The Predictive Value of Semen Analysis in the Evaluation of Stallion Fertility

The Predictive Value of Semen Analysis in the Evaluation of Stallion Fertility

B Colenbrander1, BM Gadella2 and TAE Stout1

1Departments of Equine Sciences and 2Biochemistry and Cell Biology, Faculty of Veterinary Medicine,Utrecht University, Utrecht, The Netherlands

Contents

Pregnancy rates in managed horse populations depend on theinnate fertility of the mares and stallions involved and on thequality of breeding management. Of course, because a singlestallion usually mates many mares, stallion fertility is a criticalfactor in the overall success of a breeding program. Unfortu-nately, accurate evaluation of stallion fertility per se requires alarge number of normal mares to be mated and is necessarilyretrospective. Rather, the ideal is to predict fertility in advanceof the stallion’s breeding career, and this is currently attemptedby way of a thorough physical examination and a routineanalysis of semen quality. However, while such a ‘breedingsoundness examination’ identifies stallions that clearly lack thecapacity for adequate fertility, it is of limited use for predictingthe level of fertility and fails to identify some seriously sub-fertile animals. Similarly, while various sperm function tests(e.g., sperm head morphometry, the hypoosmotic swelling test,glass wool-sephadex filtration, progesterone receptor expo-sure) have been shown to correlate fairly well with fertility inthe field, most examine only a single or a narrow range of theattributes that a sperm must possess if it is to fertilize an oocytein vivo, and are thus more useful for identifying specific causesof sub-fertility than for predicting the level of fertility. On theother hand, combining the results of the various spermfunction tests does improve the reliability of fertility estimationand current research is therefore concentrated on identifying arange of tests that covers as many important sperm attributesas possible but that can be performed rapidly and cheaply. Inthis respect, flow-cytometry has proven to be an ideal toolbecause it allows the objective, rapid and simultaneousanalysis of a number of properties in a large number ofsperm. Moreover, stains are available for an increasing rangeof sperm characteristics including viability, capacitation andacrosome status, mitochondrial activity and chromatin integ-rity. Flow-cytometric analysis of sperm with appropriateprobes thus offers considerable promise for the prediction ofstallion fertility.

Introduction

Evaluating the fertility or ‘fertility potential’ of a stallionis an important part of sire selection and of breedingmanagement. In addition, knowledge of past fertilityand semen quality can be invaluable in the investigationof problems, or suspected problems, with fertility. Ofcourse, the true indices of fertility are the pregnancy andfoaling rates, however both are retrospective and areinfluenced dramatically by factors extrinsic to thestallion, such as mare quality and breeding management(Sullivan et al., 1975; van Buiten et al., 1998; Morrisand Allen, 2002). Furthermore, in many circumstances,a prospective test is desired so that likely (sub)-fertilitycan be identified before a stallion embarks on hisbreeding career. Within this context of selecting stallionsfor ‘breeding soundness’, it has become accepted that a

combination of a thorough physical examination andconventional semen evaluation provides a useful alter-native to actual fertility data (Kenney et al., 1983;British Equine Veterinary Association, 1991). However,while poor semen quality is a good indicator of sub-fertility, good semen quality (in terms of sperm number,motility and morphological normality) is no guaranteeof acceptable fertility. For this reason, considerableeffort is being invested in identifying markers forfunctional sperm capacity that can more accuratelypredict a stallion’s fertility. This is no easy task giventhat any one test is likely to measure only one, or alimited range, of the many attributes that a sperm mustpossess if it is to fertilize an oocyte. Nevertheless, manyrecently developed tests offer promise in identifyingabnormalities inconsistent with ‘normal’ fertility, and acombination of the available tests should identify mostsubfertile stallions, and may eventually be fine-tuned togive a true prospective index of fertility (Graham, 2001).This review aims to summarize the most appropriateindices of stallion fertility and to examine the utility ofstandard or newly developed semen quality tests foridentifying subfertility or for predicting the level offertility in stallions.

Measuring Stallion Fertility

Measuring stallion fertility is not an exact science and allof the indices used have obvious shortcomings. Forexample, while the per season and per cycle foaling rates(i.e. the number of foals produced as a percentage of themares mated) are often considered the ultimate meas-ures of stallion fertility, they are influenced strongly bynon-stallion factors such as the age and reproductivestatus of mated mares and the intensity and quality ofveterinary management (Sullivan et al., 1975; van Bui-ten et al., 1998; Morris and Allen, 2002). Using the firstcycle foaling rate instead of the all cycles or seasonalrates, will remove some of the bias attributable to maresubfertility, but the foaling rate remains a retrospectivemeasure for which data are not available until a yearafter the stallion has begun to mate. A quicker way ofassessing fertility is to examine the pregnancy rate (firstcycle or per cycle), which has the advantages of a shorterinterval until figures are available and of giving a‘rolling’ indication of fertility during the breedingseason; this can be a useful early warning system for adeveloping fertility problem. However, if the figures areto give an accurate indication of fertility, data must becollected from a relatively large number of normalyoung mares. The day 14–18 pregnancy rate will alsoexclude later pregnancy losses where it is possible, if

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unusual, for a stallion to be a major source of pregnancyloss because of sperm chromosome or chromatinabnormalities (Kenney et al., 1991). The ‘non-return’percentage is another parameter often used as an earlyindex of fertility in farm animal species in whichpregnancy examination can only be performed reliablyafter the time of the next expected oestrus, and in horseswhen mares are not routinely returned for early preg-nancy diagnosis (e.g. Shetland ponies: van Buiten et al.,1999). The non-return values suffer from the obviousdisadvantage that mares may not be returned for matingfor reasons other than becoming pregnant. In otherrespects, the non-return percentage gives similar infor-mation to the pregnancy rate.

Stallion Fertility in Practice

Stallions are selected for breeding primarily on the basisof their pedigree and their athletic prowess, or otherphenotypic characteristics. Fertility or fertility potentialare usually at best secondary considerations, and inmost cases assessment is limited to selecting out stallionsthat clearly do not possess the characteristics necessaryfor reasonable fertility. In short, the animals rejected asunsatisfactory breeding prospects are mostly those thathave or would have ‘selected themselves out’ by failingto get mares pregnant at an acceptable rate. By contrast,in other domestic livestock species the ultimate goal isproductivity and, because reproductive performance isan integral part of productivity, selection of males forgood fertility is much more rigorous. This is reflected inthe high and consistent per cycle fertility figures seen forrams (per cycle pregnancy rates of 80–90%; Menzies,1999) and boars (per cycle pregnancy rates of 85–90%;Colenbrander et al., 1993). Per cycle pregnancy rates forstallions tend to be both lower (43–60%; Woods et al.,1987; Bruck et al., 1993; Morris and Allen, 2002) andconsiderably more variable between individuals(35–90%; Morris and Allen, 2002). And while experi-mental studies demonstrate that fertilization rates canreach high levels in mares mated at the appropriate timewith semen from a stallion of proven fertility (>90%;Ball et al., 1989), the discrepancy between experimentalfertilization rates and pregnancy rates in the fieldappears to be explained primarily by deficiencies installion fertility and management, and only partially bypregnancy loss (Jasko et al., 1992).That stallions with relatively poor inherent fertility

are tolerated is because of two major factors; the goalsof horse breeding and our ability, at least in some cases,to improve fertility by more intensive veterinary man-agement. While the ideal is to get a mare pregnant asearly in the season as possible, the bottom line is toestablish pregnancy within the season and to produce afoal the following year; even then, relatively low live foalrates may be accepted if the stallion is considered worthpersevering with by dint of an exceptional (and herit-able!) phenotype. In general, an end of season preg-nancy rate of 80% and a foaling rate of 70–80% areconsidered satisfactory, and can be achieved within fourcycles per mare by a stallion with a per cycle pregnancyrate as low as 35%, assuming normal rates of pregnancyloss. Management also plays a critical part in reproduc-

tive performance and good, intensive management cansubstantially improve the ‘apparent’ fertility of manypoorly performing stallions, primarily by ensuringinsemination closer to the time of ovulation. We arewell acquainted with the need to inseminate frozen-thawed stallion semen as close as possible to the time ofovulation to compensate for its reduced longevity(Samper, 2001) and a similar approach appears to benecessary to achieve acceptable pregnancy rates withfresh semen of certain stallions. On the other hand, forother stallions, pregnancy rates with frozen-thawedsemen can be just as good when AI is repeated at 24 h(Vidament et al., 1999) or 48 h (Knaap et al., 1999)intervals during oestrus instead of attempting toinseminate as close as possible to ovulation. All inall, it is increasingly clear that the general guidelines ofup to 48 h (fresh semen) and 12 h (frozen semen)insemination-to-ovulation intervals and 300–500 mil-lion motile sperm (Householder et al., 1981) per AIdose, do not fully take account of the great inter-stallion variation in requirements for good fertility. Ofcourse, it is impractical to try to determine themaximum insemination-to-ovulation interval and mini-mum sperm number needed for good fertility for everystallion. Nevertheless, retrospective analysis of preg-nancy rates as a factor of the insemination-to-ovulationinterval can provide valuable information for futurebreeding management.

The Breeding Soundness Examination as a ProspectiveIndicator of Fertility

The aim of the breeding soundness examination (BSE) isto determine whether a stallion shows the requirementsnecessary for ‘adequate’ fertility. As such, the BSE isusually performed before the start of a stallion’sbreeding career and covers all aspects of potentialfertility including libido, mating ability, testicle size,sperm production capacity and semen quality (motilityand morphological normality) and, where available,past reproductive performance (Colenbrander et al.,1992). As it appears that semen quality in stallions isheritable (Parlevliet et al., 1994), it should be possible toimprove average semen quality by adherence to strictselection criteria.In terms of the ability of the standard BSE to predict

stallion fertility, while sperm production capacity doesnot correlate closely with fertility (Parlevliet andColenbrander, 1999), it does give valuable informationwith regard to the number of mares a stallion can cover,or insemination doses he can produce, per day.Exceeding these limits, will negatively affect apparentfertility. Jasko et al. (1992) performed the most com-prehensive study of the relationship between conven-tional semen quality parameters and fertility and,although they found reasonable correlations betweenthe percentages of motile (r ¼ 0.40), progressively motile(r ¼ 0.46) and morphologically normal (r ¼ 0.36) spermwith fertility, they also reported that variation in thesecharacteristics accounted for only 20% of the totalvariation in fertility. Thus, the standard BSE remainsstrictly a means for selecting out obviously unsatisfac-tory breeding prospects. In this respect, the general rule

306 B Colenbrander, BM Gadella and TAE Stout

of thumb used is that fertility will definitely becompromised when levels of progressive motility ormorphological normality drop below 40%. Based onthis premise, the Dutch warmblood studbook setsthreshold values of 50% for both of these parameters;the fact that only 8% of stallions are classified asunsatisfactory breeding prospects using these figures(Parlevliet et al., 1994) emphasizes that the BSEexcludes only stallions with significant problems.

New Approaches to Fertility Prediction

Given the limitations of the standard BSE in predictingfertility or even in identifying all seriously sub-fertilestallions, many different approaches have been inves-tigated in the hope of finding one relatively straight-forward and inexpensive test that correlates closelywith fertility. The limitation of this approach is thatmost tests only assess a limited number of the attributesthat a sperm must possess to fertilize an egg, and willthus tend to identify stallions with an obvious problemfor that parameter while failing to recognize stallionswith a catastrophic abnormality in another compart-ment. For this reason, combinations of tests are likelyto offer more promise for reliable identification of sub-fertile animals and for the generation of an expectedfertility level.

Computer-assisted semen motility analysis

In theory, computerized analysis of sperm motilityshould offer a more reliable, unbiased and repeatablemeans of assessing sperm motility than examination byeye. In addition, it allows analysis of additional aspectsof the way in which a sperm moves, which may relateeither to its activation status (Rathi et al., 2001) or itsability to reach and penetrate an oocyte. In practice,computer-assisted semen motility analysis (CASA) ofsperm has proven to correlate with fertility of fresh(Jasko et al., 1992) and frozen (Samper et al., 1991)semen no better than standard motility analysis. How-ever, Quintero-Moreno et al. (2003) suggested recentlythat the failure to find a significant relationship betweenCASA motility characteristics and fertility was due moreto inappropriate use of the data than actual absence of arelationship. They used multivariate analysis of CASAdata to differentiate sperm into four different sub-populations, as described previously for boar andgazelle sperm (Abaigar et al., 1999), and tentativelyconcluded that the category with the highest degree ofprogressive motility represented sperm with the highestfertilizing potential; unfortunately, data that prove arelationship between the different sperm sub-popula-tions and fertility have yet to be generated.

Computer assisted sperm head morphometry

Computerized technology can also be used to analyse theshape of sperm heads (sperm head morphometry). In thisrespect, Gravance et al. (1996) reported that subfertilestallions had a significantly lower percentage of spermwith heads that conformed to the morphometric norm(19% vs 52% for fertile stallions), and suggested that

sperm head morphometry was a useful adjunct to routinesemen analysis for the prediction of fertility.

Hypoosmotic swelling test

The hypoosmotic swelling test (HOS) is a means ofinvestigating membrane integrity in sperm and, assuch, is an alternative to supra-vital staining. In fact,the HOS test is thought to have the advantage ofindicating not only whether the membrane is intact butwhether it is osmotically active. When exposed to anhypoosmotic solution, sperm with an intact, functionalmembrane swell to establish an osmotic equilibrium,this is seen as a characteristic swelling of the sperm tail(Nield et al., 1999). The HOS test is simple to performand, for man, has been reported to correlate highlywith other predictive tests for fertility, such as hamsteroocyte penetration (Jeyendran et al., 1992) and in-vitrofertilization (IVF) results (van der Venn et al., 1986).In boars, a modified HOS test correlated fairly wellwith pregnancy rates (r ¼ 0.43); certainly better thaneither motility or acrosome integrity (Perez-Llanoet al., 2001). While the HOS test has also beenvalidated for use on stallion sperm (Nield et al.,1999; Nie and Wenzel, 2001) and has proven to besimple and repeatable, it is not yet clear how closelythe findings correlate with fertility. It seems most likelythat, as in boars (Perez-Llano et al., 2001), the HOStest will identify a small population of subfertilestallions that would not have been identified byconventional semen analysis without offering muchpredictive value in other animals. The HOS test may infact be more appropriate for predicting the fertilizingcapacity of frozen-thawed than fresh semen, given thatmembrane damage is a more important limiting factorin the former.

Capacitation and the acrosome reaction

In order to develop fertilizing capacity, ejaculatedsperm cells must, after their arrival in the femalegenital tract, be activated in a process called capacita-tion. Capacitation involves a delicate modification ofthe plasma membrane molecules that, in turn, enablesthe sperm to bind to the zona pellucida (ZP). Subse-quently, the ZP primes the sperm cell to initiate theacrosome reaction (AR), an exocytotic event that isrequired for ZP-penetration; only capacitated spermcan bind to the ZP in the fashion required to triggerthe later events of fertilization (Yanagimachi, 1994).The membrane changes that occur during capacitationin stallion sperm include changes in the lateralheterogeneity of the phospholipids, partial depletionof cholesterol (Gadella et al., 2001), scrambling ofphospholipids (Rathi et al., 2001) and progesteronereceptor exposure (Cheng et al., 1998a); all of theseevents precede and enable the AR. In essence, there aretwo ways of using capacitation and AR analysis in theprediction of fertility. The first is to assess the numberof acrosome reacted and capacitated sperm in un-treated semen; the former are no longer capable offertilization while the latter are thought to suffer fromreduced subsequent longevity (Watson, 1995). The

Semen Analysis and the Evaluation of Stallion Fertility 307

alternative approach is to examine the effect ofcapacitation or AR inducing agents, to determinewhether the sperm are able to undergo these essentialbiological changes. The latter approach seems to bemore informative with regards to prospective analysisof the fertilizing capacity of fresh semen, while theformer may be most useful in assessing the integrity offrozen-thawed semen.

(i) Chlortetracycline (CTC) staining: Chlortetracyclineis a fluorescent antibiotic that binds to the surface ofsperm cells in a calcium dependent fashion anddistinguishes three different stages of sperm activation,namely non-capacitated, capacitated acrosome-intactand capacitated acrosome-reacted. However, there issome debate as to how closely the different patternsfollow the biological events of sperm activation, e.g.CTC staining seems to identify relatively late capaci-tation-related changes (Rathi et al., 2001). Further-more, because analysis is not amenable to flowcytometric analysis, it is relatively laborious and hasbeen largely superseded by stains that track changes insperm activation status more specifically. Nevertheless,there are reports that in some species (e.g. cattle;Thundathil et al., 1999) the proportion of viable non-capacitated sperm correlates to the fertility of frozen-thawed semen, presumably because the freeze-thawprocess induces capacitation-like changes that limitsperm longevity.(ii) Flow cytometric detection of capacitation and theacrosome reaction: CTC staining has largely beensuperseded by more specific probes for capacitation(e.g. merocyanine 540; a marker of plasma membranefluidity; Harrison et al., 1996) and the AR [e.g. fluo-rescein isothiocyanate conjugated peanut agglutinin(FITC-PNA) Cheng et al., 1996] whose presence canbe analysed flow cytometrically. Flow cytometry offersthe added advantage of allowing simultaneous evalua-tion of sperm viability by inclusion of a membraneimpermeable DNA dye such as propidium iodide (Rathiet al., 2001). The combination of stains allows differen-tiation between non-activated, capacitated/acrosomeintact and capacitated/acrosome reacted, viable sperm.In addition, flow cytometry allows for the very rapidand objective discrimination of large numbers of sperm(e.g. 10 000 in a few seconds). As with CTC staining,assessing capacitation and AR status in unstimulatedsperm is likely to be most appropriate to estimating thenumber of damaged sperm in a frozen-thawed sample,and there appears to be little difference in the rate ofspontaneously acrosome reacted sperm in fresh semenfrom fertile or infertile boars (Herrera et al., 2002) orstallions (Meyers et al., 1995). On the other hand,Meyers et al. (1995) reported that fertile stallionsshowed higher rates of AR in response to progesteronetreatment than subfertile stallions. Similarly, we haveexamined a number of stallions referred for the inves-tigation of poor fertility despite apparently good semenquality; many showed a failure of the sperm to capac-itate and/or acrosome react at normal levels in responseto priming with bicarbonate and progesterone (unpub-lished observations). Thus, the ability to AR in responseto an inducing agent appears to be an important

indicator of fertilizing capacity, but it is not yet clearwhether this will serve only to identify a specificpopulation of subfertile stallions or whether it will offerfurther predictive value.

Glass wool/sephadex filtration

A further technique reported for identifying the pro-portion of sperm that are capacitated or that havesuffered membrane or acrosome damage is the use of afilter composed of sephadex gel and glass wool (Samperet al., 1991). These authors reported a high correlationbetween the percentage of sperm passing through glasswool/sephadex or just sephadex filters and the fertility offrozen (r ¼ 0.93 and 0.84, respectively) and, to a lesserextent, fresh stallion semen (r ¼ 0.86 and 0.64). As theirdata suggested, this technique is probably more appro-priate for evaluating the quality of frozen-thawed spermwhere the percentage of sperm showing advanced capaci-tation-like changes or membrane damage is likely to behigher.

Sperm mitochondrial function

Analyzing mitochondrial function offers a means ofassessing the ‘motility potential’ of sperm. This can beperformed with stains that attach preferentially to thedepolarized membrane of active mitochondria, such asrhodamine 123 and JC-1, where the latter is preferredbecause it more clearly separates sperm into levels ofmitochondrial function (Troiano et al., 1998; Graham,2001). In man, there is some evidence of a correlationbetween poor sperm mitochondrial function, diminishedmotility and reduced fertility (Troiano et al., 1998). Todate, there is very little information on the correlationbetween fertility and sperm mitochondrial activity installions, but the staining patterns can be analysed flow-cytometrically and, therefore, have the advantage thatthey can be done simultaneously with the examinationof other properties such as viability, activation statusand DNA stability.

Sperm progesterone receptor exposure

It is thought that progesterone present in follicular fluidand secreted by the cumulus oophorus is a majorinducer of the AR in stallion spermatozoa and that itexerts its effects via specific progesterone receptorsfound on the plasma membrane of the sperm (Meyerset al., 1995; Cheng et al., 1998a). Physiologically, itappears that progesterone enhances both sperm–zonabinding and ZP-mediated AR induction (Cheng et al.,1998b). Moreover, sub-fertility in some stallions hasbeen correlated to an inability of their spermatozoa toundergo the AR in response to progesterone stimulation(Meyers et al., 1995). Similarly, Rathi et al. (2000)found that the percentage of spermatozoa in anejaculate with exposed progesterone receptors on theirplasma membrane after incubation in capacitatingconditions, was highly correlated with the fertility ofthe donor stallion (r ¼ 0.73–0.84 for different indices offertility). These authors suggested that progesterone


receptor exposure after capacitation is related toZP-binding capacity and, thereby, to fertility.

The sperm chromatin structure assay

The chromatin in a sperm cell is more highly condensedthan that in the nucleus of a somatic or a spermatogeniccell, and it has been proposed that this condensation is amechanism to protect the DNA from environmentalstress and mutagenesis (Ward and Coffey, 1991). It hasalso been proposed that alterations in chromatin struc-ture may affect the rate of decondensation, a prerequis-ite for male pronucleus formation during fertilization,and thereby disrupt embryo development (Perreaultet al., 1987). Indeed, in man, reduced sperm chromatinstability has been related to recurrent abortion (Ibrahimand Pedersen, 1988) and it is possible that a similarmechanism explains why, while fertilization rates inhorses appear to be very high, pregnancy rates areconsiderably lower and the early pregnancy loss rate ishigh (Ball, 1988). DNA stability can be assessed usingthe sperm chromatin structure assay (SCSA), which usesthe metachromatic properties of acridine orange to dis-tinguish between denatured (red fluorescence ¼ singlestranded) and native (green fluorescent ¼ double stran-ded) DNA in sperm chromatin (see Evenson et al.,1999). Strong correlations have been demonstratedbetween sperm chromatin denaturation and fertilityrankings in both bulls (r ¼ 0.94: Ballachey et al., 1988)and boars (r £ 0.93: Evenson et al., 1994). With regardto the discriminative ability of the SCSA, Evenson et al.(1999) reported that men with ‡30% denatured spermDNA were subfertile/infertile; in the same study, theproportion of men predicted to have a fertility problemthat actually had one, was 52%. The SCSA has beentested on stallions; Kenney et al. (1995) reported higherrates of sperm chromatin denaturation in semen fromsubfertile than fertile stallions (32% vs 16%) and anegative correlation between denaturation score andseasonal pregnancy rate. In a group of fertile stallions,Love and Kenney (1998) were able to demonstratevariations in SCSA that correlated moderately withboth seasonal and per cycle fertility rates; they suggestedthat the SCSA could be used to prospectively rankstallion fertility and to identify changes associated withimpending fertility loss.

Sperm–zona binding (hemi-zona assay)

A further test of sperm function is the ability to bind tothe extracellular matrix of an oocyte (the ZP), preferablyfrom the same species. However, considerable differencesexist between oocytes and, therefore, a hemi-zona assayis more useful because it allows a bona fide comparisonbetween test semen and semen from an animal of knownfertility. Fazeli et al. (1995) used the hemi-zona assay todemonstrate that the number of sperm binding to a hemi-zona was significantly higher for fertile than for subfer-tile stallions, even though no significant differences inconventional semen parameters were apparent betweenthe two groups of stallions compared. On the down side,equine oocytes are relatively difficult to obtain and thehemi-zona assay is laborious, not least because the zonae

must be bisected precisely if the comparison of boundsperm numbers is to be valid.An alternative oocyte-based approach to assessing

sperm fertilizing capacity is the zona-free hamster oocytepenetration test. However, even studies that havereported an association between hamster oocyte penet-ration and stallion fertility have noted anomalies such assubfertile stallions with high penetration rates (e.g.Wilhelm et al., 1996).

Conclusions

During the last two decades, improvements in veterinarymanagement have resulted in an increase in averagefertility rates in intensively managed horse populations(Morris and Allen, 2002). Nevertheless, it is clear thatbreeding stallions vary greatly in their per cycle fertilityrates and that some achieve the target pregnancy ratesper season despite marginal pregnancy rates per matedoestrous cycle, or thanks to very intensive breedingmanagement.Stallion fertility can be measured in various ways

(foaling, pregnancy or non-return rates per cycle, seasonor first cycle), but there is still a need to develop teststhat can predict fertility with a reasonable degree ofcertainty before the stallion has begun his breedingcareer. In the past, the ‘holy grail’ was thought to be thedevelopment of a single test that correlated stronglywith in-field fertility. However, as our knowledge of thecomplexity of sperm physiology and the array of attri-butes a sperm needs to successfully reach and fertilize anoocyte has increased, it has become evident that this isprobably a futile objective. The aim of most currentresearchers is instead to identify a combination of teststhat together analyse the most important sperm functionparameters, since combining sperm function tests allowsmore accurate prediction of fertility (e.g. Wilhelm et al.,1996). Given the simultaneous need to limit thecomplexity, labour and costs of fertility prediction if itis to become practiceable in the field, flow cytometry hasemerged as a favoured tool. Stains are now available forassessing sperm viability, capacitation and acrosomestatus, chromatin stability and mitochondrial function.Moreover, a range of properties can be assessedsimultaneously in a simple, rapid and objective manner.Future research is likely to focus more on whatcombination of sperm properties correlates best withfertility, bearing in mind that this is likely to differmarkedly depending on whether the semen is to be usedfresh or after freezing and thawing.

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Submitted: 17.03.2003

Authors’ address (for correspondence): B Colenbrander, Departmentsof Equine Sciences, Faculty of Veterinary Medicine, Utrecht Univer-sity, Utrecht, The Netherlands. E-mail: [email protected]


Documents

The Predictive Value of Semen Analysis in the Evaluation of Stallion Fertility