Practical Reproduction Studies of 2015

In one study, researchers determined that reducing social stress, by keeping mares outside and in a stable herd, for instance, can increase reproductive efficiency and decrease early pregnancy loss.

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On the first day of educational sessions at the 2015 American Association of Equine Practitioners convention, held Dec. 5-9 in Las Vegas, practitioners, veterinary students, and other industry members packed the Mandalay Bay Convention Center’s ballroom to listen to a rundown of the year’s most practical publications, as selected from the fields of surgery, medicine, and reproduction. On the breeding side, researcher and Texas A&M University theriogenology professor Terry Blanchard, DVM, MS, Dipl. ACT, described 11 equine reproduction-related papers on topics ranging from prostaglandin treatment of mares to semen quality in stallions.

Decreased Blood Flow to the Uterus Might Contribute to Mare Infertility

Throughout a mare’s estrous cycle, her uterine blood flow changes frequently. University of California, Davis, researchers compared uterine blood flow and perfusion in normal, young, healthy mares to that of older subfertile mares with vascular degeneration—a condition in which the vessels of the uterine wall have degenerative, often disruptive, changes in surrounding elastic fibers (called elastosis). This vascular elastosis may be associated with infertility. They found decreased uterine blood flow and perfusion in the uterus of older subfertile mares, compared to the young fertile mares, regardless of estrous cycle stage. They also noted no increase in uterine blood flow during estrus in the subfertile mares, which should have occurred.

“Their take-home message was that decreased uterine perfusion in mares with vascular degeneration may play a role in subfertility or infertility and postulated that it could contribute to post-mating-induced endometritris (inflammation of the uterine lining), delayed uterine clearance (of semen and inflammatory debris following breeding), and possibily even early embryonic or fetal death,” said Blanchard.

Esteller-Vico A, Liu IK, Vaughan B, et al. Effects of vascular elastosis on uterine blood flow and perfusion in anesthetized mares. Theriogenology 2015 Apr 1;83(6):988-94. 

NSAID Administration Can Cause Failure to Ovulate

Veterinarians use non-steroidal anti-inflammatory drugs (NSAIDs) to treat a variety of conditions in horses. However, results from two new studies show that these drugs can negatively affect a mare’s reproductive activity. In the next study Blanchard described, Brazilian researchers evaluated how two NSAIDs affected mares’ development of pre-ovulatory follicles. They studied 11 mares over three consecutive estrous cycles: the first cycle served as the control, on the second cycle mares received a standard therapeutic dose of phenylbutazone (often referred to as Bute), and in the third a standard therapeutic dose of meloxicam (Metacam, a COX-2 inhibitor available for use in horses in Europe). The research team found that all mares ovulated as expected during the control cycle, while only one and two mares ovulated during the meloxicam and phenylbutazone cycles, respectively. With both NSAID treatments the researchers saw hemorrhagic anovulatory follicles (follicles that didn’t release eggs, and so cannot result in pregnancy) on ultrasound. In conclusion, “administration of NSAIDs may lead to formation of these hemorrhagic anovulatory follicles,” said Blanchard.

He also pointed out that in studies on lab animals, women, and some horses, the timing and dose of these drugs seem to affect the development of the anovulatory structures. “Whether stopping administration of NSAIDs during estrus would avoid this potential problem has not been studied,” he said.

Lima AG, Costa LC, Alvarenga MA, et al. Does Clinical Treatment with Phenylbutazone and Meloxicam in the Pre-ovulatory Period Influence the Ovulation Rate in Mares? Reprod Domest Anim. 2015 Oct;50(5):771-5. 

Prostaglandin Administration Might Counter Flunixin Meglumine’s Impact on Ovulation

In past studies NSAIDs have been shown to reduce prostaglandin production, which is important for preparing the follicle to ovulate and make way for a potential pregnancy. So in the next study Blanchard described, scientists in Spain sought to determine whether administering synthetic prostaglandin could reverse the NSAID effects. The team administered flunixin meglumine (Banamine) to five mares over the course of two estrous cycles. In the treatment cycle, they administered the NSAID every 12 hours post-human chorionic gonadotrophin administration (used to induce ovulation) for 36 hours.

At Hour 32, the researchers administered prostaglandins into the follicle. For the control cycle, the researchers injected water instead of prostaglandins. They inseminated mares in three control and three treatment cycles. All prostaglandin-treated mares ovulated on schedule, while only one in five water-treated mares did so. Only prostaglandin-treated mares became pregnant. The team concluded that intrafollicular treatment with prostaglandins overcame Banamine’s anovulatory effects.

Blanchard noted, however, that he doesn’t see much clinical use for this technique unless dealing with a mare that repeatedly forms anovulatory follicles. “Otherwise, by the time you detect hemorrhage, it would probably be too late to use this treatment,” he said.

Martínez-Boví R, Cuervo-Arango J. Intrafollicular treatment with prostaglandins PGE2 and PGF2α inhibits the formation of luteinised unruptured follicles and restores normal ovulation in mares treated with flunixin-meglumine. Equine Vet J. 2014 Dec 1. 

Prostaglandin Administration Timing’s Effect on Fertility

Another group of researchers from Spain and South America aimed to determine the effect of the interval from prostaglandin administration to subsequent ovulation on a mare’s fertility. They used data from two breeding farms: an embryo transfer facility in South America that reported embryo recovery rates following insemination with fresh semen, and a Standardbred farm in Europe that reported pregnancy rates following breeding with frozen semen. All mares received prostaglandin prior to artificial insemination. The team broke the data from these farms into three groups—mares that ovulated less than six days, six to eight days, and more than eight days post-prostaglandin administration—as well as controls that did not receive prostaglandin. They determined that pregnancy rate and embryo recovery rate both decreased as the interval to ovulation became shorter following prostaglandin administration, said Blanchard.

Because mares that ovulate quickly after prostaglandin administration are less likely to get pregnant, veterinarians might want to manage these mares accordingly.

Cuervo-Arango J, Mateu-Sánchez S, Aguilar JJ, et al. The effect of the interval from PGF treatment to ovulation on embryo recovery and pregnancy rate in the mare. Theriogenology. 2015 May;83(8):1272-8. 

Different Cloprostenol Doses Impact Mare Pregnancy Outcomes

In this study out of Rood & Riddle Equine Hospital, in Lexington, Kentucky, researchers grouped Thoroughbred mares by follicle size at the time of cloprostenol (a prostaglandin analog) injection to induce estrus. The veterinarians administered deslorelin (a GnRH analog to induce ovulation) to all study mares to sychronize their ovulation. The team determined that fertility decreased (to 50% pregnancy rate) in mares with large follicles when injected with cloprostenol to induce estrus for breeding.

“So, while the previous study demonstrated reduced pregnancy rates in mares returning to estrus in a shorter period after prostaglandin administration, this paper describes reduced pregnancy rates in mares administered prostaglandin when they have larger (< 30 mm) follicles present,” said Blanchard.

Agnew ME, et al. Pregnancy outcomes in Thoroughbred mares administered different doses of cloprostenol. Clinical Theriogenology. 2015

Cloprostenol Administration, Follicle Size’s Effect on Ovulation

Researchers’ objectives were twofold in a Colorado State University study focusing on cloprostenol: to assess the effect of follicle size at the time of administration on the interval until ovulation, and to determine the incidence of hemorrhagic anovulatory follicle formation after administration. No ovulatory agents were administered. The researchers found that cloprostenol administration to mares with large (at least 35 mm in diameter) follicles primarily resulted in ovulation after 48 hours accompanied by uterine edema (73.1%), with 13.4% ovulating within 48 hours with variable edema and 13.4% of the large follicles regressing without ovulation. There was a very low (2.5%) incidence of hemorrhagic anovulatory follicle formation.

“Three-fourths of mares with large follicles present at prostaglandin administration did develop edema and ovulate more than two days later,” Blanchard summarized. “Whether the uterine edema indicates that the follicle was therefore competent and capable of producing a viable pregnancy remains to be determined.”

He added that because competent follicles produce more estrogen, veterinarians might be able to use uterine edema development to predict a successful breeding.

Burden C, McCue P, Ferris R. Effect of Cloprostenol Administration on Interval to Subsequent Ovulation and Anovulatory Follicle Formation in Quarter Horse Mares. Journal of Equine Veterinary Science. 2015 June;35(6):531–535. 

Managing Mares in Low-Stress Environments Can Improve Foaling Rates

“Sometimes, some simple commonsense approaches to mare management can make a big difference in reproductive outcome,” Blanchard began. He described a study out of Brazil in which the researchers sought to determine whether management strategies centered around horse well-being do, in fact, improve foaling rates. They evaluated 1,206 barren and in-foal Thoroughbred mares over the course of 10 breeding seasons that were divided into “stressed” and “relaxed” groups. The relaxed mares lived in consistent (i.e., mares weren’t moved out of or added to the herd group) small groups outside and were not teased prior to breeding. The stressed mares were kept in boxed stalls at night, lived in larger groups on pastures, and were teased individually each day. Based on the study results, barren mares in the relaxed group had significantly higher seasonal pregnancy rates than barren mares in the stressed mares; the in-foal mares in the relaxed group had higher pregnancy rates per cycle; and both barren and in-foal mares in the relaxed groups had a lower incidence of early embryonic death. “Reducing social stress can increase reproductive efficiency and decrease early pregnancy loss,” Blanchard said.

Malschitzky E, Pimentel AM, Garbade P, et al. Management Strategies Aiming to Improve Horse Welfare Reduce Embryonic Death Rates in Mares. Reprod Domest Anim. 2015 Aug;50(4):632-6. 

Pregnancy Rates and Uterine Fluid Post-Breeding with Frozen-Thawed Semen

In the past it’s been reported that insemination with frozen-thawed semen can result in an increased incidence of post-breeding intrauterine fluid (IUF) accumulation and decreased pregnancy rates compared with inseminations using fresh or chilled semen, said the authors of this study out the U.K. So they took another look and evaluated pregnancy rate and IUF’s association with frozen semen breeding in their retrospective study of 1,023 fresh, chilled, or frozen-thawed inseminations at one facility. “All mares, including old mares, had similar pregnancy rates between frozen and chilled semen breeding,” Blanchard said. “Even the older maiden mares did quite well with frozen semen breeding and had less intrauterine fluid accumulation following frozen semen breeding than with chilled or fresh semen.

“The authors concluded that frozen breeding can be suitable for older mares, including older maiden mares, and might not require the increased post-breeding management we sometimes expect to be necessary,” he said.

Lewis N, Morganti, M, Collingwood, F, et al. Utilization of One-Dose Postovulation Breeding With Frozen-Thawed Semen at a Commercial Artificial Insemination Center: Pregnancy Rates and Postbreeding Uterine Fluid Accumulation in Comparison to Insemination With Chilled or Fresh Semen. Journal of Equine Vet Sci. 2015 Nov-Dec.;35(11-12):882–887.e1 

Stallion Sperm Still Viable After Death

Finally, in this study out of Italy, researchers evaluated whether severe illness—in this case colic—would affect a stallion’s epididymal semen quality and freezability. They looked compared the epididymal sperm of five stallions that died within 12 to 36 hours after colic surgery and compared it to that of healthy stallions post-castration. The deceased stallions’ sperm motility parameters were initially lower than the healthy stallions’, but their sperm membrane integrity values were essentially the same. After processing to prepare for freezing, or after freezing and thawing, epididymal semen quality parameters did not differ between the two groups. Thus, frozen semen can still be good-quality after severe illness resulting in death, the researchers determined.

“While motility might be poor initially, if the membrane integrity is high, then we have a much better chance that it’s going to survive the freezing process,” Blanchard explained. “Now that we have the Nucleocounter (which also allows evaluation of sperm membrane intactness or viability) we can make those assessments quite rapidly and not waste our clients’ money on the freezing process if chances are poor that the sperm will survive freezing/thawing.”

Gloria A, Carluccio A, Petrizzi L, et al. Characteristics of frozen epididymal spermatozoa from stallions that died 12 to 36 hours after colic surgery. Theriogenology. 2015 Sep 11. pii: S0093-691X(15)00477-X. 

About the Author

Alexandra Beckstett, The Horse Managing Editor

Alexandra Beckstett, Managing Editor of The Horse and a native of Houston, Texas, is a lifelong horse owner who has shown successfully on the national hunter/jumper circuit and dabbled in hunter breeding. After graduating from Duke University, she joined Blood-Horse Publications as Assistant Editor of its book division, Eclipse Press, before joining The Horse.

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