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AUTOMATED AND STANDARDIZED
ANALYSIS OF EQUINE SEMEN AND
INFLUENCES OF CENTRIFUGATION
ON EQUINE SEMEN PRESERVATION
Maarten Hoogewijs
Faculty of Veterinary Medicine
Department of Reproduction, Obstetrics and Herd Health
Salisburylaan 133 – 9820 Merelbeke – Belgium

I am among those who think that science has great beauty.
A scientist in his laboratory is not only a technician:
he is also a child placed before natural phenomena
which impress him like a fairy tale.
Marie Curie
Printed by: Ryhove Plot-it
Foto kaft: Johan Jacobs - Droomwereld

AUTOMATED AND STANDARDIZED ANALYSIS OF EQUINE
SEMEN AND INFLUENCES OF CENTRIFUGATION ON EQUINE
SEMEN PRESERVATION
Thesis submitted in fulfillment of the requirements for the degree of Doctor in Veterinary Sciences
(PhD), Faculty of Veterinary Medicine, Ghent University, 2010
Maarten Hoogewijs
Faculty of Veterinary Medicine
Department of Reproduction, Obstetrics and herd Health
Salisburylaan 133 – 9820 Merelbeke – Belgium
Promoters:
Prof. Dr. Aart de Kruif
Prof. Dr. Ann Van Soom
Prof. Dr. Sarne De Vliegher

LIST OF ABBREVIATIONS
TABLE OF CONTENTS
PREFACE 1
CHAPTER 1 GENERAL INTRODUCTION 3
1.1 Semen analysis 5
1.2 Collection and processing of equine semen 27
1.3 Shortcomings in current practice 49
CHAPTER 2 SCIENTIFIC AIMS 71
CHAPTER 3 ANALYSIS OF EQUINE SEMEN – IS AUTOMATION (THE) KEY TO STANDARDIZATION? 75
3.1 Influence of technical settings on CASA motility parameters of frozen-thawed stallion
semen 77
3.2 Counting chamber influences the CASA motility outcomes of equine semen analysis 85
3.3 Validation and usefulness of the SQA-Ve (1.00.43) for equine semen analysis 109
3.4 Need for further improvement of SQA-Ve (1.00.61) for a univocal analysis of the
quality of frozen-thawed equine semen 123
CHAPTER 4 DIFFERENT CENTRIFUGATION TECHNIQUES – TO WHAT EXTENT DO THEY AFFECT QUALITY AND
PRESERVATION OF EQUINE SEMEN 135
4.1 Influence of different centrifugation protocols on equine semen preservation 137
4.2 Sperm selection using single layer centrifugation prior to cryopreservation can
increase post-thaw sperm quality in stallions 157
CHAPTER 5 GENERAL DISCUSSION 177
SUMMARY 201
SAMENVATTING 205
DANKWOORD 211
CURRICULUM VITAE 215
BIBLIOGRAPHY 219
ADDENDUM I FUNDAMENTAL ASPECTS OF CRYOPRESERVATION 225
ADDENDUM II DETAILED LOADING TECHNIQUE OF THE DIFFERENT TYPES OF COUNTING CHAMBERS USED TO ANALYZE
EQUINE SEMEN SAMPLES WITH CASA 231
i

A Area
AI Artificial Insemination
ALH Amplitude of the Lateral Head displacement
AO Acridine Orange
AR Acrosome Reacted
ASMA Automated Sperm Morphology Assessment
AV Artificial Vagina
BCF Beat Cross Frequency
CASA Computer Assisted Sperm Analysis
CC Coefficient of Correlation
CG Cover Glass
ConA Concovalin A
CONC Concentration
CP Centrifugation Protocol
CPA Cryoprotective Agent
CSU Colorado State University
CTC Chlortetracycline
CV Coefficient of Variation
D Depth
DGC Density Gradient Centrifugation
DMF Dimethylformamide
DMSO Dimethyl Sulfoxide
FACS Fluorescence Activated Cell Sorter
FITC Fluorescein Isothiocyanate conjugated
HGLL Hank’s salt solution supplemented with Hepes, glucose and lactose
HOST Hypo Osmotic Swelling Test
LF Leucosorb® Filtration
LIN Linearity
LIST OF ABBREVIATIONS
iii

LIST OF ABBREVIATIONS
LM Light Microscopy
MF Methylformamide
MORF Percentage spermatozoa with normal Morphology
NPPC Native Phosphocaseinate
PAP Papanicolaou
PBS Phosphate Buffered Saline
PI Propidium Iodide
PM Progressive Motility
PMS Progressive Motile Spermatozoa
PNA Arachis hypogea (Peanut) agglutinin
PSA Pisum sativum agglutinin
PVP Polyvinyl Pyrrolidone
Rapid Percentage Rapid Spermatozoa
SCD Sperm Chromatin Dispersion
SCSA Sperm Chromatin Structure Assay
SD Standard Deviation
SLC Single Layer Centrifugation
SQA Sperm Quality Analyzer
SS Segre Silberberg
STR Straightness
TM Total Motility
TUNEL Terminal deoxynucleotidyl transferases dUTP end labeling
V Volume
VAP Average Pathway Velocity
VCL Curved Line Velocity
VEL Velocity
VSL Straight Line Velocity
WBFSH World Breeding Federation for Sport Horses
WHO World Health Organization
iv

PREFACE
The first recorded case of artificial insemination (AI) in horses goes back to the Arabs in 1322,
when a chief decided to steal semen from a rival’s stallion to use for one of his own mares (Bowen,
1969). Still, it wasn’t until 1677 that spermatozoa were actually visualized by van Leeuwenhoek and
Ham using one of van Leeuwenhoek’s first microscopes. These so called “animalcules” were
described as living creatures with a tail and have been studied ever since. In 1781, Spallanzani
described the influence of temperature on the motility of these animalcules. He found that cooling in
ice reduced their motility, and after subsequent warming the animalcules became motile again
(Gonzalès, 2006).
Between the first reported AI from the Arabs and the research during the late 19 th century by
Sir Walter Heape, equine AI languished in silence. Early research in reproductive work was done by
Heape, a student from the University of Cambridge and a pioneer in embryo transfer in the rabbit
(Heape, 1890). He also collected semen from a stallion followed by successful inseminations in mares
(Heape, 1898). Continuing research in horse reproduction and in utero development was performed
by Sir John Hammond, who carried out reciprocal inseminations of Shire mares with Shetland stallion
semen and vice versa to examine the maternal effect on growth and conformation (Walton and
Hammond, 1938). Unfortunately, most of the early work which was done by the Russians and the
Chinese between 1930 and 1960 remained unpublished, hidden by the Iron Curtain and language
barriers (Allen, 2005). In the late 1960s, equine AI revived with the work of B.W. Pickett and his large
group of co-workers who worked on the collection, dilution, cooling, freezing, and insemination of
stallion semen. Much of this work is summarized by Squires et al. (1999).
The acceptance of AI by the majority of horse breed registries and the possibility to transport
cooled and frozen semen, have changed the entire horse industry and equine reproduction in
particular. This widespread use of “processed” semen does not only increase the breeder’s
possibilities but also surfaces challenges for the researchers and veterinarians harvesting and
preparing these AI doses. First of all, there is the never ending quest to increase the pregnancy rates
(requiring improved processed semen), and secondly, following human andrology, standards need to
be formulated when examining that semen, so the quality can be assessed worldwide using the same
criteria.
1

PREFACE
Allen W.R., 2005: The development and application of modern reproductive technologies to horse
breeding. Reproduction in Domestic Animals, 40:310-329.
Bowen J.M., 1969: Artificial insemination in the horse. Equine Veterinary Journal, 1:98-110.
Gonzalès J., 2006: Histoire du spermatozoïde et mobilité des idées. Gynécologie Obstétrique &
Fertilité, 34:819-826.
Heape W. 1890. Preliminary note on the transplantation and growth of mammalian ova witin a
uterine foster-mother. Proceedings of the Royal Society of London series B 48:457-458.
Heape W. 1898. On the artificial insemination of mares. Veterinarian 71:202-212.
Squires E.L., Pickett B.W., Graham J.K., Vanderwall D.K., McCue P.M., Bruemmer J.E. 1999. Cooled
and frozen stallion semen. Animal Reproduction and Biotechnology Laboratory Bulletin N°9 –
Colorado State University, Fort Collins.
Walton A., Hammond J. 1938. The maternal effects on growth and conformation in Shire horse-
Shetland pony crosses. Proceedings of the Royal Society of London series B 125:311-335.
2

GENERAL INTRODUCTION
CHAPTER 1
3

Semen analysis
1.1
5

1.1.1. Introduction
CHAPTER 1.1
The widespread increased use of artificial insemination (AI) in domestic animal reproduction
implies a growing awareness concerning the male’s fertilizing potential. Indeed, the fertility of one
individual male can have an enormous impact on the pregnancy outcome of hundreds of females. As
such, the economic importance of male fertility in breeding animals has increased proportionally.
Focusing on livestock, the financial interests are equally important for business both for
owners of male and female breeding animals. For the male side, more insemination doses represent
a higher income for the breeder or AI company, but the downside can be that lowering the dose
might result in a decreased conception rate (Chenoweth et al., 2010). This will affect the production
results and might lead to law suits against the AI companies that are involved. International
guidelines on the minimal quality requirements for an AI dose would protect the owners of the
female animals but could also provide insurance for the male counterpart. However, before those
uniform international guidelines for different animal species can be established, a consensus is
required on techniques and settings used for semen analysis. After all, methodology of analysis has a
major influence on the outcome parameters.
In many aspects the equine breeding industry is comparable with the production animal
breeding industry, although there are some striking differences. In the latter, the importance of a
given positive trait (e.g. faster growing rate, higher milk production) might be less attractive for
selection when associated with markedly decreased fertility. This is in sharp contrast with equine
breeding, where selection is most often based on breed characteristics such as phenotype or sports
performance. Consequently, a possible reduced fertility outcome becomes only important in the long
run. Additionally, a superior performing stallion with decreased fertility might even be more
attractive to breeders of sport horses, as breeding with this stallion will not only lead to offspring
with a high performance expectation, but due to its subfertility it will also result in a low number of
foals. The game of supply and demand will thus increase the value of the offspring even more.
Due to great demands of AI doses from famous, popular stallions, normal fertile stallions
might seem subfertile. At a given time a (too) popular stallion might breed more mares than there is
semen available, which will very often result in more AI doses containing lower numbers of sperm.
Eventually, this might lead to a decreased conception rate and increased costs for the mare owners.
Therefore, regardless of the intrinsic fertility of a stallion, minimum requirements for an AI dose
concerning number and quality of the sperm might provide an assurance for the breeders.
7

CHAPTER 1.1
8
In the next paragraphs, the state-of-the-art concerning the analysis of (equine) semen is
reviewed. Where possible, the comparison is made with the guidelines according to the World Health
Organization (WHO). The WHO laboratory manual for the examination of human sperm and sperm-
cervical mucus interaction was published for the first time in 1980. This guideline was developed in
response to a growing need for the standardization of procedures for the examination of human
semen. The manual has ever since provided global standards for human semen analysis and has been
updated on numerous occasions based on new understandings and developments. In 2010 the fifth
edition was published.
1.1.2 Gross evaluation of equine semen quality
Following ejaculation the gel is separated from the remainder of the semen in the laboratory
if no inline filter was applied during collection. The color and consistency of the semen are recorded.
Normal semen should be white, grayish-white to slightly cream coloured and this provides a rough
estimate of sperm concentration. Usually, larger volumes are more watery instead of a creamy
consistency and this will be reflected in low sperm concentrations. A deviant color of the ejaculate
may indicate the presence of admixtures, such as blood, urine or purulent material (Estrada and
Samper, 2007; Binsko et al., 2011). The conventional semen analysis consists of the evaluation of
semen volume, spermatozoal concentration, morphology and motility. Additional evaluations using
more recent techniques might improve the predictive value of the examination (Varner, 2008).
1.1.3. Volume
The volume of an ejaculate is most often determined by pouring the semen from the
collection bottle into a tilted, pre-warmed graduated cylinder (Jasko 1992; Picket, 1993; Squires et al.,
1999). This is in sharp contrast with human andrology, where it is advised to weigh the empty and
filled container in order to calculate the volume based on the density of semen (WHO, 2010). For
human samples, it is not recommended to aspirate or to decant the sample into a graduated cylinder.
Anyhow, by decanting a part of the sample will get lost and sample losses have been reported to vary
between 0.3 and 0.9 mL (Brazil et al., 2004; Iwamoto et al., 2006; Cooper et al., 2007). Obviously,
these losses weigh heavily when handling small volume samples in human andrology with a total
volume of about 4.0 mL, when compared to equine ejaculates in which a 10 times larger volume is
most often present (Picket, 1993; Squires et al., 1999). In species with lower volume ejaculates (bulls,
rams,…) losses due to decanting will be more important.

1.1.4. Concentration
CHAPTER 1.1
According to the WHO manual (WHO, 2010), determination of the concentration of a human
semen sample is preferably done using a 100 µm deep haemocytometer. The improved Neubauer
haemocytometer is most commonly used. These evaluations are rather time consuming and as such,
not easily applicable in production settings of animal AI companies where numerous ejaculates are
processed. Hence, automated devices are commonly used in animal andrology, which is in contrast
with practices in human andrology laboratories.
Photometers
Photometers (Fig. 1a) are probably the most frequently used devices for estimating
concentration. Determination of concentration is based on the absorbance of a part of a light beam
by the particles (spermatozoa) in a suspension. The alteration in light transmitted through a sample
corresponds to the concentration of particles (Squires et al., 1999). As such, these instruments allow
rapid measurements of concentration with a good accuracy as long as the concentration is within
range (>100 and

CHAPTER 1.1
a.
10
b.
Fig. 1. (a) photometer used to assess sperm concentration quickly of a raw equine semen sample by
means of density measurement (picture provided by IMV), (b) NucleoCounter SP100 to
analyse sperm concentration of any kind of sample, based on fluorescent microscopy using
propidium iodide imbedded in the analysis cassette (picture provided by Chemometec).
Computer Assisted Sperm Analysis
A number of scientific studies have been performed using computer assisted sperm analysis
(CASA) systems to determine sperm concentration. One of the major concerns is the use of the
shallow disposable counting chambers, which typically consist of capillary-loaded slides of
approximately 20 µm depth (Douglas-Hamilton et al., 2005a). This chamber depth needs to be
limited in order to obtain a clear, sharp image necessary for sperm recognition. Sperm cells are
counted per unit area in this monolayer, and concentration can be calculated (Douglas-Hamilton et
al., 2005a). However, the distribution of particles is not uniform throughout the chamber due to the
dynamics occurring when filling such a shallow chamber. This phenomenon is known as the Segre-
Silberberg (SS) phenomenon and has been described extensively (Douglas-Hamilton et al., 2005b). A
mathematical model has been determined which can be used to correct for the underestimation of
the concentration (Douglas-Hamilton et al., 2005a), based on the viscosity of the sample and the
coherent filling time of the chamber. Although this correction is possible, it was shown that the use
of 20 µm deep capillary-loaded slides for the determination of sperm concentration is not compatible
with the requirement for high-standard quality assurance in the andrology laboratory (Björndahl and
Barratt, 2005). Hansen et al. (2006) found varying repeatability for CASA systems, depending on
manufacturer, and a large difference between the evaluated systems.

Fluorescent Activated Cell Sorter
CHAPTER 1.1
A fluorescent activated cell sorter (FACS) has been evaluated to determine the concentration
of boar semen. The FACS showed a very good correlation and the highest repeatability (Hansen et al.,
2006). This is not surprising since this flow cytometric technique uses an internal standard of
fluorescent beads with every analysis.
Haemocytometer
Since the accurate and precise devices (NucleoCounter SP-100 and FACS) are all rather
expensive, it is not useful to propose them as the “gold standard”. Therefore, the haemocytometer is
most likely the best alternative as standard for concentration determination, in concordance with
human andrology. The counting chamber type is known to have a major influence on the outcome of
sperm concentration. In shallow chambers, loaded with capillarity, the SS effect has an influence on
concentration (Douglas-Hamilton et al., 2005a, 2005b). In the Makler chamber (Fig. 2a), a reusable
chamber with a 10 µm depth, the cover glass is placed upon the loaded area, the time interval
between loading and applying the cover glass is a major factor influencing the obtained
concentration (Matson et al., 1999). The Makler chamber has been documented as having a low
precision (Christensen et al., 2005), and the time interval before applying the cover slide might
attribute to this imprecision (Makler, 2000). Different haemocytometers result in different outcomes,
especially when chambers with different depths are used. Therefore, it might be advisable to
recommend the use of a standard counting chamber. In accordance with the WHO, the improved
Neubauer haemocytometer (Fig. 2b) could be the chamber of preference. This chamber was also
described for use in horses (Vanderwall, 2008). Alternatively, a Bürker haemocytometer can be used
as well (Kuisma et al., 2006). Before filling a haemocytometer, correct placement of the cover slip can
be verified by looking for the presence of iridescence (multiple Newton’s rings) between the two
glass surfaces (WHO, 2010). Following a 1:100 dilution (Vanderwall, 2008) of the semen with a
fixative to immobilize the spermatozoa, the well mixed sample is loaded into the chamber. The filled
chamber should be allowed to rest horizontally (>4 min) at room temperature in a humified chamber
to prevent evaporation. The immobilized cells will sediment onto the grid and this will facilitate
counting. When assessing concentration using a haemocytometer, it is advised to fill and count
duplicate chambers to reduce the variation (Freund and Carol, 1964). For assessing concentration
using a haemocytometer the use of cover slips with a thickness of 400 µm is advised (WHO, 2010).
11

CHAPTER 1.1
Fig. 2. Different counting chambers for assessing sperm concentration: (a) 10 µm deep Makler
chamber, and (b) 100 µm deep improved Neubauer haemocytometer with a 400 µm thick
cover slide.
1.1.5. Motility
12
Subjective motility analysis
b.
Spermatozoal motility can either be estimated subjectively or assessed objectively with
automated devices. Subjective motility analysis is known to be inaccurate and imprecise (Davis and
Katz, 1993), with the greatest variation caused by differences between examiners (Katila, 2001). In
order to obtain an estimate as accurate as possible, environmental conditions should be
standardized and optimized for semen (Katila, 2001), which means that all materials that can have
contact with the semen should be stored in an incubator at 37°C and the stage of the light
microscope should also be warmed to this temperature. The sample should be properly extended to
a range of 25 to 50 × 10 6 sperm/mL using an extender that does not alter the motility, since it is
known that there is a strong relationship between sperm concentration and subjective appraisal of
motility (Van Duijn and Hendriske, 1968). Another critical factor in motility analysis is the depth of
the sample. A depth of more than 20 µm causes a less clear view, since sperm will move in and out
the microscopic plane of view. Shallow depths of 10 µm allow for a good visualization, however,
motility may be altered because the sperm head is restricted in its free rotation (Jasko, 1992). The
semen sample should be prepared in order to obtain a monolayer, so sperm cells can be properly
identified and motility pattern is not altered due to spherical limitations. Extra attention is required
when not using fixed depth chambers. In the fifth edition of the WHO laboratory manual (WHO,
2010), a special section is dedicated to the depth of wet preparations. Briefly, the depth of a

CHAPTER 1.1
preparation (D, µm) is obtained by dividing the volume of the sample (V, µL = mm³) by the area over
which it is spread (A, mm²). Consequently, when using a 18 mm × 18 mm or 22 mm × 22 mm
coverslip, a 6.5 µl or 10 µl sample should be used, respectively. In veterinary andrology the
importance of sample depth for motility analysis is clearly not well known. Very often the for motility
analysis prepared slides have very limited sample depths of 8.7 µm (Akcay et al., 2006).
Objective motility analysis: Sperm Mobility Assay
Inaccuracies and subjectivity in motility analyses can be avoided using automated techniques.
Multiple techniques are available to analyze and record sperm motility in an objective way. One of
the less familiar techniques is the sperm mobility assay. This technique was first described by McLean
and Froman (1996), who found that spermatozoa from subfertile roosters were characterized by
decreased sperm motility when sperm suspensions were overlaid upon 6% (w/v) Accudenz®. The
motile sperm enters the Accudenz® solution by their own power, as such increasing the optical
density of the solution which can be measured with a photometer (Froman, 2007). Absorbance is
measured after a 5 min incubation interval (at 41°C for fowl sperm), and higher absorbance is
associated with higher motility and higher fertility (Froman and McLean, 1996). Although the test
was validated as a sperm motility assay (Froman and McLean, 1996), a key distinction was made: the
assay estimates the mobility (= gross movement) of a sperm population, rather than sperm motility (=
individual movement) per se (Froman, 2007). This way of analysis has been well documented for fowl
sperm, and was more recently validated for stallion and boar sperm (Vizcarra and Ford, 2006).
Objective motility analysis: Sperm Quality Analyzer
Another alternative for automated motility analysis is the sperm quality analyzer (SQA),
which analyzes total and progressive motility and sperm motility of a sperm sample, loaded in a
special designed capillary, by passing a light beam through the sample. The motile sperm cells create
disturbances in the light beam, which are converted into electrical signals by a motility detector and
translated into a numerical output. The latest version has a separate channel for simultaneously
analyzing the concentration, based on light absorbance. A similar device was already described in
1981 (Bartoov et al., 1981), and ever since, this device has been updated, adapted and studied. The
first reports using the SQA concerned the analysis of human semen (Bartoov et al., 1991; Makler et
al., 1999; Martinez et al., 2000). Later on, the human devices have also been tested for different
animal species [dogs (Iguer-Ouada and Verstegen, 2001; Rijsselaere et al., 2002), turkeys (Neuman et
13

CHAPTER 1.1
al., 2002), boars (Maes et al., 2003; Vyt et al., 2004) rams (Fukui et al., 2004), bulls (Hoflack et al.,
2005), and mice (Tayama et al., 2006)]. The newest version of the SQA (version V) was initially
developed for human samples as well and later on adapted for different animal species, namely bulls,
boars, stallions and turkeys. Specific devices for rams and roosters are in development. The SQA is
ready to use after delivery and does not require (or allow) user specific settings, as such reducing a
large potential source of bias (Hoflack et al., 2005).
14
Objective motility analysis: Computer Assisted Sperm Analysis
The best known way to objectively analyze sperm motility is analysis by means of a CASA
system. The first CASA system was introduced over 30 years ago, and has been used ever since in
human and veterinary andrology laboratories. By equipping a microscope with a camera, the sperm
cells are visualized and the actual sperm tracks can be analyzed after sperm cell recognition. The
latter can be achieved either based on the number of pixels and intensity or, in newer versions, by
means of fluorescent dyes. This technique facilitates sperm recognition and allows CASA analysis of
(post thaw) samples containing egg yolk particles (Tardif et al., 1998) or debris for example after
obtaining epididymal sperm in cats (Filliers, personal communication). The major advantages of CASA
systems are, amongst others, the good repeatability and the absence of subjectivity. Nevertheless,
standards for analysis need to be established first, since different technical settings and sample
preparations have been shown to influence the outcome of CASA analysis (Table 1). The impact of
different technical settings (frame rate and number of frames analyzed) and procedures
(temperature, concentration, diluents and chamber) have been studied for different species (Contri
et al., 2010; Lenz et al., 2011; Rijsselaere et al., 2003). Also, the motility settings may have an
influence on the CASA outcome as well. Based on low and medium average pathway velocity (VAP)
cut-off values and on straightness (STR), spermatozoa are graded. In literature, a plethora of settings
is used (Table 2). Not only is there a lack of agreement in the cut-off values used by different
research groups, also the description of these values is not uniform for CASA systems from different
companies.
A comparable lack of standardization can be found in the chambers used to analyze the
motility of a semen sample. Although the effect of the counting chamber was already described in
2001 (Iguer-ouada and Verstegen, 2001), different brands and types of chambers are used. The most
frequently used chambers are a 20 µm deep Leja chamber (Waite et al., 2008; Ortega-Ferrusola et al.,
2009; Len et al., 2010), a 20 µm deep Cell-Vu (Almeida and Ball, 2005; Glazar et al., 2009; Spirizzi et
al., 2010) and the 10 µm deep reusable Makler chamber (Kavak et al., 2003; Johannisson et al., 2009).

CHAPTER 1.1
These chambers are frequently loaded with a different volume of sperm, sometimes only the depth
of the chamber is mentioned without the brand name (Pagl et al., 2006; Aurich and Spergser, 2007),
or only the volume used is mentioned (Quintero-Moreno et al., 2003; Price et al., 2008) and
occasionally nothing is mentioned at all (Love et al., 2004; Macia-Garcia et al., 2009; Ponthier et al.,
2009). In three recent studies, the influence of different chambers on motility was evaluated. For bull
semen, the Makler chamber resulted in higher total and progressive motility compared to 20 µm
deep Leja chambers (Contri et al., 2010; Lenz et al., 2011) but results were not different compared to
these obtained with the WHO prepared slide (Lenz et al., 2011). The effect of different counting
chambers when analyzing equine semen needs to be analyzed.
Table 1. Influence of technical settings and sperm preparation on outcome motility parameters generated with
a CASA system (tested variables are listed between brackets).
Setup Rijsselaere et al., 2003 Contri et al., 2010
Species Dog Cattle
Type of CASA Hamilton-Thorne
CEROS 12.1
Frame rate Influence
(15-30-60Hz)
Number of frames Limited influence
(30-60)
Concentration Influence
(100 – 50 – 25 × 10 6 )
Advised concentration: 50 × 10 6
Diluent Influence
(physiological saline – prostatic fluid –
Hepes-TALP-medium –
egg-yolk-Tris extender)
1.1.6. Morphology
Hamilton-Thorne
IVOS 12.3
Influence
(30-60Hz)
No influence
(30-45)
Influence
(100 – 50 – 30 – 20 – 10 – 5 × 10 6 )
Advised concentration: 20 × 10 6
Influence
(physiological saline – PBS –
Bio-excell)
In human andrology, three different stainings are recommended by the WHO (2010), namely
the Papanicolaou (PAP), the Shorr and the Diff-Quick stain which can all be interpreted using
brightfield optics. With these staining procedures, the sperm head is stained pale blue in the
acrosome region and dark blue in the post-acrosomal region. The midpiece may show some red
staining and the tail is stained blue or reddish. Excess residual cytoplasm is stained pink or red using
the PAP stain or reddish-orange in case of the Shorr stain (Boersma et al., 2001; WHO, 2010). The use
of rapid staining methods such as eosin-nigrosin staining, is not recommended by the WHO because
15

Table 2. Type of counting chamber used and technical settings [average pathway velocity (VAP) and straightness (STR)] in combination with computer
assisted sperm analysis in horses as reported in literature, blank fields indicate that the information was not available in the paper.
STR
(%)
medium VAP
cut-off (µm/s)
low VAP cutoff
(µm/s)
Frames
acquired
Frame
rate (Hz)
Counting
Chamber
Study
Albrizio et al., 2010 Leja (20 µm) 60 45 20 50 75
Aurich and Spergser, 2007 20 µm
Ball and Vo, 2001
Baumber et al., 2002
Brinsko et al., 2000a
Glazar et al., 2009 Cell-Vu (20 µm) 60 45 20 50 75
Kavak et al., 2003 Makler (10 µm) 32 15 10 25
Loomis and Graham, 2008
60 40 20 50 75
Macias Garcia et al., 2009 Leja (20 µm)
10 15 45
Melo et al., 2007 Makler (10 µm) 60 30 30 70 80
Miro et al., 2005
25 16
Nascimento et al., 2008 Leja (20 µm) 80 30 20 70 60
Ortega-Ferrusola et al., 2009 Leja (20 µm) 25
10 15 45
Pagl et al., 2006 20 µm
Papa et al., 2008
Squires et al., 2004 Cell-Vu (20 µm)
20 20 25 80
Taberner et al., 2010
10 90 75
Vidament et al., 2009
60 30 20 40 80
Waite et al., 2008 Leja (20 µm) 60 45 15 30 50
Webb et al., 2009 Leja (20 µm) 60 30 20 50 75

CHAPTER 1.1
it is not possible to observe the details necessary for the morphological classification when
spermatozoa are not equally distributed using the smearing technique (WHO, 2010).
The PAP stain is almost a synonym for sperm morphology assessment according to WHO
standards. However, the major downside of this stain, is the time consuming procedure, which
involves 20 processing steps using more than 12 different chemical solutions. Due to the long
staining procedure, the PAP staining is not frequently used for morphological analysis of animal
semen. However, a rapid PAP stain procedure has been developed which has been used in
combination with automated sperm morphology assessment (ASMA) (Boersma et al., 2001).
The importance of the staining procedure for morphology analysis is well known. Staining
techniques affect the morphometric dimensions of the sperm head, most likely due to differences in
osmolarity in fixatives and staining solution. Most of these substances are not iso-osmotic in relation
to the sperm (Marree et al., 2010). For instance, after staining sperm with PAP, the heads were
shrunk when compared to fresh sperm. A new developed stain, SpermBlue®, is iso-osmotic in
relation to human semen (van der Horst and Maree, 2009) and has been demonstrated not to
influence the morphometric dimensions of the human sperm head (Marree et al., 2010). Additionally,
the entire fixation and staining process requires only 25 min.
Animal semen samples are often analyzed as wet mounts without staining (Estrada and
Samper, 2007), after fixing in buffered formol saline or buffered glutaraldehyde solution (Brinsko et
al., 2011). Depending on the laboratory, animal semen is stained with specific sperm stains [e.g.,
Williams stain (Williams, 1950) and Casarett stain (Casarett, 1953)], general purpose stains (e.g.,
Wright’s, Giemsa, Hematoxylin-Eosin) or background stains (e.g., eosin-nigrosin, India ink) (Barth and
Oko, 1998; Varner, 2008).
Despite the already mentioned associated disadvantages, eosin-nigrosin is probably the most
frequently used staining method for animal semen because of its simplicity. Besides, this staining
method gives good results for routine morphology evaluations. The eosin component of the stain will
penetrate the damaged sperm membrane, causing the cell to turn pink, while sperm with an intact
sperm membrane will remain white against the dark background provided by the nigrosin (Barth and
Oko, 1989). Based on this principle, the eosin-nigrosin stain can also be used to assess the
percentage of live, acrosome intact sperm, since stained sperm are considered to be dead or to have
lost the acrosome (Brinsko et al., 2011). However, artefactual changes can occur simply from cold
and osmotic shock which will lead to an increased percentage of stained sperm. Therefore, the
17

CHAPTER 1.1
staining procedures should be followed exactly, i.e. the semen, stain and slides should be warm (37°C)
and the preparation should be thin allowing the smear to dry rapidly (Fig. 3). For preparing an eosin-
nigrosin stain, the following procedure is recommended (Bart and Oko, 1989):
18
a. Place a drop of warm stain near the frosted end of a warm microscope slide.
b. Place a drop of warm semen near the stain and mix the two on the slide (the ratio stain to
semen depends on the concentration of the semen sample).
c. To make a smear, a second slide held at a 30-40° angle, is pushed against the drop of
stained semen and pulled back slowly as shown in Fig. 3.
d. Dry the smear quickly by blowing air across it.
Fig. 3. Method for making a sperm smear using eosin-nigrosin stained sperm.
Sperm morphology is classified in different categories based on the origin of the abnormality
or based on the specific morphological defects. Based on origin, morphological defects are classified
as primary, secondary or tertiary abnormalities. Primary defects, of testicular origin, originate during
spermatogenesis. Secondary abnormalities are associated with the excurrent duct system
(epididymal origin) while tertiary defects are considered artefactual defects caused during sperm
collection and preparation. On the other hand, the classification by the specific morphological
appearance might be preferable since it provides information about the actual defect instead of the
presumptive origin. After all, a specific defect might have different causes. Therefore, sperm can be
classified as either normal or with abnormalities i.e. in the head, neck and midpiece, or tail, and
sperm with excess residual cytoplasm. Within any category, subdivisions are made. In Fig. 4, an

CHAPTER 1.1
overview of common abnormalities in equine spermatozoa morphology is given as viewed by
differential-interference contrast microscopy of fixed and unstained wet-mount sperm.
1.1.7. Optional procedures
Although highly specialized, a spermatozoon can be considered as a relative simple structure.
Due to the dramatic reduction in cellular organelles when compared to somatic cells, a
spermatozoon is composed of only a head and a tail and can be subdivided into: (1) a nucleus, (2) an
overlaying acrosome, (3) the fibrous and microtubular components of the flagellum, (4) a
mitochondrial sheath, and (5) an enveloping plasma membrane (Varner, 2008). A large number of
tests is available to evaluate these different compartments.
Membrane integrity
The plasma membrane of a sperm cell is semi-permeable and surrounds the entire sperm cell
and plays an important role in its function. The integrity of this membrane can be evaluated using
different techniques. Most methods rely on the evaluation of the ability of the membrane to exclude
extracellular membrane-impermeable dyes. For this purpose eosin as well as fluorescent dyes such
as PI, bis-benzimide (Hoechst 33258), YO-PRO®-1, TOTO®-1 and ethidium homodimer-1 can be used
(Varner, 2008). Some of these membrane impermeable dyes can be combined with membrane
permeable dyes to provide a more accurate reflection of the membrane integrity. For instance, PI is
frequently combined with SYBR®-14, which results in three different staining patterns representing
three classes of sperm, i.e. green cells (SYBR®-14 stained) have an intact membrane, red cells (PI
stained) are membrane damaged and double stained cells are moribund cells (Fig. 5) (Garner and
Johnson, 1995).
An alternative approach to evaluate the integrity and functionality of the sperm plasma
membrane is analyzing the response of the sperm cells when placed in hypotonic medium. This hypo
osmotic swelling test (HOST) was first reported by Drevius and Ericksson (1966) and has been used
for both human and domestic animal semen (Jeyendran et al., 1984 ; Neild et al., 2000). Spermatozoa
with a functional membrane in a hypo-osmotic environment respond with different kinds of swelling.
A schematic overview of the different swelling patterns is shown in Fig. 6.
19

CHAPTER 1.1
Fig. 4. Drawing of equine spermatozoa as viewed by differential-interference contrast microscopy
of fixed and unstained wet-mount sperm. (A) Spermatozoa with normal morphology in
dorsolateral (A1, A2) and (A3) side view. Abaxial midpieces (A1) are considered
morphologically normal in stallion spermatozoa. (B) Spermatozoa with different head
abnormalities; (B1) macrocephaly, (B2) microcephaly, (B3) nuclear vacuoles, (B4) tapered
20

CHAPTER 1.1
head, (B5) pyriform head, (B6) constricted head, and (B7) degenerated head. (C) Acrosomal
defects (knobbed head) in (C1, C2) dorsolateral and (C3) side view. (D) Residual cytoplasmatic
droplets, located (D1) proximally or (D2, D3) distally. (E) Abnormal midpieces; (E1) segmental
aplasia of the mitochondrial sheath, (E2) roughed midpiece from uneven distribution of
mitochondria, (E3) enlarged mitochondrial sheath, (E4, E5, E6) bent midpieces, and (E7)
double midpiece/double head. (F) Bent tail or hairpin tail, (F1-F4) involving the midregion of
the principal piece, or (F5) with a singular bend or (F6) proximal bend involving the midpieceprincipal
piece junction. (G) Coiled tail, with (G1) tail tightly encircling the head or (G2,G3) tail
coil not encircling the head. (H) Fragmented sperm; (H1) detached or tailless heads, and (H2)
fragmentation at the level of the annulus. Fragmentation can also occur at different sites (F4).
(I) Premature germ cells with (I1) a single nucleus or (I2) multiple nuclei (from Varner, 2008 –
Image provided by Dr. Varner and used with permission).
Fig. 5. Fluorescent image of equine sperm cells stained with SYBR®-14/PI for analysis of the plasma
membrane: green sperm cells (1) are membrane intact, red cells (2) have a damaged
membrane and dual stained cells (3) are moribund.
21

CHAPTER 1.1
22
Defoin et al. (2005) developed a variation to HOST where semen was mixed with solutions of
different osmolarity. The cellular response to these solutions was analyzed by adding PI to the
samples and assessing the percentage of PI positive cells in the population. Since PI+ spermatozoa
included the fluorescent probe, they can be considered membrane damaged.
Fig. 6. Schematic representation of the morphological changes in spermatozoa subjected to
hypoosmotic stress (a = no change, b-g = different types of tail changes, swelling is indicated
by the grey area) (reproduced from Jeyendran et al., 1984).
Capacitation and acrosome status
Ejaculated sperm cells achieve the potential to fertilize once they are activated in the so
called capacitation process. Capacitation involves delicate changes in the plasma membrane enabling
sperm cells to bind to the zona pellucida. This binding initiates the acrosome reaction, which is an
exocytotic event required for the penetration of the zona pellucida (Colenbrander et al., 2003). This
indicates the importance of the acrosome in the whole of fertilization.

CHAPTER 1.1
The acrosome can be evaluated using different techniques including various staining
techniques for light microscopic evaluation. For this purpose, Chicago sky blue and Giemsa staining
(Kútvölgyi et al., 2006) and Coomassie blue (Brum et al., 2006) are described as specific stainings to
assess the acrosome. However, most often fluorescent staining techniques are used to analyze the
acrosomal status. The stained samples can be analyzed with fluorescent microscopy or with flow
cytometry. The latter enables the objective analysis of a large number of sperm cells.
Chlortetracycline (CTC), an antibiotic with fluorescent characteristics, binds to the surface of
sperm cells in a calcium dependent matter. Three different staining patterns can be distinguished,
depending on the stage of sperm activation, i.e. non-capacitated, capacitated acrosome-intact and
capacitated acrosome-reacted sperm (Colenbrander et al., 2003). Unfortunately, this CTC staining
cannot be used in combination with flow cytometry and additionally, CTC detects changes in
capacitation status more slowly when compared to the merocyanine 540/Yo-Pro®-1 staining. The
latter is capable of detecting early capacitation changes in combination with vitality and allows for a
flow cytometric approach (Rathi et al., 2001). Analysis of the proper acrosome status using
fluorescent dyes is commonly done by means of different fluorescein isothiocyanate conjugated
(FITC) lectins, such as concovalin A (FITC-ConA: Blanc et al., 1991), Pisum sativum agglutinin (FITC-PSA:
Farlin et al., 1992) and Arachis hypogea agglutinin (FITC-PNA: Cheng et al., 1996). The binding of FITC-
PSA to the acrosome has been confirmed by electron microscopy (Casey et al., 1993; Meyers et al.,
1995). Using this technique, only two staining patterns are achieved, namely cells with an intact
acrosome and cells missing the acrosome (Fig. 7) (Meyers et al., 1995), and additionally, PSA binds to
the equine zona pellucida (Cheng et al., 1996) rendering this technique useless when assessing the
acrosome during the sperm-oocyte interaction. On the other hand, the acrosome can be classified in
4 groups when using FITC-PNA staining: 1) intact outer acrosomal membrane, 2) vesiculation and
breakdown of the acrosomal membrane, 3) residues of the outer acrosomal membrane and 4)
complete loss of the outer acrosomal membrane. In contrast with PSA, PNA does not stain the
equine zona pellucida (Cheng et al., 1996).
23

CHAPTER 1.1
Fig. 7. Equine spermatozoa labeled with the fluorescent staining FITC-PSA with (a) representing an
intact acrosome (green fluorescence over the entire acrosomal region) and (b and c) reacted
acrosomes ( b = no green fluorescence over the acrosomal region, c = thin band of green
fluorescence over the equatorial region of the sperm head) .
24
The capacitation and acrosome status analysis can be applied to predict the fertility in two
possible ways. Firstly, by looking at the percentage of acrosome reacted and capacitated sperm in
the sample, since acrosome reacted sperm can no longer fertilize an oocyte and capacitated sperm
has a reduced longevity (Watson, 1995). Therefore, their numbers in a sperm sample should be low.
Secondly, the ability of sperm to undergo capacitation and the acrosome reaction in vitro as a
response to stimulation can be evaluated. Since the latter approach simulates essential functions of
sperm in order to achieve fertilization, this may be a more valuable approach. However, the
induction of capacitation and acrosome reaction has been described using different techniques, such
as using HCO3-/CO2-free Tyrodes medium with addition of Ca2+ ionophores (Rathi et al., 2001) or by
procaine treatment (McPartlin et al., 2009), nevertheless, these methods lack a good reproducibility.
DNA analysis
The value of DNA analysis in predicting fertility is not uniformly accepted, particularly since
contradictory results can be found in literature. However, DNA integrity of the sperm may be more
important than previously thought and may be equally important as classical sperm parameters since
“in contrast to the traditional measures of viability, morphology and motility that examine the carrier,
DNA tests assess the content of the package” (Makhlouf and Niederberger, 2006). A wide range of
diagnostic tests is available for analyzing the sperm’s DNA, and these different tests measure
different aspects of DNA damage. The DNA breaks can be analyzed either directly, or alternatively,
the susceptibility of the DNA to denaturation can be analyzed.

CHAPTER 1.1
The sperm chromatin structure assay (SCSA) is perhaps the most frequently used test to
analyze the DNA content of sperm. This assay measures the susceptibility of DNA to denaturation
when exposed to a low pH. The SCSA test was introduced by Evenson in 1980 (Evenson et al., 1980)
and can be used for analyzing a number of species, including horses (Evenson et al., 1995). The test is
based on the metachromatic properties of acridine orange (AO), which is a fluorescent dye that shifts
fluorescence from green to red when associated with double or single stranded DNA, respectively
(Evenson and Jost, 2000). Normally, AO does not penetrate well into the sperm chromatin, so
nucleoproteins must be decondensated first using a low pH solution (Schlegel and Paduch, 2005).
The chromatin susceptibility to denaturation, as measured with the SCSA, is correlated with the level
of actual DNA strands breaks (Evenson et al., 1995). These lesions may induce post-fertilization
embryonic failure (Fatehi et al., 2006), which explains the clinical relevance since this represents a
potential noncompensible defect (Varner, 2008). This means that for spermatozoa with
noncompensible defects increasing the insemination dose will not improve pregnancy rate since the
percentage of noncompensable defects will remain proportionally equal in the increased
insemination dose.
Besides the SCSA, the terminal deoxynucleotidyl transferases dUTP end labeling (TUNEL)
assay is frequently used to assess the DNA integrity of sperm. This assay analyses DNA breaks directly
and the original technique has been used in human and veterinary medicine to analyze sperm from
different species (Waterhouse et al., 2006; Filliers et al., 2008; Purdy, 2008). However, recent work
from the group of Dr. Aitken (Mitchell et al., 2011) proved that the original TUNEL assay is not
suitable for analyzing DNA strand breaks in (human) spermatozoa. They found that the assay was
insensitive and unresponsive to DNA fragmentation induced in spermatozoa when exposed to
Fenton reagents (H2O2 and Fe 2+ ). Additionally, they demonstrated that it is important to assess the
viability of sperm simultaneously when analyzing DNA breaks. Moreover, they proved that DNA
fragmentation can appear as a cause as well as a consequence of cell death. Based on these findings,
the protocol for TUNEL assay of sperm has been modified to the new sperm TUNEL assay which
combines a vitality stain (LIVE/DEAD Fixable Dead Cell Stain (far red) from Molecular Probes) (Fig. 8)
with a 45 min exposure to 2mM dithiothreitol, allowing for a correct assessment of DNA damage in
live cells (Mitchell et al., 2011). So far, this adapted TUNEL protocol has not been used to analyze
equine sperm. An additional advantage of this assay is the possibility to combine it with either a
flowcytometer as well as with fluorescence microscopy.
25

CHAPTER 1.1
26
Several other techniques can be used to analyze the DNA status of sperm. The sperm
chromatin dispersion (SCD) assay and the Comet Assay (a single-cell electrophoresis) also evaluate
the susceptibility of DNA to denaturation, while an in situ nick translation (NT) assay directly analyzes
DNA strand breaks (Makhlouf and Niederberger, 2006; Varner, 2008).
Fig. 8. Fluorescence microscopy images of the modified TUNEL staining in combination with the far
red viability stain [from left to right; LIVE/DEAD stain alone showing a non-viable, red stained
cell, TUNEL stain alone from the same TUNEL positive (green) cell, LIVE/DEAD and TUNEL
stains combined and the corresponding phase contrast image (scale bar = 5 µm)] (Mitchell et
al., 2011) (Image used with the permission of Dr. Aitken and Wiley Press)
Mitochondrial sheath
The mitochondrial sheath can be analyzed using mitochondria selective fluorescent markers.
Most common are Rhodamine 123, a variety of MitoTracker® probes and 5,5’,6,6’-tetrachloro-
1,1’,3,3’-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Due to its photoinstability and
tendency to lose retention in the mitochondria following a loss of membrane potential, Rhodamine
123 has largely been replaced by the other markers. Some of the MitoTracker® probes only fluoresce
when oxidized, allowing for an easy assessment of the respiration rate of the mitochondria. Probably,
JC-1 may be the best suited probe to evaluate the energy state of mitochondria since it produces a
membrane potential-sensitive shift in emission pattern, resulting in a change of the color from green
to red-orange as the membrane polarization increases (Baumber-Skaife, 2011; Garner and Thomas,
1999; Varner, 2008).

Collection and processing of equine semen
1.2
27

1.2.1. Semen collection
CHAPTER 1.2
The first sperm collections of stallions were performed using an intravaginal sponge, which
was placed inside the vagina of the mare prior to natural mating. The amount as well as the quality of
the thus obtained semen was poor. The admixture of vaginal secretions with the semen raised
concerns about the possibility of spreading diseases and led to the development of different
techniques. In 1934, Roemmele and Milovanov reported the use of a rubber condom inserted into
the mare’s vagina for the collection of sperm (Love, 1992). This technique often led to a
malpositioning of the rubber sperm collector resulting in the subsequent loss of the ejaculate. If the
sperm collection was successful, the ejaculate was prone to contamination due to close contact with
the penis. Consequently, artificial vaginas (AV) in different sizes and shapes were developed from
which 4 types are now commonly used throughout the world.
The first and probably best known model is the Colorado State University (CSU) AV (Fig. 9a),
which is a large, robust and rather heavy model (up to 5 kg), measuring 59 cm in length with an
internal diameter of 15 cm. It requires filling with 4.5 L of heated water of 60°C in order to obtain the
desired internal pressure and temperature of 46°C to stimulate ejaculation in most stallions. The big
advantages of this type are the good acceptance by most stallions and the good retention of heat
because of the large volume of hot water (Allen, 2005). The major drawbacks are the heavy weight
which makes it cumbersome to handle, and the fact that the stallions ejaculate in the heated water
portion of the AV. Following ejaculation, the semen must drain over the heated surface into the
recipient during which heat shock may occur. After all, heat damage in spermatozoa can easily occur
at temperatures above 43°C (Love, 1992).
The Missouri AV (Fig. 9b) consists of two heavy-duty latex rubber liners sealed together and
supported by a leather casing to create support to the water filled liner which is as such responsible
for the pressure. This light weight, easy-to-use model is well accepted by stallions and allows
protrusion of the glans penis beyond the inner water jacket, hereby reducing the likelihood of heat
damage of the sperm. However, this type of AV tends to lose heat rapidly (Allen, 2005; Love, 1992).
The design of the Missouri model AV allows the placement of a towel between the water jacket and
the leather case at the proximal end of the AV. When this towel is saturated with hot water, it can be
applied as a hot compress at the base of the penis to provide additional stimulation and base
pressure for stallions that are reluctant to ejaculate. Care must be taken to avoid contamination of
the ejaculate with water dripping from the towel (Brinsko, 2011).
29

CHAPTER 1.2
30
(a) Colorado State University AV (b) Missouri AV
(c) Hannover AV (d) Krakow AV
Fig. 9. Images of different models of artificial vaginas (AV) and schematic presentation of the relative
position of the penis at the time of ejaculation; (a) Colorado State University AV, (b) Missouri
AV, (c) Hannover AV and (d) Krakow AV (images of the CSU and Hannover AV provided by
Minitüb, image of the Missouri AV provided by IMV).

CHAPTER 1.2
The Nishikawa model AV and the Hannover Model AV (Fig. 9c) consist of an aluminum or
plastic outer casing, respectively, with a large proximal diameter and a small distal luminal diameter,
preventing the penetration of the glans penis beyond the heated portion. Although these types are
accepted well by most stallions, they are liable to cause heat damage to the spermatozoa.
The Krakow model (or adapted Cambridge model) (Fig. 9d) is a short version of the CSU AV
which allows the penis to thrust right through the length of the AV. The Krakow model is often used
as an open ended AV to allow the stallion to ejaculate into mid-air, which makes it possible to collect
individual pulses of semen and as such, allows for collection of the sperm rich fraction without the
excessive seminal plasma or gel fraction. Since contact with the glans penis is avoided, a clean
collection is performed using this AV (Allen, 2005).
Different modifications of the described AVs are being used worldwide combining the best
features of different models and avoiding the disadvantages associated with each specific type of AV.
Immediately before semen collection, the AV is prepared by filling the water jacket with hot
water providing an internal temperature of 44-48°C. Using an AV temperature above body
temperature seems to stimulate the penis and facilitates ejaculation. Some stallions require even
higher AV temperature (up to 55°C) for semen collection. In those cases, contact of the sperm during
collection with the warm water jacket must be avoided (Brinsko et al., 2011). The inner surface of the
AV is usually lubricated with a nonspermicidal lubricant. According to some authors, the use of
petroleum-based lubricants should be avoided due to sperm toxicity (Love, 1992) while others favor
vaseline to water soluble lubricant for its osmotic inactivity (Devireddy et al., 2002). The collection
receptacle should be warmed and kept at body temperature to prevent cold shock during and
immediately after collection. Usually, an isolating bag which also protects the sperm from light is
used. An inline filter can be fitted between the collection bottle and the AV to minimize trapping of
sperm in the gel fraction due to close contact after collection. This technique is superior to filtering
the whole ejaculate after collection or to aspirating the gel with a syringe. Nylon micromesh filters
are described to be superior to polyester matt filters because they are nonabsorptive, hence fewer
sperm cells are trapped. Following collection, the filter containing the gel should be removed
instantly to avoid leaking of gel into the gel-free sperm fraction.
Most often, sperm collection is performed on a dummy mare (phantom) or mount mare.
Alternatively, stallions can be trained for a ground collection, for example stallions which are unable
to mount or to maintain the mounting position. These ground collections can be accomplished by
using an AV or a plastic bag attached to the penis. In the latter case, ejaculation can be elicited either
by placement of warm (45-50°C) towel compress on the glans and base of the penis (Love, 1992) or
31

CHAPTER 1.2
can be induced pharmacologically with a combination of imipramine and xylazine hydrochloride
(McDonnell, 2001), or with xylazine hydrochloride alone, preferably following sexual prestimulation
(McDonnel and Love, 1991).
32
In conclusion the final goal for semen collection is to obtain a complete ejaculate with a
single mount and minimal sexual stimulation of the stallion before collection of semen, hereby
optimizing the potential to obtain an ejaculate with relatively low volume and high sperm
concentration (Loomis, 2006).
1.2.2. Preparation of cooled semen
As soon as an ejaculate is collected, the semen should be transported to the laboratory
taking care of minimizing physical trauma, exposure to light, cold shock or excessive heat. In the lab,
the raw, undiluted semen should be processed in an incubator using only materials preheated to
body temperature (37-38°C). If no inline filter was used during the collection, the semen should be
filtered immediately through a non-toxic, sterile filter to remove debris and gel admixtures. The gel
fraction can also be careful aspirated with a syringe. These two methods are associated with a higher
sperm loss in comparison to the use of inline nylon micromesh filters. Subsequently, volume,
concentration, color and possible admixtures should be registered. Independent of the following
procedures, the semen should be mixed with an appropriate preheated extender as soon as possible
following collection to maximize sperm longevity. At this point, a dilution ratio of 1:1 to 1:2 is
appropriate. Stallions with semen that is extremely sensitive to cold shock might benefit from adding
the same volume of preheated extender as the expected volume of semen to the collection bottle
prior to collection (Pickett, 1993; Squires et al., 1999; Brinsko et al., 2011).
Influence of sperm collection
Excessive sexual stimulation prior to ejaculation must be avoided due to two reasons. Firstly,
the increased volume of the ejaculate following excessive stimulation is associated with a reduced
sperm concentration, meaning that the total sperm output is not affected. Secondly, large volumes
of seminal plasma have a negative effect on semen quality after 24h of cooled storage (Sieme et al.,
2002). Moreover, multiple mounts in the same AV without changing the liner or the collection vessel,
will increase the contaminants and the amount of presperm fraction in the collected semen. So if

CHAPTER 1.2
multiple mounts are required for ejaculation, the collection receptacle should be emptied or
preferably be replaced together with the inner liner (Loomis, 2006).
Sperm concentration and seminal plasma during storage
In general, it is accepted that equine semen needs to be diluted at least 3:1 in order to dilute
the seminal plasma to levels ≤ 25% (Varner et al., 1988). Additionally, for optimal preservation, it is
advised to dilute the semen to a concentration of 25 to 50 × 10 6 /mL (Varner et al., 1987; Jasko et al.,
1991, 1992). If the initial sperm concentration is too low to use an appropriate dilution and maintain
an acceptable concentration at the same time, centrifugation is advised. Standard centrifugation
protocols for equine semen consist of 400× to 600× g-force for 10 to 15 minutes (Loomis, 2006;
Aurich, 2008), and rely on a 75% sperm recovery after aspirating the supernatant and resuspending
the sperm pellet. Prior to centrifugation, semen should be extended at least to a 1:1 ratio. Seminal
plasma acts like a double edge sword on fertility: it is detrimental for stallion spermatozoa during
storage, but at the other hand it shortens the duration of the postbreeding induced endometritis
caused by the inflammatory response triggered by the spermatozoa (Troedsson et al., 2000, 2005).
With most extenders, it is advisable to retain 5 to 20% seminal plasma following centrifugation (Jasko
et al., 1991). However, DNA integrity was maintained best in complete absence of seminal plasma,
whereas DNA damage increased as seminal plasma levels increased (Love et al., 2005a).
The effect of removal of seminal plasma in stallions whose sperm responds poorly to cooling
(to which we will further refer as “poor cooling” stallions) remains a topic of discussion. Brinsko et al.
(2000a) found that partial removal of seminal plasma after centrifugation had a positive effect on
spermatozoal motility following 48 of cooled storage. A more recent study, using more stallions,
demonstrated no effect of seminal plasma removal of “poor cooling” stallions on motility after
cooled storage. Only the percentage of membrane intact spermatozoa was higher for the samples
devoid of seminal plasma (Barrier-Battut et al., 2010).
Centrifugation of sperm
Normally, a 75% sperm recovery rate, obtained after conventional centrifugation (400× to
600× g for 10 to 15 min) is reported (Loomis, 2006; Aurich, 2008). Contradicting results however are
also present in literature, with recovery rates exceeding 98% using the same centrifugation protocol
(Weiss et al., 2004). Losses of about 25% are agreed on, but can be further reduced by increasing
33

CHAPTER 1.2
centrifugation time and force. However, this usually results in decreased sperm quality (Loomis,
2006). In 1984, Cochran et al. described a technique where a dense solution was layered below the
extended semen at the bottom of a centrifugation tube. This dense solution could serve as a cushion
for the spermatozoa during centrifugation (Cochran et al., 1984). Initially, a glucose-EDTA cushion
was used and later replaced by an egg yolk containing extender cushion supplemented with 4%
glycerol (Amann and Pickett, 1987). All these initial reports, except Volkman and van Zyl (1987),
showed a beneficial effect on sperm motility following centrifugation. Subsequently, a 60% iodixanol
solution was placed beneath extended semen followed by centrifugation for 25 min at 1000 × g. This
cushion prevented sperm from compacting at the bottom of the tube despite the high g-force used
and resulted in 30% more normal living sperm (Revell et al., 1997). More recently, high sperm yields
following high speed centrifugation without impairing sperm quality were reported using 3.5mL or
5mL of cushion fluid in a 50 mL centrifugation tube (Ecot et al., 2005; Knop et al., 2005). A variation
to these techniques has been described in which a small volume (30 µL) of cushion solution is placed
at the bottom of a special designed nipple centrifugation tube, followed by centrifugation at 400 × g
for 20 min. This technique has slightly lower sperm yields and slightly improved in vitro quality
characteristics, and has the additional advantage that it does not require aspiration of the cushion
following centrifugation (Waite et al., 2008).
34
Alternatives to centrifugation
As alternative for centrifugation to increase the sperm concentration and reduce the amount
of seminal plasma, fractionated semen collection can be used. Using a Krakow AV or a modified open
ended Missouri AV, it is possible to collect only the sperm rich fraction of the ejaculate. An
automated phantom (Equidame®) can also be used. This device provides semen samples with a lower
bacteriological colony count, and with a similar motility compared to semen collected using a
Missouri AV (Lindeberg et al., 1999). However, it is important to realize that fractionated collection
using the Equidame® does not correspond completely with the fractionated collection described
above. Separation using the Equidame® is not in concordance with the jets ejaculated by the stallion,
but is performed based on adjusted volume separation in the semen collection cups. Once a cup is
filled with the preset volume, a following cup is filled (Lindeberg et al., 1999). Sperm originating from
sperm rich fractions had higher motility at 12h and 24h post collection than sperm from total
ejaculates (Varner et al., 1987). However, seminal plasma from sperm rich fractions was found to be
more deleterious on sperm motility compared to seminal plasma from sperm poor fractions (Akcay
et al., 2006). Indicating that centrifugation might still be advisable, especially for prolonged storage.

CHAPTER 1.2
Recently, a new technique for concentrating equine semen was described. Based on a filter
with a hydrophylic synthetic membrane that does not allow sperm passage. Following filtration, up
to 95% of sperm cells could be recovered without affecting sperm motility and viability (Alvarenga et
al., 2010).
Selection of spermatozoa
Different techniques are available for separation and selection of spermatozoa. Separation is
the most straightforward procedure, in which the only objective is to separate the spermatozoa from
the seminal plasma. This is done by centrifugation and can be achieved as described above (Henkel
and Schill, 2003). In contrast, selection aims at simultaneously separating the spermatozoa from the
seminal plasma and selecting a sperm subpopulation based on sperm quality characteristics.
Migration, filtration and colloid centrifugation are three techniques available for sperm selection
(Morrell and Rodriguez-Martinez, 2009).
→ Migration
Sperm selection by migration relies on the ability of motile spermatozoa to move from one
suspension into a medium of a different composition. The original sperm population (diluted semen
or a washed sperm pellet) can be either underneath, on top of or beside the migration medium.
During an incubation stage the spermatozoa migrate actively into the selection medium (Morrell and
Rodriguez-Martinez, 2009) indicating this technique selects spermatozoa based on motility rather
than on morphology, chromatin integrity, viability and acrosome integrity (Somfai et al., 2002). The
major disadvantage of migration is the low recovery rate, rendering it impractical for clinical use for
preparing AI doses (Morrell and Rodriguez-Martinez, 2009).
→ Filtration
Filtration is achieved by the interaction of spermatozoa with the different filter substances.
Non-viable spermatozoa adhere more to the filter substrate compared to motile spermatozoa, as
such increasing the quality of the sample following filtration (Bussallou et al., 2008). Filtration allows
for processing of relative large volumes, but leukocytes and debris also pass by the filtration process.
Additionally, the sperm remains suspended in the same volume containing the seminal plasma, as
such requiring an additional centrifugation step (Henkel and Schill, 2003), or possibly a filtration to
increase the concentration (Alvarenga et al., 2010).
35

CHAPTER 1.2
36
→ Colloid centrifugation
Centrifugation of semen through (one or more) layers of colloid can be used to separate
spermatozoa from seminal plasma and to select a subpopulation of spermatozoa with good motility,
viability and chromatin integrity (Pertoft, 2000). The cells move to the point in the gradient which
matches their density, i.e. the isopycnic point, during centrifugation (Fig. 10) (Pretlow and Pretlow,
1989), rather than actively swimming through the colloid (Mortimer, 2000). After colloid
centrifugation, the sperm pellet is resuspended in fresh extender (washing medium) and washed by
centrifugation. Percoll® was the first colloid used for the selection of spermatozoa. In 1996, Percoll®
was restricted for non-clinical use only, due to possible toxicity of the product (Avery and Greve,
1995; Mortimer, 2000). Different colloids have been developed, and this has also resulted in species
specific products. Until recently, the major disadvantage of colloid centrifugation was the limitation
in volume that could be processed per centrifugation tube, and the time consuming practice of
carefully preparing the different layers of the Density Gradient Centrifugation (DGC) (Morrell and
Rodriguez-Martinez, 2009).
Fig. 10. Schematic presentation of spermatozoa following colloid centrifugation with location of the
spermatozoa according to their differences in isopycnic point based on their morphology.
Single Layer Centrifugation (SLC) was described as an alternative to DGC for processing
animal semen, resulting in comparable sperm yield and similar sperm quality characteristics (Morrell
et al., 2008; Thys et al., 2009). Additionally, SLC can be used to process larger volumes of extended
semen (Morrell et al., 2009) per centrifugation tube, allowing for the processing of entire stallion
ejaculates.

Semen extenders
CHAPTER 1.2
Many extenders for equine semen have been developed and most of them include egg yolk,
milk, milk by-products or chemicals to regulate osmolarity and pH (Pickett and Amann, 1987). The
most popular extenders worldwide are similar in composition to the original recipe published by
Kenney et al. in 1975. These extenders are inexpensive, easy to prepare and can be stored in frozen
form (Aurich, 2008). In the early days of modern AI, milk was used as a semen extender after heating
it. Heating raw milk was necessary to inactivate lactenin, so that sperm could survive in it for a
prolonged time (Thacker and Almquist 1951, 1953; Flipse et al., 1954). Ever since, people have been
refining the diluters and developing media to diminish detrimental components (Aurich, 2008).
Battelier et al. (1997) investigated the ability of different purified milk fractions to preserve equine
sperm and found very differing protection levels, varying from harmful to sperm to supporting a
better survival when compared to commercial heated milk. Native phosphocaseinate (NPPC) and β-
lactoglobulin were both protective for sperm storage, without synergetic effect between them, NPPC
afforded higher protection to spermatozoa. When NPPC was added to Hank’s salts solution
supplemented with Hepes, glucose and lactose (HGLL), it was equally capable of maintaining
spermatozoal motility compared to INRA82, a classical skimmed milk diluter which was commonly
used in Europe (the composition of INRA82 is presented in table 3c). However, higher fertility results
were obtained using this NPPC-HGLL extender in comparison to INRA82, (Batellier et al., 1997). The
discovery of this protective effect of NPPC, which is a direct effect since there is no evidence of its
binding to sperm membranes (Battelier et al., 2000), led to the development of INRA96, a chemically
defined diluter containing Hank’s salts, Hepes, glucose, lactose and NPPC. Another chemically
defined extender, EquiPro, contains a combination of defined caseinates and whey proteins. When
compared to a classical skim milk-glucose extender (Kenney extender), EquiPro was capable of
preserving the in vitro assessed quality of equine semen in a comparable (24h) or even better (≥48h)
way than the skim milk extender (Pagl et al., 2006). Finally, a defined extender containing soybean
lecithin has been developed in order to prevent possible disease transmission by animal proteins
(AndroMed ® , Minitüb) and can also be used for cooled storage of equine semen (Aurich, 2005; Aurich
et al., 2007).
Cooling of equine semen
Generally, it is believed that equine semen is best preserved when chilled at 4 -5 °C (Varner
et al., 1988; Aurich, 2008) in absence of air (Douglas-Hamilton et al., 1984), and in combination with
a slow cooling rate (Province et al., 1985; Varner et al., 1988). Storage at ambient temperature is only
advised if insemination can occur within 12h following collection (Varner et al., 1988). Diluted semen
37

CHAPTER 1.2
of a stallion stored for 24h at either 5°C or 20°C gave an identical pregnancy outcome, however, all
motility parameters tested decreased significantly more when stored at 20°C in comparison with
storage at 5°C (Varner et al., 1989). Storage using a synthetic extender like HGLL-BSA, is preferably
done at 15°C under aerobic conditions (Magistrini et al., 1992), which was confirmed by Battelier et
al. (1997) for HGLL-NPPC. Semen preserved in HGLL-NPPC at 15°C under aerobic conditions showed a
markedly better motility compared to storage at 4°C under anaerobic conditions. In a more recent
study (Chanavat et al., 2005), semen storage at 15°C in INRA96 resulted in lower in vitro quality,
whereas fertility in vivo was the same as compared to storage at 4°C. When using EquiPro as defined
extender, no difference was present for semen stored under aerobic or anaerobic conditions, nor at
15°C nor at 5°C. The latter temperature, however, resulted in a better motility for samples stored up
to 3 or 4 days when no antibiotics were added to the extender (Price et al., 2008).
38
The storage of equine semen at 15°C in INRA96 under aerobic atmosphere did not only result
in an excellent motility in vitro, it also yielded good in vivo fertility results, even after long term
storage (72h) at this temperature. In fact, it was better when compared to storage at 4°C in INRA82
(Battelier et al., 1998).
1.2.3. Transport of semen
As described above, the ideal temperature for preserving equine semen might differ
depending on the type of extender used. Semen is generally best preserved at 4-5°C in anaerobic
conditions using any extender, mainly skimmed milk extenders, except when using INRA96, storage
at 15°C in aerobic conditions is preferable. A wide variety of passive cooling devices, for cooling and
storage to 4-5°C, is available. These devices have variable cooling rates since the cooling process
slows down as the internal temperature is reduced (Brinsko et al., 2000b). The Equitainer I and II
are the best known passive cooling devices for gradual slow cooling and transport of equine semen.
A first version of the Equitainer, made by Douglas-Hamilton et al. (1984), was highly successful in
obtaining a slow cooling curve leading to a plateau at a constant temperature of 5°C. Furthermore,
the Equitainer is extremely durable and designed for reuse. However, the initial cost of the shipping
container and the inevitable charges for return shipping, are the major disadvantages of this device.
Other systems, which are disposable and cheaper, are available as well (Fig. 11). All of these systems
fail under extreme temperatures in keeping the samples at 4-5°C, however, most of them provide an
adequate protection when the container is kept at 22°C. A higher ambient temperature might
influence the core temperature of the sample, nevertheless, in vitro semen characteristics are not
affected in most systems. Lower temperatures might be more cumbersome, especially if they are

CHAPTER 1.2
extreme or last for a long time. So far, the Equitainer remains the most suited cooler available
(Katila et al., 1997; Malmgren, 1998; Brinsko et al., 2000b).
Fig. 11. Passive cooling and transport containers for equine semen; (A) the Equitainer I which is
able to cool and maintain semen up to 70h and provides protection against x-rays and (B) a
disposable neopor semen shipper from Minitüb.
The use of INRA96 at 15°C to enhance fertility (in “poor cooling” stallions) poses problems
due to the lack of appropriate passive cooling devices which can cool to 15°C and maintain this
temperature for a longer time (Batellier et al., 2001). However, in Europe, companies are specialized
in transporting equine semen by car. It should be possible to equip these cars with portable semen
storage units which are frequently used for storage and transport of porcine semen. These devices
can be preset to the desired temperature and run on the car’s battery. Alternatively, the Max Semen
Express (Agrofarma, Brazil) could perhaps be used as a passive cooling device since that box was
reported to maintain temperature around 16°C (Melo et al., 2006). The use of one frozen ice brick
and one ice brick at room temperature in an Equitainer has also been proposed to maintain semen
at 15°C (Clulow et al., 2008).
39

CHAPTER 1.2
1.2.4. Cryopreservation of equine semen
40
History of semen cryopreservation
The early history of cryopreservation goes back to 1789, when Spallanzani reported the
fertilization of frog eggs with frozen thawed frog spermatozoa (Squires et al., 1999). Still, it was not
until 1949, with the accidental discovery of the cryoprotective properties of glycerol, that the era of
cryopreservation of gametes took a start (Polge et al., 1949; Amann and Picket, 1987). Soon, the first
results on cryopreservation of equine spermatozoa were obtained. In one of the earliest papers, it
was demonstrated that 25% of equine spermatozoa survived a process of freezing to -79°C and
thawing, after initially removing the seminal plasma and resuspending the sperm pellet in a buffer
containing glucose and glycerol (Smith and Polge, 1950). The first report of the birth of a foal
conceived with frozen thawed equine sperm followed in 1957 (Barker and Gandier, 1957). Ever since,
a lot of research on this topic was performed, especially since frozen semen was accepted as a
method to produce registered foals by the American Quarter Horse and American Paint Horse
Association (Loomis, 2001).
Equine semen can be frozen using different protocols, which are all using the same principle:
following collection, the semen is diluted in an appropriate extender and centrifuged in order to
concentrate the sperm and remove most of the seminal plasma and initial extender, subsequently
the sperm pellet is resuspended in an extender containing a cryoprotectant after which the semen is
gradually cooled and then frozen using liquid nitrogen (Palmer, 1984; Loomis et al., 1983).
Fundamental aspects of cryopreservation
A detailed description on the changes that occur during cooling and freezing does not lie
within the scope of this paper. However, in the addendum an overview of these changes is presented.
Packaging of semen for cryopreservation
Historically, a wide variety of packaging systems for freezing semen have been used. Initially,
semen was frozen in pellets (Merkt et al., 1975), by placing the semen in cylindrical notches in blocks
of dry ice. Following freezing, the sperm pellets were stored in tubes in liquid nitrogen. The lack of
proper identification and the potential risk for contamination were two major problems associated
with pellet frozen sperm (Pickett et al., 1978). These problems were avoided when semen was frozen

CHAPTER 1.2
in flattened aluminum packs, which were frozen either above the surface of liquid nitrogen (Tischner,
1979) or in copper containers that were immersed in liquid nitrogen (Love et al., 1989). These large
volume flat packs (20-25 mL and 10-12 mL) were replaced by large volume straws (4 – 5 mL) (Martin
et al., 1979; Love et al., 1989; Samper and Morris, 1998), which could be used as single insemination
dose straws. The straw volume further decreased to 1.0 mL (Cochran et al., 1983) and 0.5 mL (Loomis
et al, 1983). Using these smaller straws, the temperature was more uniform for the whole sample
during the freezing process and cooling was achieved more rapidly (Loomis et al., 1983). Following
successes in human medicine when freezing semen in cryotube vials, 0.5 mL straws were compared
to cryotubes (3.6 mL of extended semen) for freezing equine semen. However, using these cryotubes,
post-thaw motility was significantly reduced (Kozink et al., 2006). On the other hand, a big
disadvantage associated with French 0.5 mL straws, is the necessity to use multiple straws for one AI
dose. To reduce the risk of making errors when handling frozen 0.5 mL straws, Love et al. (2005b)
investigated the impact of freezing multiple straws in one globlet, in order to reduce post freeze
handling of straws. However, post-thaw semen quality was hampered and the semen quality for
individual straws varied depending on their localization in the globlet during freezing. The use of
smaller 0.25 mL straws (frequently used for freezing bull sperm) for freezing stallion sperm does not
appear to influence post thaw-sperm quality, when compared to 0.5 mL straws (Nascimento et al.,
2008).
Sperm concentration
Generally, the semen is centrifuged after the first dilution in order to have sufficient sperm
numbers per straw. The second dilution is determining for the final sperm concentration at which
straws will be filled and frozen. It has been reported that this sperm concentration during freezing
has an influence on sperm quality of post-thaw samples. As such, sperm concentrations exceeding
400 × 10 6 /mL could negatively influence post-thaw motility (Heitland et al., 1996). However, these
results were not confirmed by Leipold et al. (1998), who found no decreased post-thaw motility
when semen was frozen at 1600 × 10 6 /mL compared to 400 × 10 6 /mL. The major difference in their
experimental setup was the glycerol concentration. In the latter study, adjustment of the final
glycerol concentrations to 4% was done for both groups. On the other hand, the semen was
progressively further diluted in the first study, resulting in lower glycerol concentrations for high
concentrated samples, and vice versa. Clulow et al. (2007) observed a reduced post-thaw motility for
semen diluted to 20 × 10 6 /mL compared to 200 × 10 6 /mL. In this study the confounding factor was
that semen at 20 × 10 6 /mL was frozen in 0.25 mL straws while semen at 200 × 10 6 /mL was frozen in
41

CHAPTER 1.2
0.5 mL straws, and glycerol concentration was not corrected for the different concentrations. Later
on, Clulow et al. (2008) froze equine semen at 40 × 10 6 /mL and 400 × 10 6 /mL, and adjusted this time
the glycerol levels for each group to 4%. Immediately following thawing, the low concentration
yielded a superior motility, however, after incubation for 3h, motility was clearly higher for the
higher concentration. Semen frozen in BotuCrio® showed better motility characteristics for semen
frozen at 100 × 10 6 /mL, and 200 × 10 6 /mL was better than 400 × 10 6 /mL (Nascimento et al., 2008).
In this study, no correction for cryoprotectant concentration was made. In conclusion, the influence
of concentration on post-thaw semen quality is not uniformly elucidated, however, high
concentrations might negatively influence the outcome of cryopreservation. Interactions between
the spermatozoa and the different components of the diluter cannot be excluded and as such might
be determinant for the optimal sperm concentration for cryopreservation.
42
Glycerol as cryoprotectant
The first developed freezing media were rich in egg yolk or milk and contained glycerol as
cryoprotective agent (CPA). After some experimental modifications, following media were developed
and frequently used: (a) modified lactose EDTA (Martin et al., 1979), (b) lactose EDTA egg yolk
(Tischner, 1979), and (c) INRA82 with egg yolk and glycerol (Palmer, 1984); the exact composition of
these extenders is presented in table 3. Although glycerol is still the most frequently used
cryoprotectant, little uniformity exists concerning the glycerol concentration which is used. The
optimal glycerol concentration differs depending on the extender composition and might be
influenced by the egg yolk concentration (Ecot et al., 2000). Nevertheless, glycerol has a clear
negative effect on fertility, since not only post-thaw sperm motility is affected (Burns and Reasner,
1995) but it will also influence female fertility. This effect is well known in hens, although the
contraceptive effect has also been documented in equine species (Bedforf et al., 1995; Vidament,
2005), and is most pronounced in asine species (Vidament, 2005). In literature, a wide variety in
glycerol concentration is found in equine freezing extenders, i.e., 2.5% (Palmer, 1984; Vidament et al.,
2000, 2001), 3% (Wilhelm et al., 1996a), 3.5% (Tischner, 1979), 4% (Leipold et al.,1998, Wilhelm et al.,
1996b), 5% (Martin et al., 1979) and 7% (Pace and Sullivan, 1975). It is advised that glycerol
concentration in frozen stallion semen should not exceed 3.5% in order to avoid negative influences
on fertility (Vidament et al., 2005). However, the final glycerol concentration is often not available
since the glycerol concentration is indicated on the freezing extender but not for the processed
semen, where it is variable depending on the dilution ratio used to resuspend the sperm pellet
following centrifugation (Vidament, 2005). The final glycerol concentration can accurately be

Table 3. Composition of different media for centrifugation, preservation and freezing of equine semen.
a) modified lactose EDTA b) lactose EDTA egg yolk
c) INRA82 for freezing
d) Modified INRA82
(Martin et al., 1979)
(Tischner, 1979)
(Palmer, 1984)
(Magistrini et al., 1992)
Glucose 6.0 g Lactose 11 g
Glucose 2.5 g Glucose 2.5 g
100
150
Na2-EDTA 370 mg EDTA
Lactose
Lactose 150 mg
mg
mg
150
Na-citrate 375 mg Na-citrate 89 mg
Raffinose
Raffinose 150 mg
mg
Na-bicarbonate 120 mg Na-bicarbonate 8 mg Na-citrate 30 mg Na-citrate (5.5 H2O) 30 mg
Streptomycin 50 mg Streptomycin 50 mg K-citrate 41 mg K-citrate (H2O) 41 mg
INRA82
Penicillin 50 IU Penicillin 50 IU Gentamycin 5 mg Hepes 476 mg
Centrifugation medium
Distilled water 100 mL Egg yolk 1.6 g Penicilin 500 IU Gentamycin 50 IU/mL
Lactose (11%, w/v) 50 mL Glycerol 3.5 mL Distilled water 50 mL Penicillin 50 IU/mL
Distilled water 50 mL
50 mL
UHT sterilized
skimmed milk
100
mL
Distilled water
25 mL
Centrifugation
medium
UHT skimmed milk 50 mL
INRA82 + 2% egg yolk
Centrifugation
medium
Freezing
medium
Egg yolk 20 mL
pH 7.1
INRA82 + 2% egg yolk +
2.5% glycerol
OrvusEs paste 0.8 mL
Freezing medium
Glycerol 5 mL

CHAPTER 1.2
determined if the final dilution is done in freezing extender without glycerol, followed by adding
glycerol until the desired concentration (Knop et al., 2005).
44
Alternative cryoprotective agents
It is known that glycerol is an important, though frequently discussed compound of freezing
extenders. Hence, a lot of efforts have been made to find alternative CPA’s. When the cryoprotective
properties of glycerol, ethylene glycol, dimethyl sulfoxide (DMSO) and propylene glycol were
compared, it was found that DMSO improved the post-thaw semen characteristics for a subgroup of
stallions who consistently failed to achieve ≥ 30% post-thaw motility with glycerol. Semen frozen in
ethylene glycol and propylene had lower post-thaw characteristics in comparison to DMSO and
glycerol (Chenier et al., 1998). Reports of Alvarenga et al. (2000), Graham (2000) and Squires et al.
(2004) revealed the cryoprotective capacities of ethylene glycol, methylformamide (MF) and
dimethylformamide (DMF) for freezing equine semen. A French study did not observe any additional
value of either DMF alone or in combination with different concentrations of glycerol in comparison
to glycerol alone (Vidament et al., 2002). These results are in sharp contrast with a Brazilian report
describing the superiority of DMF alone or in combination with glycerol when compared to glycerol
alone (Medeiros et al., 2002). The observed differences could be explained by differences in semen
freezability from these stallions when using glycerol. It has been observed that DMF was superior for
stallions from whom the semen displayed a poor freezability when using glycerol (Alvarenga et al.,
2003). Furthermore, differences between breeds were noticed, i.e. post-thaw quality was better
using DMF for Warmblood, Quarter Horse, and especially Mangalarga Marchador Breed stallions.
Fertility trials using semen frozen with DMF showed equal (Vidament et al., 2002) or higher fertility
(Alvarenga et al., 2005) when compared to glycerol. These finding from the Brazilian researchers led
to the development of BotuCrio®, a skimmed milk-egg yolk based extender containing 1% glycerol
and 4% methylformamide as CPAs (Alvarenga, personal communication; Melo et al., 2007; Terraciano
et al., 2008)
Egg yolk in freezing extenders
The egg yolk traditionally used in semen extenders is derived from chicken eggs. Alternative
egg yolk sources have been tested for their protective properties. As such, it has been demonstrated
that duck egg yolk protected the in vitro characteristics of equine semen during cryopreservation
more when compared to chicken egg yolk. However, in vivo fertility trials need to confirm this

CHAPTER 1.2
superiority (Clulow et al., 2007). On the other hand, quail egg yolk resulted in a higher post-thaw
motility compared to chicken egg yolk for cryopreserving Poitou jackass semen, which can probably
be explained by the different fatty acid composition. The optimal concentration of quail egg yolk was
found to be 10% (Trimeche et al., 1997).
Commonly, the chicken egg yolk in extenders is used as whole egg yolk. The clarification of
egg yolk by ultracentrifugation is already known for a long time (Pace and Graham, 1974), but an
early study on the use of clarified egg yolk in freezing extenders preferred the use of whole egg yolk
instead of using clarified egg yolk (Cristanelli et al., 1985). In this report, following ultracentrifugation,
the bottom sediment was discarded as well as the top lipid layer, while in more recent reports only
the bottom sediment was discarded (Pillet et al., 2010). In 1992, a modified Kenney’s extender for
the cryopreservation of stallion sperm was presented which contained clarified egg yolk solution
prepared by ultracentrifugation (10000 × g for 15 min) of a volume of egg yolk mixed with an equal
volume of centrifugation extender (3 g glucose, 5 g sucrose, 1.5 g BSA, 100000 IU penicillin, distilled
water q.s. 100 mL) (Burns, 1992). Only a few other reports describe the use of extenders containing
clarified egg yolk (Torres-Boggino et al., 1995) despite their advantage of the clear aspect when
evaluating the semen microscopically. For many years, a ready to use skimmed milk extender
containing clarified egg yolk has been commercially available, both for fresh and cooled use as well as
for cryopreservation (5% glycerol), namely the Ghent extender.
Egg yolk has been replaced by soybean lecithin for cryopreservation of bull semen (Aires et
al., 2003). Incorporated in an extender for equine semen (AndroMed ® ), soybean lecithin was also
used with good results for cooled storage of equine semen (Aurich, 2005; Aurich et al., 2007).
However, soybean lecithin can also replace egg yolk in BotuCrio® for the cryopreservation of equine
semen. Papa et al. (2010) found no difference in post-thaw motility and viability between the egg
yolk and soybean lecithin treatment. However, fertility was reduced for semen frozen in the soybean
lecithin extender. This could have been caused by irreversible binding of the lecithin lipids to the
sperm membrane, which might interact with the capacitation process (Papa et al., 2010). Soy
phosphatidylcholine has been used as well and is equally capable as egg yolk for protection of
spermatozoa during cryopreservation (Ricker et al., 2006).
45

CHAPTER 1.2
46
Chemically defined extenders in cryopreservation
Two recent studies were performed to evaluate the use of INRA96 as base for freezing
equine semen. As such, the addition of Cryoguard® (Minitube, Verona, Wi, USA) to INRA 96 resulted
in a higher post-thaw semen quality compared to a skimmed milk glucose extender containing
Cryoguard® (Scherzer et al., 2009). On the other hand, lower motility parameters for semen frozen in
INRA96 based freezing extender were found when compared to INRA82 based extender although
membrane integrity was better for semen frozen in INRA96. Fertility trials showed much higher
pregnancy rates for INRA96 frozen semen, clearly demonstrating the value of the INRA 96 diluter for
cryopreservation as well (Pillet et al., 2008a). In an additional study, comparable conflicting in vitro
quality results were found, such as a lower lipid peroxidation but a higher percentage of acrosome
damaged sperm for semen frozen in INRA96 (Pillet et al., 2008b). These studies led to the
development of a ready to use freezing extender for equine semen, INRA-Freeze which is a INRA96
based extender with 2.5% glycerol and 2% sterilized egg yolk plasma added. The egg yolk plasma is
prepared by ultracentrifugation and resulted in comparable in vitro characteristics and in vivo fertility
when compared to whole egg yolk (Pillet et al., 2010).
Cooling and freezing rate
Over the years, a huge variety of cooling and freezing curves has been described. A long term
follow-up study using the same freezing method and the same extenders has been performed.
Initially, semen was processed and frozen as described by Palmer (1984). Briefly, the initial protocol
was as follows. In a first step semen was diluted in INRA82 + 2% egg yolk at 32°C, followed by cooling
and subsequently centrifuging the semen at 4°C. After resuspension of the sperm pellet in INRA82 +
2% egg yolk + 2.5% glycerol at 4°C and semen was left to equilibrate for 1h at 4°C, next straws were
filled and frozen 4 cm above liquid nitrogen (Palmer, 1984). This protocol was refined by changing
every step one by one in order to obtain the best freezing procedure using these diluters (Vidament
et al., 2000, 2001). As such, it was demonstrated that centrifugation is better performed at 22°C
instead of at 4°C and that a moderate cooling rate is preferred to a slow cooling rate. Additionally,
the first cooling from 37°C to 22°C can be done rapidly au bain-marie by plunging the tubes
containing the semen in a 22°C water bath. These results confirm previous reports by Kayser et al.
(1992). The resuspension of the sperm pellet is preferably done at 22°C after which the semen is
allowed to cool and equilibrate for 80 min at 4°C. After filling, the 0.5 mL straws are subsequently
frozen by placing them 4 cm above liquid nitrogen or in a programmable freezer at -60°C/min until
-140°C, there after the straws are plunged and stored in liquid nitrogen.

CHAPTER 1.2
The actual freezing technique i.e. above liquid nitrogen or in a programmable freezer should
not only be decided on from a mere economical point of view. The cooling curve of straws placed at
a fixed distance above liquid nitrogen, has been recorded in order to measure the exact temperature
changes during cryopreservation. The resulting curve has been entered into a programmable freezer
and ejaculates were frozen using a split design. The semen frozen using the programmable freezer
resulted in better post-thaw in vitro semen characteristics, which is likely due to the more uniform
freezing rate (Clulow et al., 2008). However, these findings are in contrast with a previous report
where no differences were noticed between semen frozen above liquid nitrogen and semen frozen in
a programmable freezer (Cristanelli et al., 1985).
The use of alternative CPAs instead of glycerol might influence the optimal cooling and
freezing curve. As such, sperm that was cooled to 5°C for 1h or without cooling, followed by slow
(-20°C/min) or fast (-70°C/min) freezing rates using MF or DMF as CPA, has been compared. Post-
thaw sperm motility was not affected by the freezing rate, however, cooling prior to freezing resulted
in better post-thaw sperm characteristics (Alvarenga et al., 2005). According to the manufacturer, in
order to obtain an optimal freezing process when using BotuCrio®, it is advised to cool the semen to
5°C in 20 min after resuspending the sperm pellet, after which the straws are frozen 6 cm above
liquid nitrogen or at -30°/min in a programmable freezer. The equilibration time using BotuCrio® is
clearly much shorter compared to other extenders. Increasing this equilibration to 60-80 min using
BotuCrio® has a clear negative impact on post-thaw semen quality (Hoogewijs, unpublished data). A
faster freezing rate of -70°C/min, i.e. 4cm above liquid nitrogen, results in lower post-thaw motility
compared to a moderate freezing rate (Terraciano et al., 2008)
Thawing of frozen semen
Semen can be thawed using quite some different protocols (Vidament et al., 2001). In the
protocol which is commonly used, sperm is thawed during 30s in a 37°C water bath. Thawing of
semen in a 75°C water bath for 10s resulted in a slightly higher motility, however, timing is critical
since 15s at 75°C resulted in a zero motility. In Brazilian studies, semen frozen in BotuCrio® is
consistently thawed at 46°C for 20s (Melo et al., 2008; Pinheiro et al., 2008; Pucci et al., 2008; Papa
et al., 2010).
47

CHAPTER 1.2
48
Cryopreservation – trial and error
It is clear that cryopreserving equine semen is far more complex than most owners imagine.
An optimal freezing protocol varies between laboratories and is determined by practical implications,
limited by the infrastructure. But above all, the intrinsic quality and characteristics of an ejaculate is
by far the most determining factor that will decide on the outcome after thawing. So after all, the
stallion determines the cryopreservation protocol, and test freezing ejaculates using different
extenders and cooling/freezing rates is often rewarding.

Shortcomings in current practice
1.3
49

1.3.1. Standards for equine AI doses
CHAPTER 1.3
In literature, actual guidelines concerning the minimal requirements for an AI dose of equine
semen are missing. The semen used for AI can be divided, based on preparation, into fresh/diluted
semen, cooled (transported) semen and frozen-thawed semen. The World Breeding Federation for
Sport Horses (WBFSH) has clear minimal requirements for each type of semen used for AI, based on a
minimal percentage of progressive motility (PM) combined with a minimal dose of progressive motile
spermatozoa (PMS) (Table 4).
Table 4. Minimal requirements for a dose of equine semen following the WBFSH guidelines (source:
http://www.wbfsh.com/files/Semen%20standards.pdf, website last visited November 23 rd 2010).
Semen type
Fresh semen ≥ 300
Progressive Motile
Spermatozoa (× 10 6 )
Time between
collection and AI
Cooled semen ≥ 300 at portioning < 12h 35
Cooled transported
semen
≥ 600 at portioning < 36 h 35
Frozen semen ≥ 250 35
Progressive Motility (%)
In former days, 500 × 10 6 PMS was commonly recommended as standard AI dose for fresh
semen (Picket et al., 1975), whereas more recent studies advise on a standard AI dose using fresh
semen of 300 × 10 6 PMS (Vidament et al., 1997; Gahne et al., 1998). Nevertheless, a large variety of
studies present good fertility results with lower doses. Doses of 100 (Rigby et al., 1999), 50 (Sieme et
al., 2004), and 25 × 10 6 total spermatozoa (Woods et al., 2000), respectively, can give good fertility
results. Based on these data some authors suggest to reevaluate the sperm dose recommendations
(Sieme et al., 2004). The findings of Rigby et al. (1999) imply that fertility status of the stallion might
be more important than merely the numbers of sperm or PMS inseminated. However, the only
possibility to report on a stallion’s fertility status to date is based on findings from in vivo fertility
observations. In order to bypass this problem the semen can be evaluated after collection and
following an in vitro storage, a predictive value can be obtained.
51

CHAPTER 1.3
1.3.2. Dose and dose makes two
52
Without a uniform method to analyze an equine semen sample, it will remain very difficult to
determine the optimal sperm dose to maximize the chance to obtain pregnancy. An insemination
dose is in most cases expressed as being equivalent to a particular number of motile spermatozoa. As
such , an inseminate presumed to contain 300 million PMS as assessed with subjective motility
analysis is very likely to actually contain fewer progressive motile cells when the analysis would be
performed using a CASA system implemented with very strict motility criteria. As such the motility of
the spermatozoa in the same inseminate might be different depending on the type of analysis used.
Animal andrology uses the same techniques available as human andrology. However,
veterinary andrology analyses are done in a stochastic, almost randomized way when comparing
different laboratories. This hampers or even renders comparison of results between laboratories
impossible. This lack of analysis standards does not only slow down the scientific progress as far as
research is concerned; it also interferes with business, as the reliable trading of frozen AI doses in
particular is impeded.
The main problems that occur when performing light microscopic evaluations are subjectivity
and variability (Davis and Katz, 1993). Variations of 30 to 60% in sperm motility are reported for
subjective motility analysis of the same ejaculate by different observers (Verstegen et al., 2002).
Objective methods are available, but this objective analysis is not all standardized. Different settings
can be found in literature (Table 2), however, with the current knowledge it is unclear to what extent
these different motility setting affect the analysis outcome. Additionally, a large variety in different
counting chambers is available to use in combination with CASA systems. Different chambers clearly
influence motility parameters of dog and bull spermatozoa (Iguer-ouada and Verstegen, 2001; Contri
et al., 2010; Lenz et al., 2011). To which extent the motility of stallion semen is affected by these
apparent unimportant utensils remains unclear. This is in clear contrast with human andrology where
the preparation of a motility slide should be done following strict criteria (WHO, 2010).
On the other hand, the wide variety of procedures that exist to prepare AI doses provides the
liberty of choosing and adapting protocols to the specified needs of any given stallion. The
abundance in processing procedures requires a uniform way to objectively determine the best
suitable protocol for “problem stallions” and to chase off the ghost stories once and for all. One of
these techniques, frequently used when processing equine semen is centrifugation. Although
frequently used, the influence on equine semen requires further elucidation since there is quite
some misunderstanding and different opinions concerning the sperm losses accompanying aspiration
of supernatant (Weiss et al., 2004; Ecot et al., 2005; Knop et al., 2005).

CHAPTER 1.3
The use of density gradient centrifugation to select a superior sperm population was first
described in the early 1980’s. This technique gained interest due to the increased use of many of the
specialized reproductive techniques. The (clinical) use in domestic animal reproduction remained
minimal because of the inability to process large volumes of semen. More recently, this technique
has been modified to a single layer centrifugation so larger volumes of semen could be selected,
enabling the processing of entire stallions’ ejaculates in a limited number of tubes (Morrell et al.,
2009). This selection technique might prove valuable when processing a suboptimal semen sample.
53

CHAPTER 1
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69

Scientific Aims
CHAPTER 2
71

CHAPTER 2
As evidenced in the general introduction, little or no standardization is present in equine
(animal) andrology, which is in sharp contrast with human andrology. Even the use of computer
assisted sperm analysis (CASA), an excellent tool for objectively reporting on semen motility, can so
far not achieve uniformity in equine semen analysis.
Therefore, the general aim of the present thesis was to evaluate to which extent motility is
influenced during analysis with an objective analysis system like CASA or to find an alternative
objective analytical method that is not influenced by the operator. Additionally, the muddled
perspective on sperm losses following centrifugation was analyzed combined with the impact of
different centrifugation protocols on sperm quality. A recently developed technique to select large
volumes of semen (Single layer Centrifugation using Androcoll-E) was evaluated for its possibilities to
improve the post-thawed sperm quality.
Consequently, the specific aims of this thesis were:
• To determine the effect of technical settings and utensils on the reported motility
parameters when using CASA systems for equine semen analysis (CHAPTER 3.1 – 3.2).
• To validate the “Sperm Quality Analyzer – V equine” for analyzing equine semen
(CHAPTER 3.3 – 3.4).
• To evaluate the effect of centrifugation procedures (classical centrifugation and
single layer centrifugation) on the quality of processed equine semen (CHAPTER 4.1 –
4.2).
73

CHAPTER 3
Analysis of Equine Semen – Is automation (the) key to
standardization?
75

3.1
Influence of technical settings on CASA motility
parameters of frozen-thawed stallion semen
Adapted from: Hoogewijs M., Govaere J., Rijsselaere T., De Schauwer C.,
Vanhaesebrouck E., de Kruif A., De Vliegher S. 2009 Proceedings of
the 55 th annual convention of the American Association of Equine
Practitioners – Las Vegas - USA 55:336-337.
77

3.1.1. Abstract
CHAPTER 3.1
The repeatability of the analysis of frozen-thawed equine semen using a CASA system is high.
However, the settings used to analyze the semen samples influence the results to a large extent.
Total and progressive motility differed on average 7% between two different settings, although
results were highly correlated. We concluded that settings used in different scientific studies and for
semen reports should be described clearly, facilitating comparison of results.
3.1.2. Introduction
The widespread use of frozen-thawed semen in the equine breeding industry creates
opportunities for breeders as well as stallion owners. Semen can be shipped all over the world so
that any given stallion may become available for any given mare. Inseminations with frozen-thawed
semen, however, are less successful compared to inseminations with cooled and especially fresh
semen. In order to optimize pregnancy outcome, frozen-thawed semen should fulfill a few criteria
such as a minimal total number of spermatozoa per dose and minimal progressive motility. E.g., the
insemination dose for conventional artificial insemination (AI) using frozen-thawed semen should at
least contain 240 × 10 6 progressively motile spermatozoa (PMS) (Loomis, 2001). The PM should at
least be 30% (Loomis, 2001) whereas the World Breeding Federation for Sport Horses advocates a
PM of even 35% (WBFSH, 2010).
Computer assisted sperm analysis (CASA) is accepted as an accurate tool for motility analysis.
A number of motility settings for CASA have been described. In this paper we investigated the
repeatability of CASA. Furthermore the influence of two different CASA motility settings on an
number of sperm parameters when analyzing frozen-thawed semen was investigated.
3.1.3. Materials and Methods
The semen used in this experiment was frozen in 0.5 mL straws at two European approved AI
centers in Belgium. Individual straws of different Warmblood and Arabian stallions (n=63) were
thawed in a water bath of 37 - 38°C for 30s, dried and emptied in a small cup. An aliquot of 200 µL
was diluted with INRA96 ® (IMV-technologies, L’Aigle, France), a good quality semen extender free of
debris when visualized microscopically to a final concentration of 35 - 40× 10 6 /mL. The diluted
samples were incubated at 37-38°C for 10 minutes prior to analysis. Each sample was analyzed with
79

CHAPTER 3.1
a CEROS (Hamilton-Thorne Inc., Beverly, USA) CASA system using two different settings (CASA1 and
CASA2, respectively; see further). Settings for CASA analysis were obtained from established semen
processing laboratories as routinely applied to fresh, cooled and frozen-thawed semen samples.
80
The major settings used for CASA1 were straightness (STR) threshold for progressive motility:
75%; average path velocity (VAP) threshold for progressive motility 50 µm/s; VAP threshold for static
cells: 20 µm/s (Loomis and Graham, 2008). These settings were first obtained after a personal
communication (Loomis P., personal communication) and settings for cell detection were adapted
for optimal sperm recognition. The major settings used for CASA2 were STR threshold for
progressive motility: 50%; VAP threshold for progressive motility 30 µm/s; VAP threshold for static
cells: 15 µm/s, settings for cell detection are listed as well (Waite et al., 2008).
Six µL of diluted semen was loaded into a 20 µm Leja counting chamber (Leja, Nieuw-Vennep,
The Netherlands), placed on a minitherm stage warmer (Hamilton-Thorne Inc., Beverly, USA) during
the analysis, to maintain the sample at 37.5°C. Five different fields were analyzed 4 times to obtain a
total of 20 fields in order to obtain a minimum of 1500 cells analyzed. For both CASA settings this
was done twice per sample (T1 and T2).
distributed).
The parameters analyzed were percentage total motility (TM), PM and PMS (normally
To assess repeatability each sample was analyzed twice within one minute. First, it was
tested whether the parameters changed over time (T1 vs. T2), using a paired-samples t-test. Second,
scatter plots were produced, where the value obtained at T1 was plotted against the value obtained
at T2. The line of perfect agreement (hatched line at 45° and intercept zero) was integrated in each
plot as comparison to the regression line to fit the data (Arunvipas et al., 2003). Third, Bland and
Altman plots were produced. This method plots the difference of the paired measurements (y-axis)
against their mean (x-axis) (Bland and Altman, 1986). Finally, for each parameter, the coefficient of
variation (CV) was calculated per sample and reported as mean CV over all samples.
To assess agreement, the values obtained at T1 were used for comparison, and observations
obtained with CASA1 were compared with these obtained with CASA2. Statistical analysis was done
as described for repeatability using a paired samples t-test, scatter plots and Bland and Altman plots.
Statistical analysis was done using SPSS (version 17.0, SPSS Inc., Chicago, Illinois, USA).

3.1.4. Results
CHAPTER 3.1
Analyzing the semen samples using CASA1 and CASA2 was highly repeatable as the averages
at T1 were not significantly different from T2 (Table 1). The scatter plots and Bland-Altman plots
show a very good repeatability (Fig. 1), and the mean CVs were

CHAPTER 3.1
Progressive Motility CASA1 T2 (%)
Fig. 1. Scatter plots and corresponding Bland-Altman plots for analysis of repeatability of
progressive motility obtained with CASA using settings 1 (CASA1, see text details) and
settings 2 (CASA2, see text for details), respectively at time 1 (T1) vs. time 2 (T2) for frozenthawed
equine semen ( line of perfect agreement, regression line fit the actual
data).
82
60
40
20
0
0 20 40 60
Progressive Motility CASA1 T1 (%)
Progressive Motility CASA2 T2 (%)
60
40
20
0
0 20 40 60
Progressive Motility CASA2 T1 (%)

Totoal Motility CASA2 (%)
100
80
60
40
20
0
CHAPTER 3.1
Fig. 2. Scatter plots and corresponding Bland-Altman plots for agreement of total and progressive
motility obtained with CASA using settings 1 (CASA1, see text for details) vs. settings 2
(CASA2, see text for details) for frozen-thawed equine semen ( line of perfect
agreement, regression line fit the actual data).
3.1.5. Discussion
0 20 40 60 80 100
Total Motility CASA1 (%)
The importance of indicating which CASA settings are used when assessing the quality of
frozen-thawed semen is clearly demonstrated. The settings used here resulted in differences with a
potential important clinical and financial impact. Still, even more extreme settings have been
reported in literature (Ortega-Ferrusola et al., 2009; Vidament et al., 2009). However, results
obtained with both settings showed a very good agreement. This might enable laboratories to relate
their own findings with other reports. Depending on the settings used, an ejaculate might be
discarded or accepted for use in an AI program. So if the same standards are not uniformly applied,
Progressive Motility CASA2 (%)
80
60
40
20
0
0 20 40 60 80
Progressive Motility CASA1 (%)
83

CHAPTER 3.1
than at least they should be shared. After all, measuring by two standards might result in conflicting
situations.
84
In conclusion, different technical settings results in different CASA motility outcomes,
although results correlate well.
References
Arunvipas P., VanLeeuwen J.A., Dohoo I.R., Keefe G.P. 2003.Evaluation of the reliability and
repeatability of automated milk urea nitrogen testing. Canadian Journal of Veterinary
Research 67:60-63.
Bland J.M., Altman D.G. 1986. Statistical methods for assessing agreement between two methods of
clinical measurement. Lancet 1:307-310.
Loomis P.R. 2001. The equine frozen semen industry. Animal Reproduction Science 68:191-200.
Loomis P.R., Graham J.K. 2008. Commercial semen freezing: individual male variation in cryosurvival
and the response of stallion sperm to customized freezing protocols. Animal Reproduction
Science 105:119-128.
Ortega-Ferrusola C., Macías García B., Suárez Rama V., Gallardo-Bolaños J.M., González-Fernández L.,
Tapia J.A., Rodriguez-Martinez H., Peña F.J. 2009. Identification of sperm subpopulations in
stallion ejaculates: changes after cryopreservation and comparison with traditional statistics.
Reproduction in Domestic Animals 44:419-423.
Vidament M., Vincent P., Martin F.X., Magistrini M., Blesbois E. 2009. Differences in ability of jennies
and mares to conceive with cooled and frozen semen containing glycerol or not. Animal
Reproduction Science 112:22-35.
Waite J.A., Love C.C., Brinsko S.P., Teague S.R., Salazar J.L. Jr., Mancil S.S., Varner D.D. 2008. Factors
impacting equine sperm recovery rate and quality following cushioned centrifugation.
Theriogenology 70:704-714.
WBFSH. 2010 http://www.wbfsh.com/files/Semen%20standards.pdf.

3.2
Counting chamber type influences equine semen CASA
motility outcomes
Hoogewijs M., De Vliegher S., Govaere J., De Schauwer C., de Kruif A.,
Van Soom A. submitted
85

3.2.1. Abstract
CHAPTER 3.2
Sperm motility is one of the key features of semen analysis. Nowadays, motility analysis is
frequently performed using computer assisted sperm analysis (CASA). We hypothesized that type of
counting chamber used may influence the outcomes. Therefore, the effect of chamber type on
concentration and motility of equine semen assessed with CASA was studied. Frequently used
disposable Leja chambers of different depths were compared with disposable and reusable ISAS
chambers, a Makler chamber, and a motility slide prepared according to the guidelines of the World
Health Organization (WHO). Both concentration as well as motility parameters were significantly
influenced by the chamber type used. The correlation coefficients for concentration between the
different evaluated chambers and the NucleoCounter as gold standard were low for all chambers,
except for the 12 µm deep Leja chamber. Filling a chamber using capillary forces resulted in a lower
observed concentration and in reduced motility parameters. All evaluated chambers had significant
lower progressive motility when compared to the WHO prepared slide, except the Makler chamber
which resulted in a slightly higher but statistically significant progressive motility. In conclusion,
CASA provides only a rough estimate of the sperm concentration and is prone to overestimation
when drop-filled slides using a coverslip are used. Motility outcomes determined by CASA are highly
depending on the counting chamber type used, so complete descriptions of the utensils need to be
mentioned in a semen report and in materials and methods sections of scientific papers.
3.2.2. Introduction
Sperm motility is still considered as one of the most important sperm features when
analyzing a semen sample. Subjective microscopic measurement of motility is known to be
inaccurate, imprecise and susceptible for subjectivity. An objective evaluation of motility can be
performed using computer assisted sperm analyzers (CASA) since these systems provide precise and
accurate information on different sperm motion characteristics (Holt & Palomo, 1996). However, the
use of a CASA system to report on motility does not necessarily allow for comparison of the obtained
results since several factors influence the outcome of CASA analysis and aggravate the complex
comparison between studies. As such, the influence of technical settings, frame rate and number of
analyzed frames has been previously evaluated (Contri et al., 2010; Rijsselaere et al., 2003) as well as
the influence of motility settings (Hoogewijs et al., 2009). The sperm concentration and the used
semen diluents can affect sperm motility outcomes as well (Contri et al., 2010; Rijsselaere et al.,
2003).
87

CHAPTER 3.2
88
In addition, it has been demonstrated that chamber depth influences human sperm motility
parameters (Le Lannou et al., 1992). Also in veterinary medicine, the effect of the chamber type has
been analyzed for a few chambers (Contri et al., 2010; Iguer-ouada and Verstegen, 2001; Lenz et al.,
2011). The Makler chamber was associated with higher motility outcomes compared to the Leja and
Cell-vu chambers (Contri et al., 2010; Iguer-ouada and Verstegen, 2001; Lenz et al., 2011), but was
comparable to the WHO slide (Lenz et al., 2011). In literature, the depth of the sperm suspension for
motility analysis is variable and not seldom left unmentioned which is in sharp contrast to human
andrology where the WHO laboratory manual for the examination and processing of human semen
(WHO, 2010) clearly defines how a slide for sperm motility analysis should be prepared. This is
particularly important since it is known that a chamber depth of less than 20 µm constrains the
rotational movement of human spermatozoa (Kraemer et al., 1998; Le Lannou et al., 1992). Ishijima
and co-workers (1986) showed that the flagellar beating of a spermatozoa was three dimensional
rather than planar and results in a sperm rotation on a helical trajectory. As such, a shallow chamber
of 10 µm depth prevents the development of highly curved flagellar beats; as such the trajectory
remains straight and the rate of progressive hyperactivated motions increases (Le Lannou et al.,
1992). The differences in width of sperm heads between human and animal species (Table 1) might
affect motion characteristics to a different extent.
Table 1. Dimensions of the sperm head of different mammalian species.
Human 1
Equine 2
Bovine 3
Canine 4
Porcine 5
Length (µm) 4.1 5.49 8.67 6.65 8.12
Width (µm) 2.8 2.65 4.55 3.88 4.07
1 WHO laboratory manual for the examination and processing of human semen, 2010
2 Gravance et al., 1996
3 Gravance et al., 2009
4 Rijsselaere et al., 2004
5 García-Herreros et al., 2007
Literature on equine semen reports a wide variety both in CASA motility settings (Loomis
and Graham, 2008; Ortega-Ferrusola et al., 2009; Vidament et al., 2009; Waite et al.,2008) as well as
in the use of different chambers in combination with CASA systems. The chambers most frequently
used are a 20 µm deep Leja chamber (Hoogewijs et al., 2010; Len et al., 2010; Ortega-Ferrusola et al.,
2009; Waite et al., 2008), the 20 µm deep Cell-vu chamber (Almeida and Ball, 2005; Glazar et al.,
2009; Spizziri et al., 2010) and the 10 µm deep Makler chamber (Johannisson et al., 2009; Kavak et
al., 2003). These chambers are frequently loaded with a different volume, and sometimes only the

CHAPTER 3.2
depth but not the chamber brand is mentioned (Aurich and Spergser, 2007; Pagl et al., 2006), or only
the volume used (Price et al., 2008; Quintero-Moreno et al., 2003), and sometimes, nothing at all is
mentioned (Love et al., 2005; Macís-García et al., 2009; Ponthier et al., 2009).One might conclude
that objective semen analysis with CASA systems is not at all standardized in horses.
Also the determination of concentration by means of CASA is not indisputable. Especially the
use of capillary filled 20 µm disposable chambers has been subject of debate after discrepancies
with haemocytometer counts have been described (Kuster, 2005). The variation between these two
techniques has been attributed to the Segre-Silberberg (SS) effect, which describes flow dynamics in
capillary filled chambers resulting in a concentration differential for particles in laminar flow. Based
on a mathematical model, corrections can be made (Douglas-Hamilton et al., 2005a).
In the veterinary clinic of the Department of Reproduction, Obstetrics and Herd Health of
Ghent University, we noticed a huge discrepancy when analyzing an equine semen sample from the
same aliquot with both a Makler chamber and a 20 µm Leja chamber. The obtained concentrations
and motility parameters differed so extremely, that it became clinically relevant. For instance, an
analysis using the Leja chamber could lead to rejection of a sample, ejaculate or stallion, whereas
using the Makler chamber would have resulted in a more favourable outcome.
The major aim of this study was to evaluate the difference between a number of counting
chamber types on equine semen motility parameters. Additionally, the differences in concentration
depending on the chamber type used were scrutinized.
3.2.3. Materials and Methods
3.2.3.1. Semen and preparation for analysis
Fifty straws of frozen semen from 50 different stallions, frozen in two European recognized
artificial insemination centres were used in this experiment. Straws were thawed in a 37°C water
bath for 30 sec and subsequently dried and emptied in a 1.5 mL vial (eppendorf). Concentration was
determined using the NucleoCounter SP-100 (ChemoMetec, A/S, Allerød, Denmark) as described
earlier (Hansen et al., 2006; Morrell et al., 2010). Thawed semen was diluted using INRA96 (IMV,
L’Ailgle cedex, France) at 37°C to obtain a final concentration of 25 – 35 × 10 6 sperm /mL based on
the findings of the NucleoCounter, after which the samples were incubated at 37°C for 5 min prior to
analysis. The dilution ratios varied between 1:7 to 1:14.
89

CHAPTER 3.2
90
3.2.3.2. Quality analysis
Objective motility assessment was performed using CASA (Hoogewijs et al., 2010). Briefly, a
Ceros 12.3 CASA system (Hamilton Thorne Inc. Beverly, MA, USA) was used. The chambers were
loaded with semen and maintained at 37 °C using a Tokai Hit thermoplate (Tokai Hit CO., Ltd,
Shizoka-ken, Japan). Five randomly selected microscopic fields, evenly distributed over the slide,
were scanned 5 times each, obtaining 25 scans of every semen sample. The mean of the 25 scans for
each microscopic slide was used for the statistical analysis. The software settings of the HTR 12.3,
based on Loomis and Graham (2008), are summarized in Table 2. Following parameters recorded by
CASA were used for statistical analysis: concentration (CONC), percentage progressive motility (PM),
percentage rapid sperm cells (Rapid), average pathway velocity (VAP, µm/s), straight line velocity
(VSL, µm/s), curved linear velocity (VCL, µm/s), amplitude of the lateral head displacement (ALH,
µm), beat cross frequency (BCF, Hz), straightness (STR, %), and linearity (LIN, %). Every sample was
analyzed using ten different chambers. To avoid a possible influence from an increased incubation
time, the first chamber used for analysis changed in alternating way for the different samples, so
every chamber was equally used as a first and as a consecutive chamber. After each analysis in any
type of chamber, concentration was recorded and an additional dilution was performed if this
concentration obtained with CASA was higher than 35 × 10 6 sperm /mL, in order to obtain a
concentration within the 25 – 35 × 10 6 sperm /mL range for all motility analyses.
Table 2. Software settings of the Hamilton Thorne Ceros 12.3 used in this study.
Parameter Value
Frames acquired 30
Frame rate (Hz) 60
Minimum contrast 60
Minimum cell size (pixels) 6
Minimum static contrast 25
Straightness cut-off (%, STR) 75
Average-path velocity cut-off PM (µm/s,VAP) 50
VAP cut-off static cells (µm/s) 20
Cell intensity 100
Static head size 0.55 – 2.04
Static head intensity 0.45 – 1.70
Static elongation 11 - 99

3.2.3.3. The different chambers and loading technique
CHAPTER 3.2
The different types of counting chambers with their specific characteristics used in this study
are presented in Table 3. Disposable chambers are marked with a “d” following the depth in µm
while reusable chambers are marked with an “r”. The ISAS20r chamber was filled both with
capillarity (C) as well as using the drop filled cover slide technique (M). A detailed description of the
loading technique per chamber is presented in addendum II. Briefly, chambers filled with capillarity
(Leja10d, Leja12d, Leja20d, ISAS12d, ISAS16d, ISAS20d and ISAS20rC) were loaded by placing an
abundance of semen in the loading area. When the semen reached the opposite air valve, the excess
of semen was carefully removed by means of a Kimtech Science ® tissue (Kimberly-Clark, Surrey, UK).
Drop filled chambers (ISAS20rM, Makler10r and WHO) were filled by placing the exact volume on
the designated place, followed by immediate application of the coverslide. All chambers were left to
rest (at 37°C) prior to analysis until drifting of the cells ceased (approximately 30s).
Table 3. Characteristics of the different counting chambers used to analyze equine semen with
CASA.
Name
Chambers/
slide
Depth
(µm)
Volume/
chamber (µL)
Way of
filling
Volume
loaded (µL)
Reusable -
Disposable
Leja10d 4 10 1 capillarity 2.5 disposable
Leja12d 2 12 3 capillarity 5 disposable
Leja20d 4 20 2 capillarity 5 disposable
ISAS12d 4 12 - capillarity 5 disposable
ISAS16d 4 16 - capillarity 5 disposable
ISAS20d 4 20 - capillarity 5 disposable
ISAS20rM 2 20 - drop filled 5 reusable
ISAS20rC 2 20 - capillarity 5 reusable
Makler10r 1 10 - drop filled 5 reusable
WHO 1 20.6 - drop filled 10 reusable
3.2.3.4. Experimental design
The fifty straws were thawed individually, concentration was determined using the
NucleoCounter and the semen was diluted within the above mentioned range and loaded in a
chamber. A random rotation with the 10 viewing chambers was done to avoid time effects. After
every scan with the CASA system, the playback function was used to ensure the correct reading of
the sample. If a given sample loaded in a certain chamber yielded a concentration out of range for
that chamber, an additional dilution was prepared based on the later results to ensure a motility
analysis within the designed concentration range.
91

CHAPTER 3.2
92
Preliminary findings indicated that loading a chamber with capillary force might have an
influence on CONC. Therefore, it was decided not to use a classical haemocytometer as gold
standard for CONC. The NucleoCounter has been shown to be highly repeatable and to have very
good correlation with sperm concentration (Hansen et al., 2006). As such, the concentration as
determined with the NucleoCounter was used as gold standard. Motility outcomes obtained with
the WHO prepared slide were used as gold standard for motility.
3.2.3.5. Statistical Analysis
The data obtained for CONC showed a left skewed distribution. A normal distribution was
achieved using the natural log transformation (lnCONC). Progressive motility (PM) and percentage
rapid sperm (Rapid) were transformed by dividing them by 100 followed by an arcsine of the square
root of that value, in order to obtain a normal distribution. The other motility parameters, VAP, VSL,
VCL, ALH, BCF, STR, and LIN were normally distributed.
First, it was analyzed whether the averages obtained with the different chambers were
different. Mixed models (including stallion as a random effect) were fit with chamber and capillarity
(1 or 0) as fixed effects, respectively, to analyze chamber effect and effect of loading [filled with
capillarity (=1) vs. drop filled with cover slide (=0)]. LSD was used as post hoc tests to compare the
different chambers with the gold standard.
Secondly, scatter plots were produced for CONC and PM, where the value obtained with the
gold standard was plotted against the corresponding value obtained with the different chambers
(Microsoft Office Excel 2007). Ideally, this should result in the line of perfect agreement, which is the
hatched line at 45° and intercept zero. In each plot, this line of perfect agreement was drawn in
comparison to the regression line fit to the data (Arunvipas et al., 2003).
Third, to assess agreement between the values for CONC and PM obtained using the gold
standard with the values obtained using different chambers, Bland-Altman plots were generated.
This method plots the difference of the paired measurements (y-axis) against their mean (x-axis)
(Bland and Altman, 1986). The 95% limits of agreement were computed as the mean difference plus
or minus 1.96 times the standard deviation (SD) of the difference.

CHAPTER 3.2
Finally, Pearson coefficients of correlation (CC) were calculated between the different
chambers and the gold standard.
3.2.4. Results
Statistical analyses were done using SPSS (Version 17.0, SPSS Inc, Chicago, Illinois, USA).
3.2.4.1. Concentration
The counting chamber used had a significant effect (p

CHAPTER 3.2
Fig. 1. Average concentration (± standard error of the mean) of 50 frozen-thawed equine semen
samples from different stallions (n=50) assessed with computer assisted sperm analysis using
different chambers in comparison to concentration assessed with the NucleoCounter (NC) as
gold standard [bars with an asterisks are statistically different from the gold standard (black
bar), *p

90
80
70
60
50
40
30
20
10
0
drop filled with coverslip filled with capillarity
Conc PM Rapid
CHAPTER 3.2
Fig. 2. Influence of loading the chamber with frozen-thawed equine semen on concentration (CONC,
×10 6 /mL), progressive motility (PM, %) and percentage rapid spermatozoa (Rapid, %)
determined with computer assisted sperm analysis, presented as averages ± standard error
of the mean (all differences were significant, p

Leja20d Leja20dSS ISAS20d
60
60
50
50
60
50
40
40
40
30
30
30
20
Conc ISAS20d (x10 6 /m)
20
20
10
Conc Leja20dSS (x10 6 /m)
10
Conc Leja20d (x10 6 /m)
10
0
0
0
0 10 20 30 40 50 60
Conc NucleoCounter (x106 /m)
0 10 20 30 40 50 60
Conc NucleoCounter (x106 /m)
0 10 20 30 40 50 60
Conc NucleoCounter (x106 /m)
Fig. 3. Scatter and corresponding Bland-Altman plots for agreement between the NucleoCounter, as gold standard, and different chambers [Leja20d (20µm
depth, disposable), Leja20dSS (the same Leja20 chamber, corrected for Segre Silberberg effect) and ISAS20d (20 µm depth, disposable)] to analyze
the concentration (×10 6 /mL) of frozen-thawed equine semen samples. ( line of perfect agreement, regression line fit the actual data)

ISAS20rM Makler10r WHO
120
120
120
100
100
100
80
80
80
60
60
60
40
Conc WHO (x10 6 /m)
40
40
20
20
Conc Makler10r (x10 6 /m)
20
Conc ISAS20rM (x10 6 /m)
0
0
0
0 10 20 30 40 50 60
Conc NucleoCounter (x106 /m)
0 10 20 30 40 50 60
Conc NucleoCounter (x106 /m)
0 10 20 30 40 50 60
Conc NucleoCounter (x10 6 /m)
Fig. 4. Scatter and corresponding Bland-Altman plots for agreement between the NucleoCounter, as gold standard, and different chambers [ISAS (20µm
depth, reusable, drop filled with coverslide), Makler10r (10µm depth, reusable) and WHO prepared slide] to analyze the concentration (×10 6 /mL) of
frozen-thawed equine semen samples. ( line of perfect agreement, regression line fit the actual data)

CHAPTER 3.2
Table 4. Pearson correlation coefficient (CC) between the gold standard [NucleoCounter (NC)] and
the different chambers for assessment of concentration (CONC) and progressive motility
(PM) (after transformation to obtain normal distribution, for details see text) using
computer assisted sperm analysis.
98
NC
WHO
Leja10
Leja12
Leja20
Leja20SS
CONC
CC 1 0.31 0.34 0.74 0.42 0.44 0.33 0.38 0.38 0.17 0.21 0.42
pvalue
PM

60 PM Rapid
50
40
30
20
10
0
CHAPTER 3.2
Fig. 5. Average percentage of progressive motility (PM – dark grey) and percentage of rapid sperm
(Rapid – light grey) (± standard error of the mean), respectively, of 50 frozen-thawed equine
semen samples from different stallions (n=50), assessed with computer assisted sperm
analysis using different chambers in comparison to the WHO prepared slide as gold standard
(bars with one or more symbols are statistically different from the gold standard (darker
bars), *, ♦ p

Leja20d ISAS20d Makler10r
100
100
100
80
80
80
60
PM Makler10r (%)
60
PM ISAS20d (%)
60
40
40
40
20
20
20
PM Leja20d (%)
0
0
0
0 20 40 60 80 100
0 20 40 60 80 100
0 20 40 60 80 100
PM WHO (%) PM WHO (%) PM WHO (%)
Fig. 6. Scatter and corresponding Bland-Altman plots for agreement between the WHO prepared slide, as gold standard, and different chambers [Leja
(20µm depth, disposable), ISAS (20 µm depth, disposable), Makler (10µm depth, reusable)] to analyze the progressive motility (PM) (%) of frozen-
thawed equine semen samples. ( line of perfect agreement, regression line fit the actual data)

Table 5. Averages (± standard error of the mean) of different motility parameters from frozen-thawed equine semen obtained with computer assisted
sperm analysis using different chambers, compared with the WHO slide, which was used as the gold standard, analysis done using mixed models
with LSD post hoc tests. Values within a column with asterisks are significantly different from the gold standard (* p≤0.05, ** p

CHAPTER 3.2
102
In veterinary medicine, a number of studies have focussed on counting chambers in
combination with CASA devices. Discrepancies between CASA estimates and the Bürker
haemocytometer have been described for different species (Rijsselaere et al., 2003; Vyt et al., 2004).
To obtain a sharp image of the sperm cells when performing the motility analysis, CASA devices are
primarily used in combination with shallow chambers to keep the moving sperm within focus plane
of the microscope. Therefore CASA systems are frequently used in combination with 20 µm deep
disposable counting chambers. These chambers are most often loaded by capillary force, and such a
capillary flow in a 20 µm chamber follows the laminar Poiseuille flow (Douglas-Hamilton et al.,
2005a). This leads to a transverse lifting force on suspended particles and results in an unevenly
concentration throughout the sample as described by Segre and Silberberg and is, as such, known as
the SS effect (Douglas-Hamilton et al., 2005b). The influence of this SS effect on the distribution
throughout the slide depends on the viscosity of the sample. Correction factors are available and are
based on the time it takes for a chamber to be filled, and as such determined by viscosity of the
loaded sample (Douglas-Hamilton et al., 2005a). Although the SS effect is well documented in
literature, interpretation of the results is not always performed correctly. For example, in a recent
study (Maes et al., 2010), two types of 20 µm Leja slides were compared, where one of the slides
was especially developed to be able to correct for the SS effect. Unfortunately, the results obtained
with the two chambers were compared as such, without applying the correction factor. Therefore it
was faulty concluded that the newly shaped counting chamber was not able to correct for the SS
effect.
CASA instruments were developed to provide a solution for problems linked with subjective
motility analysis. Indeed, subjective assessment of motility is known to be inaccurate, imprecise and
subject to variability (Davis and Katz, 1993; Matson, 1995). However, in order for CASA systems to
contribute to a standardized and objective analysis, uniformity in protocols and settings is required.
In literature, a large variation in technical settings can be found as well as a lack in uniformity for
preparing a sample for motility analysis. It has been demonstrated that the large variety in motility
settings used influences motility outcomes significantly (Hoogewijs et al., 2009). The chambers used
in combination with CASA are very diverse and quite often not clearly described. As such, it is nearly
impossible to compare results between different studies.
The big difference in loading technique and motility outcomes may be attributed by physical
forces on the spermatozoa during loading as postulated by Lenz et al. (2011). When a semen sample
is placed on the loading area of a capillarity filled chamber, the flow that occurs might damage the

CHAPTER 3.2
sperm (tail) which could result in decreased motility, however, these hypotheses remain yet to be
tested.
The pressing question for standardization becomes more alive when one considers the
approval and distribution of equine semen by the dose and not by the pregnancy. Indeed, the
widespread use of frozen-thawed semen resulted in increased trading of semen. Mare owners buy
semen without the assurance of obtaining a pregnancy, so they tend to make demands on semen
quality. When using the following criteria as a minimum for post-thaw equine semen; a post-thaw
PM ≥30% and a minimum dose of 240×10 6 progressive motile spermatozoa per insemination dose
(Loomis, 2001), the insemination dose from the same ejaculate analyzed in this study can vary from
2 straws (CASA analysis using a Makler chamber) to 10 straws (CASA analysis using Leja10).
Comparable results can undoubtedly also be found for other species, where the economic
consequences are even more important. This might encourage researchers to get together and start
developing a comparable manuscript as the “WHO laboratory manual for the examination and
processing of human semen”, so that semen samples from the different domestic species can be
analyzed using the standards for that given species.
In conclusion, automated semen analysis using highly specialized CASA systems does not
produce easily comparable motility outcomes. The influence of the chamber used for analysis on
concentration and motility is not minor. As such a complete and accurate description of the
“materials and methods” used is of utmost importance, and without it, the correctness of a semen
report can at least be considered as questionable.
103

CHAPTER 3.2
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Davis R.O., Katz D.F. 1993. Operational standards for CASA instruments. Journal of Andrology 14:385-
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tolerance limits and membrane permeability characteristics of stallion spermatozoa treated
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Hoogewijs M.K., Govaere J.L., Rijsselaere T., De Schauwer C., Vanhaesebrouck E.M., de Kruif A., De
Vliegher S. 2009. Influence of technical settings on CASA motility parameters of frozen
thawed stallion semen. Proceedings of the . 55 th annual convention of the American
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Hoogewijs M., Rijsselaere T., De Vliegher S., Vanhaesebrouck E., De Schauwer C., Govaere J., Thys M.,
Hoflack G., Van Soom A., de Kruif A. 2010. Influence of different centrifugation protocols on
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system” for dog semen analysis. Theriogenology 55:733-749.
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Evaluation of cryopreserved stallion semen from Tori and Estonian breeds using CASA and
flow cytometry. Animal Reproduction Science 76:205-216.
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(after cooling) effects of centrifugation on equine sperm. Theriogenology 73:225-231.
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semen analyzer. Journal of Animal Science 89:383-388.
Loomis P.R., Graham J.K. 2008. Commercial semen freezing: individual male variation in cryosurvival
and the response of stallion sperm to customized freezing protocols. Animal Reproduction
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Loomis P.R. 2001. The equine frozen semen industry. Animal Reproduction Science 68:191-200.
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Love C.C., Brinsko S.P., Rigby S.L., Thompson J.A., Blanchard T.L., Varner D.D. 2005. Relationship of
seminal plasma level and extender type to sperm motility and DNA integrity. Theriogenology
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106

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107

3.3
Validation and usefulness of the SQA-Ve (1.00.43) for
equine semen analysis
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194.
109

3.3.1. Abstract
CHAPTER 3.3
Routine semen analysis includes evaluation of concentration combined with seminal volume,
morphology and motility. Subjective analysis of these parameters is known to be inaccurate,
imprecise and subject to variability. Automated semen analysis could lead to an increased
standardization in and between laboratories but for that to happen automated devices need to be
validated. A new device, the sperm quality analyzer V equine (SQA-Ve) version 1.00.43, was
evaluated for its repeatability and agreement with light microscopy (LM), for raw and extended
equine semen. Results were compared with computer assisted sperm analysis (CASA), which was
also tested for its repeatability and agreement with LM. The SQA-Ve showed a good repeatability
and fine agreement for assessing sperm concentration of raw semen based on scatter and Bland-
Altman plots. This was in contrast with the motility parameters, which had a low repeatability.
Morphology assessment with SQA-Ve was poorly repeatable as well as in poor agreement with LM.
For extended semen, the findings were comparable. The SQA-Ve did well for concentration, whereas
for the motility parameters repeatability was only just acceptable, with however, no agreement with
LM. This sharply contrasted the CASA findings that were highly repeatable and almost in perfect
agreement with LM. Based on these findings, the tested version of the SQA-Ve is insufficiently
accurate to be used for analyzing raw or extended equine semen.
3.3.2. Introduction
Although a plethora of specialized tests is available for analyzing semen samples (Varner,
2008), a routine semen analysis includes the evaluation of concentration, seminal volume (Perreault,
2009), morphology and motility (Varner, 2008). Microscopical measurements of sperm numbers,
motility and morphology have been shown to be inaccurate and imprecise (Davis and Katz, 1993)
and subject to high subjectivity (Matson, 1995). In order to overcome these inaccuracies, automated
systems have been developed. In 1981, the Sperm Motility Analyzer was described by Bartoov et al.
(1981) as a practical tool to evaluate overall sperm quality. This device, in later articles called the
Sperm Quality Analyzer (SQA), registers fluctuations in optical density of light passing through a
capillary which contains the semen sample. These fluctuations are registered by a photometric cell
and converted to a numerical output. As such, the SQA does not recognize the actual sperm cells
during the analysis.
Computer-assisted semen analysis (CASA) was introduced both for humans and animal
species about two decades ago (Verstegen et al., 2002). Using the combination of a microscope and
111

CHAPTER 3.3
a camera, the sperm cells are visualized and the actual sperm tracks analyzed. The proper sperm cell
recognition is determined by software settings. These systems produce accurate and highly
repeatable data of different semen-motility parameters in different species (Verstegen et al., 2002).
However, handling procedures and parameter settings still differ between laboratories, making it
difficult to compare results (Davis and Katz, 1993; Hoogewijs et al., 2009; Verstegen et al., 2002).
112
Unlike CASA systems, the SQA does not require parameter settings, hence reducing a large
source of potential bias (Hoflack et al., 2005), making this a ‘practical’ device which might be a
bridge between subjective light microscopic assessments and highly specialized CASA interpretations.
The aim of this study was to investigate the agreement and repeatability of two different
automated semen analyzers, the SQA-Ve and CASA, to assess equine semen quality using
conventional subjective light microscopy (LM) as the gold standard.
3.3.3. Materials and methods
3.3.3.1. Stallions and semen preparation
For this study, a total of 60 ejaculates (six ejaculates per stallion, 5 Shetland ponies and 5
Warmblood stallions, respectively) was collected. After collection, semen was filtered through sterile
gauze to remove the gel fraction and debris. For 30 ejaculates (3 per stallion), one part of the raw
ejaculate, approximately 5 mL of fresh semen, was transferred into a sample container and placed in
the preheated block heater. The rest of the semen was diluted 1:5 with INRA 96 and transferred in a
sample container and placed in the heating block. The other 30 ejaculates were, after filtration,
processed in the latter way, i.e. no analyses were performed on the undiluted semen.
3.3.3.2. Analysis
Light microscopy
Subjective analysis of motility was performed as described by the World Health Organization
(WHO, 2010), and considered to be the gold standard for this study. Briefly, a fixed volume of 10 µL
of preheated (37°C) diluted semen was placed on a preheated clean glass slide and covered with a
22 mm × 22 mm coverslip and transferred to the warmed (37°C) stage of the phase contrast
microscope. At least five microscopic fields were assessed to classify 200 spermatozoa. Individual

CHAPTER 3.3
morphological abnormalities were noted after eosin-nigrosin staining (Barth and Oko, 1989)
according to their location (head, midpiece or tail). The concentration was determined by use of a
Bürker hemocytometer after diluting the raw semen 1:10 with 1M HCl.
SQA-Ve
Prior to the first analysis on any given test day, the SQA-Ve (Medical Electronic Systems,
Caesarea, Israel) (Fig. 1a) was turned on and the unit was allowed to complete the self-test. The
incorporated software version of the SQA-Ve at the time of the experiments was version 1.00.43.
a.
b.
Fig. 1. (a) The latest model of the Sperm Quality Analyzer, the SQA-V equine, for the analysis of
equine semen, and (b) the CEROS computer assisted sperm analyzer from Hamilton Thorne.
Raw semen
Prior to any analysis the block heater was preheated for at least 15 min, set at 40°C,
according to the operating instructions, to ensure a sample and capillary temperature of 37°C. The
preheated block was loaded with clean, empty capillaries. The samples were incubated in the block
heater for 5 minutes prior to testing. The SQA-Ve was prepared for analyzing raw samples following
the onscreen instructions. A warm capillary was loaded according the description manual and
inserted into the SQA-Ve. The capillary was then pre-heated by the SQA-Ve, after which the analysis
began automatically. After each first analysis (T1), the measurement was repeated on the same
capillary (T2). Retesting was done without an additional pre-heating cycle. The parameters recorded
by the SQA-Ve that were used for statistical analysis, were concentration (CONC) (×10 6 /mL),
113

CHAPTER 3.3
percentage total motility (TM), percentage progressive motility (PM), percentage morphological
normal sperm cells (MORF), and velocity (VEL) (µm/s).
Extended semen
114
Extended samples were not kept in cooling conditions. Analysis was done according to the
onscreen instructions in a similar way as described for the raw semen to obtain a measurement at
T1 and T2 using the same capillary. The parameters for statistical analysis of extended semen
recorded by the SQA-Ve were CONC, TM, PM, and VEL. No information was obtained concerning the
morphology since the software algorithms only calculates morphology on raw semen samples.
Computer assisted sperm analysis
Analysis with CASA was performed as described by Hoogewijs et al. (2010) using the same
settings. Briefly, a Ceros 12.3 CASA system (Hamilton Thorne Inc., Beverly, MA, USA) (Fig. 2b) was
used. For each analysis, 5 µL of extended semen was mounted on a disposable 20 µm deep Leja
counting chamber (Orange Medical, Brussels, Belgium) and maintained at 37 °C using a minitherm
stage warmer. Five randomly selected microscopic fields in the center of the slide were scanned 5
times each, obtaining 25 scans of every semen sample. The mean of the 25 scans for each
microscopic slide was used for the statistical analysis. Immediately after the first analysis (T1), the
same counting chamber was reanalyzed in the same way (T2). The parameters recorded by CASA
that were used for statistical analysis were CONC, TM, PM, and average pathway velocity (VAP). The
velocity as recorded by the SQA-Ve is comparable with the average pathway velocity recorded by
CASA, according to the manufacturer. As such VAP is also assigned as VEL. The software version used
on the CASA system cannot assess sperm morphology.
3.3.3.3. Experimental design
Due to the frequent collisions between the sperm cells and the difficulties to individually
visualize the sperm cells, no subjective motility analysis could be performed in raw semen, therefore
only CONC and MORF could be assessed. The sample from each ejaculate was analyzed twice using
the SQA-Ve to analyze repeatability (using data at T1 and T2). Due to limitations of CASA systems,
the raw samples could not be analyzed using CASA because CONC was too high.

CHAPTER 3.3
The extended semen was analyzed with light microscopy for TM and PM, and with both SQA-
Ve and CASA and output data were obtained for CONC, TM, PM and VEL. Each sample was analyzed
twice to evaluate the repeatability.
3.3.3.4. Statistical analyses
For each type of semen (raw and extended), repeatability of methods (SQA-Ve and CASA)
was assessed by comparing the observations at T1 with T2. Next, agreement was analyzed using the
data obtained at T1 only, comparing SQA-Ve and CASA with the gold standard (LM), respectively.
For CONC, the data obtained were transformed using a natural logarithm transformation to obtain a
normal distribution (lnCONC).
Repeatability
First, it was tested whether the parameters changed over time. Therefore, mixed models
were fitted in MlwiN 2.02 (Centre for Multilevel Modeling, Bristol, UK) for the different parameters
(CONC, MORF, TM, PM, and VEL when available of raw and extended semen, respectively) with
“Time” (T1 vs. T2) as fixed effect and “Ejaculate” and “Stallion” as random effects, the latter to
adjust for clustering of repeated measurements within ejaculate and of ejaculates within stallion,
respectively.
Second, scatter plots were produced for each parameter, where the value at T1 was plotted
against the value at T2 (Microsoft Office Excel 2007). Ideally, this would result in the line of perfect
agreement, which is the hatched line at 45° and intercept zero. In each plot, this line of perfect
agreement was drawn in comparison to the regression line fit to the data (Arunvipas et al., 2003).
Third, to assess agreement between the values obtained at T1 with the values at T2, Bland
and Altman plots were produced. This method plots the difference of the paired measurements (y-
axis) against their mean (x-axis) (Bland and Altman, 1986). The 95% limits of agreement were
computed as the mean difference plus or minus 1.96 times the standard deviation (SD) of the
difference. Analyses were done using SPSS software package (version 15.0, SPSS Inc., Chicago, IL).
Finally, for each parameter, the coefficient of variation (CV) per sample was calculated and
reported as mean CV over all samples (Arunvipas et al., 2003).
115

CHAPTER 3.3
116
Agreement
First, it was tested whether outcomes of results of parameters differed between methods of
analysis. Therefore, mixed models were fitted in MlwiN 2.02 for the outcome variables lnCONC and
MORF with “method of analysis” (SQA vs. LM) as fixed effect and “Stallion” as random effect, the
latter to adjust for clustering of ejaculates within stallion. For extended semen, mixed models were
fitted for the outcome variables TM and PM with “method of analysis” (CASA vs. LM and SQA vs. LM,
respectively) as fixed effect and “Stallion” as random effect, the latter to adjust for clustering of
ejaculates within stallion.
Second, scatter plots plotting the values of SQA-Ve and CASA against LM, respectively, were
created, as well as the corresponding Bland-Altman plots.
3.3.4. Results
3.3.4.1. Raw semen
Repeatability of SQA-Ve
None of the parameters changed significantly over time (Table 1). The repeatability of
lnCONC was excellent. The linear regression line fit to the data approached the line of perfect
agreement and the corresponding Bland-Altman plot had narrow 95% limits of agreement (Fig. 2).
Repeatability for MORF and the three motility parameters on the other hand was rather poor as
shown in Fig. 2 for MORF and PM. All CVs were acceptable (

CHAPTER 3.3
Table 1: Average values of concentration (CONC), morphology (MORF), total (TM) and progressive
motility (PM), and velocity (VEL) of equine semen at time 1 (T1) and time 2 (T2) obtained with light
microscopy (LM), SQA-Ve, and CASA. Within devices (SQA-Ve and CASA only) values at T1 and T2
were compared. Values with the same superscript ( a,b,c ) are significantly different. For agreement,
values (at T1 only) between SQA-Ve and LM, and CASA and LM, respectively, were compared. Values
with the same superscript ( ♠,♣,♥,♦ ) are significantly different.
Raw semen Extended semen
LM SQA-Ve LM SQA-Ve CASA
T1 T1 T2 T1 T1 T2 T1 T2
CONC (×10 6 /mL) 317 289.9 288.1 - 31.8 31 39.8 39.1
lnCONC 5.66 5.55 5.55 - 3.33 3.29 3.60 a
MORF (%) 67.8 ♠
74.6 ♠
TM (%) - 74.6 73.1 80.2 ♣,♥
3.58 a
73.7 - - - - -
73.1 ♣
72.8 75.8 b,♥
PM (%) - 66.5 63.4 47.9 ♦ 48.7 47.4 39.8 ♦ 39.4
VEL (µm/s) - 110.4 108.2 - 80.6 78.9 99.6 c
c, ♠,♣,♥,♦ p

Repeatability Agreement
lnCONC MORF (%) PM (%) lnCONC MORF (%)
MORF SQA-Ve (%)
lnCONC SQA-Ve
T2
T2
T2
T1 T1 T1 lnCONC LM MORF LM (%)
Fig. 2. Scatter plots and corresponding Bland-Altman difference plots for repeatability of (lnCONC), morphology (MORF) (%) and progressive motility (PM)
(%) obtained with the SQA-Ve for raw equine semen and for agreement of lnCONC and MORF obtained with light microscopy (LM) and the SQA-Ve
for raw equine semen. ( line of perfect agreement, regression line fit the actual data)

Repeatability Agreement
SQA-Ve PM (%) CASA PM (%) SQA-Ve PM (%) CASA PM (%)
PM CASA (%)
PM SQA-Ve (%)
T2
T2
T1 T1 PM LM (%) PM LM (%)
Fig. 3: Scatter plots and corresponding Bland-Altman difference plots for repeatability of progressive motility (PM) (%) obtained with the SQA-Ve and CASA
for extended equine semen and for agreement of PM (%) between light microscopy (LM) and the SQA-Ve, and between LM and CASA for extended
equine semen. ( line of perfect agreement, regression line fit the actual data)

CHAPTER 3.3
3.3.5. Discussion
120
In this study, a new device for analyzing equine semen was evaluated. The SQA-Ve was highly
repeatable when assessing sperm concentration, with a good agreement with the gold standard. The
other SQA-Ve outcomes however, were less accurate. CASA proved to have a good repeatability as
well as a good agreement with LM.
The density of the extender might be an explanation for the lower repeatability when
analyzing the concentration of extended semen compared to raw semen. The low repeatability for
TM and PM obtained with the SQA-Ve and the poor agreement between LM and the SQA for these
parameters clearly demonstrate that the tested version (1.00.43) of the SQA-Ve is, with its current
algorithms, not suitable for the analysis of motility of raw or extended equine semen. Once more,
these experiments prove the agreement between CASA and LM observations. This is as expected
since the motility setup for CASA analysis was made in order to correspond with light microscopical
findings. In conclusion, the SQA-Ve software version 1.00.43 can be used with good results to
determine stallion sperm concentration. This is especially true for raw semen and to a lesser extent
for extended semen. However, repeatability for SQA-Ve was poor when analyzing motility, and not in
agreement with the gold standard, making the device insufficiently accurate. This is in contrast with
CASA, which is highly repeatable and agrees well with the gold standard when assessing TM and PM
of extended stallion semen.

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CHAPTER 3.3
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121

3.4
Need for further improvement of SQA-Ve (1.00.61) for
a univocal analysis of the quality of frozen-thawed
equine semen
Hoogewijs M., De Vliegher S., Govaere J., De Schauwer C.,
Vanhaesebrouck E., de Kruif A., Van Soom A. in preparation
123

3.4.1. Abstract
CHAPTER 3.4
In this study, two software versions of the sperm quality analyzer V equine were tested for
analyzing frozen-thawed equine semen. The first version (1.00.43) was poorly repeatable and agreed
poorly with CASA. A newer software version with improved algorithms (1.00.61) was capable of
analyzing the total motility in a more acceptable way although further improvements are mandatory
before this device could serve as a diagnostic tool.
3.4.2. Introduction
Frozen equine semen is very often sold by the dose without any guarantee of producing a
pregnancy. It is to be preferred to provide the mare owner with some quality assurance when selling
the semen although one realizes sperm quality assessment remains subject for discussion. At least,
subjective estimation of sperm motility is inaccurate and imprecise particularly if performed by
inexperienced personnel (Davis and Katz, 1993). Nevertheless, motility remains one of the most
important parameters when determining semen quality (Varner, 2008). Objective analysis using
computer assisted sperm analysis (CASA) has a reputation of providing repeatable results that are
however significantly influenced by motility settings (Hoogewijs et al., 2009).
For human andrology research an automated device which does not require motility settings,
the sperm quality analyzer (SQA, Medical Electronic Systems, Caesarea, Israel), has been developed
in the 1980s (Bartoov et al., 1981). Recently, other versions of the SQA have become available for
veterinary use. One of those, the SQA-Ve was especially designed for analyzing equine semen. This
device (version 1.00.43) has been tested previously for analyzing raw and extended semen
(Hoogewijs et al., 2010) and showed a good repeatability and good agreement when assessing
concentration. Motility determined using the SQA-Ve, however, was poorly repeatable with a poor
agreement to the gold standard. In that study, the device was not tested for its capability to analyze
frozen-thawed semen.
The aims of this study were to evaluate the repeatability of the SQA-Ve (1.00.43) and the
agreement of two different SQA-Ve software versions (1.00.43 and 1.0061) with CASA for the
analysis of frozen-thawed equine semen.
125

CHAPTER 3.4
3.4.3. Materials and Methods
126
3.4.3.1. Stallions and semen preparation
The semen used in this experiment was frozen in 0.5 mL straws at two European approved AI
centers in Belgium. The straws were concentrated to contain about 300 × 10 6 spermatozoa per mL.
Four straws per stallion, from the same ejaculate, were thawed in a water bath of 38°C for 30s, dried
and pooled in a small cup, and kept at 38°C. An aliquot of 200µL was diluted tenfold with INRA96
(IMV-technologies, L’Aigle, France), a clear semen extender free of debris when visualized
microscopically. The samples, diluted and undiluted, were incubated at 38°C for 10 minutes prior to
analysis.
3.4.3.2. Analysis with the SQA-Ve
The frozen-thawed semen was analyzed after proper incubation without diluting the thawed
semen according to the manufacturer ‘s guidelines. The semen was aspirated into the specifically
designed capillaries, wiped clean and inserted into the device following onscreen instructions. Prior
to analysis, the SQA-Ve preheats the sample once more during 60 seconds after which the analysis
starts automatically. The SQA-Ve does not require parameter settings, the software version used was
1.00.43 for experiment 1, and 1.00.61 for experiment 2.
When total motility (TM) was equal to or lower than 30%, no actual value is being displayed,
resulting in lack of progressive motility (PM) data. However, the number of progressive motile
spermatozoa (PMS) per mL remains available. As soon as TM is lower than 10%, PMS is not reported
either. As such, analysis with SQA-Ve can either result in actual values (complete data), in limited
data (TM≤30%, and actual value for PMS), or no data at all (TM

CHAPTER 3.4
therefore in these experiments CASA was used as gold standard for comparison with both versions of
the SQA-Ve.
3.4.3.4. Experimental design
Experiment 1
For experiment 1, straws from 46 stallions were used. For motility assessment using CASA
and SQA-Ve, each sample (diluted and undiluted, respectively) was analyzed twice (T1 and T2) within
one minute with each device, so repeatability could be assessed (Fig. 1a).
For agreement, the corresponding values at T1 obtained with each device were compared.
Fig. 1. Overview of the experimental design and the number of analyses resulting in (in)complete
data.
Experiment 2
For experiment 2, straws from 50 stallions were used. For motility assessment using CASA
and SQA-Ve, each sample (diluted and undiluted, respectively) was analyzed once with each device,
to assess agreement (Fig. 1b).
127

CHAPTER 3.4
128
3.4.3.5. Statistical analyses
The outcome variables analyzed were percentage TM and PM. To assess repeatability of
CASA and SQA-Ve (1.00.43) (experiment 1 only) each sample was analyzed twice within one minute.
First, it was tested whether the parameters changed over time (T1 vs. T2), using a paired-samples t-
test. Second, scatter plots were produced, where the value obtained at T1 was plotted against the
value obtained at T2. The line of perfect agreement (hatched line at 45° and intercept zero) was
integrated in each plot as comparison to the regression line to fit the data (Arunvipas et al., 2003).
Third, Bland and Altman plots were produced. This method plots the difference of the paired
measurements (y-axis) against their mean (x-axis) (Bland and Altman, 1986). Finally, for each
parameter, the coefficient of variation (CV) was calculated per sample and reported as mean CV over
all samples.
To assess agreement (experiment 1 and 2, respectively) observations obtained with CASA
were compared with these obtained with SQA-Ve. Statistical analysis was done as described for
repeatability using a paired samples t-test, scatter plots and Bland and Altman plots.
To be able to analyze all available observations, data reported by CASA were categorized
using the abovementioned SQA-Ve cut-off criteria (≤30% and >30%) and analyzed on contingency
tables using McNemar test (paired samples).
Statistical analysis was done using SPSS (version 17.0, SPSS Inc., Chicago, Illinois, USA).
3.4.4. Results
3.4.4.1. Experiment 1
For each of the different methods (CASA and SQA-Ve) 92 analyses were performed. This
yielded complete data for all observations using CASA. Analysis with SQA-Ve resulted in complete
data from 76 (82.6%) observations, incomplete data for 15 (16.3%) observations, and no data for 1
(1.1%) observation (Fig. 1). Complete data were available at T1 and T2 from 34 stallions and used for
further statistical analysis. Data were normally distributed. Averages did not differ significantly at T1
and T2 (Table 1). Analysis with CASA had mean CV’s

CHAPTER 3.4
The averages obtained at T1 with CASA and SQA-Ve differed significantly (Table 2). The TM
and PM obtained with CASA and SQA-Ve were not significantly correlated (Table 2). The scatter plot
and corresponding Bland-Altman plots showed a very poor agreement between CASA and the SQA-
Ve (Fig. 3).
The categorized data for CASA and SQA-Ve (1.00.43) are summarized in table 3. The
distribution of values ≤30% and >30% between the two devices did not differ significantly (p=0.20).
Table 1. Averages of total (TM) and progressive motility (PM) obtained from frozen-thawed equine
semen following analysis with CASA and with the SQA-Ve (version 1.00.43) at time 1 (T1)
and time 2 (T2), the correlation coefficient (CC) between the values at T1 and T2, and the
mean coefficient of variation (details see text), to assess repeatability of measurements.
CASA SQA-Ve
T1 T2 CC† CV T1 T2 CC‡ CV
TM (%) 39.3 38.1 0.92 6.4 56.1 59.4 0.41 16.9
PM (%) 22.8 22.4 0.95 8.2 45.5 48.8 0.40 16.9
Pearson’s correlation coefficients at a level of † p

CHAPTER 3.4
Fig. 2. Scatter plots and corresponding Bland-Altman plots for repeatability of total motility
obtained with CASA, and SQA-Ve (version 1.00.43) at time 1 (T1) and time (T2) for frozenthawed
equine semen ( line of perfect agreement, regression line fit the actual
data).
Table 3. Distribution of all observations for total motility obtained with SQA-Ve (version 1.00.43 and
1.00.61, respectively) and CASA, categorized based on SQA-Ve cut-off (≤30% and >30%); in
every cell the average of the corresponding values obtained with SQA-Ve (S) and CASA (C) is
given as well.
CASA
130
Total Motility CASA T2 (%)
80
60
40
20
0
0 20 40 60 80
Total Motility CASA T1 (%)
Total Motility SQA-Ve T2 (%)
SQA-Ve (1.00.43)
SQA-Ve (1.00.61)
≤30 >30 ≤30 >30
≤30 5
S≤30
C=20.8
19
S=52.0
C=23.4
≤30 2
S≤30
C=20.5
2
S=42.9
C=24.5
>30 11
S≤30
C=38.6
57
S=57.3
C=42.0
>30 2
S≤30
C=43.5
44
S=54.9
C=51.4
CASA
80
60
40
20
0
0 20 40 60 80
Total Motility SQA-Ve T1 (%)

3.4.4.2. Experiment 2
CHAPTER 3.4
For each of the analysis methods (CASA and SQA-Ve) 50 analyses were performed. Complete data
were obtained for all CASA observations, whereas SQA-Ve resulted in complete data for 46 (92%)
observations and incomplete data for 4 (8%) observations (Fig. 1). Data were normally distributed.
The averages obtained with either one of the analyses differed significantly (Table 4). The TM
obtained with SQA-Ve showed significant correlation with the TM obtained with CASA, however, with
a low CC. The PM obtained with both analyses were not correlated (Table 3).
The scatter plot and corresponding Bland-Altman plots showed a rather poor agreement
between CASA and the SQA-Ve (Fig. 3).
The categorized data for CASA and SQA-Ve (1.00.61) are summarized in table 3. The
distribution of values ≤30% and >30% between the two devices was not significantly different
(p=1.00).
Table 4. Averages of total (TM) and progressive motility (PM) obtained from frozen-thawed equine
semen following analysis with CASA and with the SQA-Ve (version 1.00.61), and the
correlation coefficients (CC) between the results obtained with CASA and SQA-Ve.
CASA SQA-Ve
CASA - SQA-Ve
CC p
TM (%) 50.3 a 54.3 a 0.52 0.001
PM (%) 22.4 b 44.0 b 0.22 0.14
a,b Values with different superscript within row, differ significantly ( a p

CHAPTER 3.4
132
Total Motility SQA-Ve (%)
80
60
40
20
0
SQA-Ve version 1.00.43 SQA-Ve version 1.0061
Fig. 3. Scatter plots and corresponding Bland-Altman plots for agreement of total motility obtained
with CASA and SQA-Ve (version 1.00.43 and 1.00.61, respectively) for frozen-thawed equine
semen ( line of perfect agreement, regression line fit the actual data).
The algorithms used for motility analysis cannot be compared with the previous version to
explain the differences. With further improvements, the SQA-Ve might be able to analyze motility in
a more reliable way.
0 20 40 60 80
Total Motilty CASA (%)
In conclusion, with the improved algorithms included in the newer software, the SQA-Ve is
able to analyze equine semen quality in a more acceptable way. However, the need remains for
further improvements before this device can univocally report on thawed equine semen quality.
Total Motility SQA-Ve (%)
80
60
40
20
0
0 20 40 60 80
Total Motility CASA (%)

References
CHAPTER 3.4
Arunvipas P., VanLeeuwen J.A., Dohoo I.R., Keefe G.P. 2003. Evaluation of the reliability and
repeatability of automated milk urea nitrogen testing. Canadian Journal of Veterinary
Research 67:60-63.
Bartoov B, Kalay D, Mayevsky A. 1981. Sperm motility analyzer (SMA), a practical tool of motility and
cell concentration determinations in artificial insemination centers. Theriogenology 15:173-
182.
Bland J.M., Altman D.G. 1986. Statistical methods for assessing agreement between two methods of
clinical measurement. Lancet 1:307-310.
Davis R., Katz D. 1993. Operational standards for CASA instruments. Journal of Andrology 14:385-394.
Hoogewijs M., De Vliegher S., De Schauwer C., Govaere J., Smits K., Hoflack G., de Kruif A., Van Soom
A. 2010. Validation and usefulness of the Sperm Quality Analyzer V equine for equine semen
analysis. Theriogenology in press.
Hoogewijs M., Govaere J., Rijsselaere T., De Schauwer C., Vanhaesebrouck E., de Kruif A., De Vliegher
S. 2009. Influence of technical settings on CASA motility parameters of frozen thawed stallion
semen. Proceedings of the 55 th annual convention of the American Association of Equine
Practitioners – Las Vegas, USA 55:333-337.
Loomis P.R., Graham J.K. 2008. Commercial semen freezing: individual male variation in cryosurvival
and the response of stallion sperm to customized freezing protocols. Animal Reproduction
Science 105:119-128.
Varner D.D. 2008. Developments in stallion semen evaluation. Theriogenology 70:448-462.
133

CHAPTER 4
Different centrifugation techniques – To what extent
do they affect quality and preservation of equine
semen?
135

4.1
Influence of different centrifugation protocols on
equine semen preservation
Hoogewijs M., Rijsselaere T., De Vliegher S., Vanhaesebrouck E., De
Schauwer C., Govaere J., Thys M., Hoflack G., Van Soom A., de Kruif A.
2010 Theriogenology 74:118-126.
137

4.1.1. Abstract
CHAPTER 4.1
Three experiments were conducted to evaluate the impact of centrifugation on cooled and
frozen preservation of equine semen. A standard centrifugation protocol (600 × g for 10 min = CP1)
was compared to four protocols with increasing g-force and decreased time period (600 × g, 1200 × g,
1800 × g and 2400 × g for 5 min for CP2, 3, 4 and ,5 respectively) and to an uncentrifuged negative
control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by
calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on
semen quality following centrifugation was assessed by monitoring several sperm parameters
(membrane integrity using SYBR14-PI, acrosomal status using PSA-FITC, percentage total motility
(TM), percentage progressive motility (PM) and beat cross frequency (BCF) obtained with computer
assisted sperm analysis (CASA)) immediately after centrifugation and daily during chilled storage for
3 days. The use of CP1 resulted in a sperm loss of 22%. Increasing the centrifugation force to 1800 ×
g and 2400 × g for 5 min led to significantly lower sperm losses (7.4% and 2.1%, respectively; p

CHAPTER 4.1
4.1.2. Introduction
140
Numerous studies have demonstrated the beneficial effect of diluting raw semen with an
appropriate extender. As such, the negative influence of seminal plasma (SP) on the preservation of
equine sperm can be reduced. Centrifugation of diluted equine semen and subsequent resuspension
of the sperm pellet in fresh extender can even further reduce the amount of SP in stored samples. A
small proportion of SP in semen improves sperm motility after cooling and storage (Jasko et al.,
1991).
However, centrifugation itself may act like a double edged sword exerting both beneficial
and deleterious effects on sperm motility (Martin et al., 1979). Although the number of sperm cells
can be maximized by increasing centrifugation time and force, physical damage is exerted on the
sperm cells at the same time. The direct negative outcome of centrifugation on semen quality is
demonstrated by the decrease in motility and velocity in contrast to uncentrifuged controls (Jasko et
al., 1991) which indicates that the centrifugation protocol (CP) may play an important role in the
quality of the inseminate. After 24h of cooled storage the harmful effect of centrifugation is no
longer present (Jasko et al., 1991).
A CP of 600 × g for 10 min is commonly used for equine semen (Ecot et al., 2005; Knop et al.,
2005; Vidament et al., 2000; Weiss et al., 2004). In literature, data on the amount of sperm loss after
using this protocol are contradictory and vary from 1.9% (Weiss et al., 2004) to 25% (Ecot et al., 2005;
Knop et al., 2005). Nowadays, it is generally accepted that sperm losses will vary around 25% when
diluted semen is centrifuged at 400-600 × g for 10-15 min (Aurich, 2008; Loomis, 2006).
The importance of an appropriate CP has been clearly demonstrated for human sperm. The
duration of centrifugation was shown to be more important than the centrifugation force for causing
iatrogenic sperm membrane injuries which resulted in an increased formation of reactive oxygen
species (Shekarriz et al., 1995). In boars, similar findings were described: a CP with a high g-force for
a short time (2400 × g for 3 min) was used without detrimental effects on sperm yield compared to
the standard regime (800 × g for 10 min) (Carvajal et al., 2004). Additionally, a positive effect on
semen quality after cryopreservation was detected when using high g-forces for a shorter period of
time (Carvajal et al., 2004). Experiments with equine semen have been performed where semen was
centrifuged at 400 × g for an increasing period of time. Sperm loss was approximately 20%, if
samples were centrifuged for 10 min or longer, while no adverse effects on motility immediately
after centrifugation were present, unless semen was centrifuged for 20 min. However, effects on
preserved by cooling or freezing were not determined (Heitland et al., 1996). A high g-force for a

CHAPTER 4.1
short time was not detrimental for porcine sperm (Carvajal et al., 2004) and prolonged
centrifugation of equine sperm negatively influenced sperm quality (Heitland et al., 1996).
The aim of the present study was to compare high speed CPs with the standard protocol on
cooled and cryopreserved equine semen. The impact of CP on the subsequent number and quality of
sperm cells was assessed daily during 3 consecutive days of cooled preservation. Additionally, the
effect of different CPs on the quality of frozen-thawed equine sperm was investigated.
4.1.3. Materials and methods
4.1.3.1. Stallions and semen collection
For experiments 1 and 2, five ejaculates from each of five experienced Shetland ponies
(n=25), aged 3 to 6 years, were collected between August and October 2007. For experiment 3, one
ejaculate from 4 of these ponies was used. The ponies were housed in groups in a straw bedded
stable and were fed good quality hay ad libitum. Prior to the onset of the experiment, the extra-
gonodal sperm reserves were depleted with 3 collections every other day for one week. For the
experiment, the semen was collected twice a week. Semen was collected on a teaser mare using
standard procedures with a custom made open ended artificial vagina based on the Colorado State
University model as described by Pickett (1993). After collection, semen was filtered through a
sterile gauze to remove the gel fraction and debris. The gel-free volume was noted and the sperm
concentration was determined using a Bürker haemocytometer after 1:9 dilution with HCl 1M.
4.1.3.2. Media
Fresh-cooled semen was processed and stored in Gent Extender (Minitüb, Landshut,
Germany), which is a skimmed milk based diluter containing 5 % clarified egg yolk and gentamycin (1
mg/mL). If the semen was to be cryopreserved, a second dilution was performed in Gent Extender
for Freezing (Minitüb, Landshut, Germany) which has the same composition as the Gent Extender
plus 5 % glycerol.
For the fluorescent staining procedures, the samples were diluted in a HEPES buffered
solution containing 0.04% BSA.
141

CHAPTER 4.1
142
4.1.3.3. Semen processing
After determination of the initial sperm concentration, the semen was diluted to a final
concentration of 25 × 10 6 sperm /mL using Gent Extender. Six conical bottom centrifuge tubes of 15
mL (Cellstar ® , Greiner bio-one, Germany) were filled with the diluted semen (total of 375 × 10 6 sperm
cells per tube) and served as the negative control or were subjected to one of the five different CPs,
respectively. After centrifugation, 90% of the supernatant was aspirated, leaving a sperm pellet with
a volume of approximately 1.5 mL.
The concentration in the aspirated supernatant was determined using a Neubauer
haemocytometer. The obtained concentration was multiplied by the volume of aspirated
supernatant in order to calculate the total number of sperm cells lost per tube when the
supernatant was discarded. The remaining sperm pellet was resuspended in the appropriate diluter
for further processing as cooled or frozen semen.
4.1.3.4. Evaluation of sperm characteristics and staining procedures
Prior to analysis, an aliquot (500 µL) of semen was equilibrated to 37 °C. The morphology of
sperm cells was examined on eosin-nigrosin stained smears which were prepared as described by
Barth and Oko (1989). At least 200 sperm cells were evaluated and recorded per slide, individual
morphological abnormalities were noted according to their location (head, midpiece or tail).
Motility was evaluated with a computer-assisted sperm analyzer (CASA) (Hamilton-Thorne
Ceros 12.3). For each analysis, 5 µL of diluted semen was mounted on a disposable Leja counting
chamber (Orange Medical, Brussels, Belgium) and maintained at 37 °C using a minitherm stage
warmer. Five randomly selected microscopic fields in the center of the slide were scanned 5 times
each, obtaining 25 scans for every semen sample. The mean of the 5 scans for each microscopic field
was used for the statistical analysis (Rijsselaere et al., 2003). The software settings of the HTR 12.3,
based on Loomis and Graham (2008), are summarized in Table 1.

Table 1. Software settings of the Hamilton Thorne Ceros 12.3 used in this study
Parameter Value
Frames acquired 30
Frame rate (Hz) 60
Minimum contrast 60
Minimum cell size (pixels) 6
Minimum static contrast 25
Straightness cut-off (%, STR) 75
Average-path velocity cut-off PM (µm/s,VAP) 50
VAP cut-off static cells (µm/s) 20
Cell intensity 100
Static head size 0.55 – 2.04
Static head intensity 0.45 – 1.70
Static elongation 11 - 99
CHAPTER 4.1
Membrane integrity was evaluated using a fluorescent SYBR14-Propidium Iodide (PI) staining
technique (Molecular Probes cat n°: L-7011, Leiden, The Netherlands) based on a previously
described method (Garner and Johnson, 1995). Briefly, 225 µL HEPES-TALP was mixed with 25 µL of
diluted semen and 1.25 µL SYBR14 (1:50 dilution) was added. After 5 min of incubation at 37 °C, 1.25
µL PI was added and incubated for another 5 min. Two hundred cells were evaluated per slide using
a Leica DMR fluorescence microscope and three populations of sperm cells could be identified
(green = living, red = dead and orange = moribund).
Acrosomal status was determined using fluorescent Pisum Sativum Agglutinin (PSA)
conjugated with fluorescein isothiocyanate (FITC) (Sigma-Aldrich cat n°: L 0770, Bornem, Belgium).
The staining was performed in a similar way as described by Rathi et al. (2003). Briefly, 500 µL of
semen was centrifuged for 10 min at 720 × g and the pellet was resuspended with HEPES-TALP. The
semen was centrifuged again for 10 min at 720 × g, the supernatant was removed and the pellet
fixed in 100 µL absolute ethyl alcohol (Vel cat n°: 1115, Haasrode, Belgium) and cooled for 30 min at
4 °C. A drop of 20 µL semen was smeared on a glass slide and 40 µL PSA-FITC (2 mg PSA-FITC diluted
in 2 mL phosphate buffered saline (PBS)) was added. The glass slide was kept at 4 °C for 15 min,
washed 10 times with aqua bidest and allowed to air-dry in the dark. Immediately after drying, two
hundred sperm cells were evaluated per slide. The acrosomal region of the acrosome intact sperm
cells was labeled heavily green, while the acrosome reacted sperm retained only an equatorial band
of label with little or no labeling of the anterior head region.
143

CHAPTER 4.1
144
In the experiment 3, DNA integrity was analyzed using a Terminal deoxynucleotidyl
Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay. The TUNEL assay was performed using the
In Situ Cell Death Detection Kit (Boehringer, Mannheim, Germany) to detect the presence of free 3′-
OH termini in single and double-stranded sperm DNA (De Pauw et al., 2003). In short, sperm samples
were diluted with PVP solution (1 mg/mL in PBS) to a final concentration of 10 × 10 6 sperm/mL from
which a 10 μL aliquot was smeared onto a poly-l-lysine-coated microslide. After fixation with 4%
paraformaldehyde in polyvinyl pyrrolidone (PVP) solution (pH 7.4) and permeabilisation with 0.5%
(v/v) Triton X-100 in PBS, the sperm cells were incubated with the TUNEL-mixture (fluorescein-dUTP
and terminal deoxynucleotidyl transferase) for 1 h at 37 °C in the dark. Both positive (1 mg/mL
DNAse I) and negative controls (nucleotide mixture in the absence of transferase) were included in
each replicate. Hoechst 33342 was used to counter stain sperm DNA. The samples were examined by
fluorescence microscopy (Leica DMR; magnification 400×, oil immersion). At least 200 sperm cells
from each sample were analyzed randomly to evaluate the percentage of TUNEL-positive sperm cells
(bright green nuclear fluorescence) (see De Pauw et al. (2003) for further details).
4.1.3.5. Centrifugation protocols
After dilution of the raw semen with Gent Extender (at 37 °C), each tube, except for the
uncentrifuged control, was centrifuged with a Heraeus Multifuge 3 L-R (Kendro Laboratory Products,
Waltham, USA) at ambient temperature using 1 of 5 CPs. Centrifugation protocol 1 was the
reference protocol being 10 min at 600 × g (Ecot et al., 2005; Knop et al., 2005; Vidament et al., 2000;
Weiss et al., 2004); CPs 2, 3, 4 and 5 were 5 min at 600 × g, 1200 × g, 1800 × g and 2400 × g,
respectively. The deceleration speed was 1, which was the slowest possible deceleration in order to
minimize disturbance due to turbulence. Since only one centrifuge was available, the processing of
each ejaculate from the same stallion started with a different CP to eliminate influence of a
prolonged contact time with the SP. So for each stallion every ejaculate was centrifuged first with a
different alternating protocol.

4.1.3.6.Experimental design
CHAPTER 4.1
EXPERIMENT 1: influence of the centrifugation protocol on the function of fresh and cooled
sperm
Fresh semen was analyzed (concentration, CASA, eosin/nigrosin staining and SYBR14-PI)
immediately after collection (T0). The semen was diluted to 25 × 10 6 sperm cells/mL and allocated to
six 15-mL test tubes (Cellstar ® , Greiner bio-one, Germany). Five of these tubes (1 to 5) were
subjected to one of the 5 CPs whereas tube 6 served as a negative control (no centrifugation). After
centrifugation, the sperm concentration was determined in the aspirated supernatant. The sperm
pellet was resuspended in fresh diluter and an aliquot of each test tube was analyzed (T1). The test
tubes containing the processed semen were allowed to cool gradually to 5°C in 50-mL test tubes
filled with water (22°C) and placed in a 5°C refrigerator. Sperm analyses were repeated at 24h (T2),
48h (T3) and 72h (T4). PSA-FITC staining was performed immediately after centrifugation and at 72h.
EXPERIMENT 2: influence of the pre-freezing centrifugation protocol on the function of
frozen-thawed sperm
The initial semen processing was performed as described in experiment 1. After
centrifugation using each of the five CP’s, the sperm pellet was resuspended in Gent extender for
freezing to a final concentration of 50×10 6 sperm cells/mL. The extended semen was loaded into 0.5
mL straws (MRS1, L’Aigle Cedex, France) at ambient temperature, placed on racks and transported
to the freezing chamber of an automated controlled rate freezer (IceCube 14S, Neupurkersdorf,
Austria). The semen was first cooled from 22°C to 4°C in 80 min and then frozen from 4 °C to -140 °C
at 60 °C/min (adapted from Vidament et al. (2000)). At -140 °C the freezing racks were removed
from the freezing chamber and plunged into liquid nitrogen. Frozen semen was stored for at least 1
month before analysis. The samples were thawed by plunging into a 37 °C water bath for 30 s. Five
straws from the same batch (same ejaculate, same treatment) were pooled, allowed to equilibrate
for 5 min at 37 °C and analyzed once, performing the same analyses as described in experiment 1.
EXPERIMENT 3: influence of the centrifugation protocol on the DNA integrity of fresh and
cooled sperm
From four stallions, one ejaculate was collected and diluted in Gent Extender to a final
concentration of 25×10 6 sperm cells/mL. The diluted semen was subjected to three different CP’s
(CP1, CP2 and CP5) whereas one tube served as negative (uncentrifuged) control. Immediately after
145

CHAPTER 4.1
centrifugation (T1) and 72 h (T4) after cooled storage, an aliquot of each tube was examined for DNA
integrity using TUNEL assay.
146
4.1.3.7. Statistical analysis
EXPERIMENT 1: influence of centrifugation protocol on function of fresh and cooled sperm
1/ sperm loss
To evaluate whether the treatment (CP 1, 2, 3, 4 and 5) had an influence on sperm
concentration in the aspirated supernatant, a Kruskal Wallis H test was conducted (overall treatment
effect) as well as a Mann-Whitney U test (to test the difference between CP 1 versus the other
treatments, separately) using SPSS software package (version 17.0, SPSS Inc., Chicago, IL).
2/ effect of centrifugation (non CP vs CP)
Mixed models were fitted in SAS 9.1.3. (PROC MIXED) with centrifugation (non CP vs CP),
time (T1- T4) and interaction centrifugation × time included as predictor variables. Stallion (1-5) and
ejaculate (5 per stallion) were included as random effects to account for clustering of ejaculate in
stallion and the repeated measurements over time, within ejaculate (including an AR(1) correlation
structure), respectively. The outcome variables (percentage membrane intact sperm cells,
percentage acrosome intact sperm cells, BCF, TM and PM) were normally distributed based on the
inspection of QQ-plots and Kolmogorov-Smirnov tests.
3/ effect of centrifugation protocol
Mixed models were fitted in SAS 9.1.3. (PROC MIXED) with CP (1-5), time (T1- T4) and the
interaction CP × time included as predictor variables. Stallion (1-5) and ejaculate (5 per stallion) were
included as random effects to account for clustering of ejaculate in stallion and the repeated
measurements over time, within ejaculate (including an AR(1) correlation structure), respectively.
The outcome variables (percentage membrane intact sperm cells, percentage acrosome intact sperm
cells, BCF, TM and PM) were normally distributed based on the inspection of QQ-plots and
Kolmogorov-Smirnov tests.

sperm
CHAPTER 4.1
EXPERIMENT 2: influence of pre-freezing centrifugation protocol on function of thawed
Mixed models were fitted in SAS 9.1.3. (PROC MIXED) with CP (1-5), ejaculate (1-5) and the
interaction CP × ejaculate included as predictor variables. Stallion (1-5) was included as random
effects to account for clustering of ejaculate in stallion. The outcome variables (percentage
membrane intact sperm cells, percentage acrosome intact sperm cells, BCF, TM and PM) were
normally distributed based on the inspection of QQ-plots and Kolmogorov-Smirnov tests.
sperm
EXPERIMENT 3: influence of centrifugation protocol on DNA integrity of fresh and cooled
Since the percentages of DNA intact sperm cells were not normally distributed, a time effect
(T1 vs T4) was analyzed using Wilcoxon signed ranks test. Effect of treatment was determined using
Kruskal-Wallis test at T1 and T4. Analyses were done using SPSS software package (version 17.0,
SPSS Inc., Chicago, IL).
4.1.4. Results
4.1.4.1. EXPERIMENT 1: influence of the centrifugation protocol on the function of
fresh and cooled sperm
The general sperm characteristics over all the ejaculates from the 5 stallions immediately
after collection are presented in Table 2.
There was an overall effect of CP on sperm concentration in the recovered supernatant
(p

CHAPTER 4.1
(non CP vs CP) was significant for these parameters (p

35
B
100
A
33
beat cross frequency (Hz)
80
31
60
29
40
27
20
% membrane intact sperm
25
0
0 h 24 h 48 h 72 h
0 h 24 h 48 h 72 h
100
D
100
C
80
% progressive motility
80
60
60
% total motiliy
40
40
20
20
0
0
0 h 24 h 48 h 72 h
0 h 24 h 48 h 72 h
Centrifuged samples
Uncentrifuged samples
Fig. 1. Effect of centrifugation on percentage membrane intact sperm (A), beat cross frequency (B), total motility (C) and progressive motility (D) during
chilled storage for 72 hours.

35
B
80
A
33
beat cross frequency (Hz)
75
70
31
65
29
60
27
55
% membrane intact sperm
25
50
0 h 24 h 48 h 72 h
0 h 24 h 48 h 72 h
50
D
70
C
45
% progressive motility
65
40
60
% total motiliy
35
55
30
50
25
45
20
40
0 h 24 h 48 h 72 h
0 h 24 h 48 h 72 h
2400 × g 5 min
CP5
1800 × g 5 min
CP4
1200 × g 5 min
CP3
600 × g 5 min
CP2
600 × g 10 min
CP1
Fig. 2. Effect of different centrifugation protocols on percentage membrane intact sperm (A), beat cross frequency (B), total motility (C) and progressive
motility (D) during chilled storage for 72 hours.

Beat cross frequency (Hz)
40
35
30
25
20
15
10
5
0
a,b
600 × g 10min
CP1
a
600 × g 5min
CP2
b b
1200 × g 5min
CP3
1800 × g 5min
CP4
CHAPTER 4.1
Fig. 3. Effect of different centrifugation protocols on beat cross frequency of frozen-thawed stallion
sperm, error bars are SD (columns with the same character are statistically different from
the reference protocol (600 × g for 10 min), for a p

CHAPTER 4.1
152
4.1.4.2. EXPERIMENT 2: influence of the pre-freezing centrifugation protocol on the
function of frozen-thawed sperm
There was a significant effect of ejaculate for all evaluated parameters (% acrosome intact
sperm, p

4.1.5. Discussion
CHAPTER 4.1
In this study, we demonstrated that the loss of sperm after centrifugation can be minimized
by using an appropriate protocol without damaging the sperm. Centrifugation protocol had a limited
effect on in vitro sperm characteristics in our study. We specifically showed that the use of the
standard protocol (CP 1) led to an average sperm loss of 22%, resulting in a considerable reduction in
the number of AI doses obtained. If the centrifugation time was decreased while the exerted g-force
was increased up to 1800 × g or even 2400 × g, sperm losses were reduced to 7.4% and 2.1%,
respectively, without any apparent changes in the in vitro sperm quality.
If semen was cooled and stored after centrifugation, only a minimal effect of the used CP
was noticed on total motility as evaluated by CASA. Surprisingly, the motility was reduced in the
group where the lowest centrifugation force was used for the shortest time (600 × g for 5 min). The
sperm pellet probably was very soft which allowed the most motile sperm cells to swim up
immediately after centrifugation had ceased. As a consequence, these highly motile sperm may be
discarded during aspiration of the supernatant. This was confirmed in a preliminary experiment (n=4)
by analyzing the supernatant of semen samples centrifuged according to CP1, 2 and 5 (data not
shown). The average TM and PM in the supernatant was 41.3% and 23.5%, 54.8% and 31.8% and 8.3%
and 4.0% for CP1, 2 and 5, respectively, suggesting that the motility of the discarded sperm cells in
the supernatant is much higher when low centrifugation forces are used. Therefore, application of a
low g-force for a short time leads to important losses of top quality sperm cells and must definitely
be avoided.
Additionally, we found a significant effect on BCF for sperm samples cooled and stored after
centrifugation. The BCF of the sperm subjected to CPs 2 and 4 was reduced compared to the
standard protocol. Moreover, the quality of the frozen-thawed samples after being subjected to one
of the tested CPs did not differ significantly for any of the tested quality parameters except for BCF.
Protocols 2 and 4 yielded the highest BCF values after thawing, which was in contrast with our
observations for cooled preservation. However, the actual impact of BCF on fertility is difficult to
predict since few data are available in literature (Gil et al., 2009). BCF is a parameter indicative for
spermatic vigor. Changes in BCF under capacitating conditions in vitro are thought to be related to
sperm hyperactivation that occurs in vivo and might favor the penetration of oocytes (Gil et al.,
2009).
The results of the experiment 3 showed a decrease of DNA integrity over time, although
centrifugation did not influence DNA integrity. These findings are contradictory with the study of
153

CHAPTER 4.1
Love and coworkers (2005) where increased DNA damage immediately after centrifugation was
reported. These authors showed that complete removal of SP after centrifugation and subsequent
resuspension of the sperm pellet in fresh diluter resulted in better sperm quality (motility and DNA)
compared to samples in which SP was still present after simple dilution of the semen. Therefore the
impact of the centrifugation on DNA integrity needed to be further examined. In our study, no
difference in DNA damage was present immediately after centrifugation which might be explained
by the method used to evaluate DNA integrity. The study of Love et al. (2005) evaluated DNA
integrity using the sperm chromatin structure assay (SCSA). In comparison to the TUNEL assay, with
the SCSA a larger number of sperm cells is analyzed by a flow cytometer based on the colour of the
metachromatic dye acridine orange (Evenson and Wixon, 2006). Therefore it might be advisable to
measure the direct impact of different CPs with flow cytometry (SCSA or TUNEL) to get a more
accurate impression of the possible side effect of centrifugation on DNA integrity.
154
Cushioned centrifugation techniques have previously been described as an answer to reduce
sperm damage while maximizing the sperm harvest. A variety of dense solutions acting as a cushion
have been described (Cochran et al., 1984). The frequently used iodixanol was first reported for
stallion sperm cells in 1997 (Revell et al., 1997). Very high recovery rates have been described (Ecot
et al., 2005; Knop et al., 2005) using such a commercially available cushion, when centrifuging at
1000 × g for 20 min. However, the most critical step is carefully removing the cushion material after
centrifugation. Indeed, it is critical to aspirate as much cushion fluid as possible without aspirating
sperm cells from the adjacent band to avoid possible deleterious effects of the iodixanol solution
(Waite et al., 2008). The use of very small amounts (30 µL) of cushion without removal was proven
to be harmless. However, these small volumes require specially designed, custom-made nipple
centrifugation tubes which are not readily available (Waite et al., 2008). Because of these important
drawbacks, cushioned centrifugation is not commonly used in practice. Therefore, the impact of CP
on sperm quality remains an important subject to examine.
In summary, sperm losses can be reduced substantially by centrifuging stallion semen for 5
min at 1800 or 2400 × g without a cushion and without impairing the in vitro sperm function after
cooled or frozen storage. This leads to an increase in the number of AI doses produced per ejaculate.
It can easily result in an additional two AI doses from an average ejaculate of 10 billion sperm.
Nevertheless, the effects of these CPs on DNA damage requires further investigation as well as the
impact on in vivo fertility.

References
CHAPTER 4.1
Aurich C. 2008. Recent advances in cooled-semen technology. Animal Reproduction Science 107:268-
75.
Barth A.D., Oko R.J. 1989. Abnormal morphology of bovine spermatozoa. Ames, Iowa: Iowa State
University Press.
Carvajal G., Cuello C., Ruiz M., Vázquez J.M., Martínez E.A., Roca J. 2004 Effects of centrifugation
before freezing on boar sperm cryosurvival. Journal of Andrology 25:389-96.
Cochran J.D., Amann R.P., Froman D.P., Pickett B.W. 1984. Effects of centrifugation, glycerol level,
cooling to 5°C, freezing rate and thawing rate on the post-thaw motility of equine sperm.
Theriogenology 22:25-38.
De Pauw I.M.C., Van Soom A., Mintiens K., Verberckmoes S., de Kruif A. 2003 In vitro survival of
bovine spermatozoa stored at room temperature under epididymal conditions.
Theriogenology 59:1093-107.
Ecot P., Decuadro-Hansen G., Delhomme G., Vidament M. 2005. Evaluation of a cushioned
centrifugation technique for processing equine semen for freezing. Animal Reproduction
Science 89:245-8.
Evenson D.P., Wixon R. 2006. Clinical aspects of sperm DNA fragmentation detection and male
infertility. Theriogenology 65:979-91.
Garner D.L., Johnson L.A. 1995. Viability assessment of mammalian sperm using SYBR-14 and
propidium iodide. Biology of Reproduction 53:276-84.
Gil M.C., García-Herreros M., Barón F.J., Aparicio I.M., Santos A.J., García-Marín L.J. 2009.
Morphometry of porcine spermatozoa and its functional significance in relation with the
motility parameters in fresh semen. Theriogenology 71:254-63.
Heitland A.V., Jasko D.J., Squires E.L., Graham J.K., Pickett B.W., Hamilton C. 1996 Factors affecting
motion characteristics of frozen-thawed stallion spermatozoa. Equine Veterinary Journal
28:47-53.
Jasko D.J., Moran D.M., Farlin M.E., Squires E.L. (1991) Effect of seminal plasma dilution or removal
on spermatozoal motion characteristics of cooled stallion semen. Theriogenology 35:1059-67.
Knop K., Hoffmann N., Rath D., Sieme H. 2005. Effects of cushioned centrifugation technique on
sperm recovery and sperm quality in stallions with good and poor semen freezability. Animal
Reproduction Science 2005;89:294-7.
Loomis P.R. 2006. Advanced methods for handling and preparation of stallion semen. Veterinary
Clinics of North America: Equine Practice 22:663-76.
Loomis P.R., Graham J.K. 2008. Commercial semen freezing: individual male variation in cryosurvival
and the response of stallion sperm to customized freezing protocols. Animal Reproduction
Science 105:119-28.
155

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Love C.C., Brinsko S.P., Rigby S.L., Thompson J.A., Blanchard T.L., Varner D.D. 2005 Relationship of
seminal plasma level and extender type to sperm motility and DNA integrity. Theriogenology
63:1584-91.
Martin J.C., Klug E., Günzel A.-R. 1979. Centrifugation of stallion semen and its storage in large
volume straws. Journal of Reproduction and Fertility Supplement 27:47-51.
Pickett B.W. 1993. Collection and evaluation of stallion semen for artificial insemination. In: Equine
Reproduction, McKinnon AO, Voss JL (Ed.), Williams & Wilkins, pp.705-14.
Rathi R., Colenbrander B., Stout T.A., Bevers M.M., Gadella B.M. 2003. Progesterone induces
acrosome reaction in stallion spermatozoa via a protein tyrosine kinase dependent pathway.
Molecular Reproduction and Development 64:120-8.
Revell S.G., Pettit M.T., Ford T.C. 1997. Use of centrifugation over iodixanol to reduce damage when
processing stallion sperm for freezing. Journal of Reproduction and Fertility, Abstr series n°19,
p38.
Rijsselaere T., Van Soom A., Maes D., de Kruif A. 2003 Effect of technical settings on canine semen
motility parameters measured by the Hamilton-Thorne analyzer. Theriogenology 60:1553-68.
Shekarriz M., DeWire D.M., Thomas A.J. Jr., Agarwal A. 1995 A method of human semen
centrifugation to minimize the iatrogenic sperm injuries caused by reactive oxygen species.
European Urology 28:31-5.
Vidament M., Ecot P., Noue P., Bourgeois C., Magistrini M., Palmer E. 2000 Centrifugation and
addition of glycerol at 22 degrees C instead of 4 degrees C improve post-thaw motility and
fertility of stallion spermatozoa. Theriogenology 54:907-19.
Waite J.A., Love C.C., Brinsko S.P., Teague S.R., Salazar J.L. Jr., Mancil S.S., Varner D.D. 2008. Factors
impacting equine sperm recovery rate and quality following cushioned centrifugation.
Theriogenology 70:704-14.
Weiss S., Janett F., Burger D., Hässig M., Thun R. 2004. The influence of centrifugation on quality and
freezability of stallion semen. Schweizer Archiv für Tierheilkunde 146:285-93.
156

4.2
Sperm selection using single layer centrifugation prior
to cryopreservation can increase post-thaw sperm
quality in stallions
Hoogewijs M. 1 , Morrell J.², Van Soom A. 1 , Govaere J. 1 , Johannisson
A.³,Piepers S. 1 , De Schauwer C. 1 , de Kruif A. 1 , De Vliegher S. 1
submitted.
1 Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine,
Ghent University, Merelbeke, Belgium
² Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural
Sciences, Uppsala, Sweden
³ Department of Anatomy, Physiology & Biochemistry, Swedish University of Agricultural Sciences,
Uppsala, Sweden
157

4.2.1. Abstract
CHAPTER 4.2
The increasing use of modern reproductive techniques in human medicine has led to a
higher demand for isolation of motile sperm. Several of these isolation techniques have been
adapted for veterinary use and can be applied for the selection of a superior sperm sample from
stallion semen. Until recently the major downside of such isolation techniques was the limitation in
sperm volume that could be handled. Androcoll-E had been shown to be successful for processing
large volumes of equine semen but few data substantiate the potential beneficial effect of freezing
an Androcoll-E selected equine sperm sample to obtain a higher post-thaw quality. In this study, the
effect of Androcoll-E treatment of sperm prior to cryopreservation was compared with cushioned
centrifugation using ejaculates from eight different stallions with different freezability. Androcoll-E
treated sperm had increased quality parameters prior to freezing. The only downside of Androcoll-E
treated sperm was the markedly lower yield following centrifugation when compared to the cushion
centrifuged control group (50.9% vs. 97.1%, respectively). Post-thaw quality analysis showed an
overall improved sperm quality for Androcoll-E treated samples. E.g., post-thaw progressive motility
(PM) was on average 41.6% compared to 30.5% for the cushion centrifuged group. Androcoll-E
treatment increased the likelihood that straws were accepted based on post-thaw PM (≥30%). In
conclusion, Androcoll-E can be used with good results to select a superior sperm population prior to
cryopreservation, in order to produce good quality frozen thawed semen.
4.2.2. Introduction
With the introduction of clinical assisted reproductive techniques in human reproductive
medicine the requirement emerged for isolating the more motile spermatozoa. Isolation techniques
evolved from simple washing procedures to actual separation based on different principles such as
migration, filtration or density centrifugation (Henkel and Schill, 2003). In human andrology these
techniques have been used also to select a superior sperm sample prior to freezing, in order to
obtain a post-thaw sample with improved in vitro characteristics. The so-called swim-up procedure
was proven to be successful to obtain a higher quality post-thaw sperm sample (Pérez-Sánchez et al.,
1994; Esteves et al., 2000). Sperm cells after selection were equally susceptible to the stress induced
by the freezing and thawing process as unselected sperm cells, indicating post-thaw quality
improvement was probably only due to the initial better quality of the pre-freeze sample (Pérez-
Sánchez et al., 1994). A discontinuous density gradient centrifugation prior to cryopreservation
159

CHAPTER 4.2
resulted in an improvement of sperm motility for up to 24 hours after thawing (Sharma and Agarwal,
1996).
160
Different sperm selection techniques were compared such as the swim-up procedure and
the Percoll density gradient centrifugation which could process only limited volumes of semen,
whereas glass wool Sephadex filtration (GWS) and Leucosorb ® filtration (LF) allowed filtration of
larger volumes (Sieme et al., 2003). The latter two were evaluated when used prior to
cryopreservation. An increased post-thaw progressive motility (PM) for the GWS and LF treated
samples was reported, but since Percoll centrifugation could only be used to process small volumes
of 1 mL, the effect of density gradient centrifugation prior to cryopreservation was not evaluated. In
another study, glass beads column separation resulted in an increased post-thaw sperm quality
compared to unselected samples (Klinc et al., 2003). However, this technique requires specific
material not readily available.
The main drawbacks of gradient centrifugation can now be remedied since the technique
has been simplified to a single layer centrifugation (SLC) protocol using Androcoll-E with comparable
results for sperm yield and quality as for gradient centrifugation (Morrell et al., 2009a). Moreover, a
technique using Androcoll-E-Large allows processing of 15 mL of extended semen per centrifugation
tube (Morrell et al., 2009b), hence the problem of small volumes has been solved as well.
Not all stallions produce semen that can be cryopreserved with good results. In a large
French field study freezability of stallion semen was calculated by the ratio of the number of
selected ejaculates (PM>35%) over the total number of ejaculates frozen (Vidament et al., 1997).
Over a total of 427 stallions, it was found that about 50% of the stallions were non freezable or poor
freezers , about 25% were intermediate freezers and 25% were good freezers. The implementation
of Androcoll-E in the freezing protocol might be a solution to improve sperm processing of poor
freezing stallion semen.
The aim of this study was to evaluate equine sperm selection using Androcoll-E-Large in a
SLC prior to cryopreservation, by comparing this method with a cushioned centrifugation freezing
protocol. The latter has consistently yielded the best results for freezing equine semen to date,
making it an obvious point of comparison. In addition, it was evaluated whether the effect of the
treatment (Androcoll-E-Large versus cushioned centrifugation) was modified by the freezability of
the stallion as determined by number of ejaculates with post-thaw PM ≥30%.

4.2.3. Materials and methods
4.2.3.1. Stallions and semen collection
CHAPTER 4.2
Ejaculates (n=22) from 8 warm blood stallions of different freezability (7×3 ejaculates and
1×1, respectively), aged 3 to 14 years, were collected between March and May 2010. The stallions
were housed individually in straw bedded stallion boxes and were fed good quality hay ad libitum
and 2 kg of concentrates twice daily. Semen was collected using standard procedures as described
by Pickett (1993). After collection, semen was filtered through a sterile gauze to remove the gel
fraction and debris. The gel-free volume was recorded and the sperm concentration was determined
using the Nucleocounter SP-100 (ChemoMetec, A/S, Allerød, Denmark) as described earlier (Hansen
et al., 2006; Morrell et al., 2010).
4.2.3.2. Semen processing
Following collection, the ejaculates were processed as follows (Fig. 1). The raw semen was
diluted to a final concentration of 100 × 10 6 sperm /mL using INRA96 (IMV, L’Aigle cedex, France) at
37°C. After gentle homogenization of the diluted semen, 30 mL was transferred into a 50 mL conical
bottom centrifuge tube (Cellstar ® , Greiner bio-one, Germany). Subsequently, 3.5 mL of MAXIFREEZE
(IMV, L’Aigle cedex, France) was layered underneath the extended semen using a spinal needle and
5-mL syringe (Waite et al., 2008). The tubes were centrifuged (Universal 320R, Hettich, Beverly, MA,
USA) at 1000 × g for 20 min at ambient temperature. After centrifugation, the supernatant was
removed to the 5-mL mark (the top 5 mL of supernatant was kept separately in a two-part 5-mL
syringe) and the majority of the cushion medium was removed by aspiration. Subsequently, the
sperm pellet was resuspended in BotuCrio (Criovital, Gründau, Germany) to a final concentration of
100 × 10 6 sperm /mL as determined with the NucleoCounter. The diluted semen was loaded into 0.5
mL straws, placed on racks and transported to the freezing chamber of an automated controlled rate
freezer (IceCube 14S, Neupurkersdorf, Austria). The semen was first cooled from 22°C to 5°C in 20
min and then frozen from 5 °C to -150 °C at 30 °C/min. At -150 °C the freezing racks were removed
from the freezing chamber and plunged into liquid nitrogen.
161

CHAPTER 4.2
Fig. 1. Schematic presentation of the processing of the ejaculates using the cushioned
centrifugation and the Androcoll-E selection, respectively
162

CHAPTER 4.2
For the SLC group, 15 mL of the diluted semen was gently layered on top of 15 mL Androcoll-
E-Large (patent applied for) in a 50 mL conical bottom centrifuge tube. Two tubes were prepared as
such and centrifuged (Heraeus Multifuge 3 L-R, Kendro Laboratory Products, Waltham, USA) for 20
min at 300 × g. After centrifugation, the supernatant was removed as gently as possible without
disturbing the sperm pellet and leaving as little colloid as possible. The sperm pellet was
resuspended in 5 mL of INRA96 and washed by centrifugation for 10 min at 500 × g. Following
washing, the sperm pellet was resuspended to a final sperm concentration of 100 × 10 6 sperm /mL,
in 1.5 mL of supernatant from the cushioned group (which was filtered through tandem 5 µm and
1.2 µm sterile nylon syringe filters to remove any residual spermatozoa (Rigby et al., 2001)) and
BotuCrio. Adding back the supernatant ensured that the seminal plasma content of cushioned and
SLC treated samples was comparable. The diluted semen was loaded into 0.5 mL straws and frozen
as described above.
Fig. 2. Picture of equine semen diluted in INRA96® upon Androcoll-E for single layer centrifugation
(a) before and (b) after centrifugation (300 × g for 20 min). The picture on the right clearly
shows the selected sperm pellet.
163

CHAPTER 4.2
164
4.2.3.3.Evaluation of sperm characteristics and staining procedures
Motility analysis
Objective motility evaluation was performed using CASA as described previously (Hoogewijs
et al., 2010) except a Tokai Hit thermoplate was used to evaluate the semen at 37°C rather than a
minitherm stage warmer. The parameters recorded by CASA available for statistical analysis were
total motility (TM) and PM. Ejaculates with a post-thaw PM of ≥30% were classified as freezable as
described by Loomis (2001) using the same strict CASA settings (Loomis and Graham, 2008).
Freezability was calculated as described above (Vidament et al., 1997). If a stallion produced 2 or 3
out of 3 ejaculates with a post-thaw PM of ≥30%, that stallion was classified as a good freezing
stallion. If ≤1 of the ejaculates were freezable, the stallion was classified as a poor freezer.
Morphology
The morphology of the sperm cells was examined on eosin-nigrosin stained smears which
were prepared as described by Barth and Oko (1989) using the ‘feathering’ technique (WHO, 2010).
At least 200 membrane intact (unstained) sperm cells were evaluated and recorded per slide using
×1000 magnification with oil immersion. The stained smears were identified with a number and
analyzed by one person, who was unaware of the identity of the samples. Individual morphological
abnormalities were noted according to their location. The proportion of sperm with normal
morphology (NM) and the proportion of sperm with abnormal head morphology (AH) were available
for statistical analysis.
Chromatin structure
The chromatin structure was evaluated using the sperm chromatin structure assay (SCSA).
This assay, developed by Evenson et al. (1980), assesses the susceptibility of sperm DNA to acid
induced denaturation by using the metachromatic dye acridine orange (AO). In this test the ratio of
denaturated, single stranded DNA (red fluorescence) to total DNA (stable, double stranded DNA
(green fluorescence) + single stranded DNA) is calculated and expressed as the DNA fragmentation
index (DFI, %). The procedure as well as the buffers and solutions used in the current study has been
described in detail earlier (Januskauskas et al., 2001; Januskauskas et al., 2003; Evenson and Jost,
2000). Briefly, straws containing the frozen semen were thawed in a water bath (37°C) for 30
seconds. Immediately post thawing, an aliquot of semen was diluted in TNE buffer to a final sperm
concentration of approximately 2 × 10 6 sperm/mL. An aliquot (100 µL) of the TNE diluted sperm was
mixed with 200 µL of acid-detergent solution. Exactly 30 seconds later, the sample was stained by

CHAPTER 4.2
adding 600 µL of AO staining solution. The stained samples were analyzed within 3-5 minutes of OA
staining using a FACStar Plus flow cytometer (Becton Dickinson, San José, CA, USA) with settings and
software as described by Morrell et al. (2008). The proportion DFI was available for statistical
analysis.
Membrane integrity
Membrane integrity was evaluated using a fluorescent SYBR14-Propidium Iodide (PI) staining
technique (Molecular Probes cat n°: L-7011, Leiden, The Netherlands) based on a previously
described method (Garner and Johnson, 1995). Briefly, after thawing in a 37°C water bath for 30s,
the straws were emptied and 25 µL of semen was mixed with 225 µL HEPES-TALP and 1.25 µL
SYBR14 (1:50 dilution) was added. After 5 min of incubation at 37 °C, 1.25 µL PI was added and
incubated for another 5 min at 37°C. Two hundred cells were evaluated per slide using a Leica DMR
fluorescence microscope and the proportion of membrane intact (MI) sperm cells was calculated
and used for statistical analysis.
Acrosomal status
The acrosomal status was determined using fluorescent Pisum Sativum Agglutinin (PSA)
staining conjugated with fluorescein isothiocyanate (FITC) (Sigma-Aldrich cat n°: L 0770, Bornem,
Belgium). The staining was performed in a similar way as described by Rathi et al. (2003). Briefly,
after thawing in a 37°C water bath for 30s, the straws were emptied and 400 µL of semen was
centrifuged for 10 min at 720 × g after which the pellet was resuspended with HEPES-TALP. The
semen was centrifuged again for 10 min at 720 × g, the supernatant was removed and the pellet was
fixed in 100 µL absolute ethyl alcohol (Vel cat n°: 1115, Haasrode, Belgium) and cooled for 30 min at
4 °C. A drop of 20 µL semen was smeared on a glass slide and 40 µL PSA-FITC [2 mg PSA-FITC diluted
in 2 mL phosphate buffered saline (PBS)] was added. The glass slide was kept at 4 °C for 15 min in
the dark, washed 10 times with aqua bidest and allowed to air-dry in the dark. Immediately after
drying, two hundred sperm cells were evaluated per slide. The acrosomal region of the acrosome
intact sperm cells was labeled heavily green, while the acrosome reacted (AR) sperm retained only
an equatorial band of staining with little or no labeling of the anterior head region. The percentage
of AR sperm was used for statistical analysis.
165

CHAPTER 4.2
166
4.2.3.4. Experimental design
Each of the 22 ejaculates was processed as described above. After re-dilution in BotuCrio,
the sperm yield expressed as % was calculated [(final volume of in BotuCrio extended semen ×
100×10 6 sperm/mL) divided by 3000×10 6 initial sperm per treatment multiplied by 100] and an
aliquot of semen from each treatment was analyzed with CASA and a morphology smear was
prepared prior to cryopreservation. Frozen semen was thawed and TM and PM were analyzed using
CASA, while DFI, MI, AR and morphology were determined as well.
4.2.3.5. Statistical analysis
sperm quality parameters
All data were percentages and were transformed by dividing them by 100 followed by
calculating the arcsine of the square root of that value, in order to obtain a normal distribution as
assessed by QQ-plots. The possible effect of “Treatment” (cushion vs. Androcoll-E) as the main
variable of interest, on the different parameters (TM, PM, NM, AH, DFI, MI, AR, and yield,
respectively) was studied. Therefore, multilevel models were fitted in MlwiN 2.02 (Centre for
Multilevel Modeling, Bristol, UK) with the eight different parameters as outcome variables and
“Treatment” as fixed effect and “Stallion” as random effect to adjust for clustering of ejaculates
within stallion. As well, “Freezability” (good vs. poor, see above) and the interaction term
“Treatment” × “Freezability” were included in the model. Non-significant terms (p>0.05) were
excluded from the full models.
accepted straws and number of AI doses
The association between “Treatment” and “Number of accepted straws” was studied using
Chi 2 on 2×2 tables for poor and good freezing stallions using Win Episcope 2.0 (CLIVE, Edinburgh, UK).
Breslow-Day test indicated interaction was present between “Freezability” and “Treatment”
allowing only for stratum-specific (poor vs. good freezing stallions) conclusions. Odds ratios with 95%
confidence interval were calculated to visualize the strength of the associations. As well, Chi² on 2×2
table was used to test whether “Number of doses” was related to “Treatment” for good and poor
freezing stallions. Significance was set at p

4.2.3. Results
4.2.3.1. Sperm quality parameters
CHAPTER 4.2
Four stallions were considered to have a good freezability whereas the other four stallions
were poor freezers. Good freezers had a significant higher pre-freeze TM and PM, and a higher post-
thaw TM and MI compared to poor freezers (Table 1). The other parameters did not differ
significantly between good and poor freezers.
100
80
60
40
20
0
A. Pre-freeze progressive motility (%) B. Yield (%)
poor freezing
stallions
Fig. 2. Pre-freeze progressive motility and yield (with SD-bars) of stallion sperm following cushioned
centrifugation or selection through Androcoll-E (good freezing vs. poor freezing vs. all
stallions) of the stallion’s ejaculates.
Analysis of the pre-freeze samples showed a significant improvement in sperm quality
parameters for Androcoll-E treated sperm, although this was borderline non-significant for TM
(Table 1). However, sperm yield was markedly lower compared to the cushioned group. Analysis of
the post thawed samples revealed an overall increase in sperm quality for the Androcoll-E treated
samples (Table 1).
Cushion Androcoll-E
good freezing
stallions
all stallions
120
100
80
60
40
20
0
poor freezing
stallions
Cushion Androcoll-E
good freezing
stallions
all stallions
167

Table 1. Average sperm quality parameters (raw data, ± SD) and related factors of 22 different split ejaculates from 8 stallions pre- and post freezing
following cushioned centrifugation vs. Androcoll-E selection prior to cryopreservation.
Treatment Freezability Treatment × Freezability
Cushion Androcoll-E p 1 p p
Pre-Freeze Total Motility (%) 87.4 ± 9.9 91.1 ± 7.8 0.055 < 0.05 NS 2
Progressive Motility (%) 52.8 ± 14.5 64.9 ± 10.2

CHAPTER 4.2
The aforementioned “Treatment” effect was comparable for poor and good freezers except
for pre-freeze PM and yield as visualized by the significant interaction terms between “Treatment”
and “Freezability” in Table 1.. For pre-freeze PM, poor-freezing stallions benefitted more from
Androcoll-E treatment than good-freezing stallions. The yield for poor-freezing stallions was more
reduced following Androcoll-E treatment in comparison to good freezing stallions (Fig. 2).
4.2.3.1. Accepted straws and number of AI doses
The number of freezable ejaculates, the number of accepted straws and the AI doses for
good and poor-freezing stallions are presented in Table 2. The effect of “Treatment” on number of
straws accepted (from ejaculates with post-thaw PM ≥30%) was different for good freezers vs. poor
freezers (p

Table 2. Descriptives of the different stallions (n=8) used in the study based on ejaculates (n=22) frozen following cushioned centrifugation or Androcoll-E
selection prior to cryopreservation.
Poor Freezer Good Freezers
Treatment Total
1 5 7 8 Subtotal 2 3 4 6 Subtotal
Number of freezable 1 Cushion 11/22 55% 1/3 0/3 0/3 0/1 1/10 3/3 2/3 3/3 2/3 10/12
ejaculates
Androcoll-E 17/22 77% 3/3 0/3 2/3 0/1 5/10 3/3 3/3 3/3 3/3 12/12
Number of accepted 2 Cushion 620 43 0 0 0 43 171 104 175 127 577
straws
Androcoll-E 519 70 0 59 0 129 89 111 106 84 390
Cushion 58 3 0 0 0 3 13 11 21 10 55
Number of AI doses
Androcoll-E 54 5 0 4 0 9 9 12 14 10 45
1
Ejaculates were classified as freezable if post-thaw progressive motility was ≥30%
2 Accepted straws were all the straws that originated from freezable ejaculates

CHAPTER 4.2
Sperm yields markedly differed between the techniques. This is not unexpected since both
techniques have different aims. Cushioned centrifugation allows the use of an increased g-force for a
prolonged time, as such maximizing sperm yields (Ecot et al., 2005; Knop et al., 2005). On the
contrary, the objective of sperm selection is to eliminate sperm with inferior structure and function,
based on differences in size, shape and density properties (Pertoft, 2000). The high yields reported
here using the cushioned technique are comparable with other findings in literature (Ecot et al., 2005;
Knop et al., 2005; Waite et al., 2008). Using the SLC procedure as described here, the average sperm
yields were slightly higher compared with previous findings (Morrell et al., 2009b). Sperm yield after
selection depends on the initial quality of the sperm sample. The intent is to select the sperm
population with a superior morphology and higher motility. The findings of the pre-freeze sperm
quality are comparable with those of previous reports (Morrell et al., 2009a, 2009b; Morrell et al.,
2010).
Cryopreserved semen, with a post-thaw PM of ≥30%, is considered good for use in a
commercial breeding program, while semen with a PM

CHAPTER 4.2
subpopulation of superior sperm is cryopreserved. These sperm are more likely to withstand the
cryopreservation protocol; additionally the harmful effect of seminal plasma is eliminated since
almost all seminal plasma is discarded during the selection procedure and the subsequent washing of
the pellet. It is likely that some poor freezing stallions might have experienced additional benefit if
heterologous seminal plasma from a good freezing stallion is used instead of the homologous
seminal plasma that was added back in the present study, this was however not the intention of the
present study. Nevertheless it is clear that Androcoll-E can be beneficial to successfully cryopreserve
stallion semen. Stallion selection however, plays a key role in the eventual outcome.
172
In conclusion, sperm selection using Androcoll-E prior to cryopreservation resulted in an
overall increased post-thaw sperm quality. Stallions that produce ejaculates with low post-thaw PM
might benefit from sperm selection prior to cryopreservation. As such only the best population of
sperm is cryopreserved, which could result in good frozen semen. Androcoll-E can be beneficial to
successfully cryopreserve stallion semen, but stallion freezability plays a role in the eventual
outcome. Additional experiments need to be performed to evaluate the in vivo fertility of frozen
thawed equine semen, selected with Androcoll-E prior to cryopreservation.

References
CHAPTER 4.2
Barth A.D., Oko R.J. 1989. Abnormal morphology of bovine spermatozoa. Ames, Iowa: Iowa State
University Press.
Ecot P., Decuadro-Hansen G., Delhomme G., Vidament M. 2005. Evaluation of a cushioned
centrifugation technique for processing equine semen for freezing. Animal Reproduction
Science 89:245-8.
Esteves S.C., Sharma R.K., Thomas Jr. A.J., Agarwal A. 2000 Improvement in motion characteristics
and acrosome status in cryopreserved human spermatozoa by swim-up processing before
freezing. Human Reproduction 15:2173-2179.
Evenson D, Jost L. 2000. Sperm chromatin structure assay is useful for fertility assessment. Methods
in Cell Science 22:169-189.
Evenson D.P., Darzynkiewicz Z., Melamed M.R. 1980. Relation of mammalian sperm chromatin
heterogeneity to fertility. Science 240:1131-1133.
Garner D.L., Johnson L.A. 1995. Viability assessment of mammalian sperm using SYBR-14 and
propidium iodide. Biology of Reproduction 53:276-84.
Hansen C., Vermeiden T., Vermeiden J.P.W., Simmet C., Day B.C., Feitsma H. 2006 Comparison of
FACSCount AF system, Improved Neubauer hemocytometer, Corning 254 photometer,
SpermVision, UltiMate and NucleoCounter SP-100 for determination of sperm concentration
of boar semen. Theriogenology 66:2188-2194.
Henkel R.R., Schill W.-B. 2003. Sperm preparation for ART. Reproductive Biology and Endocrinology
1:108-130.
Hoogewijs M., Rijsselaere T., De Vliegher S., Vanhaesebrouck E., De Schauwer C., Govaere J., Thys M.,
Hoflack G., Van Soom A., de Kruif A. 2010. Influence of different centrifugation protocols on
equine semen preservation. Theriogenology 74:118-126.
Januskauskas A., Johannisson A., Rodriguez-Martinez H. 2001. Assessment of sperm quality through
fluorometry and sperm chromatin structure assay in relation to field fertility of frozenthawed
semen from Swedish AI bulls. Theriogenology 55:947-961.
Januskauskas A., Johannisson A., Rodriguez-Martinez H. 2003. Subtle membrane changes in
cryopreserved bull semen in relation with sperm viability, chromatin structure and field
fertility. Theriogenology 60:743-758.
Klinc P., Kosec M., Majdic G. 2005. Freezability of equine semen after glass beads column separation.
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sperm recovery and sperm quality in stallions with good and poor semen freezability. Animal
Reproduction Science 89:294-7.
173

CHAPTER 4.2
Loomis P.R. 2001. The equine frozen semen industry. Animal Reproduction Science 68:191-200.
Loomis P.R., Graham J.K. 2008. Commercial semen freezing: individual male variation in cryosurvival
and the response of stallion sperm to customized freezing protocols. Animal Reproduction
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Morrell J.M., Johannisson A., Dalin A.-M., Hammar L., Sandebert T., Rodriguez-Martinez H. 2008.
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relationship to pregnancy rates. Acta Veterinaria Scandinavica doi: 10.1186/1751-0147-50-2.
Morrell J.M., Dalin A.-M., Rodriguez-Martinez H. 2009a. Comparison of density gradient and single
layer centrifugation of stallion spermatozoa: yield, motility and survival. Equine Veterinary
Journal 41:53-58.
Morrell J.M., Johannisson A., Dalin A.-M., Rodriguez-Martinez H. 2009b. Single-layer centrifugation
with Androcoll-E can be scaled up to allow large volumes of stallion ejaculate to be processed
easily. Theriogenology 72:879-884.
Morrell J.M., Johannisson A., Juntilla L., Rytty K., Bäckgren L., Dalin A.-M., Rodriguez-Martinez H.
2010. Stallion sperm viability, as measured by the Nucleocounter SP-100, is affected by
extender and enhanced by Single Layer Centrifugation. Veterinary Medicine International
doi:10.4061/2010/659862.
Pérez-Sánchez F., Cooper T.G., Yeung C.H., Nieschlag E. 1994. Improvement in quality of
cryopreserved human spermatozoa by swim-up before freezing. International Journal of
Andrology 17:115-120.
Pertoft H. 2000. Fractionation of cells and subcellular particles with Percoll. Journal of Biochemical
and Biophysical Methods 44:1-30.
Pickett B.W. 1993. Collection and evaluation of stallion semen for artificial insemination. In: Equine
Reproduction, McKinnon A.O., Voss J.L. (Ed.), Williams & Wilkins, pp.705-14.
Rathi R., Colenbrander B., Stout T.A., Bevers M.M., Gadella B.M. 2003. Progesterone induces
acrosome reaction in stallion spermatozoa via a protein tyrosine kinase dependent pathway.
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Rigby S.L., Brinsko S.P., Cochran M., Blancard T.L., Love C.C., Varner D.D. 2001. Advances in cooled
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of Urology 156:1008-1012.
Sieme H., Martinsson G., Rauterberg H., Walter K., Aurich C., Petzoldt R., Klug E. 2003. Application of
techniques for sperm selection in fresh and frozen-thawed stallion semen. Reproduction in
Domestic Animals 38:134-140.
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174

CHAPTER 4.2
Waite J.A., Love C.C., Brinsko S.P., Teague S.R., Salazar Jr. J.L., Mancill S.S., Varner D.D. 2008. Factors
impacting equine sperm recovery rate and quality following cushioned centrifugation.
Theriogenology 70:704-714.
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human semen. 5th ed. Geneva, Switzerland, WHO Press.
175

General discussion
CHAPTER 5
177

CHAPTER 5
This thesis originated from the practical challenges faced when optimizing cryopreservation
of equine semen at our clinic. Our original cryopreservation protocol was based on old-fashioned
handiwork using the same protocol for all stallions. Gradually, the cryopreservation protocol evolved
and reached new levels, until one realized what it actually is: “the art of freezing”. As with any form
of art, or science, the correct nomenclature plays a key role in discussing the topic, and a Babel-like
confusion of tongues was and is present amongst scientists dealing with animal semen quality and
cryopreservation. Unlike human andrology, anarchy rules in this veterinarian kingdom. Hence this
anarchy needs to be replaced by a code in which scientists have agreed upon : (1) criteria and
settings used for sperm analysis in order to gain consensus between different laboratories ; (2)
standards for sperm manipulation procedures, and (3) definitions for semen quality parameters in
different domestic species. In this thesis, we have taken the first two steps towards the generation of
such a code by describing how settings for sperm quality assessment and how manipulation of
semen can affect the eventual outcome of a sperm quality assessment in the horse. I hope that more
studies of this kind, in the horse but also in other species, can lead to the generation of a code or a
manual describing guidelines and strict criteria for sperm quality assessment in domestic animals, as
it has already been done for human in the WHO Manual. In the next paragraphs, the importance of
using the right nomenclature, applying standardized equipment and manipulating semen using strict
criteria, is discussed.
179

5.1
Semen (motility) Analysis
180
Spermatozoal motility is likely to remain one of the hallmarks of semen evaluation (Varner,
2008). Still, the wide variety in settings and the use of many different chambers described in
literature clearly indicates that objective motility analysis does not equal standardized analysis.
5.1.1. Technical settings in CASA systems
The results from this thesis clearly state the influence of different motility settings when
using computer assisted sperm analysis (CASA) for the analysis of equine semen motility (Chapter
3.1). Although this influence had not been described previously, these findings are not surprising and
can be easily explained. Analysis by means of CASA is based on the relative position changes of the
head of a sperm cell as illustrated in Fig.1. As such, different trajectory parameters are calculated,
which are used to determine a sperm cell as being motile, progressive motile or static. It is logical
that changing the cut-off values (low and medium VAP cut-off and STR) will result in changes of the
outcomes. After all, a sperm cell with a VAP of 30µm/s and a STR of 50% is progressive motile
according to the settings of Varner’s lab (Waite et al., 2008), whereas according to the settings of
Loomis and Graham (2008) this same sperm cell would be assigned as motile. For analysis of equine
semen, more than ten different motility settings have been reported (reviewed in the general
introduction). For other species the variety in technical settings is as wide. The importance of
agreement of settings will become more pressing when global guidelines on AI doses would be
established.
Additionally, it is not completely clear how CASA systems from different manufacturers calculate
motility, progressive motility and subdivide sperm populations into rapid, medium and slow (WHO,
2010). Two different CASA systems (Ceros Hamilton-Thorn and ISAS Proiser) reported slightly
different results, although analyses of the same samples agreed well when using the same settings
and counting chamber (Hoogewijs, unpublished data). It should not be difficult to persuade the
industry to imply the same mathematical calculations in their device, once the andrologists have
come to an agreement. After all, non-conformity could result in non-approval of the devices from
that company.

CHAPTER 5.1
Fig. 1. Schematic presentation of the trajectory of a sperm cell as recorded by a computer assisted
sperm analysis system, the green line represents the curved line velocity (VCL), the red line is
the average pathway velocity (VAP and the blue line is the straight line velocity (VSL).
Straightness (STR) and linearity (LIN) can be calculated by the presented formula.
The uniformity in settings used is mere a matter of agreement, and once the debate has
resulted into a consensus, standardized procedures can be performed worldwide. In the previous
edition of the WHO manual (WHO, 1999), defined grading categories were made based on velocities
and trajectories to classify human sperm into one of four categories (Table 1). In order to be
considered rapid progressive motile, a sperm cell’s velocity should be ≥25 µm/s. Whereas if the
velocity was

CHAPTER 5.1
Table 1. Criteria for grading of spermatozoa into motility categories based on the 4 th WHO manual
(WHO, 1999)
Grading Category Velocity requirements
a rapid progressive motile ≥25 µm/s
b slow progressive motile < 25 µm/s and ≥5 µm/s
c non-progressive motile

CHAPTER 5.1
higher than the current costs for any laboratory, general acceptance of a more expensive chamber is
not likely to be guaranteed. Furthermore, it is important to realize that not every andrology lab is
using a CASA system, and to maximize agreement between subjective and CASA motility analysis , it
might be advisable to use the same chamber, i.e. the WHO prepared slide (namely: an exact droplet
of 10 µL of semen on a microscopical slide, covered with a 22 mm by 22 mm cover glass).
Fig. 2. (A) Image of a 20 µm deep disposable Leja® chamber as used for motility analysis using CASA;
the red circles indicate the loading area in which the sample placed so it can enter the
chamber by capillary forces, and (B) schematic drawing of the same Leja® chamber, the black
arrow indicates the flow of the sample taking the spermatozoa under the cover glass (CG)
and the red arrow indicates the possible cause of damage to a spermatozoa when it is pulled
against the CG.
The different motility outcomes yielded between chamber types can be explained by the
differing filling technique and chamber depth, and the interaction with the walls limiting the
chamber.
183

CHAPTER 5.1
184
The filling technique of a chamber is the first factor influencing sperm motility . A sperm
sample analyzed using the same chamber, resulted in differing motility outcomes when the chamber
was loaded with capillarity rather than drop filled with a cover slide (Chapter 3.2). This might be due
to forces acting on the sperm during loading which can either have a direct damaging effect on the
spermatozoa. It is however more likely that the tail of spermatozoa get damaged during the flow of
the suspension when touching the fixed cover glass as they are forced under the cover by the flow, as
illustrated in Fig. 2., Spermatozoa that are damaged in this way might exhibit reduced motility and
might be prone to accelerated cell death. A possible way to verify this theory is loading the chamber
with a sample of Sybr-14/PI stained sperm, and analyzing the proportion of membrane intact and
membrane damaged spermatozoa immediately after loading with a follow-up over prolonged time. It
is hypothesized that spermatozoa that get damaged during loading, will take up PI faster.
Depth of the preparation is another important factor influencing sperm motility. This depth
should allow good visualization, preventing that spermatozoa can move in and out of the focus plane
of the microscope. However, if the depth is too narrow it might interfere with the three dimensional
movement of the sperm. Very shallow chambers might prohibit free rotation of the sperm head and
as such reduce their motility. In slightly deeper chambers where free rotation is possible, the motility
might be artefactually enhanced because the spermatozoa are forced to move in only two directions
instead of displaying normal movement in three dimensions. This might result in higher (progressive)
motility, as observed when using the 10 µm Makler chamber. Not only is the percentage PM higher,
the velocity parameters (VAP, VCL and VSL) are also higher in the Makler chamber. Therefore, the
use of this chamber might be contra-indicated. On the other hand, WHO prepared slides are not as
user-friendly when compared to the Makler chamber which is more easily prepared without air
bubbles in the sample. A valid alternative might be the use of a modified version of the Makler
chamber that will be available shortly. This chamber, the SpermTrack-20® (Proiser, Valencia, Spain),
has a loading area that is comparable with the classic Makler but the depth of the new chamber is 20
µm rather than 10 µm. This chamber needs to be evaluated before it can be promoted as chamber of
reference, but based on our findings in Chapter 3.2, this chamber might provide the ease of use of
the Makler chamber without affecting the motility pattern. In the meanwhile however, the WHO
prepared slide might be the best solution for motility analysis in combination with CASA.
Finally, the sperm cells’ motility patterns might be influenced by diverse physical forces
acting on (moving) spermatozoa inside a chamber due to the differences in the “walls limiting the
chamber”. Sperm kinematics differ significantly for different chambers with the same depth
(Spiropoulos, 2001; Hoogewijs, unpublished data). The flagellar movement has been studied in detail
and mathematical models for flagellar beating in fluids have been developed (Brokaw, 2002; Dillon et

CHAPTER 5.1
al., 2007) showing a clear three dimensional movement of the flagella. The exact forces acting on
moving spermatozoa inside different types of chambers have, so far, not been studied. However, the
differences in motility outcomes between chambers of comparable depths indicate that spermatozoa
are influenced to a large extent. If a diluter with small particles is used for motility analysis, the
movement of (the tail of) the spermatozoa makes these particles move over a wider surface through
initiation of waves, rather than through direct contact with the tail only. These waves might interfere
more with motility in disposable chambers with fixed walls when compared to the menisci that
create the border in disposable chambers or the WHO slide. This phenomenon can be easily
visualized when looking at speedboats on rivers with natural banks rather than canals with concrete
banks. The latter will result in more reflection of the waves, as such disturbing the water surface to a
bigger extent when compared to smooth natural banks (Fig. 3). It is hypothesized the same principle
occurs in disposable chambers with fixed wall (total reflection as in Fig. 3A), and as such interfere
with motility, whereas in chambers with a loose cover slip the reflection might be minimized by the
menisci creating the border (as in Fig. 3B). In this aspect, disposable chambers might cause different
forces in the fluid filled chamber in comparison to the reusable chambers or the WHO prepared slide,
in which the fluid menisci bordering the surface might partially absorb these “waves”.
A.
B.
Fig. 3. Schematic presentation of a virtual speed boat (proxy for sperm cell) and the waves as they
are (A) completely reflected by concrete borders, and (B) only partially reflected by the
natural borders of a river absorbing a big part of the waves before reflection.
185

CHAPTER 5.1
5.1.3. SQA-Ve in standardized semen analysis
186
The sperm quality analyzer (SQA) is not susceptible to similar operational variations as CASA
since the device uses integrated algorithms that cannot be changed by the users, and only allow
insertion of the tailor-made capillary. As such, the above mentioned issues of settings and counting
chambers will not manipulate results, reducing potential sources of bias (Hoflack et al., 2005).
Despite these advantages the present versions of the SQA-Ve should not be used for
analyzing equine semen samples. So far, as shown in Chapter 3.3 and 3.4, the device is not precise
enough and it lacks accuracy to report on equine semen quality. The improvements in the agreement
of the SQA-Ve version 1.00.61 with CASA, as evidenced in Chapter 3.4, indicates that with further
optimization of the algorithms the SQA might eventually be able to univocally report on equine
semen quality.
5.1.4. Standardized semen analysis in relation to fertility
So far, sperm motility only correlates poorly with stallion fertility (Jasko et al., 1992; Kuisma
et al., 2006). Of course, a sperm cell needs more traits to fertilize, besides being motile. A
spermatozoon should also be capable of initiating capacitation and the acrosome reaction, and
should have intact DNA in order to be fertile. Mare factors interfere equally with early conception,
and affect fertility chances as well. The introduction of actual standards in semen analysis might
enable scientists all over the world to assess semen in the same way. As such more and more
uniform data will come available which might lead to additional value of in vitro parameters in
predicting in vivo fertility.
Based on in vivo fertility data of the previous breeding season, a breeding stallion can be
classified as being highly fertile, fertile or sub-fertile. For each of these categories, a different artificial
insemination (AI) dose of progressive motile sperm might be required to maintain at least a
comparable fertility level. Following every season, the fertility status should be recalculated and used
during the following breeding season. The AI dose for stallions of unknown fertility (such as young,
novice stallions) can be arbitrary set at the same dose as for fertile stallions. As soon as actual fertility
data are available, true fertility should be calculated.
Much can be debated about the fertility status of a stallion, but at the end fertility is
determined by the interaction of stallion, mare and management factors (Fig. 4). Influence of stallion
and mare on fertility is evident, but “management” factors such as cycle control, timing of AI,

CHAPTER 5.1
treatment strategy for endometritis, semen handling and so on will also interfere with the final
fertility. In this thesis, the focus was partially on the quality of the semen and how it can be assessed
(stallion factor influenced by management), and partially on preparation of AI doses (management
factor influenced by the stallion and his initial semen quality).
Fig. 4. Schematic presentation of the different factors affecting equine fertility. The proportions of
the different factors are arbitrary. However, note how semen quality (in green as determined
by the stallion) is influenced by management when performing the analysis (pink border).
Vice versa, semen handing to prepare the AI doses is influenced by the initial sperm quality
(smaller volumes of highly concentrated semen are easier to process).
The influence of “management” on determination of the sperm quality should be minimal
and standardized, facilitating comparison. Correct analysis of the semen will also point out the
weaknesses in the semen preparation more rapidly, which will result in increased sperm
management. This will be discussed in the next section, where we evaluated the effect of
centrifugation on semen quality in horses.
Mare
Management
Semen handling
Semen quality
Stallion
187

Centrifugation technique
188
5.2
Centrifugation is one of the common procedures when processing equine semen. Currently,
centrifugation is mandatory when subjecting an equine ejaculate to cryopreservation. Alternative
approaches such as fractionated collection are not as successful (Sieme et al., 2004) or are still in its
infancy (sperm filters – Alvarenga et al., 2010).
The influence of centrifugation on spermatozoa is not completely understood, or at least the
principle(s) by which spermatozoa are influenced due to centrifugation remain(s) unclear. A first
theory is based on alterations in the shape of cells submitted to centrifugation. Using an atomic force
microscope, fitted in a large centrifuge, Van Loon et al. (2009) were able to visualize mouse
osteoblasts during centrifugation. The osteoblasts were markedly reduced in height during
centrifugation at only 3 × g. Although the structure of a spermatozoon is completely different from
an osteoblast, especially because of the dramatic reduction in the cytoplasm of a mature sperm cell,
it is likely that centrifugation at much higher speeds also affect the structure of a sperm cell. Further
refinement of this technique by Van Loon et al. (2009) and research on different cell types is
necessary in order to accurately describe what happens with spermatozoa during centrifugation.
Another explanation could act at the level of the sperm membrane. Like any plasma
membrane, a sperm membrane consists of a liquid phospholipid bilayer (Amann and Pickett, 1987).
Different domains of the plasma membrane all contain different concentrations and distributions of
intramembranous particles, with different, specific functions in the process of fertilization (Flesch
and Gadella, 2010). These domains undergo redistribution following capacitation (Gadella et al.,
1994). Perhaps centrifugation acts on this liquid lipid layer and disturbs as such its function by
rearranging the distribution of the intramembranous particles. Excessive centrifugation forces induce
even more damage, and provoke a compact sperm pellet that is difficult to resuspend (Webb and
Dean, 2009).
In order to identify the effects of centrifugation force on sperm damage, we investigated the
impact of increasing forces (centrifugation protocols from 600 × g to 2400 × g) on stallion semen for
a reduced period of time (5 min) (Chapter 4.1). Additionally, we investigated sperm loss when
aspirating 90% of the supernatant following centrifugation using the different forces. Not all the
spermatozoa were pelleted at the bottom of the tube, but a part remained in suspension in the

CHAPTER 5.2
supernatant allowing sperm yield to be calculated. This technique of centrifugation only results in
separation of the spermatozoa from the seminal plasma. Alternatively, the forces originating during
centrifugation can also be used to select the superior sperm fraction from a sample when low g-
forces are combined with a colloid (Chapter 4.2).
5.2.1. Centrifugation to concentrate equine semen samples
Much uncertainty exists on how spermatozoa react on a cellular level to centrifugation. The
influence of centrifugation on sperm yields and semen characteristics is controversial as well.
However, much of the variation in study results can be explained by differences in experimental
design. Extender type (Cochran et al., 1984), volume per centrifugation tube (Cochran et al. 1984,
Webb et al., 2009), dimensions of the centrifugation tube (Webb and Dean, 2009), and of course
centrifugation time and force (Cochran et al., 1984; Weiss et al 2004; Len et al. 2008, 2010; Webb et
al., 2009; Hoogewijs et al., 2010) are all factors influencing yield and quality of equine semen after
centrifugation.
Our findings (Chapter 4.1) on sperm yields were consistent with the general trends in
literature. Using the standard protocol (600 × g for 10 min) 78% of the initial number of spermatozoa
were maintained. Sperm quality was not influenced largely. Immediately following centrifugation
motility and membrane integrity were only slightly reduced compared with non-centrifuged samples.
The use of higher forces for a shorter period of time reduced sperm losses without affecting the in
vitro sperm quality following cooled storage or cryopreservation.
When looking at the variations in the above mentioned studies on sperm losses, it is obvious
there might be a stallion effect as well. Some stallions have semen that sediments easier during
centrifugation, and some have spermatozoa that are more prone to damage caused by centrifugation
compared to other stallions (personal observations). So individual centrifugation protocols might be
necessary for some stallions, and if corrections for sperm loss following centrifugation are made, it is
important to re-assess the actual sperm number following centrifugation and not to use a fixed
correction factor since sperm loss is influenced by so many parameters.
189

CHAPTER 5.2
A.
Fig. 5. Different cushioned centrifugation techniques: (A) conventional cushioned centrifugation of
semen extended in an opaque extender (INRA96®) using a conical 50 mL centrifugation tube
with 3.5 mL of clear MAXIFREEZE® directly underneath the sperm pellet (green arrow), and (B)
Glass nipple-bottom tube following centrifugation of semen extended in an optically clear
extender, with 30µL of clear cushion directly underneath the sperm pellet (red arrow) (Waite
et al., 2008).
190
B.
Some of the downsides of centrifugation can be circumvented by the use of a cushion. This
technique, in which a protective fluid is placed at the bottom of the centrifugation tube underneath
the extended semen, was described over 25 years ago (Cochran et al., 1984), but has been refined
more recently (Ecot et al., 2005; Knop et al., 2005; Waite et al., 2008). The cushion fluid evolved from
high concentrated glucose solutions (Cochran et al., 1984), over egg yolk extenders containing
glycerol (Amann and Pickett, 1987) to iodixanol solutions (Revell et al., 1997). Various amounts of
these cushion solutions are applied, but with the introduction of commercially available cushions,
reports mention the use of 3.5 (Fig. 5A) up to 5mL of cushion fluid (Ecot et al., 2005; Knop et al., 2005;
respectively). The use of these cushions allows for longer centrifugation times and higher g-forces (20
min, 1000 × g). These so-called cushions are fluids with a higher density than the sperm cells so that
they are not compacted against the wall of the centrifuge tube (as with normal centrifugation, Fig.
6A); instead they are layered on the cushion. Since fluids are nor easily compressed, the high forces
(from the high speed centrifugation) will force the spermatozoa into the top layer of the cushion, as

CHAPTER 5.2
such keeping the pellet soft (Fig. 6B). Using the cushioned technique, higher sperm yields are
obtained without impairing sperm quality, which results in a 30% increase of produced semen doses
per ejaculate (Delhomme et al., 2004). The major downside of the cushioned centrifugation is the
necessity to remove the majority of cushion fluid at the bottom, underneath the sperm pellet
following aspiration of the supernatant. The cushion is aspirated with a syringe by placing a blunt
tipped spinal needle gently through the pellet to the bottom of the tube, as indicated in Fig. 1 from
Chapter 4.2. This additional step requires some technical skills, and might explain the lack of
popularity of cushioned centrifugation in practice. This downside was remedied by Waite et al. (2008)
who adapted the protocol and used only 30 µL of cushion fluid (Fig. 5b). However, this technique
requires special designed glass nipple-bottom centrifugation tubes with matching adapters.
Unfortunately, the glass tubes are rather fragile and do not withstand high centrifugation forces,
implying the use of severely reduced centrifugation forces (400 × g), which inevitably results in minor
reductions in sperm yield. Whether the modified cushioned centrifugation will result in increased use
in practice remains questionable. The requirement of the custom made, fragile glass nipple tubes
might prove to be a larger obstacle compared to cushion aspiration when using the regular approach.
A.
Fig. 6. Schematic representation of (A) classical centrifugation where the rather compacted sperm
pellet is located at the tip of the conical centrifuge tube, and (B) cushioned centrifugation
where the spermatozoa are located on top of and in the upper layer of the cushion, which
results in less compaction of the sperm pellet.
B.
191

CHAPTER 5.2
5.2.2. Selection of spermatozoa
192
The possibility to perform sperm selection using a single layer centrifugation (Morrell et al.,
2008) and the adaptation to scale up the volume per centrifugation tube (Morrell et al., 2009)
extends its practical use in equine andrology. This technique was used to select a superior sperm
population prior to cryopreservation and obtained post-thaw samples with increased overall sperm
quality (Chapter 4.2). The sole intention of the cryopreservation protocol we used was to analyze the
effect of cryopreservation of a superior sperm population. It is not surprising to find an overall
increased sperm quality after cryopreservation. Dead, damaged, or spermatozoa with a dysfunctional
motility are very unlikely to survive the freeze-thaw cycle, or to have a sufficient post-thaw quality.
Until recently, it was accepted that reactive oxygen species (ROS) in (human) semen were
almost exclusively produced by leucocytes. This is true for fertile men, although in oligozoospermic
patients the spermatozoa themselves were identified as a second major source of ROS (Aitken et al.,
1992). Equine semen is normally devoid of leucocytes in contrast to human semen, but equine
spermatozoa are equally capable of generating ROS, since damaged spermatozoa or morphological
abnormal spermatozoa generated significantly greater amounts of ROS than live, morphologically
normal spermatozoa (Ball et al., 2001). The presence of ROS impairs in vitro motility of equine
spermatozoa (Baumber et al., 2002) and promotes DNA fragmentation of equine spermatozoa
( Baumber et al., 2003).
We hypothesize that the increased post-thaw sperm quality we have noticed following pre-
freeze SLC selection could perhaps also be partially attributed to reduced levels of ROS. The use of
Androcoll-E might remedy these ROS related problems by working on two levels. First, due to the
selection procedure, the few leucocytes that are present are being held back from the sperm pellet.
Second, fewer abnormal spermatozoa (dead spermatozoa or with excess residual cytoplasm) are
present in the sperm pellet. As such the ROS content in the selected subpopulation of spermatozoa
should be lower than in the “full” sperm pellet, reducing the ROS related sperm damage.
The effect of sperm selection prior to cryopreservation could even be further increased if
stallion-adapted cryopreservation protocols would be used, fitting specific needs . Additionally,
sperm selection using Androcoll-E and subsequent washing of the sperm pellet removes all seminal
plasma. In our study seminal plasma from the same stallion and the same ejaculate was added back
to rule out any possible effect of presence or absence of seminal plasma on the post-thaw results.
Seminal plasma from stallions with poor freezing sperm was shown to play an important role in the
poor freezability (Aurich et al., 1996). The use of seminal plasma of stallions with good freezability
improved the post-thaw quality of stallions with poor freezability (Aurich et al., 1996). The

CHAPTER 5.2
components of the seminal plasma that are associated with fertility are not yet completely
categorized. Jobim et al. (2005) described the absence of protein 19 and higher levels of protein 17 in
the seminal plasma of low fertile stallions. The involvement of other seminal plasma components on
fertility requires further research.
The use of heterologous seminal plasma implies some risks. First of all the seminal plasma
should be guaranteed free of spermatozoa, to avoid faulty paternity. A combination of high speed
centrifugation (1000 × g for 10 min) of raw semen followed by passage of the supernatant through
tandem 5 µm and 1.2 µm nylon syringe filters should be a safe procedure to avoid spermatozoa
contamination (Rigby et al., 2001). Equally important are the risks of disease transmission through
the seminal plasma of the donor. This donor should at least have the same sanitary status as the
stallion whose spermatozoa should be frozen, and even so legislative limitations might occur.
A modified protocol in which the best diluter for a given poor freezing stallion is used in
combination with seminal plasma from a good freezing stallion might further increase post-thaw
semen quality, and enable us to freeze semen from stallions previously deemed unfreezable. Further
determination of the different components of the seminal plasma and the identification of their
function might enable researchers to isolate those proteins which positively influence
cryopreservation. These proteins could then be added to extenders to form an optimized defined
cryopreservation diluter.
Applying sperm selection prior to cryopreservation offers great advantages compared to
post-thaw selection of semen, as is frequently done in combination with assisted reproductive
technologies such as in vitro fertilization or intracytoplasmic sperm injection. First of all, the ROS are
removed prior to freezing, reducing the exposure of spermatozoa to the toxic influence. Besides that,
application of sperm selection techniques after thawing requires some level of experience and the
presence of laboratory facilities in order to process the semen, which factors are very often not
present in practice. Selection prior to cryopreservation bypasses these problem since it delivers
ready-to-use frozen semen.
193

5.3
Concluding remarks
forward:
194
Based on the results from this thesis and literature, the following conclusions can be put
♦ When performing computer assisted sperm analysis, the motility settings will influence the
motility outcomes to a great extent.
♦ The type of counting chamber used in combination with computer assisted sperm analysis has
an important effect on the concentration and motility results. Capillarity used for filling a
chamber restricts sperm motility severely.
♦ The present version of the SQA-Ve is not accurate nor precise enough to univocally report on
equine semen quality.
♦ Centrifugation protocols have a major influence on sperm yield. High centrifugation forces (up
to 2400 × g) for a short time (5 min) will substantially reduce sperm losses without apparent
effect on in vitro sperm characteristics.
♦ Sperm selection using Androcoll-E in a single layer centrifugation technique prior to
cryopreservation, results in an overall increased sperm quality. This technique is promising for
clinical use because it enables cryopreservation of a subgroup of stallions previously deemed
as unfreezable.

Final reflections
5.4
Actual changes in sperm quality can easily be missed or misinterpreted with the current
conceptions in animal andrology because of the lack of uniformity. Andrologists might have reached
the point where the disorderly analyses are left behind and a consensus is put forward to achieve
uniformity.
Which setting of all the reported ones should be agreed on for global use is of minor
importance compared to the actual implementation of the setting. In order to be able to distinguish
between stallions with variable semen quality, it might be advisable not to use the extremes. The
same principle should apply when deciding the counting chamber of preference. In our opinion,
capillary filled chambers should be avoided as well as chambers influencing motility. With the current
knowledge, the WHO prepared slide is in the lead to become the international chamber of reference.
An international group of animal andrologists should come forward and crack the nut of
standardization, allowing laboratories throughout the world to analyze and report on semen quality
in a (more) uniform way. This could be combined with a classification of stallions into one of three
fertility classes (high fertile, normal fertile or sub-fertile) based on their proven in vivo fertility. The
optimal sperm dose for each class should be determined, in order to serve as minimal requirements
for AI doses. Stallions of unknown fertility are classified as fertile, and fertility status should be
reassessed based on actual fertility of the preceding breeding season.
Subfertile stallions could be uniformly categorized and perhaps used for breeding in a more
controlled manner, where appropriate processing (sperm selection) might play a key role in obtaining
better pregnancy rates.
195

CHAPTER 5
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199

SUMMARY
Horse breeding has changed dramatically over the last century. Equine reproduction is no
longer dominated by natural breeding. All kinds of assisted reproductive techniques are now being
used for procreation of horses. To date, the most important of these techniques is artificial
insemination (AI), used for both cooled and frozen-thawed semen. Unlike human andrology, where
strict guidelines are followed concerning the handling, preparation and examination of an ejaculate,
veterinary andrology is characterized by a general lack of uniformity. This represents an important
problem for veterinarians involved in animal breeding, since every semen collection should be
followed by a standardized analysis of the quality of the ejaculate prior to the processing and
preparation of AI doses, to ensure the quality of the final AI dose.
So far, one of the most important in vitro sperm quality parameters remains sperm motility.
The subjective analysis of sperm motility highly depends on the operators’ experience and is very
often subject for discussion. Objective sperm motility analysis using computer assisted sperm
analysis (CASA) is well known and appreciated for its precision. As such, results of CASA motility
analyses should be comparable between laboratories, but since all laboratories perform CASA
analysis in a different way the opposite is true. Therefore, the first aim of this thesis was to
characterize and analyze the effect of technical motility settings and utensils on the outcome of CASA,
since no uniformity is present.
Additionally, when processing equine semen for either cooled storage or cryopreservation,
centrifugation is a frequently used technique in order to increase the relative sperm concentration
and to remove seminal plasma. Centrifugation, however, acts clearly as a double edged sword for the
two intended goals, namely to produce high yields of sperm combined with limited side effects for
sperm quality. Alternatively, centrifugation can also be used in combination with colloids to increase
the quality of a sperm sample by removing inferior sperm cells from the original aliquot. This superior
subsample might withstand cryopreservation in a superior way.
The general aim of this thesis was to identify how CASA results are influenced by settings and
utensils (Chapter 3.1 and 3.2) and by trying out a new device supposedly capable of analyzing equine
semen accurately and precise (Chapter 3.3 and 3.4). Additionally, the influence of different
centrifugation protocols on sperm yield and quality was analyzed (Chapter 4.1). A new selection
201

SUMMARY
technique that enables processing of larger semen volumes was tested as well when applied prior to
cryopreservation (Chapter 4.2).
202
Sperm motility is one of the key features of semen analysis. Nowadays, motility analysis is
frequently performed using CASA. The repeatability of the analysis of frozen thawed equine semen
using such a system is high. However, the settings used to analyze a semen sample influence the
results to a large extent (Chapter 3.1). The use of different CASA settings yields significantly different
motility outcomes. Depending on the settings used, total and progressive motility may differ to a
large extent although results correlate well. Therefore, when reporting the motility of a sperm
sample it is important to state the settings that were used. Preferably, CASA users throughout the
world should use the same settings.
Another frequently encountered problem during CASA analysis are the discrepancies that are
present amongst different laboratories concerning the type of counting chamber used. Therefore,
the effect of chamber type on concentration and motility of equine semen assessed with CASA was
studied (Chapter 3.2). Motility analysis using CASA is always done in association with a counting
chamber which provides a “monolayer” of sperm cells, so sperm recognition is facilitated. A wide
variety of chambers is available, varying in depth and in loading technique. The most frequently used
chambers are a disposable 20 µm deep chamber and a 10 µm reusable Makler chamber. In our study,
we compared frequently used disposable Leja® chambers of different depths with disposable and
reusable ISAS chambers, a Makler chamber, and a WHO prepared motility slide. Concentration as
well as motility parameters were significantly influenced by the chamber type that was used. The
correlation of sperm concentration as assessed with the different chambers and the NucleoCounter®
(which was used as gold standard) was low for all chambers, except for the 12 µm deep Leja®
chamber. This emphasizes the shortcomings of CASA systems when determining sperm
concentration. Motility parameters were equally influenced by the chamber type. Not only did the
use of different chambers result in important difference in motility outcomes, motility was
influenced as well when using the same chamber loaded with a different technique. In comparison
with the WHO prepared motility slide, i.e. a 10 µl drop of semen covered with a 22 mm by 22 mm
cover glass, loading a chamber by means of capillary forces will reduce sperm motility.
The sperm quality analyzer (SQA) is an alternative device to objectively measure the motility
of a sperm sample. Until recently, only human versions of the SQA were available. Analysis of motility
by SQA is based on disturbance of a light beam due to movement of the spermatozoa which will
scatter the light in different directions. These fluctuations are translated into a numerical motility
value. Since human spermatozoa, and those from different animal species, have differently sized

SUMMARY
sperm heads, the introduction of species-specific devices sounds logical. In a first experiment
(Chapter 3.3) the SQA-Ve software version 1.00.43 was analyzed for its repeatability and agreement
with light microscopy when analyzing raw and diluted equine semen. Analyses with SQA-Ve were
highly repeatable and agreed well with haemocytometer findings for concentration. However, the
analysis of motility parameters and of the percentage of morphologically normal spermatozoa were
poorly repeatable and did not agree with light microscopical evaluation. Light microscopy and CASA
showed a good agreement and CASA analyses were once more highly repeatable. However, CASA
was not able to analyze raw semen samples since concentration was out of range. Based on these
findings, we concluded that the tested version (1.00.43) of the SQA-Ve is insufficiently accurate and
precise to analyze raw nor extended equine semen.
In a next study (Chapter 3.4), two different software versions of the SQA-Ve were tested for
analyzing frozen-thawed equine semen. The first tested version (1.00.43) showed a poor
repeatability and a poor agreement with CASA. A newer software version (1.00.61) with improved
algorithms was capable of analyzing the total motility in a more acceptable way. However, further
improvements are mandatory before this device could serve as a diagnostic tool in equine
spermatology. An additional downside at the current configuration of the SQA-Ve is the inability to
report on motility if the samples total motility is equal to or below 30%. Under these circumstances,
no actual motility information is provided.
In literature, the reports on sperm losses associated with centrifugation are contradictory
and may vary from less than 2% till up to 25% using the same protocol. A standard centrifugation
protocol (600 × g for 10 min) was compared to four protocols with increasing g-force for a decreasing
period of time (Chapter 4.1). Sperm losses differed tremendously (22% for the standard regime), but
losses could be reduced by increasing g-force and reducing the time without affecting the apparent
in vitro sperm quality. Preservation of semen was not affected following high speed centrifugation
protocols, nor for cooled stored samples, neither for cryopreserved semen.
In human reproductive medicine, several selection techniques are used to isolate a superior
sperm population from the initial sample. These techniques only allow for processing of small volume
of semen, rendering them rather useless for processing the larger stallion ejaculate. However,
recently a new technique was developed which enabled us to increase the volume that can be
processed. Single layer centrifugation through a colloid (Androcoll-E) can be used to process up to 15
mL of diluted semen per tube. This procedure does not only increase the sperm sample immediately
following centrifugation, it also increases the post-thaw sperm quality from semen processed prior to
cryopreservation (Chapter 4.2). This technique can be very useful when trying to cryopreserve semen
203

SUMMARY
from stallions previously classified as unfreezable. Androcoll-E can be used with good results to select
a superior sperm population prior to cryopreservation, in order to produce good quality frozen
thawed semen.
204
Based on the results presented in this thesis and reported data in literature, the following
conclusions can be put forward:
♦ Motility settings, necessary when performing computer assisted sperm analysis, influence the
motility outcomes to a great extent.
♦ The type of counting chamber used in combination with computer assisted sperm analysis has
an important effect on the concentration and motility results. Capillarity used for filling a
chamber restricts sperm motility severely.
♦ The present versions of the SQA-Ve are inefficient to univocally report on the quality of equine
semen. The device has a poor repeatability and very poor agreement with gold standard
methods to analyze sperm motility.
♦ Centrifugation protocol has a major influence on sperm yield. High centrifugation forces (up to
2400 × g) for a short time (5 min) can substantially reduce sperm losses without apparent
effect on in vitro sperm characteristics.
♦ Sperm selection using Androcoll-E in a single layer centrifugation technique prior to
cryopreservation, results in an overall increased sperm quality. This technique is promising for
clinical use because it enables cryopreservation of a subgroup of stallions previously deemed
as unfreezable.

SAMENVATTING
De voorbije decennia heeft er zich binnen de paardenfokkerij een ware evolutie voltrokken.
Diverse geassisteerde reproductie technieken hebben hun intrede gedaan waardoor de voortplanting
bij het paard niet langer gedomineerd wordt door natuurlijke dekking. Zo wordt tegenwoordig veel
gebruik gemaakt van kunstmatige inseminatie (KI) met gekoeld of met diepvries sperma.
In tegenstelling tot de humane geneeskunde waar strikte richtlijnen bestaan voor de analyse
en het verwerken van sperma, wordt de veterinaire andrologie gekenmerkt door een gebrek aan
eenduidige richtlijnen. Normaliter zou na elke sperma afname de kwaliteit ervan moeten
gecontroleerd worden, waarna het ejaculaat verwerkt wordt en verschillende KI dosissen bereid
worden. Dit is echter niet het geval. Tot op heden blijft de beweeglijkheid van de spermatozoa het
belangrijkste in vitro kenmerk om de spermakwaliteit te beoordelen. De subjectieve bepaling ervan is
sterk afhankelijk van de ervaring van de onderzoeker en is vaak onderhevig aan discussie. Met
behulp van computer geassisteerde sperma analyse (CASA) kan de beweeglijkheid echter objectief en
met een goede herhaalbaarheid geanalyseerd worden.
De algemene doelstelling van dit proefschrift is de beweeglijkheid van hengstensperma op
een uniforme en gestandaardiseerde manier te onderzoeken. Hiertoe werd zowel de invloed van
verschillende CASA instellingen als van het gebruik van verschillende telkamers, op de beweeglijkheid
van het sperma na gegaan (Hoofdstuk 3.1 en 3.2). Ook werd een nieuw toestel voor de analyse van
hengstensperma als alternatief voor de CASA uitgetest (Hoofdstuk 3.3 en 3.4). Daarnaast werden de
opbrengst van sperma en de spermakwaliteit na verschillende centrifugatieprotocollen onderzocht
(Hoofdstuk 4.1). Als laatste werd de mogelijkheid van een nieuwe techniek na gegaan waarbij het
sperma voor het invriezen opgezuiverd wordt met als doel de kwaliteit van het sperma na het
ontdooien te verbeteren (Hoofdstuk 4.2).
Zoals reeds vermeld is het bepalen van de beweeglijkheid van sperma één van de
belangrijkste onderdelen van het spermaonderzoek. Hiervoor wordt meer en meer gebruik gemaakt
van CASA toestellen die een goede herhaalbaarheid hebben met betrekking tot de analyse van o.a.
ontdooid hengstensperma. Bovendien zouden de resultaten van de CASA tussen verschillende
laboratoria kunnen vergeleken worden, ware het niet dat elk laboratorium zijn eigen analysetechniek
heeft. Verschillen tussen diverse CASA instellingen zoals de minimale en medium afkapwaarden voor
snelheid (VAP) en de rechtlijnigheid (STR), hebben een grote impact op de bekomen waarde voor de
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SAMENVATTING
beweeglijkheid (Hoofdstuk 3.1). Zo zal zowel de totale als de rechtlijnige beweeglijkheid van
eenzelfde spermastaal sterk verschillen wanneer verschillende instellingen gebruikt worden. Er is
echter wel een sterke correlatie tussen deze resultaten. Daarom zouden de instellingen van het
gebruikte CASA toestel telkens moeten weergegeven worden bij het vermelden van sperma analyse
resultaten. Idealiter zouden deze CASA instellingen moeten gestandaardiseerd worden waardoor alle
instellingen wereldwijd hetzelfde zijn.
206
Verder is het belangrijk bij het beoordelen van de spermakwaliteit met de CASA, een
telkamer te gebruiken waarbij de spermacellen in één enkele laag gebracht worden. Hierdoor
worden de spermacellen vlot herkend door de computer en kan het traject dat een spermacel aflegt,
worden gevolgd en zijn beweging worden geanalyseerd. Er bestaan echter diverse telkamers met
zowel verschillen in manieren waarop het sperma in de kamer kan gebracht worden, als in diepte van
de kamer. Verschillende laboratoria gebruiken verschillende telkamers. De meest frequent gebruikte
telkamers zijn enerzijds wegwerpkamers met een diepte van 20 µm en anderzijds de herbruikbare
Makler telkamer met een diepte van 10 µm. In dit onderzoek werden Leja telkamers van
verschillende diktes vergeleken met wegwerp en herbruikbare ISAS telkamers, met de Makler kamer
en met een preparaat zoals het aangeraden wordt door de wereld gezondheidsorganisatie (WHO).
Zowel de bekomen waarden voor concentratie als beweeglijkheid waren sterk afhankelijk van de
gebruikte telkamer (Hoofdstuk 3.2). Behalve de 12 µm diepe Leja kamer vertoonden alle telkamers
een zeer slechte correlatie met de concentratie die bepaald was met de Nucleocounter, die in deze
studie beschouwd werd als de goudstandaard. Hiermee wordt bevestigd dat CASA toestellen niet
geschikt zijn om de spermaconcentratie te bepalen. De beweeglijkheid van het sperma werd niet
alleen beïnvloed door het type telkamer maar ook door de manier van laden. Als de kamer geladen
werd met behulp van capillaire krachten, werd de beweeglijkheid sterk verminderd in vergelijking
met het WHO preparaat, die in deze studie ook gebruikt als de goudstandaard. Een WHO preparaat
bestaat uit een druppel sperma van exact 10 µL op een draagglaasje bedekt met een dekglaasje van
22mm op 22mm.
Als alternatief voor de CASA kan de Sperm Quality Analyzer (SQA) gebruikt worden. Bij deze
techniek wordt een spermastaal belicht met een lichtstraal en op basis van de verstrooiing en de
schommeling van de lichtstraal, die ontstaat door het bewegen van de spermacellen, kan de
beweeglijkheid objectief geanalyseerd worden met behulp van wiskundige algoritmes. Tot voor kort
was dit toestel enkel beschikbaar voor het beoordelen van humaan sperma. Gezien de verschillen
tussen de mens enerzijds en de verschillende diersoorten anderzijds wat de afmetingen van de kop
van een spermacel betreft, was het noodzakelijk om per diersoort specifieke toestellen te ontwerpen.
In een eerste experiment werd de SQA bestemd voor het analyseren van hengstensperma, met name

SAMENVATTING
de SQA-Ve met software versie 1.00.43, getest en de herhaalbaarheid van de metingen geëvalueerd
alsook de overeenkomst met de klassieke lichtmicroscopische analyse (Hoofdstuk 3.3), en dit zowel
voor het analyseren van vers als van verdund sperma. De SQA-Ve had een goede herhaalbaarheid en
een goede overeenkomst met de haemocytometer voor het bepalen van de concentratie. De
weergegeven waarden voor de beweeglijkheid en voor het percentage morfologisch normale
spermacellen waren echter weinig herhaalbaar en hadden bovendien een slechte overeenkomst met
de lichtmicroscopische bevindingen. Deze laatste bevindingen vertoonden daarentegen wel een
goede overeenkomst met de goed herhaalbare CASA waarnemingen. Uit dit onderzoek kan
geconcludeerd worden dat de geteste versie van de SQA-Ve (versie 1.00.43) onvoldoende
nauwkeurig is voor de analyse van zowel vers als verdund sperma.
Voor het analyseren van ontdooid hengstensperma werd dezelfde versie van de SQA-Ve, met
name de 1.00.43, alsook een recentere versie met vernieuwde software algoritmes (1.00.61) getest
(hoofdstuk 3.4). Ook voor diepvries sperma had de eerste versie een slechte herhaalbaarheid en een
zeer slechte overeenkomst met de CASA waarnemingen. De vernieuwde versie daarentegen, was
duidelijk beter geschikt om hengstensperma te analyseren, al zijn er nog bijkomende aanpassingen
aan de software algoritmes nodig om betrouwbare resultaten te verkrijgen. Een bijkomend nadeel
aan de SQA-Ve is dat de ondergrens voor het weergeven van de beweeglijkheid ligt op 30% hetgeen
concreet betekent dat waarden gelijk aan of kleiner dan 30% niet weergegeven worden.
Na het beoordelen van de kwaliteit moet het sperma verwerkt worden. Om de concentratie
aan spermacellen te verhogen, wordt bij de verwerking van hengstensperma vaak gebruik gemaakt
van centrifugatie, zowel bij de gekoelde bewaring als bij het invriezen. De spermaverliezen die
gerapporteerd worden na centrifugatie, zijn zeer variabel en gaan van minder dan 2% tot 25%
wanneer gebruik gemaakt wordt van eenzelfde standaardprotocol (600 × g gedurende 10 min). In
hoofdstuk 4.1 werd dit protocol vergeleken met andere protocollen waarbij het sperma gedurende
een kortere tijd werd gecentrifugeerd aan een hogere kracht. De bekomen verliezen waren sterk
verschillend naargelang het gebruikte protocol, met 22% verlies voor het standaardprotocol. Bij een
verhoogde centrifugatiekracht waren de verliezen aanzienlijk minder zonder aantasting van de
spermakwaliteit. Noch bij gekoelde bewaring noch bij het invriezen van het sperma werd een
duidelijke invloed waargenomen van het centrifugeren aan een hogere kracht gedurende een
beperkte tijd.
Door het sperma te centrifugeren wil men een maximale opbrengst van sperma verkrijgen
zonder het echter te beschadigen, hetgeen in feite lijnrecht tegenover elkaar staat. Met behulp van
een alternatieve manier van centrifugeren waarbij gebruik gemaakt wordt van een colloid, kunnen
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SAMENVATTING
de spermacellen geselecteerd worden. Bij deze techniek worden de spermacellen gescheiden op
basis van hun kwaliteit, zodat de gevormde pellet na centrifugatie voornamelijk morfologisch
normaal sperma met een goede beweeglijkheid bevat. In de humane voortplantingsklinieken wordt
vaak gebruik gemaakt van dergelijke technieken waarbij uit het oorspronkelijke staal de goede
spermatozoa geselecteerd worden. Tot voor kort kon men echter enkel kleine hoeveelheden sperma
verwerken met behulp van deze technieken zodat het niet mogelijk was deze aan te wenden voor
hengstensperma. Recente aanpassingen laten echter toe om ook grotere hoeveelheden sperma te
verwerken. Met deze nieuwe techniek waarbij slechts één enkele laag van het colloid (Androcoll-E)
gebruikt wordt, is het mogelijk 15 mL sperma per centrifugeerbuis te verwerken, waardoor men
minder sperma bekomt maar van een veel betere kwaliteit. In een volgend experiment werd dit
opgezuiverde sperma ingevroren, waarbij verwacht werd dat de spermakwaliteit ook na ontdooien
beter zou zijn (hoofdstuk 4.2). Deze hypothese werd bevestigd: alle gecontroleerde parameters
waren na het ontdooien duidelijk beter in vergelijking met de niet-opgezuiverde stalen. Zodoende
kan Androcoll-E een belangrijk hulpmiddel zijn om sperma in te vriezen van hengsten die normaal
moeilijk of niet in te vriezen sperma hebben.
208
Op basis van de gevonden resultaten en gegevens uit de literatuur kunnen volgende
conclusies getrokken worden:
♦ De instellingen van een CASA toestel hebben een zeer grote invloed op de bekomen waarden
voor de beweeglijkheid.
♦ Het type telkamer dat gebruikt wordt bij het uitvoeren van een CASA analyse heeft een
belangrijke impact op de gevonden waarden voor de concentratie en de beweeglijkheid. Als
een kamer gevuld wordt door middel van capillariteit, wordt de beweeglijkheid aanzienlijk
onderdrukt.
♦ De huidige versies van de SQA-Ve zijn niet geschikt om op een eenduidige manier de kwaliteit
van hengstensperma te beoordelen. Het toestel heeft een slechte herhaalbaarheid en vertoont
onvoldoende overeenkomst met de klassieke alternatieven voor de analyse van de
beweeglijkheid.

SAMENVATTING
♦ Het gebruikte centrifugatieprotocol heeft een duidelijk effect op de sperma opbrengst. Hoge
centrifugatiekrachten (tot 2400 × g) gedurende een korte tijd (5 min) kunnen het verlies aan
spermacellen aanzienlijk beperken zonder invloed te hebben op de in vitro kwaliteit van het
sperma.
♦ Het selecteren van de populatie morfologisch goede spermacellen met behulp van
centrifugatie over een enkele laag Androcoll-E voor het invriezen, heeft een positieve invloed
op de spermakwaliteit na het ontdooien. Deze techniek is zeer beloftevol om het sperma van
hengsten dat normaal moeilijk of niet kan ingevroren worden, toch met goed gevolg in te
vriezen.
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DANKWOORD
Het obligatoire dankwoord… of is het toch meer dan dat? Uiteraard, want zonder de hulp van een massa
mensen was er van dit doctoraat niets in huis gekomen. En ere wie ere toekomt, mijn hoofdpromotor,
Professor de Kruif verdient wel een groot woord van dank! Ik kan me geen periode van langer dan een maand
voorstellen tijdens dewelke u me niet vroeg naar mijn onderzoek en hoe het met me ging. Een niet-aflatendezekerheid
was jouw geloof in het doel van mijn aanwezigheid aan de vakgroep, een doctoraat! En zeker als het
na enige tijd voor de meesten een fata morgana dreigde te worden, bleef jij overtuigd dat zelfs ik uiteindelijk
een doctoraat kon behalen. En zoals wel vaker krijg je toch weer eens gelijk… Naast jouw vertrouwen in de
goede afloop van m’n onderzoek was je ook altijd een ooggetuige van het gebeuren, of zeg maar avontuur, dat
zich op kliniek afspeelde. Je had waarschijnlijk bij alles wat ik deed wel je eigen idee en vermoedelijk zou je het
vaak zelf anders aangepakt hebben, maar net uit die vrijheid en je onvoorwaardelijk vertrouwen ben ik de
dierenarts geworden die ik vandaag ben. En dat is me minstens even veel waard als dit doctoraat.
Ann, van meet af aan werd het vrij snel duidelijk dat jij mede verantwoordelijk zou zijn om mij, een van die
kliniekmensen, in de richting van het onderzoek te trekken. Kliniek kwam wel meer dan eens tussen ons, maar
zoals je ziet ben je er toch in geslaagd om het eerste doctoraat voor de assistenten van paard in lange tijd te
helpen verwezenlijken! Zo zie je maar; als we willen, kunnen we wel… ☺ Ik heb het je als promotor vaak knap
lastig gemaakt, maar op een zeldzame bots na zijn er we er toch samen geraakt!
De meest vreemde eend in de bijt van mijn onderzoek is ongetwijfeld mijn andere promotor, professor De
Vliegher, Sarne! De wetenschappelijke samenwerking is begonnen met de statistiek van mijn eerste artikel. En
ja, jouw “less is more” is weldegelijk blijven hangen, ook al heb je het nadien nog vaak moeten herhalen. Voor
iemand die zich voornamelijk met melk bezig houdt weet je ook verdomd veel van sperma! Misschien heb ik je
dat laatste wel zelf opgedrongen, maar je hebt het toch maar gedaan! Bedankt voor het nalezen, herlezen en
steeds verder piekeren over hoe je het best kon analyseren waar ik naartoe wilde met mijn experimenten. Ik
hoop dat ik het je niet TE moeilijk heb gemaakt. Binnen een paar jaar kijkt iedereen naar België als ze over
Ryelands spreken, en uiteraard over de vruchtbare glooiingen van Zwalm met jouw bollekes als stammoeders!
Dr. Magistrini, Dear Michèle, thank you for your constructive remarks when carefully analysing my PhD
manuscript. I will always remember the ISSR in Brazil where you talked so enthusiastic about all your research
projects. Thank you once more that you were willing to be a member of my examination committee. Dr.
Morrell, dear Jane, I hope we will be able to continue some research in the future. But let me thank you first for
willing to collaborate for a part of the scientific work that is presented in this thesis. Without your help and my
visit to Uppsala for the SCSA analysis I wouldn’t have made it ? here today! Professor Vlaminck bedankt voor
het grondig nalezen van mijn doctoraat en voor de kritische opmerkingen. Beste Lieven, bedankt voor al het
praktische dat je me geleerd hebt, zowel als intern toen ik op heelkunde meeliep, maar ook later als ik weer
eens op je kennis beroep moest doen. Professor Sys, ik hoop dat mijn onderwerp je heeft kunnen boeien, maar
uitgaande van je constructieve opmerkingen weet ik zeker dat je alles met veel zorg en aandacht bekeken hebt.
Bedankt daarvoor.
Beste Erik, bedankt voor zoveel! De leerrijke gesprekken en discussies op onze buitenlandse congres- en
studiereizen, jouw gastvrijheid om mij bij je thuis te ontvangen en wegwijs te maken in jouw visie van de
voortplanting en verloskunde. Je mag dan misschien een “boerke uit de Limburg” zijn zoals je het zelf altijd zegt,
maar ik heb in elk geval al veel van je kunnen leren. Beste Adrie, het is leuk een gelijkgezinde te hebben als het
over standaardisatie van sperma analyse gaat. Jouw karrevracht aan ervaring en jouw inzicht in de materie
hebben duidelijk bijgedragen in de verwezenlijking van mijn onderzoek, waarvoor dank. Tom, in drukke havens
vaart men wel eens door andermans vaarwater, en dat heb ik dan ook ruimschoots bij jou gedaan! Ondanks dit
alles wist je altijd je kalmte te bewaren en wou je je steentje bijdragen aan mijn doctoraat. Bedankt…
Voor het eerst in jaren (of bij mijn weten voor het eerst ooit?) worden de mensen van paard op onze vakgroep
eens niet stiefmoederlijk behandeld in één klein paragraafje! Dus alle mensen van paard, bedankt voor alles.
Maar misschien beginnen bij Jan, nu kliniekhoofd (dus mijn baas!) maar ooit een intern en resident… Toen was
je al een bron van wijsheid en in de loop der jaren ben je naast een praktische mentor ook een vriend
geworden. We houden er misschien een andere pedagogische techniek op na, maar van de jouwe ben ik in elk
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DANKWOORD
geval overtuigd dat hij werkt of toch zeker kan werken… Stress gegarandeerd als jij in Kemmel zat en ik alvast
begon aan mijn eerste torsie! Maar gedraaid is ze toch. Als het (weer) eens een beetje (veel) minder ging op ’t
werk en daarbuiten kon ik steeds op je rekenen, maar wat je bezielde in het voorjaar van 2008 is me nog steeds
een raadsel. Volgaarne ben ik dan ingegaan op je vraag om peter te worden van je jongste, ik hoop dat je je het
nog niet (te veel) beklaagd hebt. Ik ben alvast fier dat ik je spruiten van zo dicht bij mag zien opgroeien. Het zijn
super kinderen, elk met hun eigen trekjes, maar ze vrolijken me altijd op! Geen flauw idee hoe je Greet zo ver
gekregen hebt om je te volgen in dat waanzinnige voorstel, maar ook jij bedankt voor de voorbije jaren Greet.
En Jan, nog een laatste detail! Denk ook eens aan je eigen onderzoek, ik zal het komende seizoen proberen
zoveel mogelijk op mij te nemen, beloofd!
Meerdere paardenmensen zijn sinds mijn komst al de revue gepasseerd op de kliniek, maar slechts weinigen
zijn er ook lang gebleven. Catharina, sinds meer dan 6 jaar ben je nu al een vaste waarde op kliniek. Hopelijk
ben je toch een beetje tevreden over je “voortgezette opleiding”… Ondertussen ben je al veel meer dan een
collega en dat maakt het vaak nog complexer dan het zou moeten! Maar ja, moeilijk kan ook, niet bi? Probeer
in elk ook maar werk te maken van je doctoraat en je overblijvende energie in je vakdierenartsopleiding
steken… en ik beloof je, hoewel ik meer kliniek ga doen zal ik je je baanwerk laten, ok? Bedankt voor alles!
Emilie, lang gedacht dat jij het ging volhouden bij ons keikoppen op de kliniek, maar jij weet als geen een hoe
moeilijk wij het elkaar kunnen maken. In elk geval bedankt voor de vele hulp in ’t spermalabo. En toch ben je er
sterk(er) uitgekomen. In elk geval hoop ik dat je het je verder voor de wind gaat… Al zal het voorlopig dan wel
niet in Ierland zijn. En wees gerust, we zien elkaar nog veel, zelfs al is het aan de andere kant van de wereld
maar dichter bij is ook goed! Je moet het echt zo ver niet gaan zoek als ons Katrientje! Smitsie, daar zo ver in
Aruba, bedankt voor je nuchtere kijk op het leven als je nog bij ons was. Je buro blijft voorlopig “leeg”, ttz
gevuld met papier van mij, maar off limit voor een ander. In mijn hoofd ben jij nog altijd mijn buurmeisje!
Kim, je weet ongetwijfeld al een beetje wat je te wachten staat, maar nu ik meer naar beneden kom zal ’t wel
weer wat anders zijn. In elk geval bedankt voor de hulp en we maken er nog minstens één geslaagd seizoen van!
En James, baasje was akkoord, echt waar… dus word nu maar snel groot. Sarah, jouw eerste seizoen staat voor
de deur, maar dat zal wel loslopen! En alle andere mensen met wie ik ooit “op paard” heb mogen
samenwerken, Lotte, Ines, Rita en Cabron bedankt!
Maar onze kliniek zou lang geen kliniek zijn zonder de hulp van vele andere mensen die vaak achter de
schermen veel werk verzetten. Marnik en Veronique, bedankt voor de vele hulp! Het klutsen van eitjes op de
onmogelijkste uren om weer eens een verdunner te maken voor een of ander onderzoekje, het opruimen van
het vuil achter mij als ik weer eens te snel weg moest en voor zoveel meer. Marnik, nu ik weer iets meer tijd
heb gaan we terug frequenter “een ciderke drinken” he! Willy, Dirk S en Wilfried, jullie ook bedankt… voor
alles! Chef voor het delen van zijn rijke ervaringen in de schapenhouderij en al het bijhorende werk ☺. Dirk, jij
ook bedankt voor het leiden van mijn hengsten, tot in het weekend toe (Sorry Ilse) en ja Dirk, nog een extra
bedankt voor je hulp bij de afname van Atomic enkele jaren terug! Als jij er niet was geweest, dan was ik er
geweest.
De DI08 is veel meer dan alleen paard, dus ga ik nu proberen de anderen ook allemaal te bedanken. Maar als je
er als ik ZO lang over doet dan zie je veel mensen komen en gaan dus hoop ik van harte dat ik niemand vergeet,
en als dat toch zo zou zijn, dan alvast mijn excuses. Davy, alvast bedankt voor al je boerenwijsheden, ik zal ze
nog hard nodig hebben in de toekomst, maar jij blijft nog dus dat zit al snor. Sebi, succes met je kalkoenen en je
weet, mijn “duiventil” in Balegem heeft altijd iets staan om dorstigen te laven. De jonge krachten op de kliniek
rund, Vanessa, Hilde, Kathelijne, Anneleen, Joren, als ge een extra handje nodig hebt… bel gerust!
De buitenpraktijk, waar is de tijd dat ik er deel van uitmaakte, eerst rund dan paard laten herrijzen! Geert O,
bedankt voor jouw steun in en bij het einde van mijn BP-carrière. De buitenpraktijk gaat hand in hand met wat
trekkers, Jef, van mijn studententijd tot nu heb ik veel van je geleerd, bedankt. Marcel, jij bent een nooit
uitdovende vlam die wakkert voor de diergeneeskunde, ongelooflijk hoe jij alles kan aanpakken. Zelfs een
foetotomie is leuk werk samen met jou, en zeer leerrijk! De BP collega’s vroeger, Geert H, Bart M, Tom VH,
Boudewijn (gelukkig ben jij in de buurt blijven wonen), Jo L, Steven V en Leen, wat een hechte groep was dat!
Stefaan het gaat je goed nu je nieuwe horizonten verkent! Hans, je verblijf bij ons was kort, maar krachtig
genoeg om vriendjes te blijven. Draag maar goed zorg voor Thijsje, en vergeet niet dat jullie nog kleine
herkauwers moeten komen scannen! Phillipe en Muriel, jullie waren onvergetelijk en Dr. Filliers een extra
“dank-u-tje” voor de manier waarop je Fari opgevolgd hebt. Ik ken 8 families waar een kleine farus loopt
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DANKWOORD
dankzij jou en ze zijn er allemaal zot van, bedankt. Buurman Miel, nu het seizoen weer voor de deur staat gaan
we hier terug gezellige nachten tegemoet! Hou je wel onze Cyrillus op het juiste spoor? Hij is nog zo jong… Met
de hulp van Iris lukt dat wel hé! Sofie, bedankt voor je hulp met Oscar en het bezoek van zijn grootvader in
Zweden. De andere BP’ers Emily, Mieke, Ellen, Ilse, Anita, nog veel succes in alles. Als hulpverleners van de BP
jullie ook bedankt voor alles Ria en Els! En Ria… ik vrees dat ons kieltjes nog wat nieuwe mouwkes kunnen
gebruiken, dus als je eens tijd hebt… Alle Melk-meisjes (en Joren) zijn al gepasseerd maar onze Lars nog niet!
Bedankt Larsje…
Tot slot van de vakgroep rest me nog de “beneden van ons gebouwtje”… Ik zeg niet dat ik me er niet thuis voel
maar zot veel ben ik er niet geweest. Desalniettemin hebben jullie ook vaak mijn fratsen in het labo moeten
verdragen, dus nog maals Isabel en Petra, mijn oprechte excuses voor de geurhinder als gevolg van het koken
van … en bedankt voor de vele hulp. Op het einde van de gang zit daar nu Josine met een nieuwe bende. Bekie,
veel succes met jouw verder onderzoek. Met Hilde en Bart L is het daar een vijandige overname van paard
geworden, ook jullie succes bij jullie onderzoek. Voor de technische ondersteuning kon ik altijd reken op een
arsenaal collega’s, Roger V en Steven B, Sandra (bedankt voor de uitbreiding van je takenpakket) en Leïla. Een
welgemeende dank ook voor Nicole die steeds vrolijk orde schiep in onze buro… waar een kat vaak haar jongen
niet terug zou vinden! Het idee van de briefjes op de deur is niet waterdicht maar wel een serieuze vooruitgang.
Hopelijk bots je dit seizoen tegen niemand die ’s morgens zich nog even draait in ons bed ☺. (Tante) Nadine,
dank om vele jaren onze buurvrouw te zijn, de reizen te regelen, klaar te staan voor een babbel en er gewoon
altijd te zijn. Het is leuk je regelmatig terug te zien op de dienst of op de faculty club. Den 29 zijn al lang geen
vreemden meer in je vrijdagavond clubje. En wanneer ga je nu ook eens mee op congres met mij?
Ook veel mensen van buiten de dienst verdienen een speciale vermelding. Zonder Dirk DD had er voor mij geen
diergeneeskunde ingezeten. Maak je geen zorgen, ik was gewaarschuwd maar doe het nog altijd met veel
plezier. En ja, sommige zaken had ik liever anders gezien… maar gedane zaken nemen geen keer. Nathalie, we
gaan nu elk onze eigen weg, en soms heb ik het daar nog moeilijk mee… Het spijt me konijn!
Iedereen van mijn familie, ook jullie bedankt voor de vele steun door de jaren heen. Zeker mijn zusje en broer
verdienen samen met hun gezin een extra woord van dank. We lopen elkaars deur zeker niet plat maar het is
toch leuk om weten dat ik altijd ergens terecht kan. Mieke, Kristof, Hebe en Rune, bedankt voor alle leuke
momenten en jullie gastvrijheid. Pieter, Melissa en Quinten hetzelfde geldt voor jullie. En Quinten, weet jij wie
de beste taart ter wereld KAN maken, maar het misschien iets te weinig doet? Ik wel…
Waar had ik vandaag gestaan zonder de onvoorwaardelijke steun van mijn ouders. Gutta cavat lapidem, non vi,
sed saepe cadendo… en zo is Colligur er gekomen na een dagje Roeselare! Dus jullie wisten misschien wel dat ik
het kon volhouden maar toch ben ik blij dat jullie me zijn blijven steunen. Mama en papa, bedankt voor alle
goede zorgen, voor alle hulp en nog zoveel meer. Ik ben blij jullie als ouders te hebben.
Bedankt,
Maarten
213

CURRICULUM VITAE
Maarten Hoogewijs werd geboren op 25 februari 1978 te Gent. Na het behalen van het
diploma van het hoger secundair onderwijs aan het Sint-Lievens College te Gent begon hij in 1996
met de studie Diergeneeskunde aan de Universiteit Gent. In 2002 behaalde hij het diploma
dierenarts met grote onderscheiding.
Op 1 oktober 2002 startte hij een roterend internship op de klinieken grote huisdieren van de
faculteit Diergeneeskunde. In oktober 2003 werd hij resident voor het European College of Animal
Reproduction. In oktober 2004 trad hij in dienst als voltijds assistent op de Vakgroep Voortplanting,
Verloskunde en Bedrijfsdiergeneeskunde. Zijn taak bestond uit het klinische werk op de Kliniek
Voortplanting en Verloskunde van de grote huisdieren en de Buitenpraktijk paard, en het opleiden
van de studenten. De eerste 5 jaar op de faculteit deed hij ook dienst op de Buitenpraktijk rund. Bij
het vele kliniekwerk was hij vooral geïnteresseerd in het mannelijke dier en stond hij grotendeels in
voor de hengsten die werden aangeboden op de dienst. Vanuit die klinische achtergrond is hij zijn
onderzoek gestart naar de standaardisatie van sperma onderzoek bij de hengst en het verbeteren
van de verwerking van sperma.
Maarten is auteur en mede-auteur van verschillende wetenschappelijke publicaties in
nationale en internationale tijdschriften en presenteerde zijn onderzoeksresultaten op meerdere
internationale congressen.
215

CURRICULUM VITAE
Maarten Hoogewijs was born on February 25th 1978 in Ghent, Belgium. After finishing
secondary school at Sint-Lievens College in Ghent (Science – Mathematics) in 1996, he started his
study in Veterinary Medicine at Ghent University. In 2002 he graduated as Veterinarian with great
distinction.
After a rotating internship he started as resident for the European College of Animal
Reproduction. In October 2004, he became assistant at the department of Reproduction, Obstetrics
and Herd Health. He was also responsible for the clinical education of the students during the clinical
and ambulatory work at the department. During his first five years as a vet, Maarten participated in
the night and weekend work of the bovine ambulatory clinic of the department. With his colleagues
from the “equine team”, he was responsible for the clinic of Reproduction and Obstetrics, including
night and weekend work. When working in the clinic he became especially interested in working with
the male animal. From this clinical perspective, he started his PhD research concerning
standardization of equine semen analysis and improvements for semen cryopreservation.
Maarten is author and co-author of multiple scientific publications in national and
international journals, and presented his research results on several international meetings.
217

PUBLICATIONS IN INTERNATIONAL PEER-REVIEWED JOURNALS
BIBLIOGRAPHY
Deprez P., Hoogewijs M., Vlamick L., Vanschandevijl K., Lefevre L., Van Loon G. 2006. Incarceration of
the small intestine in the epiploic foramen of three calves. Veterinary Record 158:869-870.
Van Brantegem L., de Cock H.E.V., Affolter V.K., Duchateau L., Hoogewijs M.K., Govaere J., Ferraro
G.L., Ducatelle R. 2007. Antibodies to elastin peptides in sera of Belgian draught horses with
chronic progressive lymphoedema. Equine Veterinary Journal 39:418-421.
Govaere J.L.J., Hoogewijs M.K., De Schauwer C., Dewulf J., de Kruif A. 2008. Transvaginal ultrasoundguided
aspiration of unilateral twin gestation in the mare. Equine Veterinary Journal 40:521-
522.
Govaere J., Hoogewijs M., De Schauwer C., Van Loon G., de Kruif A. 2008. Incomplete placentation
after twin reduction in a mare. Veterinary Record 163:747-748.
Delesalle C., Hoogewijs M., Govaere J., Declercq J., Schauvliege S., Vanschandevijl K., Deprez P. 2009.
Ultrasound-guided perivaginal drainage of abscesses associated with rectal tears in four
mares. Veterinary Record 165:662-663.
Govaere J., Hoogewijs M., De Schauwer C., Van Zeveren A., Smits K., Cornillie P., de Kruif A. 2009. An
abortion of monozygotic twins in a warmblood mare. Reproduction in Domestic Animals
44:852-854.
Smits K., Goossens K., Van Soom A., Govaere J., Hoogewijs M., Vanhaesebrouck E., Galli C., Colleoni
S., Vandesompele J., Peelman L. 2009. Selection of reference genes for quantitative real-time
PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts. BMC Research Notes
2:246.
Thys M., Nauwynck H., Maes D., Hoogewijs M., Vercauteren D., Rijsselaere T., Favoreel H., Van Soom
A. 2009. Expression and putative function of fibronectin and its receptor (integrin
alpha(5)beta(1)) in male and female gametes during bovine fertilization. Reproduction
138:471-482.
Thys M., Vandaele L., Morrell J.,M., Mestach J., Van Soom A., Hoogewijs M., Rodriguez-Martinez H.
2009. In vitro fertilizing capacity of frozen-thawed bull spermatozoa selected by single-layer
(Glycidoxypropyltrimethoxysilane) silane-coated silica colloidal centrifugation. Reproduction
in Domestic Animals 44:390-394.
Filliers M., Rijsselaere T., Bossaert P., Zambelli D., Anastasi P., Hoogewijs M., Van Soom A. 2010. In
vitro evaluation of fresh sperm quality in tomcats: A comparison of two collection techniques.
Theriogenology 74:31-39.
219

BIBLIOGRAPHY
Govaere J., Ducatelle R., Hoogewijs M., De Schauwer C., de Kruif A. 2010. Case of bilateral seminoma
in a trotter stallion. Reproduction in Domestic Animals 45:537-539.
Hoogewijs M., Rijsselaere T., De Vliegher S., Vanhaesebrouck E., De Schauwer C., Govaere J., Thys M.,
Hoflack G., Van Soom A., de Kruif A. 2010. Influence of different centrifugation protocols on
equine semen preservation. Theriogenology 74:118-126.
Hoogewijs M., De Vliegher S., De Schauwer C., Govaere J., Smits K., Hoflack G., de Kruif A., Van Soom
A. 2010. Validation and usefulness of the Sperm Quality Analyzer V equine for equine semen
analysis. Theriogenology 75:189-194.
De Schauwer C., Meyer E., Cornillie P., De Vliegher S., van de Walle G.R., Hoogewijs M., Declercq H.,
Govaere J., Demeyere K., Cornelissen M., Van Soom A. Optimization of the isolation, culture
and characterization of umbilical cord blood mesenchymal stromal cells. submitted.
Hoogewijs M., Morrell J., Van Soom A., Govaere J., Johannisson A., Piepers S., De Schauwer C., de
Kruif A., De Vliegher S. Sperm selection using single layer centrifugation prior to
cryopreservation can increase post-thaw sperm quality in stallions. submitted.
Hoogewijs M., De Vliegher S., Govaere J., De Schauwer C., de Kruif A., Van Soom A. Counting
chamber influences equine semen CASA outcomes. submitted.
Hoogewijs M., De Vliegher S., Govaere J., De Schauwer C., Vanhaesebrouck E., de Kruif A., Van Soom
A. Need for further improvement of the SQA-Ve software for a univocal analysis of the
quality of frozen thawed equine semen. In preparation.
220

PUBLICATIONS IN NATIONAL PEER-REVIEWED JOURNALS
BIBLIOGRAPHY
Hoogewijs M., Govaere J.L.J., Paijmans L., Van Hove E., Deuchande R., de Kruif A. 2004.
Tweelingdracht bij de merrie. Vlaams Diergeneeskundig Tijdschrift 73:396-406.
Paijmans L., Govaere J.L.J., Hoogewijs M., Deuchande R., de Kruif A. 2004. Endometriumcysten bij de
merrie. Vlaams Diergeneeskundig Tijdschrift 73:23-237.
Govaere J.L.J., Hoogewijs M., Paijmans L., Van Crombruggen I., Riga P., Sterckx J., de Kruif A. 2005.
Nieuwe inseminatietechnieken bij de merrie. Vlaams Diergeneeskundig Tijdschrift 74:222-
232.
De Bock M., Govaere J., Martens A., Hoogewijs M., De Schauwer C., Van Damme K., de Kruif A. 2007.
Torsion of the spermatic cord in a warmblood stallion. Vlaams Diergeneeskundig Tijdschrift
76:443-446.
Martens K., Govaere J.L.J., Hoogewijs M.K., Lefevre L., Nollet H., Vlaminck L., Chiers L., de Kruif A.
2008. Uterine torsion in the mare: a review of three case reports. Vlaams Diergeneeskundig
Tijdschrift 77:397-405.
Smits K., Govaere J., Hoogewijs M., De Schauwer C., Van Haesebrouck E., Van Poucke M., Peelman
L.J., van den Berg M., Vullers T., Van Soom A. 2010. Birth of the First ICSI foal in the Benelux.
Vlaams Diergeneeskundig Tijdschrift 79:134-138.
Vanhaesebrouck E., Govaere J., Smits K., Durie I., Vercauteren G., Martens A., Schauvliege S.,
Ducatelle R., Hoogewijs M., De Schauwer C., de Kruif A. 2010. Ovarian teratoma in the mare:
a review of two cases. Vlaams Diergeneeskundig Tijdschrift 79:32-41.
Van Loo H., Govaere J., Chiers K., Hoogewijs M., Opsomer G., de Kruif A. 2010. Hydrops uteri in a
BWB heifer combined with a vascular hamartoma of the mandible of the calf. Vlaams
Diergeneeskundig Tijdschrift 79:139-142.
221

BIBLIOGRAPHY
ABSTRACTS AND PROCEEDINGS IN INTERNATIONAL CONFERENCES
Govaere J., Hoogewijs M., De Schauwer C., De Vliegher S., Duchateau L., Van Soom A., de Kruif A.
2007. Effect of artificial insemination protocol and sperm dose on pregnancy results in mares.
Theriogenology 68:505.
De Schauwer C., Piepers S., Hoogewijs M.K., Govaere J.L.J., Rijsselaere T., Demeyere K., Meyer E.,
Van Soom A. 2008. Isolation, preservation and characterization of equine umbilical cord
blood stem cells. Reproduction, Fertility and Development 20:220.
Govaere J., Hoogewijs M., De Schauwer C., De Vliegher S., Saey V., de Kruif A. 2008. Lack of
association between low vitamin E levels and retentio secundinarum in Belgian draught
horses (BDH) and warmblood (WB) mares. 16 th International Congress on Animal
Reproduction – Budapest – Hungary.
Govaere J.L.J., Hoogewijs M.K., De Schauwer C., De Vliegher S., Van Crombruggen I., Van Molle A., de
Kruif A. 2008. Lack of association between hypocalcemia and retained placenta in Belgian
draft horses and warmblood horses. 54 th annual meeting of the American Association of
Equine Practitioners – San Diego – California – USA.
De Schauwer C., Meyer E., Hoogewijs M., De Vliegher S., Rijsselaere T., Govaere J., Demeyere K., Van
Soom A. 2009. Comparison of different methods to isolate and culture multipotent
mesenchymal stromal cells from equine umbilical cord blood. 13 th Annual Conference of the
European Society of Domestic Animal Reproduction – Ghent – Belgium.
Govaere J., Hoogewijs M., De Schauwer C., Smits K., Van Haesebrouck E., de Kruif A. 2009. Placental
characteristics in Belgian draught and warmblood horses. 13 th Annual Conference of the
European Society of Domestic Animal Reproduction – Ghent – Belgium.
Govaere J., Martens K., Van Haesebrouck E., Hoogewijs M., De Schauwer C., Smits K., Roels K.,
Vandaele L., de Kruif A. 2009. The FoalinMare: insights inside the foaling mare. 13 th Annual
Conference of the European Society of Domestic Animal Reproduction – Ghent – Belgium.
Hoogewijs M., Piepers S., Rijsselaere T., Govaere J., de Kruif A. 2009. Effect of egg yolk in skimmed
milk extender on cooled preservation of equine semen. 13 th Annual Conference of the
European Society of Domestic Animal Reproduction – Ghent – Belgium.
Hoogewijs M., Vanhaesebrouck E., De Vliegher S., Govaere J.L.J., Rijsselaere T., Van Soom A., De
Schauwer C., Smits K., de Kruif A. 2009. Effects of centrifugation protocol on sperm loss and
sperm quality of equine semen. Annual conference of the American College of
Theriogenologists – Albuquerque – New Mexico – USA.
Hoogewijs M.K., Govaere J.L., Rijsselaere T., De Schauwer C., Vanhaesebrouck E., de Kruif A., De
Vliegher S. 2009. The influence of technical settings on CASA motility parameters of frozen
thawed stallion semen. 55 th Annual meeting of the American Association of Equine
Practitioners – Las Vegas – Nevada – USA.
222

BIBLIOGRAPHY
Filliers M., Rijsselaere T., Bossaert P., Anastasi P., Hoogewijs M., Van Soom A. 2010. In vitro
evaluation of fresh sperm quality in tomcats: a comparison of collection techniques.
Reproduction, Fertility and Development 22:291.
Govaere J., Hoogewijs M., De Schauwer C., Smits K., Vandaele H., Van Loon G., de Kruif A. 2010.
Twisting of mummified remains around the umbilical cord as a potential risk of delayed twin
reduction in the mare. 14 th Annual Conference of the European Society of Domestic Animal
Reproduction – Eger – Hungary.
Hoogewijs M., Piepers S., Govaere J., De Schauwer C., Smits K., de Kruif A., Van Soom A. 2010.
Influence of extender on the storage of equine semen at different temperatures. 14 th Annual
Conference of the European Society of Domestic Animal Reproduction – Eger – Hungary.
Hoogewijs M.K., De Vliegher S., Govaere J.L., De Schauwer C., de Kruif A., Van Soom A. 2010. Need
for further improvement of SQA-Ve software for a univocal analysis of the quality of frozen
thawed equine semen. 7 th Biennial meeting of the Association for Applied Animal Andrology
– Sydney – Australia.
223

FUNDAMENTAL ASPECTS OF CRYOPRESERVATION
ADDENDUM I
Cryopreservation of equine semen is associated with a wide range of stresses. Spermatozoa
are exposed to components in the extender, cooling, intracellular changes due to dehydration, and
damage by ice crystals (Amann and Pickett, 1987).
The plasma membrane is composed of lipids and proteins, arranged as a lipid bilayer (mainly
phospholipids and cholesterol) with the hydrophilic ends of the lipids externally and the hydrophobic
fatty acyl chains internally (Fig. 1) (Robertson, 1983).This lamellar arrangement provides a
hydrophobic barrier through which water, and molecules dissolved in water pass only with difficulty.
Molecules normally pass or are transported through channels and pores formed by proteins that are
intermingled with the lipids. The different phospholipids are randomly arranged in a lamellar
structure, and move free laterally within one leaflet of the membrane bilayer (Quinn, 1981), because
membranes are “fluid” at body temperature. Temperature is a very important factor altering the
membrane fluidity. By cooling, the phase of the “liquid membrane” shifts to a crystalline state as a
consequence of alteration in shape of the phospholipid molecules (Robertson, 1983). In the
crystalline conformation the phospholipids can no longer move in a random fashion, which will result
in clustering of the integral proteins in small regions of liquid lipids. The aggregation of proteins can
result in increased membrane permeability and decreased metabolic function.
The cooling of phospholipids (normally shaped like an inverted cone) will alter their shape,
and will result in a more sharply conical shape. Groups of these phospholipids may assume a special
configuration, termed hexagonal-II phase. This is a circular arrangement with the head groups
internally and the fatty acyl chains outward (Fig. 1). This is more likely to occur with rapid reduction
in temperature and results in increased membrane permeability. This formation may not be reversed
upon reheating when reformation of a lamellar bilayer occurs (Quinn, 1981).
225

ADDENDUM I
a.
b.
Fig. 1. (a) Schematic presentation of the plasma membrane with the different components; (A)
phosholipid, (B) transmembrane protein, (C) hydrophobic region, and (D) hydrophilic region
in random arrangement in the classical lamellar bilayer, and (b) the different presentations as
response to cooling; (b1). phospholipids in crystalline domains, (b2). Hexagonal-II inverted
micelle, and (b3) aggregated proteins (adapted from Amann and Pickett, 1987).
226
When spermatozoa in a suspension are cooled below 0°C, ice crystals start to form. Initially,
ice crystals are formed in the extracellular fluid. Due to these ice crystals, the relative amount of free
extracellular water diminishes, resulting in increased concentration of salts and other osmotic active
compounds (such as sugars) and as such, resulting in a hypertonic extracellular environment. Water
within the spermatozoa does not freeze at this stage but will be transported extracellular which will
lead to a progressive dehydration (Amann and Pickett, 1987; Muldrew et al., 2004). If cooling
proceeds at a constant slow rate, the spermatozoa will remain close to the osmotic equilibrium by
losing water at the same rate that water is lost by the extracellular fluid by its conversion to ice. In
contrast a faster cooling rate will cause extracellular water to crystallize more rapidly than the cell
can lose water, resulting in an increased osmotic gradient. Cells are damaged at rapid and slow
cooling rates, so the optimal cooling rate should be in between these extremes (Mazur, 1984). Slow
cooling will induce “solution-effects” injury, caused by exposure to high concentrations of solutes
(hyper-osmolarity). Rapid cooling injury is associated with the formation of intracellular ice crystals.
Slow cooling injuries can be avoided by increasing the cooling rate, and vice versa (Mazur et al.,
1972).
The addition of cryoprotectant agents (CPAs) to the freezing extender will increase the
survival following freezing and thawing substantially. The CPAs used to cryopreserve semen are
penetrating CPAs, they diffuse through the plasma membrane and equilibrate in the cytoplasm. The

ADDENDUM I
presence of these CPAs affects the freezing point, and more importantly, they lower the
concentration of salts normally found in physiological solutions for a given temperature (Muldrew et
al., 2004). They do so by lowering the amount of ice present at a given temperature (less ice equals
more water), and they act as secondary solvents for the salts (Pegg, 1984). Penetrating CPAs can
greatly reduce slow-cooling injury, however, they provide little protection against rapid cooling injury.
Fig. 2. Schematic representation of the physical changes in equine spermatozoa and surrounding
extender during freezing. The effect of various cooling rates on the formation of ice crystals
or microcrystals (large or small stars) and the movement of solvents and penetrating solutes
(heavy or light arrows) are shown (Figure from Amann and Picket, 1987; Hammerstedt et al.,
1990).
227

ADDENDUM I
228
Slow cooling injury can be caused by solute toxicity (mechanisms not yet elucidated) or by
shrinkage of the cells resulting from the hypertonic extracellular solution. Intracellular ice formation
(rapid cooling injury) occurs when a cell is not able to maintain osmotic equilibrium with the external
environment (Fig. 2). Due to the sudden exaggerated extracellular ice formation, the increase in
solutes is so dramatically that the cell cannot respond to it with exosmosis. The cytoplasm will
consequently become increasingly supercooled, as such increasing the likelihood of intracellular ice
formation (Muldrew et al., 2004). There are multiple hypotheses that attempt to explain how
extracellular ice interacts with the plasma membrane in the initiation of intracellular ice formation,
but they are all topic of debate. The nature of cellular injury caused by freezing and thawing is very
complex.
Amann and Pickett (1987) assumed that for stallion spermatozoa, in an extender of given
composition, there should be a cooling rate that maximizes sperm survival. Figure 3 represents such
a theoretical cooling curve, depicting the sperm survival as a function of the cooling rate. Slow
cooling (pathway A to B) will cause sperm damage due to excessive dehydration (solution effect),
while rapid cooling (pathway C to D) will cause sperm damage because of intracellular ice formation.
The optimal cooling rate is between B and C, and so far this optimal cooling rate can only be
determined empirically (Amann and Pickett, 1987). A theoretical approach for determining the
optimal cooling rate for the cryopreservation of bull semen has been proposed based on the
different compositions in the extender used (Woelders and Chaveiro, 2004). So far, no such work has
been presented for stallion spermatozoa.
A very remarkable observation concerning cryo-injury was the demonstration that
membrane integrity of ram sperm during cryopreservation clearly retained throughout the freeze-
thaw procedure, and that the permeabilization occurred not only after the samples had been thawed,
but once they reached threshold temperatures (Holt et al., 2005). These findings are in contrast with
the concept that the lethality is situated in the intermediate zone of temperature (between freezing
point and -60°C) that the cells must traverse twice during the freeze and thaw cycle (Mazur, 1963).

ADDENDUM I
Fig. 3. Theoretical curve indicating the survival of cells as function of the cooling rate at the
presence (green line) or absence (red line) of glycerol in the extender (Adapted from Amann
and Pickett, 1987).
All the above clearly states that cryopreservation (of sperm) induces many important
changes with the cells. So far the knowledge concerning these changes is incomplete, and therefore
till date, semen cryopreservation is a science as well as an art.
References
Amann R.P., Pickett B.W. 1987. Principles of cryopreservation and a review of cryopreservation of
stallion spermatozoa. Journal of Equine Veterinary Science 7:145-173.
Hammerstedt R.H., Graham J.K., Nolan J.P. 1990. Cryopreservation of mammalian sperm: What we
ask them to survive. Journal of Andrology 11:73-88.
Holt W.V., Medrano A., Thurston L.M., Watson P.F. 2005. The significance of cooling rates and animal
variability for boar sperm cryopreservation: insights from the cryomicoscope. Theriogenology
63:370-382.
Mazur P. 1963. Kinetics of water loss from cells at subzero temperatures and the likelihood of
intracellular freezing. Journal of General Physiology 47:347-369.
Mazur P. 1984. Freezing of living cells: Mechanisms and implications. American Journal of Physiology
247:125-142.
Mazur P., Leibo S.P., Chu E.H. 1972. A two-factor hypothesis of freezing injury. Evidence from Chinese
hamster tissue-culture cells. Experimental Cell Research 71:345-355.
229

ADDENDUM I
Muldrew K., Acker J.P., Elliott J.A.W., McGann L.E. 2004. The water to ice transition: implications for
living cells. In: Life in the frozen state Fuller B.J., Lane N., Benson E.E. (Ed.) CRC Press, pp. 67-
108.
Pegg D.E. 1984. Red cell volume in glycerol/sodium chloride/water mixtures. Cryobiology 21:234-239.
Quinn P.J. 1981. The fluidity of cell membranes and its regulation. Progress in Biophysics and
Molecular Biology 38:1-104.
Robertson R.N. 1983. The lively membranes. Cambridge University press – Cambridge – UK.
Woelders H., Chaveiro A. 2005. Theoretical prediction of ‘optimal’ freezing programmes. Cryobiologie
49:258-271.
230

ADDENDUM II
DETAILED LOADING TECHNIQUE OF THE DIFFERENT TYPES OF COUNTING CHAMBERS
USED TO ANALYZE EQUINE SEMEN SAMPLES WITH CASA
1. Disposable Leja, 10 µm depth, 4 chambers per slide (Leja10) – Each chamber has a volume of
1 µl, the chamber was loaded with 2.5 µl of diluted semen as described by the manufacturer.
Briefly, the sample was placed carefully within the boundaries of the loading area so that the
chamber was filled by capillary force. Once the sample reached the air vent at the other end
of the chamber, the excess semen was carefully removed with a tip of a kimwipe tissue to
prevent floating of cells. The filled chamber was left to rest for 10 seconds after which
analysis was started.
2. Disposable Leja, 12 µm, 2 chambers per side (Leja12) – Each chamber has a volume of 3µl
and was loaded with 5µl as described above.
3. Disposable Leja, 20 µm, 4 chambers per slide (Leja20) – Each chamber has a volume of 2 µl
and was loaded with 5µl as described above. The filling time (time from pushing down the
piston until the sample reached the air vent at the other end of the chamber) was recorded
as described in the loading instructions, in order to be able to correct for the Segre Silberberg
(SS) effect (Leja20SS).
4. Disposable ISAS, 12 µm, 4 chambers per slide (ISAS12d) – The volume of the disposable ISAS
chambers is not mentioned on the slide or box. The chambers were loaded in a similar way
as described for the Leja slides. Briefly, the loaded tip (5µl) of the pipette was carefully
placed within the boundaries of the loading area, taking care not to touch the cover slip to
prevent premature filling. The piston was pushed down allowing the chamber to be filled
with capillary force, taking care that the sample stayed within the borders of the loading area.
Once the sample reached the opposed air vent, excess semen form the loading area was
231

ADDENDUM II
232
removed by means of a kimwipe. The filled chamber was left to rest for 10 seconds after
which analysis was started.
5. Disposable ISAS, 16 µm, 4 chambers per slide (ISAS16d) – The chamber was loaded as
described for 12µm ISAS slide.
6. Disposable ISAS, 20 µm, 4 chambers per slide (ISAS20d) – The chamber was loaded as
described for 12µm ISAS slide.
7. Reusable ISAS, 20 µm, loaded like a Makler chamber (ISAS20rM) – The chamber was loaded
with 5 µl of the sample by reverse pipetting, immediately followed by placing the special
cover slide (24mm by 24mm, and 400µm thick) and the metal weight to assure a chamber
depth of 20 µm. The loaded chamber was left to rest until the drifting had ceased (between
10 and 20 seconds).
8. Reusable ISAS, 20 µm, loaded by capillary force (ISAS20rC) – The chamber was assembled by
placing the cover slide (24mm by 24mm, and 400µm thick) and the metal weight after which
the chamber was loaded with 5 µl of diluted semen by capillary force. The filled chamber was
left to rest for about 10 seconds until drifting had stopped, after which analysis was started
9. Reusable Makler, 10 µm (Makler) – The Makler chamber was loaded with 5 µl of diluted
semen (Matson et al., 1999) by reversed pipetting and the cover glass was applied
immediately. The filled chamber was left to rest for 10 seconds after which analysis was
started
10. Slide prepared according to WHO guidelines (WHO) – A fixed volume of 10 µl of the diluted
semen was placed on a clean glass slide by reversed pipetting. A 22mm by 22mm coverslip
was applied immediately afterwards, taking care to avoid formation and trapping of air
bubbles. The slide was evaluated as soon as the contents stopped drifting (WHO manual,
2010).

In m’n hoofd is alles heel eenvoudig
In m’n hoofd valt alles op z’n plaats
Raymond van het Groenewoud

In m’n hoofd is alles heel eenvoudig
In m’n hoofd valt alles op z’n plaats
Raymond van het Groenewoud