breeding-management strategies and semen- handling ... · breeding frequency is increased (table...

Breeding-Management Strategies and Semen-Handling Techniques for Stallions—CaseScenarios

Dickson D. Varner, DVM, MS, Diplomate ACT;Charles C. Love, DVM, PhD, Diplomate ACT;Terry L. Blanchard, DVM, MS, Diplomate ACT; David L. Hartman, DVM;Shalyn B. Bliss, DVM; Shelby S. Hayden, DVM; Justin Voge, DVM;Brian S. Carroll, DVM; Mark C. Eslick, DVM; andMargo L. Macpherson, DVM, MS, Diplomate ACT

Authors’ addresses: Department of Large Animal Clinical Sciences, College of Veterinary Medicineand Biomedical Sciences, Texas A&M University, College Station, Texas 77843 (Varner, Love,Blanchard, Hartman, Bliss, Hayden, Voge); Hartman Equine Reproduction Center, 15225 U.S.Highway 377, PO Box 915, Whitesboro, Texas 76273 (Hartman, Bliss); Oklahoma City Equine Clinic,400 Northeast 70th Street, Oklahoma City, Oklahoma 73105 (Carroll); Hagyard Equine MedicalInstitute, 4250 Iron Works Pike, Lexington, Kentucky 40511-8412 (Eslick); and Department of LargeAnimal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida32610-0136 (Macpherson); e-mail: [email protected]. © 2010 AAEP.

1. Introduction

Stallions become sires based on three basic quali-ties: pedigree, performance record, and conforma-tion. Although stallions represent 50% of thebreeding equation, the decision to retire a stallion tostud duty is made with little consideration to breed-ing capability or reproductive health. As such, theequine breeding industry abounds with stallionswhose level of fertility is less than optimal.

Undesirable fertility can emanate from physical,mental, or environmental aberrations that result indisruption of mating ability and inefficient sementransfer from the stallion to the reproductive tract ofthe mare. Oftentimes, subfertility results from dis-

turbances in testicular, epididymal, or deferent ductfunction. Some forms of subfertility may have agenetic basis, as evidenced by studies involvingother species.1,2 Genetic-related causes of reducedfertility are only now being investigated in stallions.Nonetheless, subfertility is often associated with ag-ing in stallions because of the poorly understoodeffects of age on testicular and/or epididymal func-tions. Effects of long-term medications, such asprogestogens or anabolic steroids, on testicularhealth and fertility of younger sires that have re-cently retired from a performance career must alsobe considered. Other environmental effects, suchas hot environmental temperature, fever, or genital

AAEP PROCEEDINGS � Vol. 56 � 2010 215

IN-DEPTH: REPRODUCTION

NOTES

trauma, can also induce temporary or in some in-stances, permanent subfertility or infertility.

Taken together, these scenarios rationalize theneed for veterinary intervention as a means to max-imize the fertility of stallions. Although ethicsshould be considered before applying assisted repro-ductive techniques that could propagate geneticforms of subfertility in stallions, it is difficult atpresent, except in isolated circumstances, to differ-entiate between heritable and non-heritable causesof reduced fertility. This paper addresses somebreeding and semen management strategies thatcan be applied in an effort to maximize the fertilityof breeding stallions, either in natural-cover or arti-ficial-insemination programs.

2. Natural-Cover Breeding Programs

Although stallions subjected to only natural-coverconditions cannot benefit from many laboratorytechniques that would likely improve their breedingperformance, one can implement methods to criti-cally assess their fertility and devise officially au-thorized strategies to enhance their productiveness.

Assessment of Breeding RecordsAssessment of breeding records for a stallion is alogical starting point when determining causes ofsubfertility, because critical analysis of theserecords will aid one in determining specific sourcesof a fertility problem (i.e., mare-related factors,management factors, and/or intrinsic stallion fac-tors).3 In most commercial programs, each of thesefactors contributes to the perceived fertility of agiven stallion.

Actual examples of mare factors that can impact astallion’s fertility are presented in Tables 1 and 2.As noted in Table 1, the pregnancy rate for thestallion represented was higher for the first cycle ofbreeding compared with the second cycle of breed-ing. This situation suggests that the mares thatdid not become pregnant during their first breedingcycle were less fertile than the mares that did be-come pregnant. This is a common finding in thecommercial setting, and the circumstance repre-sents a potential confounding factor when one isattempting to assess the intrinsic fertility of astallion.

Similarly, Table 2 shows that mare category cannegatively impact the perceived fertility of a stal-lion. Although pregnancy rate for the stallion rep-

resented is above average when bred to maiden andfoaling (wet) mares, fertility is lower than averagewhen the stallion is bred to barren mares. Thisagain shows that mare reproductive quality can con-found one’s interpretation of a stallion’s intrinsicfertility.

Management factors in commercial programs canalso negatively impact a stallion’s perceived fertility.For instance, Table 3 represents two stallions withgood fertility. The added effects of mare factors(determined by first- versus second-cycle pregnancyrate) and reduced opportunities to breed maresthrough the season (as determined by return rate fornon-pregnant mares) can lead to reduced seasonalfertility rates in stallions with good intrinsicfertility.

Other management factors can accentuate subfer-tility problems in stallions, and breeding records canhelp delineate how management efforts can be usedto improve, rather than hinder, breeding perfor-mance. Some stallions exhibit improved fertilitywhen bred more frequently, whereas other stallionsexperience a decrease in pregnancy rate whenbreeding frequency is increased (Table 4). Closeexamination of breeding records will be able to de-marcate these differences.

Table 1. Effect of Breeding Cycle on Pregnancy Rate for an IndividualThoroughbred Stallion

Parameter Value

Number of mares bred on first estrous cycle 133First-cycle pregnancy rate 62%Number of mares bred on second estrous cycle 33Second-cycle pregnancy rate 53%

Table 2. Effect of Mare Category (Maiden, Foaling, or Barren) on Preg-nancy Rate for an Individual Thoroughbred Stallion

Parameter Value

Number of maiden mares 12Total number of maiden mare estrous

cycles 17Per-cycle pregnancy rate in maiden

mares 59%Number of foaling (wet) mares 87Total number of foaling mare estrous cycles 127Per-cycle pregnancy rate in foaling

mares 62%Number of barren mares 21Total number of barren mare estrous cycles 42Per-cycle pregnancy rate in barren

mares 43%

Table 3. The Additive Effects of Mare Fertility and Breeding Opportu-nity (Management Factor) on Pregnancy Rate for Two ThoroughbredStallions

Stallion 1 Stallion 2

Number of mares bred on 1st cycle 133 135First-cycle pregnancy rate 63% 67%Number of mares bred on 2nd cycle 38 41Second-cycle pregnancy rate 53% 68%Percentage of mares that were

open on first breeding cyclethat were bred on a secondcycle 78% 91%

Seasonal pregnancy rate 86% 93%

216 2010 � Vol. 56 � AAEP PROCEEDINGS


In Table 4, Stallion 1 experienced an apparentimprovement in fertility as breeding frequency in-creased from one to three times daily, and preg-nancy rate also tended to increase from Session 1 toSession 3 on days when three mares were covered.Conversely, pregnancy rate declined precipitouslywhen the stallion was allowed 1–2 days of sexualrest between covers. Stallions like this are com-monly encountered when evaluating breedingrecords. Such stallions typically have large testesand tend to develop stagnant stores of sperm in theextragonadal ducts when not breeding frequently.Sperm numbers in ejaculates are generally not thelimiting factor for establishment of pregnancies.Management of this type of stallion in an effort tomaximize reproductive efficiency will include cover-ing of non-commercial mares to avoid periods ofsexual abstinence that will negatively impact breed-ing performance. This breeding strategy becomesespecially important in the days and weeks beforethe onset of the commercial breeding season, be-cause these stallions are generally quite susceptibleto a condition termed “plugged ampullae” after ex-tended periods of sexual rest.4 On the contrary,

Stallion 2 in Table 4 shows that fertility of somestallions is lowered as breeding frequency increases.As is evidenced with this stallion, pregnancy ratesare often improved in the last covering session of theday when such stallions cover multiple mares in aday. Possibly, mares in the last covering session ofthe day were being covered nearer to the time ofovulation, although the precise time of ovulationwas not determined. Stallions with this breedingscenario may be managed most effectively by limit-ing the number of mares that are covered per day sothat the stallion can ejaculate the threshold numberof normal sperm necessary to achieve an acceptablepregnancy rate. Alternatively, mares might be cov-ered closer to the time of predicted ovulation in anattempt to reduce the need for extended sperm lon-gevity in the mare reproductive tract.

Reinforcement BreedingThis breeding approach is becoming more widelyused in natural-cover programs; however, the tech-nique is not authorized by certain governances.As such, the technique should only be applied afterauthorization by the governing body of any breedregistry.

The technique involves collection of semen fromthe penis immediately on dismount of a stallion froma mare after an ejaculatory mating. The protocolrecommended by the authors is to maintain the dis-mount semen sample at body temperature, strain itthrough a filter to remove extraneous debris, andthen, mix the filtered semen with a small volume(5–10 ml) of warmed good-quality semen extender.The extended semen is then loaded into an all-plas-tic syringe, and the filled syringe is affixed to astandard insemination pipette. Covered mares areimmediately placed in stocks, and the perineal areais prepared for insemination of the extended dis-mount semen sample. As the pipette and handcourse through the vagina, any fluids grossly free ofurine or blood are also aspirated into the pipette,

Table 5. Effect of Reinforcement Breeding on Per-Cycle PregnancyRate for Five Stallions at a Thoroughbred Breeding Facility

StallionReinforcement

breedingPer-cycle

pregnancy rate (%)

ANo 56/146 (38%)*Yes 46/88 (52%)†

BNo 78/137 (57%)*Yes 56/77 (73%)†

CNo 86/164 (52%)*Yes 19/25 (76%)†

DNo 108/204 (53%)*Yes 12/24 (50%)*

ENo 93/179 (52%)*Yes 16/32 (50%)*

*†Within stallion, values with different superscripts differ(p�0.05)

Table 4. Effect of Breeding Frequency on Pregnancy Rate for TwoThoroughbred Stallions

Stallion 1 Stallion 2

Number of mares covered 79 126Per-cycle pregnancy rate on days

when 1 mare covered* 39% 46%Per-cycle pregnancy rate on days

when 2 mares covered** 48% 35%Per-cycle pregnancy rate on days

when 3 mares covered*** 52% 22%Per-cycle pregnancy rate on

Session 1 when 3 marescovered† 40% 18%

Per-cycle pregnancy rate onSession 2 when 3 marescovered‡ 50% 9%

Per-cycle pregnancy rate onSession 3 when 3 marescovered§ 67% 40%

Per-cycle pregnancy ratefollowing one day of sexualabstinence 25% 71%

Per-cycle pregnancy ratefollowing two days of sexualabstinence 18% 67%

*Average per-cycle pregnancy rate on days when stallion bredonly 1 mare

**Average per-cycle pregnancy rate on days when stallion wasbred to two mares

***Average per-cycle pregnancy rate on days when stallion wasbred to three mares

†Average per-cycle pregnancy rate for the 1st breeding sessionof the day on days when the stallion was bred to three mares

‡ Average per-cycle pregnancy rate for the 2nd breeding sessionof the day on days when the stallion was bred to three mares

§Average per-cycle pregnancy rate for the 3rd breeding sessionof the day on days when the stallion was bred to three mares



and then, the contents are discharged into the uter-ine body.

Table 5 provides data regarding five commercialstallions whose semen was sometimes used for rein-forcement breeding of mares during a single season.All of the stallions were located at the same breedingfacility. Three of five stallions (60%) experiencedan improvement in fertility when reinforcementbreeding was used compared with traditional breed-ing. One stallion, Stallion A, tended to dismountmares prematurely, and dismount semen samples ofthis stallion were known to contain up to 5 billionsperm. As such, the rationale for reinforcementbreeding was apparent. Stallions B and C in Table5 did not dismount prematurely during ejaculation,and therefore, the reason for any of the beneficialeffects of reinforcement breeding remains specula-tive. Improved longevity of sperm viability may bea possibility, because metabolizable substrate (glu-cose) was available in the semen extender. It isalso possible that simple deposition of sperm intothe uterus that would otherwise be lost for breedingpurposes may have resulted in colonization of theoviducts by increased sperm numbers. Othershave reported an improvement in fertility for a ma-jority (8/13; 62%) of Thoroughbred stallions afterreinforcement breeding.5

As another case study, a 4-yr-old Thoroughbredstallion received a kick in the groin by a mare duringhis first cover of the commercial season, resulting ina basal penile hematoma with associated swelling ofthe prepuce and sheath. No scrotal edema was de-tected, but the stallion was sexually rested for 13days before resuming breeding duty. The stallion’sresulting per-cycle pregnancy rate was 27% (14/51).Examination of the stallion revealed that testicularvolume was 300 ml (normal), but the collected ejac-ulate contained only 278 million sperm, with 45%being progressively motile and 44% being morpho-logically normal. Spermatogenic efficiency (esti-mated to be 15%) and the sperm morphologic profile(26% abnormal heads, 16% abnormal midpieces, and4% round germ cells) were consistent with ongoingtesticular dysfunction.6 Reinforcement breedingwas initiated with the stallion, yielding a 67% per-cycle pregnancy rate (14/21). The attending veter-inarian then elected to discontinue reinforcementbreeding, and the resulting pregnancy rate was 10%(1/10). Reinforcement breeding was resumed, andper-cycle pregnancy rates for the next 3 mo of thebreeding season were 58% (35/60), 57% (41/72), and58% (14/24), respectively. Although additionalbreeding soundness examinations were not per-formed, the stallion apparently recovered from theeffects of the breeding accident, because he achieveda 58% per-cycle pregnancy rate when bred to 117mares during the next breeding season and a 57%per-cycle pregnancy rate when bred to 200 maresduring the following breeding season. Althoughthe beneficial effects of reinforcement breeding arecircumstantial, this case study suggests that rein-

forcement breeding improved the pregnancy rate ina stallion whose fertility was temporarily impairedby a breeding accident.

3. Artificial-Insemination Programs

Approval of artificial insemination by most horseregistries has resulted in commercial application ofa multitude of assisted reproductive technologies tomaximize the reproductive efficiency of stallions.Two relatively new strategies that are becomingmore commonplace in equine breeding programs in-volve advancements in centrifugation of semen(cushioned centrifugation and gradient centrifuga-tion) and application of deep-horn low-doseinsemination.

Cushioned Centrifugation of SemenCentrifugation of semen is oftentimes applied inequine breeding programs, and therefore, the valueof improving centrifugation procedures to maximizesperm harvest, without associated sperm injury, be-comes apparent. Development of a more efficientcentrifugation method (i.e., one that increases post-centrifugation recovery rate while maintaining se-men quality) would optimize the number of sperm inan ejaculate that are available for breeding pur-poses. One of the main concerns, when attemptingto maximize sperm recovery through centrifugation,is the adverse effect that centrifugation can have onthe integrity of sperm. Typically, an increase incentrifugation time or gravitational (g) force resultsin an increased sperm recovery rate, but it can alsolead to decreased sperm motility or quality becauseof the mechanical forces associated with centrifuga-tion and excessive packing of the sperm.7–9 Ideally,centrifugation should result in a 100% sperm recov-ery rate with no resulting damage in sperm quality.Previous studies have used a variety of centrifuga-tion forces and times in an attempt to achieve thisgoal7–14; however, such protocols can lead to a 15–20% loss of sperm that could otherwise be used forbreeding purposes.

Recently, a cushioned centrifugation procedurehas been applied to stallion semen to maximizesperm harvest without attendant injury to sperm.A non-ionic iodinated compound, iodixanol, was firstreported for density-gradient cell fractionation15–17

and has since been used as either a density gradientor a cushion for centrifugation of sperm.18–25 In-vestigations regarding cushioned centrifugation ofstallion semen with this product showed excellentyields of sperm that were undamaged by the centrifu-gation process, but an optically clear centrifugationmedium was required to reduce sperm losses.18,20,23,24

Results from our laboratory indicate that cush-ioned centrifugation of stallion semen in either con-ical-bottom tubes containing 3.5 ml of iodixanolsolution as a cushion (Fig. 1) or nipple-bottom tubescontaining 30 �l of iodixanol solution as a cushion(Fig. 2) can yield a high sperm harvest while main-taining sperm function. Additionally, an optically



opaque extender, as is typically used in the equinebreeding industry, can be used to achieve this goal.26

We recommend the nipple-bottom tubes over the50-ml conical-bottom tubes for cushioned centrifu-gation when the sperm number in ejaculates arerelatively low (i.e., less than 2–3 � 109 sperm) orwhen it is necessary to separate more seminalplasma from sperm after centrifugation than is pos-sible with cushioned centrifugation in conical-bot-tom tubes. Recently, we have found that thevolume of iodixanol solution can be reduced from 3.5to 1 ml in conical-bottom tubes without impairingsperm harvest or semen quality.b

Gradient Centrifugation of SemenBoth continuous and discontinuous density centrif-ugation gradients have been used to separate spermpopulations.27–30 Continuous centrifugation gradi-ents may be more sensitive for cell selection thandiscontinuous density gradients, but discontinuousgradients are useful when separating cell types withknown densities, such as sperm.31 Application of

discontinuous density centrifugation gradients forsperm selection has had broad clinical application inrecent years, because these gradient procedures arerelatively simple to perform and have been shown toeffectively separate sperm with various morpholog-ical features in an ejaculate.32,33

Using different concentrations of colloidal silicaparticles to form density gradients, cells are sepa-rated by specific gravity into different layers basedon isopycnic point. Density gradients are advanta-geous over other methods of sperm separation,because the media does not penetrate cell mem-branes.31 Furthermore, colloidal silica does not os-motically stress sperm when added to culturemedium; it can be formulated to create a high spe-cific gravity to separate dense cells, and it has a lowviscosity so as to not impede sperm-cell sedimenta-tion.30 The most well-known discontinuous densitygradient, commercialized as Percoll, contains colloi-

Fig. 2. Glass nipple-bottom tube (40-ml capacity) showing a spermpellet at the bottom of the tube after centrifugation. A clear ex-tender was used to aid in showing the sperm pellet. Clear iodixa-nol solution (30 �l; Cushion Fluid) is directly underneath the spermpellet.

Fig. 1. Plastic conical-bottom tube (50-ml capacity) showing asperm pellet at the bottom of tube after centrifugation. A clearextender was used to aid in showing the sperm pellet at the 5-mlmark on the centrifuge tube. Clear iodixanol solution (3.5 ml;Cushion Fluida) is directly underneath the sperm pellet.



dal silica particles coated with polyvinylpyrrolidone(PVP). PVP coating is used to protect cells from thepotentially toxic actions of colloidal silica.31 Percollwas the most widely used discontinuous density gra-dient in clinical reproductive medicine until the mid-1990s when reports of endotoxin contamination ledto removal of this product for clinical use. As aresult, alternative discontinuous gradient solutionssuch as polysucrose and iodixanol combined with(IxaPrep™c) and colloidal silanized silica particles(PureSperm™d and Isolate™e) were investigated forclinical application in assisted reproductive tech-niques.33–37 Colloidal solutions with silanized sil-ica particles have been used clinically as a human-assisted reproductive technology with successcomparable with Percoll.34

Centrifugation of equine semen through a si-lanated silica-particle solution (EquiPure™f) hasshown promise for selecting sperm with good motil-ity, morphology, and chromatin quality (Fig. 3),38,39

and enhancing the fertility of selected subfertilestallions.6 Our laboratory has found that spermrecovery rate is higher when using 15-ml capacityconical-bottom tubes compared with 50-ml capacityconical-bottom tubes,39 that use of a one-layer (Equi-Pure Bottom Layer™g) gradient yielded a highersperm recovery rate than a two-layer gradienth, andthat gradient volumes of 2, 3, or 4 ml in 15-mlcentrifugation tubes yielded similar semen qualityand sperm recovery.39

Centrifugation of semen through a silica-particlesolution, such as EquiPure™, is not a logical ap-proach for stallions with normal semen quality, be-cause a relatively high percentage of the spermpopulation can be lost after centrifugation. Be-cause this technique results in sperm separationbased on sperm buoyancy or isopycnic point, its useis most justified when an ejaculate contains a highpercentage of sperm with morphologic defects, spe-cifically sperm with abnormal heads, abnormal mid-pieces, bent midpieces, bent tails, coiled tails, orpremature (round) germ cells. However, we havefound that the technique will also improve chroma-tin quality in the recovered sperm population, re-gardless of sperm morphologic profile. Thetechnique can also be used when more completeseparation of seminal plasma from sperm is desired.

Low-Dose Insemination

The threshold number of sperm that can be used toinseminate mares and yield a commercially accept-able pregnancy rate is not known, but this number iscertainly impacted by the fertility of a given stallionand the semen-processing method that is applied(e.g., cooled storage or crypreservation) before in-semination. Using customary methods, mares areinseminated with 200–500 � 106 progressively mo-tile sperm deposited directly into the lumen of theuterine body when fresh semen is used.40–42 How-ever, one recent study revealed no difference be-

Fig. 3. Plastic conical tube (15-ml capacity) showing a spermpellet at the bottom of the tube after centrifugation through anEquiPure™ two-layer gradient. For this procedure, the centrif-ugation tube was loaded with 2 ml EquiPure™ Bottom Layerfollowed by 2 ml EquiPure™ Top Layer and 1 ml extended semencontaining approximately 500 � 106 sperm. The sperm pellet atthe bottom of the tube contains the sperm population most free ofmorphologic defects, whereas the thin white line at the interfaceof the bottom and top layers of EquiPure™ contains sperm morelikely to possess morphologic defects. The thick white line over-lying the clear EquiPure™ layers contains milk-based extenderas well as somatic cells and premature germ cells. This layermay also contain sperm with pronounced morphologic defectsthat would result in markedly reduced buoyancy. Note that thebottom sperm pellet contains fewer sperm than that in Figure4. This semen was from a subfertile stallion with poor semenquality.4



tween insemination doses of 50 or 300 � 106

progressively motile fresh sperm.43

Rigby et al.44 showed that only 0.0007% of spermthat are deposited into mare uteri actually gain ac-

cess into the oviducts and are available for fertiliza-tion of an oocyte. These same investigators,however, showed that insemination in the tip of theuterine horn ipsilateral to an ovary containing adominant follicle resulted in a greater percentage ofoviductal sperm (77%) in the ipsilateral oviduct com-pared with uterine-body insemination (54%).44

These data indicate that more sperm gain accessinto the oviduct of fertilization when the insemina-tion location is the tip of the uterine horn as opposedto the uterine body. Morris et al.45 showed thatexcellent pregnancy rates could be achieved byvideoendoscope-assisted placement of as few as 1 �106 sperm on the oviduct papilla of pre-ovulatorymares when the semen was first centrifuged thougha discontinuous Percoll density gradient.

This breeding technique, termed deep-horn low-dose insemination, has been examined in the re-search setting and applied clinically in recent years.Two techniques are most commonly used for depo-sition of an insemination dose on or near the oviduc-tal papilla: (1) use of a videoendoscope (termedhysteroscope) to visually locate the papilla and per-mit accurate placement of semen through a longcatheter passed through the biopsy channel of theinsertion tube, and (2) use of a flexible catheter(usually double-lumen) in which the catheter tip isguided to a position adjacent to the papilla by ma-nipulation per rectum before deposition of semen.The optimal method for low-dose insemination ofmares is subject to debate.40–43 One investigatorstated that hysteroscopic insemination may be jus-tified over transrectally guided insemination, unlessa stallion’s fertility was excellent or sperm numberexceeded 25–50 � 106 in the insemination dose.46

Others indicate that hysteroscopic inseminationmay yield better results than transrectally guidedinsemination if the insemination dose contains lessthan 5 � 106 progressively motile sperm.41 Recentfindings in our laboratory revealed no significantdifference between the two methods when mareswere inseminated with as little as 0.5–1.0 � 106

sperm from a stallion with known good fertilityi.Certainly, the hysteroscopic technique has the dis-advantages of increased equipment costs, laborforce, and procedure time. As such, the transrec-tally guided approach would seem to lend itself morefavorably to widespread application in the equinebreeding industry. Heightened reproductive skillof the attending veterinarian, however, is necessaryfor this latter technique to be successful.

The value of low-dose insemination for improvingsubfertility in stallions has been questioned, al-though successful results with this breeding strat-egy exist.6,47 These reports involved eitherhysteroscopic or transrectally guided approaches forlow-dose insemination.

Clinical Case 1

A 12-yr-old Quarter Horse was admitted to theTexas Veterinary Medical Center for assessment of

Fig. 4. Plastic conical tube (15-ml capacity) showing a spermpellet at the bottom of the tube after centrifugation through anEquiPure™ one-layer gradient. For this procedure, the centrif-ugation tube was loaded with 3 ml EquiPure™ Bottom Layerfollowed by 1 ml extended semen containing approximately 500 �106 sperm. The sperm pellet at the bottom of the tube containsthe sperm population most free of morphologic defects, whereasthe thick white line overlying the clear EquiPure™ layers con-tains milk-based extender as well as somatic cells, prematuregerm cells, and sperm more likely to possess morphologic de-fects. Note that the bottom sperm pellet is larger than that inFigure 3. This semen was from a fertile stallion with excellentsemen quality.



fertility and to determine if altered strategies ofbreeding management could improve pregnancyrates. According to the owner, the stallion hadbeen fertile in previous years, but per-cycle preg-nancy rate dropped to approximately 35% in theprevious season when covering approximately 90mares. Combined testicular volume48 of this stal-lion was calculated to be 184 ml. Predicted dailysperm output (DSO), as determined from testicularvolume, was 3.7 � 109 sperm, but actual DSO wasapproximately 1.7 � 109 sperm; therefore, spermat-ogenic efficiency was estimated to be less than 50%.Ejaculates contained an average of 33% morpholog-ically normal sperm and 38% progressively motilesperm. The most common morphologic defectswere bent tails (average of 35%) and abnormallyshaped midpieces (average of 33%). The averagepercentage of progressively motile sperm after 24 hof cooled storage of extended semen was 29%.

Centrifugation of extended semen was proposedas a means to increase sperm concentration in in-seminates, because sperm concentration in ejacu-lates was generally less than 100 � 106 per ml(range � 28–109 � 106/ml; median � 68 � 106/ml).Extended semen from the stallion was subjected tocushioned centrifugation for 20 min at 400 � g usingglass nipple-bottom centrifuge tubes to reduce seminalplasma concentration and increase sperm concentra-tion in resuspended sperm pellets.23 Reproductivelynormal mares were inseminated with processed se-men to evaluate the effects of sperm number, storagetime, and insemination method on fertility. Owing tothe costs associated with a breeding trial, the clientwas amenable only to insemination of a small group ofmares to determine if simple concentration of spermand transrectally guided or hysteroscopic low-dose in-semination techniques would result in pregnancyrates that would allow this stallion to be commerciallyviable (Table 6).

Although the number of mares inseminated ineach of the five treatment groups was small, theoutcome was sufficient to provide generalizationsregarding treatment strategies for the subject stal-lion. The results suggest that no difference in fer-tility existed between inseminates of 250 � 106 or500 � 106 total sperm. Cooled storage of semenhad no apparent deleterious effect on fertility, andtransrectally guided insemination of semen yielded

results that were similar to or exceeded that of hys-teroscopic insemination. A follow-up trial withcool-transported semen and transrectally guidedlow-dose insemination resulted in recovery andtransfer of five embryos from four inseminatedmares.

Semen from this stallion had not previously beensubjected to cooled storage and transport. The stal-lion was discharged with recommendations to usecushioned centrifugation of semen and transrectallyguided insemination with 250 million sperm permare initially, with the prospect that sperm numberin inseminates could be lowered if initial pregnancyresults were favorable. In addition, the owner wasinformed of the potential value of cool-stored semen.A decision was made to breed mares the next season,primarily with fresh-processed semen. A total of132 mares were bred over 170 cycles, with cool-transported semen used on only three occasions.The stallion achieved a seasonal pregnancy rate of85% with an average of 1.5 cycles per pregnancy(i.e., a per-cycle pregnancy rate of 66%).

Clinical Case 2

A 6-yr-old Quarter Horse stallion was admitted tothe Texas Veterinary Medical Center for a breeding-soundness examination and potential therapeuticapproaches to improve his fertility. The stallionwas administered an orally active progestogen, al-trenogest (Regu-Mate™j), on a daily basis during his4-yr athletic career. The dosage of altrenogest ad-ministered was unknown to the owner of the stal-lion. Only one mare was pregnant from the firsteight mares inseminated with entire ejaculates, re-sulting in a 12% per-cycle pregnancy rate.

The stallion had a total testicular volume of 134ml and a predicted DSO, based on testicular volume,of 2.5 � 109 sperm. Actual DSO for the stallion wasapproximately 1 � 109 sperm, and therefore, sper-matogenic efficiency was determined to be less than50%. Ejaculates contained an average of 15% mor-phologically normal sperm and 34% progressivelymotile sperm. The most common sperm morpho-logic defects were abnormally shaped midpieces (av-erage of 28%) and bent tails (average of 22%).

After consultation with the owner, a decision wasmade to inseminate a small group of reproductivelynormal mares by low-dose insemination techniques.

Table 6. Effects of Total Sperm Number, Insemination Volume, and Method of Insemination on Pregnancy Rate in Mares With Semen From a SingleStallion.6

Total sperm ininseminate (x106)

Storage time forprocessed semen (h)

Volume ofinseminate (ml)

Method ofinsemination

Per-cyclepregnancy rate (%)

500 0 1 Transrectally-guided 4/6 (66%)500 24 1 Transrectally-guided 4/6 (66%)250 0 0.5 Transrectally-guided 5/6 (83%)250 24 0.5 Transrectally-guided 4/6 (66%)250 0 0.2 Hysteroscopic 1/2 (50%)



Semen-processing techniques consisted of cushionedcentrifugation only or cushioned centrifugation fol-lowed by density-gradient centrifugation through asilanated silica-particle solution (EquiPure™ TopLayer and Bottom Layer; i.e., two-layer approach)for 30 min at 200 � g.33,38 The density-gradientcentrifugation procedure was used in an attempt toimprove semen quality through separation of sper-matozoal morphologic types based on isopycnic point(i.e., buoyancy). Results of the clinical trial are pro-vided in Table 7.

Pregnancy rates seemed to improve as insemina-tion dose by transrectally guided technique in-creased from 15 to 100 � 106 progressively motilesperm, suggesting a dose-dependent effect of insem-ination number on fertility. After cushioned cen-trifugation, no distinct advantage was gained byusing hysteroscopic insemination compared withtransrectally guided insemination in the tip of theuterine horn. Insemination in the tip of the uterinehorn, however, yielded a higher pregnancy rate thanstandard insemination in the uterine body when theinseminate contained 100 � 106 progressively mo-tile sperm. Further processing of semen throughEquiPure™ also seemed to improve pregnancy ratescompared with only cushioned centrifugation whenthe inseminate contained 50 � 106 progressivelymotile sperm.

The stallion was discharged from the hospitalwith instructions to breed mares with a low-doseinsemination technique using a minimum of 100 �106 progressively motile sperm after cushioned cen-trifugation or 50 � 106 progressively motile spermafter centrifugation in EquiPure™. We also pro-posed that the insemination dose with EquiPure™-treated semen might be lowered without impactingfertility negatively but that this possibility shouldbe tested before it could be recommended for com-mercial purposes. We did not test the fertility ofcool-stored semen and therefore, could not offer rec-ommendations regarding its use. The owner indi-cated that the stallion had normal fertility thefollowing breeding season when mares were insem-inated by a transrectally guided technique usingsemen previously subjected to cushioned centrifuga-tion. The precise mare book, insemination doses,

and fertility statistics were not available for inclu-sion in this communication.

Clinical Case 3

A 4-yr-old Quarter Horse stallion was admitted tothe Texas Veterinary Medical Center for evaluationof breeding soundness after his first season at studwhere he achieved a 59% seasonal pregnancy ratewhen covering 165 mares. Approximately one-halfof the mares were bred with cool-transported semen.During that breeding season, progressive sperm mo-tility was estimated to be 70% for 76 of 84 semencollections performed, and the average total spermnumber in the 84 ejaculates was approximately5.349 � 109 sperm. The sperm concentration wasestimated to be less than 100 � 106 sperm/ml for 67of 84 ejaculates collected.

The testicular volume of this stallion was deter-mined to be 225 ml, resulting in a predicted dailysperm output of 4.6 � 109 sperm assuming that thetestes were producing sperm with normal efficiency.Four ejaculates were collected from the stallion.The stallion’s actual sperm output on the fourthdaily collection was 3.67 � 109 sperm, and therefore,spermatogenic efficiency was considered to be belownormal. The percentage of morphologically normalsperm in ejaculates averaged 38%, and the percent-age of progressively motile sperm averaged 27%.The most common morphologic defects were abnor-mally shaped midpieces (average of 30%) and benttails (19%).

Semen from three ejaculates was processed bycushioned centrifugation or cushioned centrifuga-tion followed by centrifugation in EquiPure™ Bot-tom Layer. Semen was evaluated for sperm-motion characteristics immediately after eachprocessing step and after 24 h of cooled storage.The effect of seminal plasma was also evaluated.The sperm morphologic profiles of unprocessed (raw)semen and EquiPure™-processed semen were alsocompared. Data from a representative ejaculateare provided in Tables 8 and 9.

From these data, one can surmise that the Equi-Pure™ centrifugation procedure considerably im-proved the semen quality of this stallion, becausemeasures of sperm-motion characteristics and

Table 7. Effects of Semen-Processing Technique, Method of Insemination, and Number of Progressively Motile Sperm in Inseminates on PregnancyRate in Mares With Semen From a Single Stallion.6

Centrifugation technique Method of insemination

Progressively motilesperm in inseminate

(x 106)

Inseminatevolume

(mL)

Per-cyclepregnancy rate

(%)

Cushioned method Transrectally-guided 15 1 0/6 (0%)Cushioned method Transrectally-guided 50 1 2/6 (33%)Cushioned method Transrectally-guided 100 1 5/6 (83%)Cushioned method Hysteroscopic 50 0.1 3/8 (38%)Cushioned method Standard uterine body 100 1 2/6 (33%)EquiPure™ (two-layer) Hysteroscopic 50 0.1 6/8 (75%)



sperm morphology were enhanced with this proce-dure. The increase in the percentage of morpholog-ically normal sperm after EquiPure™ Bottom Layercentrifugation was primarily attributable to reducedpercentages of abnormal (irregular or bent) mid-pieces and bent tails. For this ejaculate and othersevaluated from this stallion, sperm velocity was in-creased when seminal plasma from the test stallionwas replaced with that obtained from a fertile con-trol stallion. For all treatments in Table 8, spermmotility values did not change appreciably after 24 hof cooled storage compared with values evaluatedbefore storage.

A follow-up fertility trial was conducted with se-men from this stallion to determine if inseminationof mares with EquiPure™-processed semen wouldyield commercially acceptable pregnancy rates.Ten reproductively normal mares were inseminatedin this trial; each of five mares was inseminated one

time with 100 � 106 total sperm, and each of fivemares was inseminated one time with 200 � 106

total sperm. The test stallion’s seminal plasmawas replaced with that of a fertile donor stallion.Seminal plasma from the donor stallion was pro-cured by centrifuging raw semen for 1,000 � g for 15min followed by filtration of supernate through tan-dem 5.0- and 1.2-�m pore-size nylon filters to re-move any remaining sperm from the seminalplasma. One-milliliter aliquots of seminal plasmawere frozen in vials at �80°C before use. Insemi-nate volumes ranged from 0.25 to 0.58 ml, and atransrectally guided low-dose insemination tech-nique was used. The per-cycle pregnancy rateswere 100% (5/5) for mares inseminated with 100 �106 total sperm and 4/5 (80%) for mares inseminatedwith 200 � 106 total sperm. Two mares, one ineach treatment group, experienced double ovula-tions, and each of these mares was diagnosed withtwin pregnancies. As such, pregnancy rate perovulation was 100% (6/6) for mares inseminatedwith 100 � 106 total sperm and 5/6 (83%) for maresinseminated with 200 � 106 total sperm. Based onthe post-centrifugation recovery rate of sperm inthis trial, the stallion would have had sufficient se-men to breed 17 mares per ejaculate if mares were tobe inseminated with 100 � 106 total sperm.

For the following commercial breeding season, 212mares were inseminated, primarily using freshEquiPure™-treated semen with seminal plasmafrom a fertile donor stallion. The stallion achieveda seasonal pregnancy rate of 91% in an average of1.61 cycles per pregnancy (i.e., a per-cycle pregnancyrate of 62%).

4. Summary

Equine veterinarians may encounter owners of stal-lions who are seeking methods that can improvestallion breeding performance. Breeding and se-men-manipulation strategies can be applied to max-imize the fertility of these stallions and to extendtheir productive life. Any recommended breeding

Table 8. Effects of Semen Processing Protocol (Simple Dilution, Cushioned Centrifugation, or EquiPure™ Bottom Layer Centrifugation), Source ofSeminal Plasma (Same Stallion or Fertile Control Stallion), and Storage Time (0 or 24 h) on Measures of Sperm Motility.6

Centrifugation TechniqueSource of

seminal plasma

Spermconcentration

(x106/ml)Storage Time

(h)

TotalMotility

(%)*

ProgressiveMotility

(%)*

CurvilinearVelocity(�m/s)*

Simple dilution Same 30 0 54 21 176Cushioned centrifugation Same 30 0 59 33 121EquiPure™ Same 30 0 90 77 146EquiPure™ Control 30 0 93 82 224Simple dilution Same 30 24 50 20 136Cushioned centrifugation Same 200 24 59 29 152Cushioned centrifugation Same 30 24 56 33 135EquiPure™ Same 200 24 87 72 141EquiPure™ Control 200 24 89 73 208

*Measurements for total motility (%), progressive motility (%), and curvilinear velocity (�m/s) were performed by computer-assisisted sperm motion analysis, as described previously.6

Table 9. Effect of Semen Centrifugation Through EquiPure™ BottomLayer on Sperm Morphologic Features, as Viewed by Differential-Inter-ference Microscopy at 1250 x Magnification.6

Sperm morphologicfeature (%)

Unprocessed(raw) semen

EquiPure™-processed

semen

Normal 40 76Abnormal heads 5 1Abnormal acrosomes 1 1Tailless heads 3 2Proximal protoplasmic

droplets 10 5Distal protoplasmic

droplets 13 5Abnormal (irregular)

midpieces 28 6Bent midpieces 13 3Bent tails 19 5Coiled tails 1 0Premature (round) germ

cells 1 0



modifications can oftentimes be tested outside thecommercial breeding season by conducting clinicalfertility trials. Recipient-mare herds retained byembryo-transfer facilities are an excellent source forsuch trials. The information gained provides use-ful information for the stallion owner/agent as theyprepare for a forthcoming breeding season.

Acknowledgments

Financial assistance for this study was providedby the Link Equine Endowment Fund, Texas A&MUniversity; the Azoom, Corona Cartel, and TellerCartel Syndicates (Lazy E Ranch); the ShiningSpark-Carol Rose Stallion Reproduction Fund(American Quarter Horse Association); the Mr. JessPerry Syndicate (6666 Ranch); and proceeds fromthe Legends Premier Stallion Season Auction, TexasA&M University. Portions of this manuscript werepublished previously in Journal of Equine Veteri-nary Science.

References and Footnotes1. Krausz C, Giachini C. Genetic risk factors in male infertil-

ity. Arch Androl 2007;53:125–133.2. Carrell DT. The genetics of male infertility in the era of

genomics: tools for progress. In: Carrell DT, ed. The ge-netics of male infertility. Totowa, NJ: Humana Press,2007;3–27.

3. Love CC. The role of breeding record evaluation in the eval-uation of the stallion for breeding soundness, in Proceedings.Annual Conference and Symposium of the Society for Ther-iogenology 2003;68–77.

4. Varner DD, Blanchard TL, Brinsko SP, et al. Techniques forevaluating selected reproductive disorders of stallions. AnimReprod Sci 2000;60–61:493–503.

5. Blanchard TL, Love CC, Thompson JA, et al. Role of rein-forcement breeding in a natural service mating program, inProceedings. 52nd Annual American Association of EquinePractitioners Convention 2006;384–386.

6. Varner DD, Love CC, Brinsko SP, et al. Semen processingfor the subfertile stallion. J Equine Vet Sci 2008;28:677–685.

7. Macpherson ML, Shore MD, Fernandez MH, et al. Process-ing factors which influence viability and fertility of cryopre-served equine spermatozoa. Havemeyer Found Monogr Ser2001;6:27–29.

8. Alvarez JG, Lasso JL, Blasco L, et al. Centrifugation ofhuman spermatozoa induces sub-lethal damage; separationof human spermatozoa from seminal plasma by a dextranswim-up procedure without centrifugation extends their mo-tile lifetime. Hum Reprod 1993;8:1087–1092.

9. Len JA, Jenkins JA, Eilts BE, et al. Immediate and delays(after cooling) effects of centrifugation on equine sperm.Theriogenology 2010;73:225–231.

10. Clay CM, Slade NP, Amann RP, et al. Effects of extender,storage temperature and centrifugation on stallion sperma-tozoal motility and fertility, in Proceedings. 2nd Interna-tional Congress on Animal Reproductiona and ArtificialInsemination 1984;186–188.

11. Pickett BW, Sullivan JJ, Byers WW, et al. Effect of centrif-ugation and seminal plasma on motility and fertility of stal-lion and bull spermatozoa. Fertil Steril 1975;26:167–174.

12. Samper JC, Morris CA. Current methods for stallion semencryopreservation: a survey. Theriogenology 1998;49:895–903.

13. Vidament M, Ecot P, Noue P, et al. Centrifugation andaddition of glycerol at 22°C instead of 4°C improve post-thawmotility and fertility of stallion spermatozoa. Theriogenol-ogy 2000;54:907–919.

14. Vidament M, Yvon JM, Couty I, et al. Advances in cryo-preservation of stallion semen in modified INRA 82. AnimReprod Sci 2001;68:201–218.

15. Van Veldhoven PP, Baumgart E, Mannaerts GP. Iodixanol(Optiprep), an improved density gradient medium for theiso-osmotic isolation of rat liver peroxisomes. Anal Biochem1996;237:17–23.

16. Graham J, Ford T, Rickwood D. The preparation of subcel-lular organelles from mouse liver in self-generated gradientsof iodixanol. Anal Biochem 1994;220:367–373.

17. Bendixen B, Rickwood D. Properties and applications of anew type of iodinated gradient medium. J Biochem BiophysMethods 1994;28:43–52.

18. Ecot P, Decuadro-Hansen G, Delhomme G, et al. Evaluationof a cushioned centrifugation technique for processing equinesemen for freezing. Anim Reprod Sci 2005;89:245–248.

19. Harrison K. Iodixanol as a density gradient medium for theisolation of motile spermatozoa. J Assist Reprod Genet1997;14:385–387.

20. Knop K, Hoffmann N, Rath D, et al. Effects of cushionedcentrifugation technique on sperm recovery and sperm qual-ity in stallions with good and poor semen freezability. AnimReprod Sci 2005;89:294–297.

21. Matas C, Decuadro G, Martinez-Miro S, et al. Evaluation ofa cushioned method for centrifugation and processing forfreezing boar sperm. Theriogenology 2007;67:1087–1091.

22. Revell SG, Pettit MT, Ford TC. Use of centrifugation overiodixanol to reduce damage when processing stallion spermfor freezing, in Proceedings. Joint Meeting Soc Study FertilAbstr Series No 92 1997;38.

23. Sieme H, Knop K, Rath D. Effects of duration, force ofcentrifugation and cushioned centrifugation technique onsperm recovery and sperm quality in stallions with good andpoor semen freezability. Havemeyer Found Monogr Ser2005;18:6–9.

24. Sieme H, Knop K, Rath D. Effects of cushioned centrifuga-tion on sperm quality in stallion semen stored cooled at 5°Cfor 24 h, and stored cooled for 2 or 24h and then frozen.Anim Reprod Sci 2006;94:99 –103.

25. Smith TT, Byers M, Kaftani D, et al. The use of iodixanol asa density gradient material for separating human spermfrom semen. Arch Androl 1997;38:223–230.

26. Waite JA, Love CC, Brinsko SP, et al. Factors impactingequine sperm recovery rate and quality following cushionedcentrifugation. Theriogenology 2008;70:704–714.

27. Pertoft H, Rubin K, Kjellen L, et al. Viability of cells grownor centrifuged in a new density gradient medium, Percoll.Exp Cell Res 1977;110:449–457.

28. Gorus FK, Pipeleers DG. A rapid method for the fraction-ation of human spermatozoa according to their progressivemotility. Fertil Steril 1981;35:662–665.

29. Bolton VN, Braude PR. Preparation of human spermatozoafor in vitro fertilization by isopycnic centrifugation on self-gen-erating density gradients. Arch Androl 1984;13:167–176.

30. Mortimer D. Sperm preparation methods. J Androl 2000;21:357–366.

31. Pertoft H. Fractionation of cells and subcellular particleswith Percoll. J Biochem Biophys Methods 2000;44:1–30.

32. Hall JA, Fishel SB, Timson JA, et al. Human sperm mor-phology evaluation pre-Percoll and post-Percoll gradient cen-trifugation. Human Reprod 1995;10:342–346.

33. Soderlund B, Lundin K. The use of silane-coated silica par-ticles for density gradient centrifugation in in-vitro fertiliza-tion. Human Reprod 2000;15:857–860.

34. Centola GM, Herko R, Andolina E, et al. Comparison ofsperm separation methods: effect on recovery, motility, mo-tion parameters, and hyperactivation. Fertil Steril 1998;70:1173–1175.

35. Claassens OE, Menkveld R, Harrison KL. Evaluation of threesubstitutes for Percoll in sperm isolation by density gradientcentrifugation. Human Reprod 1998;13:3139–3143.

36. Chen MJ, Bongso A. Comparative evaluation of two densitygradient preparations for sperm separation for medically as-sisted conception. Human Reprod 1999;14:759–764.



37. McCann CT, Chantler E. Properties of sperm separatedusing Percoll and IxaPrep density gradients. A comparisonmade using CASA, longevity, morphology and the acrosomereaction. Int J Androl 2000;23:205–209.

38. Macpherson ML, Blanchard TL, Love CC, et al. Use of asilane-coated silica particle solution to enhance the quality ofejaculated semen in stallions. Theriogenology 2002;58:317–320.

39. Edmond AJ, Teague SR, Brinsko SP, et al. Effect of densitygradient centrifugation on quality and recovery rate of equinespermatozoa. Anim Reprod Sci 2008;107:318.

40. Morris LHA. Low dose insemination in the mare: an up-date. Anim Reprod Sci 2004;82–83:625–632.

41. Lyle SK, Ferrer MS. Low-dose insemination—why, whenand how. Theriogenology 2005;64:572–579.

42. Brinsko SP. Insemination doses: How low can we go?Theriogenology 2006;66:543–550.

43. Sieme H, Bonk A, Hamann H, et al. Effects of differentartificial insemination techniques and sperm doses on fertil-ity of normal mares and mares with abnormal reproductivehistory. Theriogenology 2004;62:915–928.

44. Rigby S, Derczo S, Brinsko S, et al. Oviductal sperm num-bers following proximal uterine horn or uterine body insem-ination, in Proceedings. 46th Annual American Associationof Equine Practitioners Convention 2000;332–334.

45. Morris LH, Hunter RHF, Allen WR. Hysteroscopic insemi-nation of small numbers of spermatozoa at the uterotubal

junction of preovulatory mares. J Reprod Fertil 2000;118:95–100.

46. Squires EL. Integration of future biotechnologies into theequine industry. Anim Reprod Sci 2005;89:187–198.

47. Weems JS, Beyers W. How to incorporate low-dose hyster-oscopic insemination in an on-farm fresh semen program, inProceedings. 50th Annual American Association of EquinePractitioners Convention 2004;399–401.

48. Love CC, Garcia MC, Riera FR, et al. Use of testicularvolume to predict daily sperm output in the stallion, in Pro-ceedings. 36th Annual American Association of EquinePractitioners Convention 1990;15–21.

aCushion Fluid, Minitub, Tiefenbach, Germany.bBliss SB. Unpublished data, 2009.cIxaPrep, Medi-Cult, Copenhagen, Denmark.dPureSperm, Nidacom International AB, Molndal, Sweden.eIsolate, Irvine Scientific, Santa Ana, CA 92705.fEquiPure, Nidacon International AB, Molndal, Sweden.gEquiPure Bottom Layer, Nidacon International AB, Molndal,

Sweden.hEdmond AJ. Unpublished data, 2008.iHayden SS. Unpublished data, 2009.jRegu-Mate, Intervet/Schering-Plough Animal Health, Mills-

boro, DE 19966.



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