How to Breed Mares With Frozen Semen by DeepHorn Insemination

Andrew R. Schmidt, DVM, MS, Diplomate ACT

Author’s address: Wisconsin Equine Clinic and Hospital, 39151 Delafield Road, Oconomowoc, WI53066-9105; e-mail: aschmidt@wiequine.com. © 2011 AAEP.

1. Introduction

Stallion and mare owners are becoming increasinglyaware of the number of foals produced by a variety oflow-dose insemination techniques and may requestthat you, the private practitioner, breed their mareswith partial doses of frozen semen. Reproductiveveterinarians are breeding mares, including theolder less fertile mare, using frozen semen with lowsperm numbers, low volume, low progressive motil-ity, and low percentage of normal cells. Stallionsmay be unavailable due to show schedules, unable toprovide good-quality semen, frozen or cooled, or maybe deceased. Likewise, some of today’s importedsemen from popular stallions can be quite expensiveand is frequently sold by the “dose,” without a livefoal guarantee.

There are a variety of successful strategies avail-able today to help veterinarians use frozen semen.This list includes estrus synchronization, ovula-tion induction, timed insemination, endoscopic in-semination at the ostium (papilla), and deep horninsemination. All of these techniques require effec-tive participation between owners, managers, andthe veterinarian to allow access to mares for treat-ment and examination. Although frozen semenbreeding can be successful in any type of facilitywith adequate personnel, working areas, and labo-

ratory equipment, most mares being bred with fro-zen semen in equine practices will come into ahospital or clinic for at least 3 to 5 days. Whereverthe mares are bred, the overall success of any frozeninsemination is dependent on accurate induction ofovulation; proper timing of insemination; and well-developed veterinary skills, including transrectalpalpation, ultrasonography, and artificial insemina-tion techniques. Practitioners also must be famil-iar with the current methods used to properlyhandle, transfer, store, thaw, and evaluate frozensemen, as it is imperative that semen samples arenot damaged before or during the thaw process.1,2

An accurate review of the previous literature,3

and results from a large number of mares that werebred using low-dose insemination, are currentlyavailable.4 It became apparent in our practice overthe last 5 to 6 years that the timely use of deep horninsemination (DHI) could result in favorable preg-nancy and embryo recovery rates even when fewerstraws or lower numbers of sperm were available.It is certainly appropriate to use a full dose of frozensemen from any stallion using standard AI tech-niques (insemination in the uterine body) and thengradually drop the number of straws used andswitch to DHI as pregnancy rate dictates. Splittingthe dose, in most cases, will save semen and may

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NOTES

also produce less post-breeding endometritis in sus-ceptible mares. It makes sense to use as little se-men as possible, conserving semen for another cycleor another mare, particularly when the sample’squality characteristics or pregnancy rates areknown. Frozen semen has the distinct advantagethat when handling and storage are well managed,it will last for decades.

The aim of this report is to provide valuable ref-erences and descriptions of a technique, which withpractice will help veterinarians become successfulbreeding mares with lower doses of semen. It de-scribes a step-by-step technique for DHI in maresusing a disposable commercially available pipette.a

This technique allows mares to be inseminated withmultiple straws through one pipette and a reusablestainless steel styletb (empties a 0.50-cc straw at theend of the pipette and retrieves it). There is also asimilar pipette with an inner tubec to deliver semenfrom a syringe when semen is packaged in 2.5- or5-cc straws. This system does require a rectal-vag-inal procedure and consequently veterinary skills,yet it avoids the use of additional extender or anendoscope while delivering the semen efficiently andquickly.

2. Materials and Methods

The majority of mares bred by DHI in our practiceare placed in breeding stocks for the actual insemi-nation. This is ideal for the mare that will toleratebreeding stocks, as the mare remains more confinedduring the process. It is also helpful to move mareswith foals or have them stabled as close to the lab aspossible, so that insemination can be completed inan efficient manner after the semen is thawed whileallowing the foal to be contained in a safe manner.

As the mare is being prepared, the correct semenshould be located in the storage tank and an appro-priate water bath set up. Mare preparation con-sists of restraint similar to what is needed forpalpation of that mare, a rectum free of manure,then a standard perineal cleaning. This particularflexible pipettea is easiest to use when kept warm (in

an incubator if possible), because it will be morepliable and will retain the curl that is placed in itbefore introducing it through the cervix.

When the package is opened, a 3-cc syringe isattached to the external end of the pipette to avoidexcessive air from being introduced into the uterus.The veterinarian places a sterile sleeve over the armfor the insemination and puts a full circle curl in thewarm pipette (Fig. 1). The pipette is then passedtranscervically (should retain about 45 degrees ofbend) with the internal tip kept ventrally (Fig. 2).As the gloved hand is removed from the cervix andanterior vagina, the other hand should gently guidethe pipette forward. The gloved hand can then bepassed transrectally to palpate the pipette tip in theuterine body or base of the horn and assist it in a 90degree rotation, left or right, toward the appropriatehorn (Fig. 3). Transrectally, the fingers are used togently lift the uterine horn base and facilitate pas-sage of the pipette to the appropriate horn’s tip.The entire process tends to go fairly easy whenthings are in the right position. One should feel thepipette slide up the horn, the rounded tip close to theovary, and thus the shaft of the pipette is palpatedinside the chosen horn. Good palpation skills,knowledge of the mare’s uterine position, and someexperience using the technique is necessary.

Fig. 1. Placing a curl in the warm pipette.

Fig. 2. Retained curl in the pipette.

Fig. 3. Transrectal placement of the DHI pipette.

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At this time, an assistant can hold and fixate theposition of the external end with a hand against themare’s body next to the vulvar lip (Fig. 4) while thesemen is thawed. We use a portable water bath(stainless steel cup with handle, 1000 cc), with aclip-on thermometer (Fig. 5), so that the semen canbe transferred to the breeding cart next to the mareduring the thaw process. Thermometers used inthawing the semen must be accurate. Obviously,most thawing protocol times are short (20–30 sec-onds) and therefore the mare must be inseminatedquickly. Dry the straws, remove the sealed endfurthest away from the cotton plug on all strawswith a straw cutter, and then place the first strawinto the pipette (Fig. 6). The metal stylet shouldthen be inserted behind the straw and pushed all theway through the pipette, seating the open end at thenipple inside the tip and thus pushing the cottonplug through the straw depositing the semen. Thisstylet tends to work well with most 0.50-cc strawsand allows you to completely empty each straw atthe pipette tip one at a time. If only 90% of the laststraw is inseminated, then the last bit of remainingsemen can be placed on a warm slide in the lab toprovide an assessment of post-thaw progressive mo-tility (Fig. 7).

3. Results

Per-cycle success rates included in this report arefrom mares bred 12 to 48 hours after injection ofdeslorelin or HCG, using frequent ovarian palpationand ultrasonography. All were bred by DHI.Generally they were given an induction medicationbetween 6 and 8 PM and checked a minimum of every8 hours until ovulation, starting the next morning.Many of these mares were bred once during theperiod of 36 to 40 hours after the ovulation inductionagent was given, either before ovulation or shortlyafter ovulation. Mares with limited semen werechecked every 2 to 4 hours until ovulation, startingat least the morning they reached 36 hours.

Pregnancy rates in our practice over the last 4years (2007–2010), using DHI and low-dose frozeninseminations, have been encouraging. This groupof mares was an average population of fertile andsubfertile mares; there was no selection for fertility.Over the four seasons and 629 cycles that frozensemen was used, 58% (364 cycles) of the insemina-tions were performed with a partial dose of semen.Per-cycle pregnancy rates with frozen semen aver-aged 46% (291/629) for all cycles and 49% (178/364)for cycles in which less than one dose of semen wasinseminated. Low-dose breeding was generally notattempted unless results were acceptable using afull dose of semen and post-thaw progressive motil-

Fig. 4. Assistant fixating the pipette after placement.

Fig. 5. Portable water bath used next to the mare.

Fig. 6. Insemination with the stylet.

Fig. 7. Post-breeding motility evaluation.

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ity was 40% or higher. A recovered embryo at 7 to8 days after ovulation was counted as a pregnancy.

Frozen semen from many stallions in our practicehas produced pregnancies or embryos from cycles inwhich two 0.50-cc straws [one-quarter] dose) wereused once. Several pregnancies from multiple stal-lions have been obtained with a single 0.50-cc straw(8 straws per dose) in fertile mares. It appears thatquality and timing of insemination are more impor-tant than sperm numbers with some stallions.Many of these samples were processed by SelectBreeders Southwest’s mobile lab, which routinelyfreezes in 0.50-cc straws at 8 straws per dose, pro-viding 250 to 400 million progressively motile sperm(PMS) per dose (31–50% PMS post-thaw). For ex-ample, two straws at 40% post-thaw would provide80 million PMS. It appears that DHI is successfulfor low-dose and small volumes, but there may be nodifference when larger volumes are used.

4. Discussion

In review, most practitioners use one of two proto-cols for breeding frozen semen mares: timed in-semination at 24 and 40 hours after ovulationinduction, or frequent palpation and ultrasonogra-phy with insemination occurring once or twicewithin 2 to 8 hours of ovulation.

The latter lends itself to situations in which thesemen source is limited, expensive, or of poor qual-ity, yet is labor intensive. A recent report from twobreeding seasons and 251 mares shows that twodoses of frozen semen using timed insemination canresult in pregnancy rates equivalent to those ofmares bred with cooled semen.5 The timed insem-ination protocol relies on accurate ovulation induc-tion and access to plenty of semen. It could be adisadvantage if semen is in short supply, if the mare

does not respond to the ovulation induction agent, orif she is subfertile and/or susceptible to endometritisafter multiple inseminations.

Complicating either of these two protocols is thefact that many mares have erratic estrous cycles.They may show variable signs of estrus over 5 to 10days and ovulate in the last 1 to 2 days with folliclesof inconsistent sizes. Consequently, they requireroutine and frequent palpation and ultrasonographyfor effective breeding with cooled or frozen semen.

With the frozen semen technology available today,using low-dose and deep horn insemination tech-niques seems to be the wave of the future. Con-serving semen and limiting the amount of semenexposure in difficult mares has proved to be benefi-cial and rewarding in our practice.

References and Footnotes1. Loomis PR, Squires EL. Frozen semen management in

equine breeding programs. Theriogenology 2005;64:480–491.

2. Dascanio JJ, Kasimanickam R. Breeding the mare with fro-zen semen. Equine Vet Educ 2008;20:667–672.

3. Morris L. Advance insemination techniques in mares.Vet Clin North Am Equine Pract 2006;22:693–703.

4. Samper JC, Sanchez R, Gomez I, et al. Artificial insemina-tion with frozen semen: pregnancy rates after rectallyguided or endoscopic deposition, in Proceedings. Am AssocEquine Pract 2005;51:213–214.

5. Crowe CAM, Ravenhill PJ, Hepburn RJ, et al. A retrospec-tive study of artificial insemination of 251 mares using chilledand fixed time frozen-thawed semen. Equine Vet J 2008;40:572–576.

aMinitube of America, IncTM, Verona, WI; 57-cm flexible pipette(ref No. 17209/0005).

bMinitube of America, IncTM, Verona, WI; 57-cm stylet (ref No.17209/1057).

cMinitube of America, IncTM, Verona, WI; 65cm flexible pipettewith inner sheath (ref No. 17207/1165).

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