2011's Top Equine Reproduction News (AAEP 2011)

2011's Top Equine Reproduction News (AAEP 2011)

Investigators from Argentina presented lessons learned from and techniques used in a successful embryo transfer program, which included nearly 15,000 embryo flushes, nearly 8,000 established pregnancies, and 1,900 embryos transferred.

Photo: Anne M. Eberhardt/The Horse

Equine reproduction is a popular topic for researchers to study, so it comes as no surprise that hundreds of scientific articles on a variety of reproduction-related topics are published each year. During the 2011 American Association of Equine Practitioners convention, held Nov. 18-22 in San Antonio, Texas, some of the most clinically relevant studies on the broad topic were presented during the annual Kester News Hour. Patrick M. McCue, DVM, PhD, Dipl. ACT, professor in the department of clinical sciences at Colorado State University, shared his thoughts on top studies at the popular session.

Ovarian tumors can cause hormonal disturbances that lead to behavioral and reproductive problems for affected mares. Often veterinarians confirm these tumors based on finding abnormal masses when rectally palpating the reproductive tract, but researchers at the University of California, Davis, studied whether blood levels of anti-Müllerian hormone (AMH) could also be useful in diagnosing these tumors. They found that AMH levels were low in healthy mares, and did not change throughout the estrous cycle, or with season or during pregnancy . However, mares with granulosa cell tumors (one of four ovarian tumor types) had elevated AMH levels that decreased after tumor removal. Thus, the researchers concluded, AMH level is a useful biomarker for granulosa cell tumors. The assay is not yet available for clinical use.

Almeida J, Ball BA, Conley AJ, et al. Biological and clinical significance of anti-Müllerian hormone determination in blood serum of the mare. Theriogenology 2011 (in press).

Equine viral arteritis (EVA) is a highly contagious disease that can cause fever, respiratory illness, eye inflammation, edema (swelling, especially of the limbs), weakness or sickness in foals, and abortion. The virus can be transmitted via respiratory secretions or breeding (natural cover or artificial insemination). The AAEP's EVA vaccination guidelines for pregnant mares state, "The manufacturer does not recommend use of this vaccine in pregnant mares, especially in the last two months of pregnancy." However, Oklahoma State University researchers studied the safety of vaccinating in-foal mares; McCue summarized their findings as follows: "It appears to be safe to vaccinate healthy pregnant mares against equine arteritis virus up to three months before foaling and during the immediate postpartum period. Vaccination during the last two months of pregnancy was associated with a risk of abortion."

Broaddus CC, Balasuriya UB, White JLR, et al. Evaluation of the safety of vaccinating mares against equine viral arteritis during mid or late gestation or during the immediate postpartum period. J Am Vet Med Assoc 2011;238:741-750.

EVA vaccination is also recommended for stallions (an initial vaccine, then annual boosters), but researchers on a recently described study found that there is a temporary low level of viral activity in semen shortly after initial vaccination. Thus, researchers recommend not collecting or freezing any semen from a stallion for 14 days after his first EVA vaccination.

"Withhold use of semen for 14 days after first-time vaccination to minimize risk of transmission of vaccine virus and eliminate potential for vaccine virus in frozen semen," advised McCue. The good news is that there was no evidence of persistent vaccine virus in first-time vaccinated stallions' reproductive tracts, and there was no transmission of vaccine virus to in-contact horses.

Summers-Lawyer KA, Go YY, Lu Z, et al. Response of stallions to primary immunization with a modified live equine viral arteritis vaccine. J Equine Vet Sci 2011;31:129-138.

Endometritis is inflammation of a mare's uterine lining (endometrium), usually due to infectious organisms. The result is a uterine environment that's less than hospitable to a developing equine embryo and, thereby, a less fertile or infertile mare. Diagnosing the specific cause of endometritis in affected mares is essential to developing a targeted, effective treatment strategy; thus, researchers from the University of Berlin, Germany, and the University of Napoli, Italy, studied endometrial sample collection methods and the effectiveness of various diagnostic strategies for endometritis alone and in combination. They evaluated cytology (looking for and evaluating inflammatory cells in samples) and culture (looking for any microorganisms that might grow from uterine samples), and the combination of both culture and cytology.

"Cytology or culture alone were associated with a high incidence of 'false negative' results (meaning no infectious organisms were detected when they actually were present)," reported McCue. "A combination of cytology and culture improved diagnostic capability. A procedure called the cytology brush was the preferred method of sample collection. Infection with E. coli was not always associated with inflammatory changes on cytology, whereas infection with Streptococcus zooepidemicus was associated with a positive cytology in 40 % of cases."

McCue told practitioners that if they're just screening mares with cytology, a negative result doesn't mean the mare is free of E. coli infection, and interpretation of the cytology specimens (number of white blood cells/high-powered field or the ratio of white blood cells to uterine epithelial cells) might be more important than the technique used to obtain the specimen.

Overbeck W, Witte TS, Heuwieser W. Comparison of three diagnostic methods to identify subclinical endometritis in mares. Theriogenology 2011;75:1311-1318.
Cocchia N, Paciellob O, Auletta L, et al. Comparison of the cytobrush, cotton swab, and low-volume uterine flush techniques to evaluate endometrial cytology for diagnosing endometritis in chronically infertile mares. Theriogenology (in press).

Fungal endometritis (that caused by fungus-type organisms) can be difficult to diagnose with traditional culture methods, noted McCue. However, Colorado State University (CSU) researchers have found that quantitative polymerase chain reaction (qPCR) testing can now identify fungal organisms in endometrial samples.

"The qPCR for fungal DNA was determined to be rapid (within 24 hours) and able to identify fungal organisms in instances where traditional microbial culture and biochemical analysis could not," summarized McCue. "Next year, you'll be able to get genus and species identification for $85/sample in two days (from CSU)."

Ferris RA, Dern K, Veir J, et al. Development of a broad range qPCR assay to detect and identify fungal DNA in equine endometrial samples. Clin Theriogenology 2011;3:375. Abstract from Society for Theriogenology meeting.

Oviductal patency, or whether the oviduct that channels eggs from the mare's ovary to the uterus is open/unblocked, is critical to fertility. Researchers at the Hannover Foundation in Germany recently investigated two laparoscopic (minimally invasive) surgical procedures for catheterizing the oviduct to flush it and, thereby, evaluate whether it is patent (open) or blocked.

"Catheterization of the oviduct can be accomplished using a flank laparoscopic approach and the oviduct can be subsequently flushed to evaluate patency," reported McCue. "This technique can provide diagnostic information regarding oviductal blockage in subfertile mares."

Köllmann M, Rötting A, Heberling et al. Laparoscopic techniques for investigating the equine oviduct. Equine Vet J 2011;43:106-111.

Can acupuncture stimulate mares to start cycling? Researchers conducted two studies to see if dry-needle acupuncture or electroacupuncture could induce earlier cycling in winter anestrous (noncycling) mares, but they came up empty in both cases.

"Within the parameters of this study, there was no significant difference in average interval to first ovulation of the year between acupuncture-treated and control mares," said McCue. "Additional controlled studies are clearly needed to provide more information on the efficacy of acupuncture on (ovarian) follicular development and on evacuation of uterine fluid, its other common use."

Niemantsvertriet-Murton AS, Paccamonti D, Eilts BE, et al. Use of acupuncture to induce cyclicity in anestrous mares. J Equine Vet Sci 2011;31:97-102.

"Vets are often confronted with the unfortunate, untimely death of a stallion," began McCue in his review of a study evaluating the fertility of sperm collected postmortem. "Collection of epididymal sperm (from the testes) allows a final opportunity to preserve semen from that horse."

Researchers from the Universidade Estadual Paulista, in Brazil, compared the fertility of sperm harvested from the testes after castration with that of frozen or freshly collected semen. The harvesting procedure yielded an average of 25 billion sperm--compared with about 8 billion sperm per normal ejaculate--and there was no difference in motility between harvested sperm and fresh/frozen-thawed semen. Harvested sperm also yielded slightly (although not statistically significantly) higher pregnancy rates when processed immediately.

"Collection and cryopreservation of epididymal semen immediately after castration or after 24 hours of cooling can yield breeding doses for future use in the event of a catastrophic injury to a stallion," McCue summarized. "Be ready for the death or elected castration of a valuable stallion (such as for behavioral reasons). Having a game plan ahead of time can be very valuable for the client."

Monteiro GA, Papa FO, Zahn FS, et al. Cryopreservation and fertility of ejaculated and epididymal stallion sperm. Anim Reprod Sci 2011;127:197-201.

"Assessment of sperm morphology (physical characteristics) is a key component of semen analysis," noted McCue as he described a study in which researchers compared semen evaluation staining procedures for use in horses. Wet mount preparations were found to be more sensitive for detecting abnormal sperm than stained smears, while smear preparations increased the proportion of detached heads. The Papanicolaou stain (a multichromatic staining that allows scientists to differentiate cells in smear preparations; it's well-known for its use cervical cancer screening in women) was unsuitable for stallions.

"A significant difference was noted among clinicians, suggesting that consistent training is important," McCue commented.

Brito LC, Greene LM, Kelleman A, et al. Effect of method and clinician on stallion sperm morphology evaluation. Theriogenology 2011;76:745-750.

In another study of sperm quality and fertility, scientists looked at seasonal pregnancy rate, percentage of mares pregnant per cycle, and percentage pregnant on the first cycle. They found that a number of sperm abnormalities (such as coiled tails or abnormal/detached heads) unsurprisingly depressed fertility, and more fertile stallions had better sperm motility and a higher percentage of morphologically normal sperm.

"The percentage of mares pregnant on their first cycle was the only fertility measurable to discriminate among high, average, and low fertility groups (among stallions)," McCue noted.

Love CC. Relationship between sperm motility, morphology and the fertility of stallions. Theriogenology 2011;76:547-557.

Embryo Transfer and Assisted Reproduction

McCue described one report of a successful embryo transfer program as "a valuable contribution to the world of embryo transfer with mind-boggling numbers--a great data set." Investigators from Argentina presented the techniques and lessons learned from nearly 15,000 embryo flush procedures (up to 2,300 per year), nearly 8,000 established pregnancies, and 1,900 embryos transferred over a 13-year period.

"The take-home message is that a breeding soundness evaluation should be performed on each donor and recipient mare," summarized McCue. "Also, recipient mare selection and management is critical to success. Recipient mares can ovulate anywhere from the day before the donor mare ovulates until 4 days after the donor mare ovulates.

Riera F. General techniques and organization of large commercial embryo transfer programs. Clinical Theriogenology 2011;3:318-324.

"Freezing small embryos is easy, but large ones are problematic and historical failures," said McCue as he introduced a study of a new technique for optimizing freezing of large embryos by the process of vitrification. This new technique involves removing fluid from the initially hollow early-stage embryo (termed a blastocyst) to collapse it prior to vitrification. Large embryos that were intentionally collapsed prior to freezing yielded good pregnancy rates after transfer (there was a 71% pregnancy rate in this study).

"This technique appears to be a significant step forward for equine embryo transfer and cryopreservation of embryos," McCue commented.

Choi YH, Velez IC, Riera FL, et al. Successful cryopreservation of expanded equine blastocysts. Theriogenology 2011;76:143-152.

Veterinarians may biopsy embryos for genetic evaluation for a number of reasons, from determining sex to identifying inherited diseases. McCue discussed a study in which scientists defined proper embryo biopsy techniques and ways to evaluate biopsy results.

"Biopsy of large blastocyst stage embryos was possible and did not compromise embryo viability," he reported. "Genetic analysis was accurate for sex determination and is a 'work in progress' for diagnosing HERDA (hereditary regional dermal asthenia, also called hyperelastosis cutis, a skin condition) and HYPP (hyperkalemic periodic paralysis, a metabolic/muscle condition). Pre-implantation genetic diagnosis may soon become a viable clinical procedure in the horse."

Hinrichs K. Biopsy and vitrification of equine expanded blastocysts. Clinical Theriogenology 2011; 3:314-317.

Reproduction Case Reports and Abstracts

Why do some mares routinely abort foals? There are a host of reasons, but here's one you don't see every day: chromosome swap (translocation). McCue discussed a case report of a 10-year-old Thoroughbred mare that had delivered three live foals but had aborted four in prior years, in addition to having three early-term abortions in the year she was studied (between Days 20-40 of gestation). As it turns out, she had a translation of part of Chromosome 2 to Chromosome 13, explaining her subfertility.

"The take-home message here is that just because a mare has had a live foal doesn't mean she is free of the possibility of chromosomal issues," he commented. "I would have put that way down on a differential list since she had had live foals, but these abnormalities may be more common than we think. At the (University of Kentucky's) Gluck Center lab, 35-40% of samples submitted from subfertile mares have some karyotype (chromosome) abnormality. Karyotype with additional tests (roughly $200-450) may be required to identify a chromosomal problem (i.e. a translocation)."

Lear TL, Raudsepp T, Lundquist J, et al. A chromosome translocation [64,XX,t(2:13)] in a Thoroughbred mare with repeated early embryonic loss. J Equine Vet Sci 2011;31:240.

Next McCue presented a case study involving an 8-year-old mare, pregnant for the first time, that lactated (produced milk) about three months early for a month. Then the mare delivered one healthy foal and one mummified twin on time.

"Premature lactation may be associated with loss of one fetus of a twin pregnancy," he summarized. "Early pregnancy diagnosis may have resolved this issue at 14-16 days of gestation (i.e., the twin situation would have been identified and likely resolved), but no early pregnancy exam was done in this case."

Robinson KA, Manning ST. Premature lactation and retention of a mummified fetus with live birth of the co-twin in a primiparous Morgan mare. Can Vet J 2011;52:423-425.

About the Author

Christy M. West

Christy West has a BS in Equine Science from the University of Kentucky, and an MS in Agricultural Journalism from the University of Wisconsin-Madison.

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