperm

3.5.1 Abstract

The objective of the present study was to evaluate semen quality parameters of stallion sper-

matozoa that were obtained after different centrifugation methods, which were applied to se-

lect sperm of superior quality, and either subjected to cold-storage or freezing and thawing.

Sperm quality parameters that were evaluated included progressive motility, velocity parame-

ters, plasma membrane integrity, acrosomal status and chromatin integrity. In the first ex-

periment, semen was processed using three different centrifugation methods, density gradient

centrifugation using EquiPureTM Pro, cushion centrifugation using OptiPrep and a centrifuga-

tion procedure without a sperm-selective medium. In the second experiment, post- thaw

sperm quality parameters were determined for sperm that was selected using EquiPureTM Pro

and frozen after dilution with different ratios of freezing extender.

After selection of sperm via centrifugation with EquiPureTM Pro significantly higher percent-

ages of progressively motile sperm were found. This treatment also resulted in higher per-

centages of plasma membrane intact sperm, reduced percentages of sperm with a positive

acrosome membrane status and lower percentages of sperm with DNA fragmentation as com-

pared to sperm that were not centrifuged (p≤0.05). Sperm quality of cold-stored spermatozoa

was improved when semen was processed by EquiPureTM Pro procedure (p≤0.05).

A reduced percentage (p≤0.05) of sperm with positive acrosomal status and lower DFI values

(p≤0.05) were found for sperm that was frozen and thawed after EquiPureTM Pro centrifuga-

tion as compared to the two other centrifugation treatments. Furthermore, higher ratio of

freezing extender to EquiPureTM Pro treated sperm resulted in better sperm quality (p≤0.05)

after freezing and thawing.

In conclusion, density gradient centrifugation using EquiPureTM Pro results in improved

sperm quality compared to cushion centrifugation, a centrifugation procedure without sperm-

selective medium or no centrifugation after dilution in extender. This selective centrifugation

- 21 -

procedure also improved sperm quality during cold-storage and sperm quality after freezing

and thawing.

3.5.2 Introduction

In equine breeding industry, semen is predominantly transported chilled or frozen (Aurich and

Aurich 2006), because it allows for insemination of mares independent of location and avail-

ability of stallions. This development, however, is accompanied by a decrease in pregnancy

rates (Morrell et al. 2009c). The reduced pregnancy rates when using chilled or cryopreserved

stallion sperm might be caused by high variability in semen quality amongst stallions. In con-

trast to bulls and boars, stallions have not been selected for semen quality parameters and fer-

tility (Loomis 2006, Colenbrander et al. 2003).

Sperm viability during storage is improved by reducing the proportion of seminal plasma pre-

sent in the sample (Jasko 1992, Brinsko 2000). This is achieved by centrifugation directly

after the initial dilution in extender, removal of the supernatant that contains seminal plasma,

and resuspension of the sperm pellet in fresh extender. Such a semen preparation procedure,

however, does not select sperm sub-populations of superior quality.

In human assisted reproduction sperm selection techniques are used to separate the motile,

morphologically normal sperm for AI, IVF or ICSI (Mortimer, 2000). Density gradient cen-

trifugation is most commonly used for human sperm preparation and selection (Smith et al.

1997, Claassens et al. 1998, Henkel and Schill, 2003, Mousset-Simeon et al. 2004). Use of

sperm selected via density centrifugation for human assisted reproduction results in improved

fertility rates (Tomlinson et al. 2001). When using a commercially available acidic and basic

formulations of EquiPureTM for processing of stallion semen MacPherson et al. (2002) found

an improvement of stallion sperm quality as compared to standard processing protocols.

In this study, we tested EquiPureTM Top Layer and Bottom Layer (Nidacon International AB,

Sweden) as novel density gradient centrifugation solutions for processing of stallion semen,

with the aim to separate the subpopulation of motile, viable, morphological normal spermato-

zoa from the immotile, dead sperm, sperm with damaged chromatin, the seminal plasma and

microorganisms. The EquiPureTM Pro that was used in the current study contains recombinant

protease in the top layer, which is thought to have a positive influence on the enzyme system

- 22 -

of the spermatozoa (Pattinson et al. 1990, Figenschau and Bertheussen, 1999, Westhoff and

Kamp, 1997), which in turn is thought to improve motility and fertility.

We evaluated the selection efficiency of the novel double layer formulation EquiPureTM Pro,

and compared it with other sperm preparation methods. Four preparation techniques were

compared: i.) density gradient centrifugation with EquiPureTM Pro, ii.) high- speed cushion

centrifugation (Revell et al.1997, Escot et al. 2005, Sieme et al. 2006), iii.) centrifugation

without sperm-selective medium, and iv.) semen preparation without centrifugation by dilu-

tion. For each of the preparation technique, recovery rates were determined as well as sperm

quality parameters after storage for 72h at 5°C and freezing and thawing. Sperm quality pa-

rameters that were assessed included sperm motility and velocity, plasma membrane integrity,

acrosomal status, and chromatin integrity. In addition, sperm quality parameters were deter-

mined for semen that was processed using EquiPureTM Pro and frozen after dilution with dif-

ferent ratios of freezing extender.

3.5.3 Materials and Methods

In the first experiment, the effect of selective centrifugation methods on sperm quality pa-

rameters was tested. Sperm quality was assessed after cooled-storage as well as after freezing

and thawing. In the second experiment, the effects of varying freezing extender versus Equi-

PureTM Pro ratios on survival after freezing and thawing were analyzed.

3.5.3.1 Animals and semen collection

In the first experiment, three ejaculates were collected from four warmblood horses (4-7 years

old) and two riding ponies (9 years old) that participated in the AI-program of the State Stud

of Lower Saxony in Celle, Germany. Semen collections were done every other day during the

2008 breeding season. In the second experiment, two ejaculates were collected from each of

two warmblood stallions.

Semen collections were performed using an artificial vagina (Hanover model, Klug 1993)

(Minitüb, Landshut, Germany), a phantom (Model “Celle” Klug 1993) and a teaser mare. Ste-

rile gauze filter was performed to remove the gel-portion.

- 23 -

3.5.3.2 General semen processing

The volume of gel-free semen was measured using a graduated cylinder, and the sperm con-

centration was determined using a photometer (Sperma Cue; Minitüb, Tiefenbach, Germany).

Aliquots of raw semen were immediately snap-frozen in liquid nitrogen for later analysis of

the chromatin integrity via the sperm chromatin structure assay (SCSA, Evenson and Jost,

2000).

An aliquot of gel-free raw semen was diluted with INRA 82 to a sperm concentration of 25 x

106 cells/ml for direct analysis of sperm motility and velocity parameters, and plasma mem-

brane integrity, and acrosomal status. For centrifugation processing, three 30 ml samples were

prepared of 30 x 106 cells/ml in INRA 82. Centrifugation protocols are described in detail

below.

3.5.3.3 First experiment


EquiPureTM Pro centrifugation: (EPP)

The protocol described by Nidacon International AB was used in this study, with modifica-

tions. The volumes of the top and the bottom layers and extended semen were adjusted, as

well as the force and time for centrifugation, this was done based on preliminary studies. Ten

ml of bottom layer was added in a 50 ml conical plastic tube, after which 10 ml of top layer

was carefully added on top. Finally, 30 ml diluted semen was placed on top. Centrifugation

was done for 15 min at 330 x g.

Centrifugation without sperm selective medium: (WSSM)

Thirty ml semen diluted at 30 x 106 cells/ml in INRA82 was added in a 50 ml centrifugation

tube without sperm-selective medium. Centrifugation was performed for 15 min at 330 x g.

Cushion centrifugation: (CC)

OptiprepTM (Progen, Heidelberg, Germany) was used as a cushion fluid. Thirty ml semen di-

luted at 30 x 106 cells/ml in INRA82 was added to a 50 ml plastic tube, and 2 ml of cushion

fluid was layered underneath using a syringe with a sterile needle (2.00 X 80 mm). Centrifu-

gation was done for 20 min at 1000 x g according to Sieme et al. (2006).

- 24 -

Further semen processing after centrifugation:

After centrifugation with either EquiPureTM Pro or WSSM, the supernatant was aspirated,

leaving a pellet of approximately 2 ml. Following CC, the supernatant was aspirated and the

cushion fluid was removed separately with a syringe. In all cases, the pellet was transferred

into a clean tube, using a syringe with a sterile needle.

For all three centrifugation preparations, the sperm number in the pellet was determined using

a Thoma Neu counting chamber. The recovery rate was calculated by dividing the cell num-

ber recovered after centrifugation with the original cell number before centrifugation. The

concentrated sperm solution obtained after each of the centrifugation preparation, was divided

into two parts.

One part was diluted with INRA 82 to a final concentration of 25 x 106 sperms/ml and stored

at + 5°C up to 72h. The other part was diluted with INRA 82 containing 5.0 % egg yolk and

glycerol, resulting in a final concentration of 2.5 % glycerol and 100 x 106 sperms/ml. After

dilution in freezing extender, samples were equilibrated for two hours at +5°C and then pack-

aged in 0.5 ml straws that were cooled from + 5°C down to –140°C at 60°C/min in a auto-

mated cell freezer (IMV, L´Aigle, France) and stored in liquid nitrogen until further analysis.

Thawing was done in a water bath at 37°C for 30 seconds.

3.5.3.4 Second experiment

Further semen preparation after centrifugation:

The EquiPureTM Pro centrifugation protocol and recovery of the sperm pellet was similar as

described for the first experiment.

After estimation of the sperm concentration, the concentrated sperm solution obtained after

EquiPureTM Pro centrifugation was split into four equal parts. One part was diluted with

INRA 82 containing 5.0 % egg yolk and 2.5 % glycerol (freezing extender) to 100 x 106

sperms/ml, according to the first experiment. The other parts were diluted with freezing ex-

tender at a 1:1, 1:2 or 1:3 ratio (extended semen : freezing extender).

3.5.3.5 Sperm analysis

Sperm motility parameters, plasma membrane integrity, acrosomal status, and chromatin in-

tegrity were assessed prior to and directly after centrifugation protocols, as well as during

- 25 -

storage at 5°C (24h, 48h, 72h) and after dilution with freezing extender and after freezing and

thawing.

Sperm motility parameters were assessed via computer-assisted sperm analysis (CASA) using

a SpermVisionTM motility analyzer (Minitüb, Tiefenbach, Germany) and 20 µm analyzing

chambers (20 micron, SC 20-01-04-B, Leja, GN Nieuw-Vennep, NL) (Nicolae 2006). In ad-

dition to the percentage of progressively motile sperm, curvilinear velocity (VCL in µm/sec),

average path velocity (VAP in µm/sec) and the amplitude of lateral head deviation (ALH in

µm/sec) were analyzed.

Plasma membrane integrity and sperm acrosome status were evaluated using a flow cytometer

(Cell Lab QuantaTM SC, Beckman Coulter, Krefeld). Therefore, 485 µl HBS-solution was

mixed with 5 µl sperm. This mixture was stained with 2,5 µl propidium iodide (PI) and 7,5 µl

fluorescein labeled peanut agglutinin (FITC-PNA), and incubated for 15 min in the dark at

+37°C, according to Ashworth et al. (1995) and Thomas et al. (1997).

Chromatin integrity was evaluated using the SCSA-assay according to the protocols of Even-

son and Jost (2000, 2001). Using this method, a DNA fragmentation index (% DFI sperm) can

be determined that is indicative for chromatin integrity. For these measurements, a FACScan

flow cytometer was used (Becton-Dickson, Heidelberg).

3.5.3.6 Statistical analysis

The analysis of fixed and random effects and their interactions were tested with the GLM

procedure using statistical software SAS (SAS, 2008). Residuals were tested for normality

using UNIVARIATE procedure. The centrifugation method was used as a fixed effect (four

classes) whereas the stallion (six classes) and the ejaculate number (three classes) were set as

random effects. Differences in the means of the four sperm treatment methods were separated

using Tukey test comparisons (Tukey-Kramer multiple range test, Zhou, 2001). Data were

taken to be statistically significant when (p≤0.05). Correlations were analyzed with Pearson

correlation coefficient.

- 26 -

3.5.4 Results

First Experiment: Fresh semen samples

In all three centrifugation treatments a positive correlation between the raw semen sperm con-

centration and the recovery rate after centrifugation was identified (EPP: r = 0.59, CC: r =

0.51 and WSSM: r = 0.68). Semen yield was highest after CC (95.5 %) compared to WSSM

(74.0 %) and EPP centrifugation (26.6 %) (p<0.0001).

EPP treatment increased the percentage of progressively motile (p<0.0001) and plasma mem-

brane intact sperm (PMI, PI neg) (p<0.0001) while reducing the percentage of sperm with

positive acrosomal status (PAS, FITC-PNA pos) (p<0.0002) and the DFI values (p<0.0001)

compared to diluted non-centrifuged semen and semen that was centrifuged without sperm-

selective medium or with cushion.

- 27 -

Table 1: Effect of EquiPureTM Pro density gradient, cushion centrifugation and centrifugation without

sperm-selective medium on sperm quality parameters, as compared to diluted semen (means ± standard

deviation).

Centrifugation techniques

DS

25 x 106

sperm/ml

EPP

330 x g, 15

min

CC

1000 x g,

20min

WSSM

330 x g, 15

min

Sperm recovery - 26.6±11.1a 95.5±4.4b 74.0±11.1c

PMS 60.3±13.1a 80.1±5.7b 59.8±12.2a 59.3±12.0a

VCL 203.9±29. 213.9±29.3 223.1±30.6 223.8±30.9

VAP 131.1±27.3 138.7±19.4 127.0±21.8 125.9±21.4

ALH 3.9±0.5a 3.6±0.5a 4.6±0.5b 4.6±0.5b

PMI (PI neg) 69.7±11.2a 83.0±4.4b 70.4±11.4a 71.5±12.0a

PAS (FITC-PNA pos) 11.5±4.3a 4.8±1.6b 11.6±3.9a 10.3±3.6a

SCSA/DFI 10.5±6.0a 4.0±2.0b 11.2±8.4a 11.7±9.5a

(n=6 stallions per group; 3 ejaculates/stallion).

DS: diluted-non-centrifuged semen

EPP: EquiPureTM Pro centrifugation

CC: cushioned centrifugation

WSSM: centrifugation without sperm-selective medium

Sperm recovery: in %

PMS: progressively motile sperm (%)

VCL: curvilinear velocity (µm/sec)

VAP: average path velocity (µm/sec)

ALH: amplitude of lateral head displacement (µm/sec)

PMI: percentage of plasma membrane intact sperm (PI neg)

PAS: Percentage of sperm with positive acrosomal status (FITC-PNA pos)

DFI: denaturation fragmentation index of sperm DNA (%) a, b, c Values with different superscript differ significantly within rows (p≤0.05).

- 28 -

First Experiment: Cooled-stored semen samples

In Figure 3 to Figure 5 are shown the percentages of progressively motile, plasma membrane

intact sperm (PI neg) and sperm with positive acrosomal status (FITC-PNA pos) during stor-

age at 5°C for up to 72h of sperm obtained after the different preparation procedures.

Upon 72 hours of cooled-storage, spermatozoa prepared by EPP centrifugation showed a

higher percentage of progressively motile, plasma membrane intact sperm and a reduced per-

centage of sperm with positive acrosomal status compared to CC, WSSM and DS (p<0.0001).

After 48 h cooled storage, a higher percentage of progressively motile sperm and lower per-

centage of sperm with positive acrosomal status could be observed in semen samples prepared

by CC, WSSM compared to diluted non-centrifuged semen (p≤0.05).

Upon storage for 72 h, sperm VAP was found to be higher (p≤0.05) for the EPP prepared se-

men than the other treatments. For semen samples centrifuged with EPP slightly higher VCL

values were also found after 24 h compared to diluted semen, although differences between

the four treatments methods were after 48h and 72 h (p≥0.05). Furthermore, in comparison

with the velocity parameters determined direct after centrifugation (0h), semen treated with

any of the three centrifugation methods showed higher VCL as well as VAP values after 24 h.

After 24 h, ALH in the CC and WSSM samples was different (more head movement) from

diluted and EPP prepared semen (p≤0.05). Furthermore, significant differences could be ob-

served after 48 h of storage time between the semen samples treated by EPP and CC, as well

as between DS and EPP treated semen after 72 h (less movement for EPP treated semen).

- 29 -

Figure 3: Effect of preparation method on the percentage of progressively motile sperm (PMS in %) for semen samples that were stored at 5°C, for up to 72 h. Means ± standard deviation are shown for n=6 stallions per group, 3 ejaculates/stallion, DS: Diluted-non-centrifuged semen, EPP: EquiPureTM Pro cen-trifugation, CC: cushioned centrifugation, WSSM: centrifugation without sperm-selective medium,

a, b, c Values with different superscript differ significantly within days (p≤0.05).

Figure 4: Effect of preparation method on the percentage of plasma membrane intact sperm (PMI neg) (%) for semen samples that were stored at 5°C, for up to 72 h. Means ± standard deviation are shown for n=6 stallions per group, 3 ejaculates/stallion, DS: Diluted-non-centrifuged semen, EPP: EquiPureTM Pro centrifugation, CC: cushioned centrifugation, WSSM: centrifugation without sperm-selective medium centrifugation,


- 30 -

Figure 5: Effect of preparation method on the sperm with positive acrosomal status (FITC-PNA pos in %) for semen samples that were stored at 5°C, for up to 72 h. Means ± standard deviation are shown for n=6

stallions per group, 3 ejaculates/stallion, DS: Diluted-non-centrifuged semen, EPP: EquiPureTM Pro cen-trifugation, CC: cushioned centrifugation, WSSM: centrifugation without sperm-selective medium,


First Experiment: After resuspension with freezing extender

The semen quality of samples before freezing after dilution with freezing extender was as-

sessed. Progressively motility, ALH, plasma membrane and chromatin integrity were not dif-

ferent for any of the three centrifugation methods. Only semen centrifuged using EPP showed

a higher percentage of sperm with a positive acrosomal status after dilution in freezing ex-

tender (p≤0.05). In all semen samples VCL was reduced after dilution in freezing extender

(p≤0.05).

First Experiment: Frozen–thaw semen samples

Sperm quality parameters that were determined after freezing and thawing are shown in Table

2. For sperm that was selected using centrifugation with EPP reduced DFI values were found,

similar as before freezing. The percentage of plasma membrane intact and motile cells, how-

ever, were lower after freezing and thawing using EPP. WSSM also resulted in a lower per-

centage of plasma membrane intact sperm as compared to CC (p≤0.05).

- 31 -

Table 2: Effect of EquiPureTM Pro density gradient, cushion centrifugation and centrifugation without

sperm selective medium on frozen-thawed sperm quality parameters (means ± standard deviation).


DS: Diluted-non-centrifuged semen









PAS: percentage of sperm with positive acrosomal status (FITC-PNA pos)



EPP

330 x g

15 min

CC

1000 x g, 20min

WSSM

330 x g

15 min

PMS 9.7±5.0a 29.3±11.4b 25.2±9.0b

VCL 105.5±19.3a 131.8±24.8b 131.1±22.6b

VAP 77.9±13.1a 82.8±13.2a 83.9±14.1a

ALH 2.4±0.5a 3.8±0.3b 3.9±0.2b

PMI (PI neg) 29.0±8.3a 41.5±7.9b 32.1±5.7a

PAS (FITC-PNA pos) 32.5±12.5a 21.2±4.6b 23.1±6.1b

SCSA/DFI 3.1±1.6a 13.6±9.0b 14.1±8.1b

- 32 -

Second experiment: Fresh semen samples

As described for experiment 1 after centrifugation with EquiPureTM Pro, progressive motility

and plasma membrane intact sperm were increased, whereas the percentage of sperm with

positive acrosomal status and the DFI values were reduced compared to the diluted non-

centrifuged semen. However, neither these differences nor the changes in sperm velocity pa-

rameters were significant (p≥0.05).

Second experiment: Frozen–thaw semen samples

After thawing, a stepwise increase in progressive motility and plasma membrane integrity was

seen with different amounts of freezing extender (p≤0.05) (Table 3).

The % DFI values of EquiPureTM Pro treated spermatozoa after thawing were similar to the

results of the fresh semen samples independent of the dilution ratio of freezing extender.

Table 3: Effects of varying quantities of freezing extender on frozen-thawed sperm quality parameters of

EquiPureTM Pro treated semen (means ±standard deviation).


Diluted to

100 x 106

Diluted 1:1 Diluted 1:2 Diluted 1:3

PMS 19.3±9.8a 25.6±10.0a.b 27.7±10.9a.b 35.3±11.0b

VCL 81.2±7.3 87.7±5.3 91.3±5.9 97.6±7.7

VAP 54.5±12.1 62.0±10.1 63.9±7.4 70.8±13.3

ALH 2.8±0.3 2.6±0.1 2.5±0.3 2.6±0.3

PMI (PI neg) 39.9±8.3a 49.4±5.3b 51.4±9.9b 52.7±6.9b

PAS (FITC-PNA pos) 19.1±11.3 12.4±6.5 18.1±13.4 13.7±7.6

SCSA/DFI 2.5±0.7 2.7±1.1 2.4±1.1 2.7±1.4


DS: diluted-non-centrifuged semen






- 33 -



3.5.5 Discussion

Density gradient centrifugation is the most commonly used sperm selection technique to im-

prove the fertility rate of human spermatozoa. Also for stallion sperm, an improvement in

quality could be achieved by density gradient centrifugation. In this study a modification of

the original EquiPureTM Pro density gradient centrifugation protocol developed by Nidacon

International AB, Sweden was used. According to the original protocol only small volumes of

extended semen can be prepared, which makes the use in a commercial AI program difficult.

Apart from the amount of labor involved, dividing the ejaculate into small volumes would

lead to further sperm loss during the centrifugation process. The sperm yield and quality after

density gradient centrifugation is influenced by the structure of the gradient itself (density and

volume of the solution), the extender, the sperm load, the time and force of centrifugation

(Smith et al. 1997, Morrell, 2006a, Morrell et al. 2009a). The extender for dilution of semen

that was used in this study (INRA82) was shown to work well for centrifugation with Equi-

PureTM. This observation is in agreement with previous studies (Edmond et al. 2008).

In the equine breeding industry most semen processing procedures of cooled-stored and espe-

cially frozen-thawed insemination doses include centrifugation to reduce the amount of semi-

nal plasma (Graham, 1996, Brinsko et al. 2000, Moore et al. 2005). Since centrifugation can

have detrimental effects on the spermatozoa it is performed using low forces and short dura-

tion (Pickett et al. 1975), resulting in an increased loss of spermatozoa. In the present study a

high rate of sperm loss was observed during WSSM. The recovery rates after all three cen-

trifugation methods corresponded to the findings of previous studies (Delhomme et al. 2004,

Macias Garcia et al. 2009a, Sieme et al. 2006). The highest recovery rate was found after CC,

a method that aims to increase the sperm yield by high-speed (1000 x g) centrifugation. By

layering the semen samples on the cushion fluid and avoiding the formation of a pellet at the

bottom of the centrifugation tube, centrifugation can be performed at high- speed without the

deleterious effects on spermatozoa (Revell et al. 1997, Sieme et al. 2003, Matas et al. 2007,

Waite et al. 2008). Bliss et al. (2010) did not observe detrimental effects on the spermatozoa

using up to 3 billion sperm and only 1 ml of cushion fluid in 50 ml conical tubes.

- 34 -

In the present study, EPP density gradient centrifugation produced the lowest recovery rate.

The reduced sperm number after centrifugation is based on the selection process. EquiPureTM

Pro selects the motile, morphological normal sperm while other subpopulations such as dead,

immature, senescent and morphologically abnormal, with damaged chromatin were elimi-

nated from the ejaculate (MacPherson et al. 2002, Morrell, 2006a, Morrell et al. 2009a,b,c,d).

As consequence, the sperm yield after treatment is also affected by the quality of the original

ejaculate (Morrell et al. 2009a). Furthermore, the sperm yield was positively correlated to the

sperm concentration in the raw ejaculate. Similar results were reported for other spermatozoa

selective techniques like swim-up, Percoll density gradient centrifugation and glass wool fil-

tration (Sieme et al. 2003). They found higher correlations between the raw semen concentra-

tion and the yield after Percoll gradient filtration compared to the correlations in the present

study.

In accordance to previous studies, density gradient centrifugation with EquiPureTM Pro led to

an increased semen quality compared to CC or WSSM in term of progressive motility, plasma

membrane and chromatin integrity. MacPherson et al. (2002) and Morrell et al. (2009a,c)

demonstrated improvements in sperm motility and morphology by centrifugation through

siliane coated silica particles.

Reports about the improvement of semen quality by CC are contradictory. Waite et al. (2008)

found no significant differences in total motility after cushion centrifugation and significantly

lower values for VCL, VAP and VSL compared to the control samples. Matas et al. (2007)

observed a lower percentage of progressively motile (p≥0.05) and motile (p≤0.05) boar sperm

after cushion and standard (800 x g, 10 min) centrifugation compared to the pre-centrifuged

semen samples.

In accordance with Morrell et al. (2009a,c) and Johannisson et al. (2009) selection of sper-

matozoa of superior quality, and improved sperm viability upon cooled storage. Better semen

quality of selected sperm cells could not just be explained by better semen quality immedi-

ately after centrifugation. After density gradient centrifugation, the percentage of progres-

sively motile and plasma membrane intact sperm decreased to a lesser extent during cooled

storage. Furthermore, the increase in sperm with positive acrosomal status was less distinct

compared to CC, WSSM or DS. These results confirmed the observations of Smith et al.

- 35 -

(1997) who demonstrated a positive effect on sperm quality during 24 hours of cooled storage

due to pre-selection of spermatozoa. The prolonged good sperm quality after EquiPureTM Pro

centrifugation might be caused by the removal of seminal plasma. Seminal plasma is com-

posed of debris, leukocytes, epithelial cells and microbial contamination, that are sources of

reactive oxygen species (ROS) and have detrimental effects on the intracellular structure and

chromatin integrity (Baumber et al. 2003, Perez-Crespo et al. 2008,). Love et al. (2005) de-

scribed a reduction of motility and a significant increase in DFI values with increasing

amounts of seminal plasma (0, 10, 20 %). Stallion fertility correlates with sperm chromatin

integrity (Love et al. 1998, Love, 2005). In the present study, EquiPureTM Pro treatment led to

a drastic decrease in sperm with high DFI values directly after centrifugation as well as after

freezing and thawing. These results support the observations of Mancill et al. (2010) and Stoll

et al. (2010), who demonstrated significantly increased post-thaw chromatin quality in prese-

lected semen samples. The second experiment of the study, using different amounts of freez-

ing extender revealed no influence of the amount of freezing extender on chromatin integrity.

EPP selected spermatozoa showed reduced progressive motility, velocity, plasma membrane

integrity and an increase in acrosomal damage after freezing and thawing compared to CC

and WSSM. These findings are contradictory to experiences in human assisted reproduction.

Freezing of spermatozoa after density gradient centrifugation (Centola et al. 1998) is a routine

technique in semen preparation for ART, before cancer therapy or before vasectomy (Allama-

neni et al. 2005). Furthermore, studies of Morrell (2006a) showed better results with regard to

motility for selective sperm after thawing compared to non-selected stallion spermatozoa.

Mancill et al. (2010) evaluated frozen-thawed sperm quality after gradient centrifugation of

sperm from stallions that differed in fertility. When density gradient centrifugation was per-

formed before freezing, there were no difference between controls and treatments for sper-

matozoa of fertile stallions. In contrast, frozen-thawed spermatozoa from infertile stallions

had higher progressive motility, normal morphology, plasma membrane integrity and less

sperm with positive acrosomal status and better chromatin integrity when gradient centrifuga-

tion was performed before freezing. The freezing extender used in this study was INRA 82

supplemented with 5 % egg yolk and 2.5 % glycerol.

Glycerol is the most frequently used cryoprotective agent in equine sperm freezing. A concen-

tration of 2.5 -5 % led to the best post-thaw results (Samper and Morris, 1998). Egg yolk also

- 36 -

serves as a cryoprotectant, that is suggested to protect the sperm plasma membrane during

cryopreservation (Jasko et al. 1992, Crockett et al. 2001). In the present study, the concentra-

tion of the glycerol was calculated as percentage of the end-volume and was the same for all

samples, whereas the egg yolk percentage was dependent on the dilution. The effective com-

ponent in egg yolk that is thought to have a role in the protection of cellular membranes is

lecithin (Quinn et al. 1980). Lecithin can bind to membrane proteins, therewith providing pro-

tection during the cryopreservation procedure. Freezing extender can only provide its protec-

tive function if it is added to the sperm pellet prior to freezing in adequate amount. As men-

tioned above, the sperm yield recovered after EquiPureTM Pro treatment was lower than after

cushion centrifugation and centrifugation without sperm selective medium. After the centrifu-

gation step, semen was diluted to 100 x 106 sperms/ml for all three centrifugation procedures.

Due to low recovery rate after density gradient centrifugation the proportion of freezing ex-

tender that was added to achieve this concentration was much lower than for the other two

methods. The lower amount of freezing extender in the density gradient samples could ex-

plain the decreased post-thaw sperm quality in the first experiment. In the second experiment,

the effects of varying freezing extender versus EquiPureTM Pro ratios on semen quality after

freezing and thawing were analyzed. This experiment confirmed that increasing the amount of

freezing extender did result in an increase in the percentage of post-thaw progressive motility

and velocity and plasma membrane integrity of spermatozoa.

Difference in frozen-thawed sperm quality could also be associated with differences in the

removal of seminal plasma achieved by the three centrifugation methods used in this study.

Seminal plasma consists of components that can have positive (Mann, 1975) or negative in-

fluences on sperm quality parameters (Pickett et al. 1975). Semen preparations for cooled-

stored and frozen semen generally involve the reduction of seminal plasma to attenuate the

negative influence, such as ROS (Rajamannan et al. 1968, Amann et al. 1987, Brinsko et al.

2000, Moore et al. 2005, Loomis et al. 2006, Aurich, 2008). CC and WSSM reduce the

amount of seminal plasma, whereas the entire seminal plasma is removed when using Equi-

PureTM Pro treatment. For storage up to 24 h at 5°C, sperm quality was found to be improved

when samples contained 5-10% seminal plasma (Jasko et al., 1992). Ahlemeyer (1991) de-

termined optimum post-thaw sperm quality when samples contained 5% seminal plasma,

whereas more than 10-15% and less than 2% seminal plasma resulted in a decreased sperm

- 37 -

quality. Amann and Pickett (1987) recommended reducing the supernatant by 90% after cen-

trifugation and Moore et al. (2005) considered retention of 5-20% seminal plasma to be essen-

tial for survival the freezing procedure.

Apart from the observed influences of EquiPureTM Pro treatment on sperm motility parame-

ters and membrane and chromatin integrity, the effects of the serine protease treatment on

fertility needs investigation. Blevins et al. (2008) added trypsin to BoviPure and demonstrated

increased fertilization rates (88.4% to 63.1%) and a significantly higher numbers of embryos

that qualified for use for transfer (70.3 to 51.8%) as compared to the standard method.

In conclusion, the modified EquiPureTM Pro density gradient centrifugation protocol used in

this study allows for processing of large volumes of extended semen. This is essential for its

routine use in commercial AI programs. The protocol is an effective method for selecting

spermatozoa of superior quality with higher chromatin integrity and prolonged good sperm

quality during storage up to 72 h +5°C, as compared to cushion centrifugation, centrifugation

without sperm-selective medium or non-centrifuged semen samples. The study also demon-

strated positive effects of increasing proportions of freezing extender on frozen-thawed sperm

quality.

- 38 -

3.6 Sperm quality parameters of stallion spermatozoa prepared by iodixa-

nol sedimentation density gradient centrifugation of fresh, cooled-

stored and frozen-thawed semen

3.6.1 Abstract

The present study evaluated sperm quality parameters for stallion semen that was prepared via

iodixanol density gradient centrifugation (IODIX), or routine centrifugation (CENTR), as

well as for semen that was extended using EquiPro (Minitüb) (EXTEN). Sperm quality pa-

rameters that were evaluated included sperm motility and morphology, plasma membrane

integrity, acrosomal status and chromatin integrity. They were examined in samples on the

day of semen collection, during storage at 5°C for up to 72 h and after cryopreservation. Se-

men was collected from stallions known as „poor freezers“ (n=3) and „good freezers“ (n=3).

After IODIX centrifugation a recovery rate of 33.1% ± 10.9 was determined, and after

CENTR a recovery rate of 74.4 % ± 15.6 (p≤0.05). Directly after IODIX centrifugation, the

samples showed a higher progressively motility (PMS), higher percentage of plasma mem-

brane intact cells (PMI), fewer morphological abnormal spermatozoa (MAS), lower percent-

age of sperm with positive acrosomal status (PAS) and higher chromatin integrity (p≤0.05), as

compared to semen that was extended (EXTEN) or subjected to the routine centrifugation

protocol (CENTR). Sperm selected using IODIX exhibited better sperm quality (progressive

motility, velocity parameter, plasma membrane integrity, and positve acrosomal status) upon

storage at +5°C for up to 72 h, as compared to CENTR or EXTEN. Furthermore, post-thaw

sperm quality (progressive motility, plasma membrane integrity, positive acrosomal status and

chromatin integrity) was better (p≤0.05) when semen was prepared using IODIX instead of

CENTR.

In conclusion, IODIX density gradient centrifugation selects spermatozoa of superior quality,

whose quality is improved during storage at +5°C and after cryopreservation as compared to

CENTR or EXTEN semen.

- 39 -

3.6.2 Introduction

The increased use of AI has demanded the development of semen preparation methods which

provide sperm of good quality for insemination (Loomis, 2001, Aurich, 2008).

Sperm selective methods are used to provide better semen quality and minimize variation be-

tween ejaculates from the same as well as different stallions. Selection methods include: (i)

simple washing via centrifugation and resuspension of the sperm pellet in fresh extender, (ii)

adherence procedures like glass-wool, glass-beads, Sephadex and Leucosorb, (iii) sperm mi-

gration methods, such as the swim-up method, and (iv) density gradient centrifugation (for

review see Sieme et al. 2003, Mortimer, 2000). Iodixanol is one of the latter solutions intro-

duced for density gradient centrifugation in human reproduction (Harrison, 1997; Smith et al.

1997). Iodixanol is a non-ionic solution with high density (1.32 g/ml) and can be used for

different applications. Iodixanol has been used for high-speed cushion centrifugation of stal-

lion semen (Sieme et al. 2006). Whereas density centrifugation protocols exist for selecting

sperm with superior quality for bulls and humans (Smith et al. 1996; Smith, et al. 1997; Kaf-

tani, et al. 1997; Revell et al. 1997; Harrison, 1997; Van den Bergh et al. 1999) its use for

selection of stallion sperm has not been evaluated. When using iodixanol, motile, morpho-

logical normal spermatozoa will be layered within the iodixanol, instead of pelleted at the

bottom of the tube as is the case when using other density gradient protocols such as Percoll,

EquiPureTM and AndrocollTM. This avoids the deleterious effects on sperm quality caused by

pelleting of spermatozoa during centrifugation.

The purpose of this study was to determine the sperm selection efficiency of iodixanol density

gradient centrifugation, and its effects on sperm quality during storage at 5°C up to 72 h and

cryopreservation. Sperm quality parameters that were assessed included progressive motility,

sperm velocity, morphology, plasma membrane integrity, acrosomal status and chromatin

integrity.

- 40 -

3.6.3 Materials and Methods

3.6.3.1 Animals and semen collections

Three ejaculates were collected from each of six warmblood stallions (5 to 11 years old), that

participated in the AI-program of the National Stud of the State of Lower Saxony in Celle,

Germany. Prior to the onset of the study, semen was collected over a period of one week to

stabilize the extragonadal sperm reserves. The study was performed at the beginning of the

breeding season 2009.

Semen collections were done every other day, using an artificial vagina (Hanoverian model;

Minitüb, Landshut, Germany). A phantom was used, and a teaser mare was fixed in front of

the phantom. The collection device was equipped with a sterile gauze filter to remove gel.

For the second experiment, in which semen processed as described above was used for cryo-

preservation, two groups of stallions were used that differed in freezeability. Stallions were

divided into “good” and “poor” freezers (n=3 per group) according to post-thaw motility prior

to the current experiment: stallions with progressive post-thaw sperm motility ≤ 35 % in more

than 3 out of 10 ejaculates were classified as “poor freezers” and stallions above these criteria

were assigned to the “good freezers” group.

3.6.3.2 General semen processing before centrifugation

Standard spermatological characterization included determination of the volume of the gel-

free portion in a measuring cylinder and estimation of the sperm concentration via photomet-

ric measurement (Sperma Cue, Minitüb, Tiefenbach, Germany). An aliquot of the raw semen

was snap frozen in liquid nitrogen and stored for chromatin integrity analysis using the sperm

chromatin structure assay (SCSA; Evenson and Jost, 2000). The ejaculates were diluted with

EquiProTM (Minitüb, Landshut), and split into three portions. For both centrifugation meth-

ods, the concentration was adjusted to 1 x 109 sperm. A volume of 20 ml was used for iodixa-

nol density gradient centrifugation and 40 ml for routine centrifugation. Control samples were

further diluted with EquiProTM to a final concentration of 50 x 106 sperms/ml.

- 41 -

3.6.3.3 Centrifugation methods

Iodixanol density gradient centrifugation (IODIX):

The protocol for iodixanol density centrifugation of extended stallion semen, was optimized

in preliminary studies (data not shown). Iodixanol of different densities was prepared as de-

scribed in detail in section 3.2.3.6. A 10 ml top layer with a density of 1.09 g/ml was added in

a 50 ml conical plastic tube, after which 10 ml bottom layer of 1.165 g/ml was pipetted un-

derneath the top layer. The extended semen was carefully layered on top of the iodixanol so-

lutions. After centrifugation at 1000 x g for 20 minutes, spermatozoa show up as a band be-

tween the two layers. The supernatant was removed by aspiration with a syringe connected to

a sterile disposable needle. When the band that contained the sperm was reached this was

transferred into a clean tube using a new syringe.

Routine centrifugation (CENTR):

Routine centrifugation was performed by centrifuging extended semen in a 50 ml tube at 600

x g for 10 minutes, after which the supernatant was removed by aspiration with a syringe

connected to a sterile disposable needle. The resuspended sperm pellet was transferred into a

clean tube.

3.6.3.4 General semen processing after centrifugation

After centrifugation, for the resuspended pellet the number of spermatozoa was determined

using a haemocytometer (Thoma Neu, Hecht, Sontheim, Germany) and the volume was

measured, in order to determine the total sperm number and calculate the recovery rate. The

resuspended sperm pellet was divided into two parts, and one part was used for storage at 5°C

up to 72 h, whereas the other part was used for cryopreservation.

For cooled-storage, the resuspended pellet was diluted with EquiProTM to a final concentra-

tion of 50 x 10 6 sperm/ml. Sperm morphology, motility parameters, plasma membrane integ-

rity, acrosomal status and chromatin integrity were assessed immediately, as well as after 24

h, 48 h and 72 h of storage at 5°C.

For cryopreservation, the centrifuged samples were diluted with EquiProTM to a concentration

of 100 x 106 sperm/ml, after which they were diluted with an equal amount of freezing ex-

tender (EquiProTM containing 5 % glycerol). After two hours of equilibration at 5°C, the se-

- 42 -

men was packed in 0.5 ml straws, cooled from + 5°C to –15°C at 10°C/min and -15°C to –

140°C at 25°C/min in controlled rate freezer (IMV, L´Aigle, France). Straws were transferred

in liquid nitrogen, and stored there until further analysis. Thawing was done in a water bath at

37°C for 30 seconds, after which samples were analyzed for sperm motility and velocity,

morphology, plasma membrane integrity, acrosome membrane integrity and chromatin integ-

rity.

3.6.3.5 Sperm analysis

Sperm motility parameters were assessed via computer-assisted sperm analysis (CASA;

SpermVisionTM, Minitübe, Tiefenbach, Germany) using 20 µm analyzing chambers (20 mi-

cron, SC 20-01-04-B, Leja, GN Nieuw-Vennep, NL) as described by Nicolae (2006). Motility

parameters included percentage of progressively motile sperm (PMS), curvilinear velocity

(VCL in µm/sec), velocity average- path (VAP in µm/sec) and the amplitude of lateral head

deviation (ALH in µm). Morphology was evaluated for 200 eosin-nigrosin stained cells, ac-

cording to Weitze (2001). Sperm VitalStainTM was used for the eosin-nigrosin staining

(Björndahl et al. 2004).

Plasma membrane intact (PMI) and the positive acrosomal status (PAS) of spermatozoa were

evaluated using a flow cytometer (Cell Lab QuantaTM SC, Beckman Coulter, Krefeld). There-

fore, 485 µl HBS-solution was mixed with 5 µl sperm. This mixture was stained with 2,5 µl

propidium iodide (PI) and 7,5 µl fluorescein labeled peanut agglutinin (FITC-PNA), and in-

cubated for 15 min in the dark at +37°C, according to Ashworth et al. (1995) and Thomas et

al. (1997).

Chromatin integrity was assessed using SCSA-assay according to the protocols of Evenson

and Jost (2000, 2001). Using this method, a DNA fragmentation index (% DFI) can be deter-

mined that is indicative for chromatin integrity. For these measurements, a FACScan flow

cytometer was used (Becton-Dickson, Heidelberg).

3.6.3.6 Sperm selective medium

Iodixanol was used for density centrifugation; its chemical formula is: 5,5´-((2-hydroxy-1-

3propanediyl)-bis(acetylamino))bis(N,N´-bis(2,3dihydroxypropyl-2,4,6-triiodo-1,3-

benzenecarboxamide)). A 60% iodixanol solution in water with a density of 1.32 ± 0.001 g/ml

(20° C) is commercially available as OptiPrepTM (Progen, Heidelberg, Germany). Before den-

- 43 -

sity centrifugation, isoosmotic solutions with different densities were prepared fresh by dilu-

tion of the 60% solution of iodixanol with Hanks Buffered Salt Solution (Invitrogen, France),

according the recommendation of AxisShield OptiPrepTM application sheet C16. The top layer

of 1.09 g/ml is a 16% iodixanol solution and the bottom layer of 1.165 g/ml is a 30% solution.

The densities of the media were checked by measuring using bench-top refractometer (Zeiss).

3.6.3.7 Statistical analysis

Statistical analysis was done using SAS software (SAS, 2008), using the GLM procedure. The

residuals were tested for normality using the UNIVARIATE procedure. Fixed effects were

the three different semen preparation methods (IODIX, CENTR and EXTEN), whereas ran-

dom effects were stallions (six classes) and ejaculates (three classes). For cryopreserved sam-

ples, the fixed effects were the two groups “poor freezers” and “good freezers”. Differences

were considered significant at a probability level of p≤0.05. Correlations were analyzed with

Pearson correlation coefficient. Differences of means were detected using Tukey test com-

parisons, according to Zhou (2001).

3.6.4 Results

Fresh semen samples

The sperm yield was higher for the routine centrifugation protocol (CENTR), as compared to

density centrifugation using iodixanol (IODIX) (p≤0.05) (Table 4). Semen samples processed

by IODIX showed a higher percentage of plasma membrane intact sperm (PMI, PI negative)

and a reduced percentage of sperm with damaged acrosomes and lower DFI values compared

to semen that was not centrifuged (EXTEN) or processed using CENTR (p≤0.05).

- 44 -

Table 4: Effect of iodixanol density gradient centrifugation and routine centrifugation on sperm quality

parameters, as compared to extended semen (means ± standard deviation)

Group EXTEN

50 x 106 sperm/ml

IODIX

1000 x g

20 min

CENTR

600 x g

10 min

Sperm recovery - 33.1a

± 10.9

74.4b

± 15.6

PMS

46.6b

± 18.8

48.2 b

± 21.7

38.0a

± 17.3

VCL

136.4

± 24.5

134.8

± 26.0

131.8

± 23.1

VAP

95.7

± 20.5

89.5

± 19.9

93.2

± 20.3

ALH

2.8

± 0.3

2.7

± 0.4

2.9

± 0.4

PMI (PI neg) 68.9a

± 11.2

79.0b

± 10.9

60.1a

± 11.7

PAS(FITC-PNA pos) 6.8ab

± 4.1

3.5b

± 2.1

8.4a

± 4.8

SCSA/DFI

15.1a

± 9.0

4.6b

± 3.3

15.8a

± 8.9

MAS 37.7

±9.8

37.4

± 15.2

45.7

± 13.5

abnormal

acrosomes

10.5a

± 4.0

5.7b

± 3.2

12.9a

± 6.3

abnormal

heads

6.1

± 2.6

8.0

± 5.2

9.6

± 7.4

abnormal

Neck region

4.8

± 3.7

4.3

± 3.7

6.0

± 3.0

mid-piece

abnormalities

8.8

± 5.2

7.7

± 4.6

5.7

± 4.2

abnormal

principal/end piece

7.4

± 4.9

11.6

± 9.1

11.2

± 5.1


EXTEN = diluted semen

IODIX = iodixanol density gradient centrifugation

- 45 -

CENTR = routine centrifugation








DFI: denaturation fragmentation index of sperm DNA (%)

MAS: morphological abnormal sperm (%) a,b,c Values with different superscript differ significantly within rows (p≤0.05).

Cooled-stored semen samples

In Table 5, sperm quality parameters are shown as determined during cooled storage (progres-

sive motility, sperm velocity, plasma membrane integrity and positive acrosomal status) of

sperm selected via different centrifugation methods. Spermatozoa selected using IODIX

showed higher progressive motility and plasma membrane intact spermatozoa, and a reduced

percentages of sperm with positive acrosomal status during storage at +5°C for 72 hours, as

compared to semen prepared via EXTEN or CENTR (p≤0.05).

- 46 -

Table 5: Effect of different centrifugation protocols on sperm quality parameters during storage for 24 h,

48 h and 72 h at 5 °C (means ±standard deviation)

24 h 48 h 72 h

EXTEN IODIX CENTR EXTEN IODIX CENTR EXTEN IODIX CENTR

PMS

18.3b

± 15.2

35.5a

± 20.0

25.7ab

± 15.8

11.7b

± 10.4

32.3a

± 18.5

20.8ab

± 12.5

8.5b

± 8.0

28.3a

± 16.4

17.2b

± 11.4

VCL

117.6a

± 32.9

138.3a

± 34.9

124.5a

± 31.7

115.3a

± 40.2

143.6a

± 48.7

134.0a

± 39.0

97.2b

± 63.0

143.5a

± 50.6

135.7ab

± 49.0

VAP

72.1a

± 18.3

83.9a

± 18.4

81.3a

± 18.7

62.9b

± 20.5

81.1a

± 25.6

82.2a

± 18.0

50.3b

± 32.6

78.5a

± 25.6

77.3a

±25.1

ALH

2.8a

± 0.7

2.9a

± 0.6

2.7a

± 0.6

3.0a

± 1.0

3.3a

± 1.0

3.3a

± 0.6

2.6b

± 1.7

3.5ab

± 1.0

3.7a

± 1.1

PMI(PI

neg)

64.8b

± 13.2

79.2a

± 11.1

60.8b

± 11.9

62.8ab

± 14.7

74.5a

± 19.1

60.3b

± 12.1

60.9b

± 15.1

78.0a

± 10.7

59.9b

± 12.5

PAS(FITC-

PNA pos)

7.9ab

± 4.3

4.7a

± 3.8

11.3b

± 6.5

10.1b

± 4.9

5.3a

± 2.7

13.4b

± 7.0

12.0b

± 4.9

6.2a

± 3.5

14.4b

± 6.4


EXTEN = diluted semen (50 x 106 sperms/ml)

IODIX = Iodixanol density gradient centrifugation (1000 x g, 20 min)

CENTR = routine centrifugation (600 x g, 10 min)








DFI: denaturation fragmentation index of sperm DNA (%) a,b,c Values with different superscript differ significantly within days (p≤0.05).

- 47 -

Cryopreserved samples

Selection of spermatozoa by IODIX also had a positive influence on sperm quality of frozen-

thawed semen samples. Compared to CENTR, IODIX samples demonstrated a higher per-

centage of progressively motile and plasma membrane intact sperm and a reduced percentage

of sperm with positive acrosomal status, sperm with abnormal acrosomes and DFI percent-

ages (p≤0.05, Table 6). Sperm samples from “good freezers” showed a higher percentage of

progressively motile sperm and a reduced DFI percentage compared to “poor freezers” both

after CENTR (p≤0.05) and IODIX. Within both centrifugation methods the spermatozoa col-

lected from “poor freezer” stallions showed fewer spermatozoa with abnormal acrosomal or

neck regions (p≥0.05).

- 48 -

Table 6: Effect of centrifugation methods on post-thaw sperm quality parameters. The means ± standard

deviation are indicated separately for “good and poor freezers”, as well as means for all stallions together

(total).

Centrifugation Techniques

IODIX

1000 x g

20 min

CENTR

600 x g

10 min

total

good

freezers

poor

freezers

total good

freezers

poor

freezers

PMS 23.8a

± 17.7

38.6*

±10.2

9.0

± 8.3

17.0b

± 12.6

27.6*

± 8.1

6.5

± 4.4

VCL 113.2

± 20.0

126.6

±19.3

99.8

± 8.3

107.0

± 23.4

115.1

± 28.0

99.0

± 15.4

VAP

72.7

± 15.5

82.8

± 14.3

62.6

± 8.9

71.8

± 20.4

80.7

± 23.9

63.0

± 11.7

ALH

2.4

± 0.4

2.6

± 0.4

2.1

± 0.2

2.5

± 0.5

2.5

± 0.3

2.5

± 0.6

PMI (PI neg) 52.6a

± 8.4

51.9

± 8.7

53.3

± 8.6

31.9b

± 6.9

29.3

± 6.8

34.4

± 6.2

PAS(FITC-

PNA pos)

13.8a

± 6.4

12.8

± 5.3

14.9

± 7.5

22.6b

± 8.0

22.5

± 8.0

22.6

± 8.5

SCSA/DFI

5.1a

± 3.7

3.4

±1.0

6.8

± 4.7

16.5b

± 9.0

11.0*

± 2.8

22.1

± 9.7

MAS 36.0

± 14.6

31.9

± 10.8

40.1

± 17.4

42.8

± 11.6

38.06

± 4.8

47.61

± 14.6

abnormal

acrosomes 7.8a

± 4.7

9.0

± 4.8

6.6

± 4.5

12.9b

± 5.5

13.8

± 3.9

12.1

± 7.0

abnormal

heads 7.0

± 3.9

5.3

± 2.1

8.7

± 4.6

8.6

± 5.1

5.7

± 3.0

11.5

± 5.3

abnormal

neck region 5.6

± 4.6

5.8

± 5.4

5.3

± 4.0

6.0

±2.7

6.3

±2.0

5.7

±3.3

- 49 -

mid-piece

abnormalities

5.2

± 4.1

4.2

± 3.6

6.2

± 4.5

3.4

± 1.9

2.7

± 1.9

4.1

± 1.8

abnormal

principal/end

piece

10.4

± 6.3

7.7

± 3.6

13.3

± 7.4

11.8

± 4.8

9.4

± 4.1

14.2

± 4.2


EXTEN = diluted semen

IODIX = Iodixanol density gradient centrifugation

CENTR = routine centrifugation








DFI: denaturation fragmentation index of sperm DNA (%)

MAS: morphological abnormal sperm (%)

a, b= significant between the centrifugation methods (p≤0.05)

*= significant between the good and poor freezers group wihtin the treatment (p≤0.05)

3.6.5 Discussion

After the withdrawal of Percoll as density gradient solution in 1996, new products like iodix-

anol (Smith et al. 1997, Claassens et al. 1998) were introduced as semen preparation methods

in animal breeding and human assisted reproduction.

Iodixanol was used as a dense cushion solution for high-speed centrifugation of stallion se-

men (Revell et al.1997, Escot et al. 2005, Sieme et al. 2006). Compared to other solutions

used for density gradient centrifugation, such as PercollTM (Sieme et al. 2003), EquiPureTM

(MacPherson et al. 2002) and AndrocollTM (Morrell et al. 2009c), iodixanol avoids pellet for-

mation. The density of the bottom layer is higher compared to the density of viable stallion

spermatozoa, which makes that sperm are collected on top of the bottom layer. Thus, the det-

rimental effects of centrifugation and pelleting are reduced (Pickett et al. 1975, Smith et al.

1997). To date, a protocol for density gradient centrifugation using iodixanol exists only for

bull semen (application sheet C16, Axis-Shield PoC AS, Norway). The present study was

conducted to develop an iodixanol density centrifugation protocol for processing of stallion

semen, and analyze the effect of iodixanol density gradient centrifugation on stallion sperm

- 50 -

quality during storage at 5°C as well as after cryopreservation and compared to routine cen-

trifugation (600 x g, 10 min).

The sperm yield from the two centrifugation methods was similar to previous studies (Del-

homme et al. 2004, Sieme et al. 2006, Aurich, 2008, Macias Garcia et al. 2009a). Using dif-

ferent iodixanol gradients, Smith et al. (1997) reported yields of motile sperm ranging from

21.0 % (1.05 /1.15 g/ml, 3 ml of each density medium, 1500 x g, 40 min) to 77.8 % (1.05/1.15

g/ml, 1.5 ml of each density solution, 1000 x g, 30 min). In contrast, Claassens et al. (1998)

obtainted significantly lower numbers of vital and progressively motile sperm using iodixanol

gradient centrifugation as compared to silanecoated silica solutions or Percoll. Anderson and

Grinsted (1997) harvested a higher number of motile spermatozoa from iodixanol gradient

than from a Percoll gradient.

After density gradient centrifugation, the recovery rate and sperm quality of the semen sam-

ples are influenced by the compound used for density centrifugation as well as method for

retrieving the spermatozoa. As mentioned above, the morphologically normal, motile sper-

matozoa are concentrated between the two layers of the iodixanol density gradient. The sperm

between the two liquid layers need to be removed carefully to avoid contamination with parti-

cles that are present in the top and bottom layer. According to Smith et al. (1997), aspirating

the supernatant followed by recovery of the sperm using a disposable needle and syringe is an

easy-to-use method with acceptable recovery rates.

In the present study, an increase in the percentage of plasma membrane intact sperm and a

decrease percentage of sperm with abnormal acrosomes, sperm with positive acrosomal status

and reduced DFI percentage were obtained after iodixanol density gradient, whereas sperm

motility was not significantly influenced. Using a density gradient centrifugation with Andro-

coll-E Morrell et al. (2009a) observed an increase in sperm motility despite the building of a

pellet in the bottom of the tube. In accordance to our observations, Smith et al. (1997) found

no significant increase in human sperm motility after iodixanol centrifugation (81 %) com-

pared to untreated semen, and reduced motility after Percoll gradient centrifugation (78 %).

When different solutions for density gradient centrifugation were compared, Mousset-Simeon

et al. (2004) found no significant differences in the percentage of progressively motile sperm

(PMS) after Percoll centrifugation (46 %), Puresperm (45%) and iodixanol centrifugation

- 51 -

(42%). In contrast, Claassens et al. (1998) observed a significant increase of vital and pro-

gressively motile sperm (PMS) after iodixanol. This was the case for human semen with re-

duced number of sperm (vital 73.4%, PMS 71.5% ) compared to untreated semen (vital

59.8%, PMS 42.0%), as well as semen with normal sperm count (vital 80.9%, PMS 82.4%)

compared to untreated semen (vital 74.0%, PMS 58.0%). Other authors found similar good

results for motility when iodixanol was compared to Percoll (Andersen and Grinsted, 1997,

Harrison, 1997, Resende et al. 2009) and concluded that iodixanol could be used instead of

Percoll (Harrison, 1997, Smith et al. 1997). These studies, however, gave only limited infor-

mation about the fertility of sperm after density gradient centrifugation. Correlations between

sperm motility and fertility varied considerably, from a low correlation (Dowsett and Pattie

1982, Magistrini et al. 1992, Voss et al. 1981, Jasko et al. 1992, Malmgren 1997) to a high

correlation (Samper et al. 1991). Inclusions of in-vitro functionality assessments may lead to

better prediction of sperm fertility (Colenbrander et al. 2003, Morrell et al. 2008). Positive

correlations were found between fertility and motility parameters using CASA analysis (Olds-

Clarke, 1996a,b et al. 1997b, De Geyter et al. 1998), normal morphology (Bielanski, 1950,

Jasko et al. 1990, Malmgren, 1997, Rodriguez-Martinez et al. 2003), sperm concentration

(Kenney et al. 1971), chromatin integrity (Evenson et al. 2000, Love et al. 1998). In addition,

fertility rates correlate with the amount viable cells and the acrosomal status of cells in an

ejaculate, and in-vitro capacitation properties as can be assessed by flow cytometry (Wilhelm,

et al. 1996, Colenbrander et al. 2003). An improvement in sperm plasma membrane integrity,

positive acrosomal status and chromatin integrity via application of sperm selection methods

will be more important in terms of fertilizing capacity and early embryonic development than

an increase in sperm motility.

The findings in this study on the increased proportion of morphologically normal sperm are in

accordance with others (Sieme et al. 2003, Morrell et al. 2008). In the present study no sig-

nificant difference in the total count of morphologically abnormal sperms could be seen for

iodixanol treated spermatozoa compared to non-centrifuged semen. In accordance to our

study, MacPherson et al. (2002) found no significant improvement of morphologically normal

spermatozoa after density gradient centrifugation by EquiPureTM. Morrell et al. (2009d) re-

ported a significantly higher proportion of morphologically normal sperm after gradient cen-

trifugation. They saw a decrease in the number of sperm that exhibited proximal cytoplasma

- 52 -

droplets, coiled tails, sperm heads with abnormal contour, undeveloped sperm head, distal

droplets, acrosome defects and bent tail, although no significant differences were found for

detached heads, nuclear pouches, defective mid-piece, double bent tails, narrow heads, vari-

able size of heads. For other abnormalities such as pear-shaped heads and tapered heads, an

increase was seen after centrifugation. In the present study, only abnormal acrosomes were

significantly different between selected and non-centrifuged semen. Different morphology

abnormalities may influence sperm density and therefore in the ability of the gradient to re-

move them (Morrell et al. 2009d). Mousset-Simeon et al. (2004) found significantly lower

numbers of morphologically normal spermatozoa after iodixanol centrifugation compared to

Puresperm and Percoll centrifugation. In our study, there were fewer abnormal acrosomes and

abnormal necks after cryopreservation of sperm from “poor freezers” as compared to “good

freezers”. Thus, the treatment may positively influence semen of poor quality. Kuisma et al.

(2006) recorded 9.5 % major morphological abnormalities and 10.7 % minor morphological

abnormalities after thawing for routine preparation. The importance of the morphological

findings of different authors depends on whether the findings are related to fertility. Kuisma

et al. (2006) did not see a relationship between the foaling rate and morphological findings,

confirming earlier reports by Voss et al. (1981) and Dowsett and Pattie (1982). Kenney et al.

(1983) concluded that the number of morphological deviations may not be important if there

are sufficient morphologically normal spermatozoa in the insemination dose.

In the present study, the influence of centrifugation and selection of sperm on sperm longevity

during cooled storage was evaluated for semen processed using iodixanol centrifugation, rou-

tine centrifugation and extended semen samples. The most pronounced decrease of progres-

sive motility occurred in the first 24 h for all three semen processing methods. In the extended

semen samples motility decreased two times more than in the centrifuged semen samples.

Smith et al. (1997) determined a modest decline in sperm motility during the first 24 h after

IODIX, whereas Forster et al. (1983) reported decline of 70 % in the first 24 hours after Per-

coll centrifugation. Johannisson et al. (2009) also observed a greater decline in values for mo-

tility parameters within the first 24 hours after Androcoll-E centrifugation. In the current

study, both routine centrifugation samples and extended semen samples showed a district de-

cline in sperm quality between 24 hours and 72 hours of cooled storage. In contrast, IODIX

samples showed only a small decline in sperm quality. After 72 hours of cooled storage the

- 53 -

IODIX samples displayed significantly better sperm motility, plasma membrane integrity and

acrosome membrane integrity than CENTR or EXTEN samples.

Furthermore, for application for cryopreservation, spermatozoa that were prepared using IO-

DIX had significantly better post-thaw progressive motility, plasma membrane integrity, re-

duced positive acrosomal status and chromatin integrity than CENTR. Saragusty et al. (2009)

reported a protective effect of iodixanol as a cryoprotective medium for bull semen. They

used 0%, 1,25%, 2,5% and 5% OptiPrep to extended semen and compared the semen quality

parameters after freezing. The positive effect of iodixanol in the present study might be due to

the selection of spermatozoa with superior quality before freezing and the cryoprotective ef-

fect of iodixanol itself. Mancill et al. (2010) compared the influence of density gradient cen-

trifugation before freezing and gradient centrifugation after thawing and demonstrated higher

numbers of post-thaw viable acrosomal intact spermatozoa from infertile stallions if the den-

sity gradient centrifugation was applied before freezing.

In conclusion, this study evaluated the effect of density gradient centrifugation with iodixanol

on stallion sperm quality. It was shown that a large number of spermatozoa (1 x 109

sperms/ml) can be prepared with one gradient. Spermatozoa treated by IODIX demonstrated

better semen quality parameters than CENTR and EXTEN. The study found that the selected

spermatozoa showed better semen quality during cooled-storage and after cryopreservation.

Even spermatozoa from “poor freezer” stallions showed an increase in post-thaw semen qual-

ity with regard to plasma membrane integrity, positive acrosomal status and chromatin integ-

rity.

- 54 -

4 Summarizing, Discussion and Conclusions

Stallion semen shows a wide range of diversity in sperm quality amongst males and ejaculates

(Colenbrander et al. 2003). Breeding stallions are not selected for sperm parameters and fertil-

ity, and consequently subfertile stallions may be included in the breeding stock. Semen prepa-

ration methods such as diluting and centrifugation are used to prepare insemination doses for

AI. Density gradient centrifugation is a technique which allows the viable, motile, chromatin

intact spermatozoa to be separated from non-vital, chromatin damaged, dying spermatozoa,

cell debris and seminal plasma. These techniques are used to minimize the variation in semen

quality and to prepare semen of good quality for use in AI. This would improve pregnancy

rates, even for subfertile stallions. In the present two studies, two different density gradient

centrifugation protocols were tested for preparing stallion semen for cooled stored and cryo-

preserved semen doses. For density gradient centrifugation in the routine daily laboratory

work of an AI station it is desirable to process large volumes of extended semen at once. That

is why the studies described in this thesis were done using semen quantities as commonly

used. Like other sperm selective methods (swim-up, glass wool, glass wool Sephadex), den-

sity gradient centrifugation results in a reduced sperm yield compared to the semen samples

that are only diluted. Recovery rates after density centrifugation are lower as recovery rates

after cushion centrifugation (1000 x g, 20 min), routine centrifugation (600 x g, 10 min) and

centrifugation without selective medium (300 x g, 15 min) (Smith et al. 1997, Claassens et al.

1999, Sieme et al. 2003, Mousset- Simeon et al. 2004, Marcias Garcia et al. 2009a,b, Morrell

et al., 2009a). For the EquiPureTM Pro gradient centrifugation, the mean recovered sperm con-

centration was 435 x 106 sperm (ranging from 128 x 106 to 738 x 106 sperm), of which 383 x

106 sperm total motile sperm and 348 x 106 sperm progressively motile spermatozoa. For io-

dixanol centrifugation, the yield was lower. We recovered on average of 331 x 106 sperm

(ranging from 159 x 106 to 532 x 106 sperm), of which 226 x 106 sperm total motile sper-

matozoa and 160 x 106 sperm progressive motile spermatozoa. The insemination doses that

are generally recommended range between 250 – 500 x 106 progressive motile sperm (Brin-

sko, 2006). Other authors showed that doses of 50 x 106 sperm resulted in reduced fertility

rates, whereas doses of 100 x 106 sperm from fertile stallions results in good fertility rates

- 55 -

(Pickett et al. 1976a, Pickett et al. 1976b, Demick et al. 1976). Kenney et al. (1975) demon-

strated good pregnancy rates by using a dose of 100 x 106 for different stallions. In a study

conducted by Gahne et al. (2000), pregnancy rates of 75 and 64% were determined when in-

semination doses of 300 million and 500 million progressive sperms were used, respectively.

Even when using lower concentrations (20 to 100 x 106 sperms) no decrease in fertility was

reported by other authors (Woods et al. 2000, Sieme et al. 2004b, Rigby et al. 1999). Sper-

matozoa selected by density gradient centrifugation have superior quality, and therefore lower

sperm concentrations are likely to produce satisfactory pregnancy rates. Morrell et al. (2010)

reported that they recovered 900 x 106 sperm from single layer centrifugation with Androcoll-

E, which allows for three insemination doses with 250 x 106 motile sperm. Macias Garcia et

al. (2009) applied density centrifugation after thawing of cryopreserved samples and recov-

ered 20-40 % of the spermatozoa. They assumed that the preparation of 200 x 106 sperm by

density gradient centrifugation is enough for deep intrauterine insemination.

After EquiPureTM Pro gradient centrifugation, the motile sperm are pelleted at the bottom of

the centrifugation tube. After iodixanol centrifugation, sperm are layered between a top and

bottom layer. The sperm pellet can be recovered easily by complete removal of the super-

natant. This resuspended pellet can then be removed with a clean syringe. Harvesting the

spermatozoa bears the risk of recontamination, which is thought to be much more trouble-

some for iodixanol centrifugation as compared to EquiPureTM Pro gradient centrifugation. It is

thought that the supernatant including spermatozoa may be removed, or that the supernatant

that was not removed affects the sperm quality. Removal of sperm with the supernatant might

be the reason for the lower recovery rates when using iodixanol density centrifugation.

The centrifugation methods which are normally applied for semen preparation such as cush-

ion centrifugation and routine centrifugation result in all kinds of sperm subclasses appearing

in the pellet. These methods keep the motile, vital and chromatin intact spermatozoa close to

cell debris and seminal plasma which contain reactive oxygen (ROS) (Mortimer, 2000, Hen-

kel and Schill, 2003, Morrell, 2006). The ROS have a detrimental effect on the sperm and can

cause damage to sperm chromatin (Love, 2005, Morrell, 2006, Johannisson et al. 2009). In the

mare, after insemination, the motile spermatozoa are capable to swim rapidly away from the

rest of the inseminate to the site of fertilization, thus removing themselves from the seminal

plasma and cells debris (Morrell, 2006). After EquiPureTM Pro as well as iodixanol centrifuga-

- 56 -

tion, progressive motility (only for EquiPureTM Pro treatment), plasma membrane integrity,

acrosome membrane integrity and chromatin structure was improved compared to non-

centrifuged semen, cushion centrifugation, routine centrifugation and centrifugation without

sperm selective medium. In addition, semen quality parameters were improved after density

centrifugation and storage at 5°C for 72 h as compared to the diluting and centrifugation

methods. Johannisson et al. (2009) also demonstrated better survival of selected spermatozoa

over 48 h. In general, for EquiPureTM Pro selected sperm, the highest decline in survival dur-

ing storage occurred from 48 h to 72 h. This is in accordance with others (Smith et al. 1997).

In contrast, the iodixanol selected spermatozoa showed the highest decline after 24 h. Johan-

nisson et al. (2009) found the largest decline in motility within the first 24 h, whereas chroma-

tin integrity exhibited a decline after 24 h.

During cooling, freezing and thawing many changes occur in the cell which can damage the

spermatozoa (Esteso et al. 2003, Pena et al. 2003a,b). As mentioned above, the damaged cells

release factors such as ROS, that can affect the chromatin of intact cells (Perez Crespo et al.

2008). In the studies described in this thesis, the post-thaw chromatin integrity for selected

spermatozoa was superior to that of non-selected sperm. This is in agreement with findings

described by other authors (Macias Garcias et al. 2009a, Morrell et al. 2009c). Also for poor

freezers iodixanol treated semen showed significantly better chromatin integrity.

The velocity parameters obtained for fresh, cooled-stored and frozen-thawed semen appear

not to be influenced solely by the centrifugation method applied. Macias Garcia et al. (2009b)

found an increase in velocity parameters after gradient centrifugation of post-thaw semen.

They saw a significant increase in VCL, VSL and VAP. For ALH, they found a non-

significant decrease. In general, the values for VAP and VCL are thought to be the most char-

acteristic parameters that described sperm movement, that serve as an indicator for sperm

quality (Macias Garcias et al. 2008). Olds-Clark et al. (1996) determined that high VCL levels

are needed to build the reservoir of spermatozoa in the female tract and for penetrating the

zona pellucida. Holt et al. (1997b) and Silva et al. (2006) demonstrated that high velocity

parameter are essential for in-vitro and in-vivo fertility, for boars and dogs respectively.

In conclusion, EquiPureTM Pro and iodixanol gradient centrifugation are capable of selecting

spermatozoa of superior quality compared to diluted semen, routine centrifugation, cushion

- 57 -

centrifugation and centrifugation without sperm selective medium. Large volumes of up to 30

ml of semen could be processed using both EquiPureTM Pro and iodixanol density centrifuga-

tion. The selected spermatozoa showed better survival rates after cooled storage as well as

after cryopreservation. The density centrifugation methods, especially increased post-thaw

chromatin integrity.

- 58 -

5 Summary

Gesa Stuhtmann

Spermselective preparation of stallion semen by density gradient centrifugation

The objective of the two present studies was to evaluate in-vitro semen quality parameters

after density gradient centrifugation of fresh, cooled-stored and frozen-thawed semen. The

parameters assessed were motility characteristics assessed by CASA (SpermVisionTM), mem-

brane integrity, acrosome membrane integrity and chromatin structure measured by flow cy-

tometry. Examination of morphological parameters was included in the second study. The

solutions EquiPureTM Pro and iodixanol were tested for density gradient centrifugation.

In the first study, the first experiment investigated sperm quality parameters of samples after

EquiPureTMPro gradient centrifugation (EPP, 330 x g, 15 min), cushion centrifugation (CC,

1000 x g, 20 min), centrifugation without sperm selective medium (WSSM, 330 x g, 15 min),

and diluted semen (DS, INRA 82, 25 x 106 sperm/ml). The second experiment evaluated the

influence of varying proportions of freezing extender (INRA 82 containing 5 % egg yolk and

2.5 % glycerol) on the quality of frozen-thawed of EquiPureTM Pro treated semen.

In the first study, the recovery rate for EPP (26.6 %) was significantly lower than for CC

(95.5%) and WSSM (74.0%, p≤0.05). For EPP treated fresh semen, progressive motility,

membrane integrity, acrosome membrane integrity and chromatin structure were significantly

improved compared to DS, CC and WSSM (p≤0.05). The same observation could be made for

cooled-stored semen. For post–thaw spermatozoa treated by EPP, better values for chromatin

structure were seen compared to CC and WSSM (p≤0.05). In the second experiment (first

study), better progressive motility and membrane integrity (p≤0.05), acrosome membrane

integrity and chromatin structure (p≥0.05) were seen for spermatozoa diluted in higher ratios

of freezing extender.

The second study tested the effect of iodixanol density gradient centrifugation (IODIX, 1000

x g, 20 min), routine centrifugation (CENTR, 600 x g, 10 min) and extended semen (EXTEN,

EquiProTM, 50 x 106 sperms/ml) on semen quality parameters. The frozen-thawed trial in-

cluded stallions to be known as „good freezers“ and „poor freezers“. The yield of spermato-

- 59 -

zoa was 33.1 % for IODIX and 74.4 % for the CENTR method (p≤0.05). Membrane integrity,

acrosome membrane integrity, chromatin structure and percentage of abnormal acrosomal

region were better after IODIX treatment compared to EXTEN and CENTR prepared sper-

matozoa (p≤0.05, fresh semen). For motility parameters and other morphology parameters

(total morphological abnormalities, abnormal heads, abnormal neck region, abnormal mid-

piece, abnormal principal/end piece) no differences were found for any of the methods IO-

DIX, CENTR or EXTEN (p≥0.05). The IODIX treatment prolonged the longevity of the sepa-

rated spermatozoa during cooled storage compared to CENTR and EXTEN. After 72 h of

storage, there were significant differences for the progressive motility, membrane integrity

and acrosome membrane integrity (p≤0.05). Post-thaw spermatozoa showed significantly bet-

ter progressive motility, membrane integrity, acrosome membrane integrity, chromatin struc-

ture and percentage of abnormal acrosomal region for IODIX treatment compared to the

CENTR method (p≤0.05). Within the „poor freezers“ group IODIX treated semen showed

better membrane integrity, acrosome status, chromatin structure, abnormal acrosomal regions

after thawing than semen prepared by CENTR.

In summary, the EPP and IODIX preparations selected spermatozoa of better quality than DS,

CC, WSSM, CENTR and EXTEN treated semen. Under various storage conditions the den-

sity gradient selected spermatozoa showed better results than non-selected spermatozoa (DS,

CC, WSSM, EXTEN and CENTR).

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6 Zusammenfassung

Gesa Stuhtmann

Spermienselektives Aufbereitungsverfahren von Hengstsperma durch Dichtegradient-

zentrifugation

Das Ziel der beiden vorliegenden Studien war die Untersuchung der Spermaqualität von

Frisch-, Versand- und Tiefgefriersamen nach der Aufbereitung durch Zweischichtendichte-

zentrifugation.

Als Dichtezentrifugationslösungen wurden EquiPureTMPro und Iodixanol hinsichtlich ihres

Einflusses auf die mittels CASA (SpermVisionTM) analysierte Spermienmotilität, auf die

Spermienmorphologie (Versuch 2) sowie durchflusszytometrisch ermittelte Plasmamembra-

nintegrität, den akrosomalen Status und die Chromatinintegrität (SCSA-assay) untersucht.

In der ersten Studie wurden die Spermienqualitätsparameter nach Dichtezentrifugation mit

EquiPureTMPro (EPP, 330 x g, 15 min), Zentrifugation ohne Dichtezentrifugationslösung

(WSSM, 330 x g, 15 min), „Kissenzentrifugationstechnik“ (CC, 1000 x g, 20 min) und von

verdünntem Sperma (DS, INRA 82, 25 x 106 Spermien/ml) miteinander verglichen sowie der

Einfluss variierender Volumenanteile des Tiefgefrierverdünners (INRA 82 mit 5 % Eigelb

und 2,5 % Glycerin) auf die Samenqualität der EPP zentrifugierten Spermien untersucht.

Im ersten Versuchsteil war die Spermienrückgewinnungsrate nach der EPP Zentrifugation

(26,6 %) niedriger als nach CC (95,5%) und WSSM (74,0%) (p≤0,05). Frischsamenproben,

die durch EPP Zentrifugation aufbereitet wurden, zeigten im Vergleich zu DS, CC und

WSSM aufbereiteten Samenproben eine Verbesserung der Spermaqualität in Hinblick auf

Vorwärtsbeweglichkeit, Plasmamembranintegrität, akrosomalen Status sowie Chromatinin-

tegrität (p≤0,05). Ähnliche Ergebnisse wurden bei der Untersuchung gekühlt-gelagerten Sa-

mens über eine Lagerungszeit von 72 h (5°C) festgestellt. Bei der Analyse des aufgetauten

Tiefgefrierspermas, welches vor der Samentiefgefrierung mit EPP zentrifugiert wurde, konnte

eine höhere Chromatinintegrität im Vergleich zu mit CC und WSSM aufbereiteten Samens

ermittelt werden (p≤0,05). Im zweiten Teil der ersten Studie wurde ein positiver Einfluss der

Zugabe höherer Verdünneranteile auf die Vorwärtsbeweglichkeit und die Plasmamembranin-

- 61 -

tegrität (p≤0,05), sowie auf den akrosomalen Status und die Chromatinintegrität der Spermien

(p≥0,05) festgestellt.

In der zweiten Studie wurde der Einfluss der Dichtezentrifugation mit Iodixanol (IODIX,

1000 x g, 20 min) auf die Spermaqualität mit derjenigen nach Aufbereitung durch die routi-

nemäßig eingesetzte Zentrifugationsverfahren (CENTR, 600 x g, 10 min) und der Verdün-

nung von Spermien (EXTEN, EquiProTM, 50 x 106 Spermien/ml) verglichen. Die eingesetzten

Hengste konnten aufgrund ihres vorhergehenden Einsatzes in einem kommerziellen Samen-

tiefgefrierprogramms in Gruppen mit guten (durchschnittlicher Anteil von progressive vor-

wärtbeweglichen Spermien nach dem Auftauen > 35%) oder schlechten Samentiefgefrierei-

genschaft (durchschnittlicher Anteil von progressive vorwärtbeweglichen Spermien nach dem

Auftauen von < 35%) zugeordnet werden.

Nach der IODIX Zentrifugation wurde eine Spermienrückgewinnungsrate von 33,1 % und

nach der CENTR Zentrifugtion von 74,4 % ermittelt (p≤0,05).

Nach IODIX Zentrifugation wurde im Vergleich zu den herkömmlichen Samenaufberei-

tungsmethoden EXTEN und CENTR eine Verbesserung der Spermaqualität im Hinblick auf

die Plasmamembranintegrität, den akrosomalen Status, die Chromatinintegrität sowie den

Anteil an Spermien mit abgelöstem Akrosom ermittelt (p≤0.05). Ebenso konnte ein positiver

Einfluss der IODIX Zentrifugation auf die Samenqualität nach 72-stündiger, gekühlter Lage-

rung bei +5°C hinsichtlich der Vorwärtsbeweglichkeit, der Plasmamembranintegrität und des

akrosomalen Status festgestellt werden (p≤0.05). Desweiteren wurde nach dem Auftauen von

Tiefgefriersamenproben eine Verbesserung der Plasmamembranintegrität, des akrosomalen

Status, der Chromatinintegrität sowie des Anteils an Spermien mit abgelöstem Akrosom

durch IODIX Zentrifugation ermittelt werden (p≤0.05), wobei insbesondere Samen von

Hengsten mit schlechten Tiefgefriereigenschaften ein positiver Einfluss der IODIX Zentrifu-

gation im Vergleich zu herkömmlichen Zentrifugationsverfahren CENTR beobachtet wurde.

Zusammenfassend führte die Dichtezentrifugation mit IODIX und EPP, die mit einer redu-

zierten Spermienrückgewinnungsrate einhergeht, zu einer Verbesserung der Spermaqualität

im Vergleich zu den Aufbereitungsverfahren DS, CC, WSSM, CENTR und EXTEN. Die

durch IODIX und EPP selektierten Spermien wiesen sowohl am Tag der Samengewinnung,

als auch nach 72-stündiger Lagerung bei +5°C und nach Spermatiefgefrierung eine Verbesse-

- 62 -

rung in standardspermatologisch und durchflusszytometrisch ermittelten Spermienqualitätspa-

rametern auf.

- 63 -

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8 Appendix

8.1 EquiPure EquiPureTM Pro centrifugation, a method for sperm clean-

up and its effects on sperm quality of cold- stored and frozen- thawed

stallion sperm

First Experiment: Fresh semen samples

Figure 6: Effect of preparation method on the sperm curvilinear velocity (VCL, µm/sec) for semen sam-

ples that were stored at 5°C, for up to 72 h. Means ± standard deviation are shown for n=6 stallions per

group, 3 ejaculates/stallion, DS: Diluted-non-centrifuged semen, EPP: EquiPureTM Pro centrifugation,

CC: cushioned centrifugation, WSSM: centrifugation without sperm-selective medium,


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Figure 7: Effect of preparation method on the sperm average path velocity (VAP, µm/sec)

for semen samples that were stored at 5°C, for up to 72 h. Means ± standard deviation are shown for n=6

stallions per group, 3 ejaculates/stallion, DS: Diluted-non-centrifuged semen, EPP: EquiPureTM Pro cen-

trifugation, CC: cushioned centrifugation, WSSM: centrifugation without sperm-selective medium,


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Figure 8: Effect of preparation method on the sperm amplitude of lateral head displacement (ALH, µm/sec) for semen samples that were stored at 5°C, for up to 72 h. Means ± standard deviation are shown for n=6 stallions per group, 3 ejaculates/stallion, DS: Diluted-non-centrifuged semen, EPP: EquiPureTM Pro centrifugation, CC: cushioned centrifugation, WSSM: centrifugation without sperm-selective me-dium, a, b, c Values with different superscript differ significantly within days (p≤0.05).

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First Experiment: After resuspension with freezing extender

Table 7: Effect of dilution with freezing extender of sperm treated with EquiPureTM Pro density gradient,

cushion centrifugation and centrifugation without sperm-selective medium on sperm quality parameters

(means ± standard deviation).

EPP

330 x g

15 min

after

centrif.

EPP

330 x g

15 min

after

resus.

CC

1000 x g 20min

after

centrif.

CC

1000 x g 20min

after

resus.

WSSM

330 x g

15 min

after

centrif.

WSSM

330 x g

15 min

after

resus.

PMS 80.1a

±5.7

76.8a

±7.2

59.8a

±12.2

54.9a

±14.6

59.3a

±12.0

56.7a

±17.2

VCL 213.9a

±29.3

168.6b

±34.7

223.1a

±30.6

186.2b

±31.6

223.8a

±30.9

182.4b

±33.2

VAP 138.7a

±19.4

117.2b

±18.8

127.0a

±21.8

103.8b

±19.2

125.9a

±21.4

101.7b

±19.2

ALH 3.6a

±0.5

3.4a

±0.6

4.6a

±0.5

4.8a

±0.7

4.6a

±0.5

4.9a

±0.8

PMI

(PI neg)

83.0a

±4.4

81.2a

±5.8

70.4a

±11.4

69.0a

±12.0

71.5a

±12.0

70.9a

±10.7

PAS (FITC

-PNA pos)

4.8a

±1.6

8.5b

±3.5

11.6a

±3.9

13.6a

±4.5

10.3a

±3.6

11.8a

±3.4

SCSA/DFI 4.0a

±2.0

4.2a

±2.0

11.2a

±8.4

12.2a

±8.3

11.7a

±9.5

13.5a

±8.6





After centrif.: values after centrifugation

After resus.: values after resuspension with freezing extender





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PAS: Percentage of sperm with positive acrosomal status (FITC-PNA pos)

DFI: denaturation fragmentation index of sperm DNA (%) a, b, c Values with different superscript differ significantly within treatment groups (p≤0.05).

Second experiment: Fresh semen samples

Table 8: Effect of EquiPureTM Pro density gradient on sperm quality parameters (means ± standard de-

viation).

Initial value After centrifugation

Sperm recovery - 30.1±4.0

MVO 82.1±8.2a 89.0±4.0a

PMV 69.2±8.9a 76.9±6.0a

VCL 187.7±22.7a 157.3±6.0b

VAP 120.3±13.0a 109.2±3.4a

ALH 3.6±0.4a 3.4±0.6a

PMI (PI neg) 74.6±8.2a 84.4±1.3a

PAS (FITC-PNA pos) 6.1±2.2a 2.4±0.6a

SCSA/DFI 6.6±1.3a 2.4±1.2b









PAS: Percentage of sperm with positive acrosomal status. (FITC-PNA pos)


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Danksagung

An dieser Stelle möchte ich allen Personen danken, die zum Gelingen dieser Arbeit beigetra-

gen haben.

Mein Dank gilt meinem Doktorvater Herrn Prof. Dr. Harald Sieme für die Überlassung des

interessanten Themas und die Unterstützung bei der Abfassung der Dissertation.

Ein besonders großes Dankeschön geht an Frau Dr. Gunilla Martinsson für die geduldige Be-

treuung und grenzlose Unterstützung in allen Lebenslagen.

Bei Herrn Landstallmeister Dr. Axel Brockmann und allen Mitarbeitern des Niedersächsi-

schen Landgestüts Celle bedanke ich mich für die Ermöglichung der Versuchsdurchführung

und die gute Zusammenarbeit.

Ebenso möchte ich mich bei Barbara für die Hilfe bei der statistischen Auswertung und Chris-

tian für die Unterstützung bei der Formatierung bedanken.

Genauso bedanke ich mich bei Jane, Jutta, Steven und Harriette für die ständige Diskussions-

bereitschaft und Hilfe bei der Fertigstellung.

Bedanken möchte ich mich weiterhin bei Pamela, Sophie, Wasyl, David, Steffi und Gesche,

Camilla, Daphne und Christiane, die mich während der Doktorantenzeit und auf dem Landge-

stüt stets unterstützt haben.

Mein größter Dank gilt meiner Familie und Heinrich für ihre Unterstützung und den Rückhalt,

den sie mir während der ganzen Zeit gegeben haben.

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