Equine Reproduction Advances Discussed at AAEP
What was the hottest news in equine veterinary medicine in 2009? During the popular Kester News Hour session at the annual American Association of Equine Practitioners (AAEP) Convention, three top veterinarians (who focus on equine reproduction, internal medicine, and lameness/surgery) summarized the top news topics and the most significant research reports of the year for a record crowd of equine veterinarians.
The presenters during the 2009 convention, held Dec. 5-9 in Las Vegas, Nev., were:
- Scott E. Palmer, VMD, Dipl. ABVP (Equine Practice), hospital director and a staff surgeon of the New Jersey Equine Clinic in Clarksburg, N.J., and past president of the AAEP and American Board of Veterinary Practitioners;
- Margo L. Macpherson, DVM, MS, Dipl. ACT, associate professor and section chief in Reproduction at the University of Florida, and past president of the American College of Theriogenologists; and
- Bonnie R. Rush, DVM, MS, Dipl. ACVIM, professor of Equine Internal Medicine at Kansas State University, and winner of the 1996 and 2003 Carl J. Norden Distinguished Teacher Award, the 2002 Pfizer Award for Research Excellence, and the 2004 Outstanding Woman Veterinarian of the Year.
Double ovulations, hemorrhagic anovulatory follicles Investigators on one study published in the Journal of Equine Veterinary Science (JEVS) found mares were more likely to have double ovulations after receiving prostaglandin (PGF2-alpha), which is often used to manage a mare's estrous cycle. Seventeen percent of treated mares had double ovulations compared to 3% of control mares; also, 13% of treated mares vs. only 3% of controls had one ovulatory and one hemorrhagic anovulatory follicle (HAF, which grows and fills with blood, but does not release an egg, and, thus, results in infertility for that cycle unless another follicle ovulates normally).
Researchers on another study, this one from Theriogenology, investigated the incidence of HAF formation in mares given either cloprostenol (another form of prostaglandin) or human chorionic gonadotropin, or both. They showed mares treated with cloprostenol had a slightly higher risk of HAF (8%) than mares given human chorionic gonadotropin (hCG).
"Practitioners should be aware of potential ramifications for development of twins or hemorrhagic anovulatory follicles when using prostaglandin, particularly in mares at that have had HAF previously," commented Macpherson.
Ginther, OJ.; Al Mamun, M. Increased Frequency of Double Ovulations after Induction of Luteolysis with Exogenous Prostaglandin F2[alpha]. Journal of Equine Veterinary Science 2009; 29(7):581-583.
Cuervo-Arango, J.; Newcombe, J.R. The effect of hormone treatments (hCG and cloprostenol) and season on the incidence of hemorrhagic anovulatory follicles in the mare: A field study. Theriogenology 2009; 72(9):1262-1267.
hCG antibodies and dosage levels Macpherson discussed two studies on the hormone hCG, which is commonly used to manage estrous cycles in mares. In the first, published in Reproduction of Domestic Animals (RDA), researchers studied mares that had antibodies in their bloodstreams against hCG and mares that did not. They found mares with antibody-positive follicles had lower blood flow to the follicular wall, fewer mature oocytes, and no hCG detectable in the bloodstream 30 hours after administration (mares without antibodies still had high hCG levels at 30 hours).
Antibodies might have neutralized the hCG and affected ovarian function, said Macpherson. "We may need hCG protocols that stimulate lower antibody formation; this may be of particular relevance for assisted reproductive techniques such as oocyte retrieval," she said.
Researchers on a second hCG study, this one on dosage levels, found a lower dose (750 IU) was just as effective as a higher one (1500 IU) at stimulating ovulation within 48 hours, and it was also the same in terms of number of multiple ovulations and pregnancy rate.
"A lower dose is as effective at inducing ovulation ... and might have the added benefit of inciting a smaller antibody reaction," commented Macpherson.
Siddiqui, M.A.R.; Gastal, E.L.; Gastal, M.O.; Beg, M.A.; Ginther, O.J. Effect of hCG in the Presence of hCG Antibodies on the Follicle, Hormone Concentrations, and Oocyte in Mares. Reproduction of Domestic Animals 2009; 44(3):474-479.
Davies-Morel, M.C.G.; Newcombe, J.R. The efficacy of different hCG dose rates and the effect of hCG treatment on ovarian activity: Ovulation, multiple ovulation, pregnancy, multiple pregnancy, synchrony of multiple ovulation; in the mare. Animal Reproduction Science 2008; 109(1-4):189-199.
Temperature, exercise, and embryo recovery Macpherson discussed two studies on the effects of exercise and moderately high ambient temperatures (around 86°F) on mare reproductive performance. Mares bred via artificial insemination each went through one study phase on a forced exercise regimen (30 minutes per day on a mechanical exerciser) and one phase without forced exercise. When exercised, the mares' body temperatures increased by about 2°C, and they exhibited decreased follicular size, increased time between ovulations, fewer embryos recovered (34% of exercised mares vs. 63% of controls), and 50% fewer Grade 1 embryos.
"Exercise is associated with increased rectal temperatures and may have a negative impact on reproductive function," noted Macpherson. "Is this due to exercise, heat, or both?" Researchers continue to work on finding the answer.
In a third study, published in JEVS, investigators used a novel temperature-measuring device, the iButton, to measure uterine temperature under different housing conditions (pasture vs. stalls with fans) and exercise. Measurements were compared with rectal temperatures and temperatures from microchips inserted in the neck. Macpherson reported the iButton proved to be an accurate measurement of uterine temperature, and temperatures were lowest in mares kept in stalls with fans, rather than mares at pasture or stalled mares that were exercised.
"With a problem mare, one might consider housing her under controlled temperature conditions using a stall with a fan. For the athletic mare, a rest from exercise might be best when breeding," she added.
Mortensen, C.J.; Choi ,Y.H.; Hinrichs, K.; Ing, N.H.; Kraemer, D.C; Vogelsang, S.G.; et al. Embryo recovery from exercised mares. Animal Reproduction Science 2009; 110(3-4):237-244.
Kelley, D.E.; Gibbons, J.R.; Pratt, S.E.; Smith, R.L.; Mortensen, C.J. Exercise lengthens the interovulatory interval in mares. Journal of Equine Veterinary Science 2009; 29(5):337-338.
Commaille, L.; Vogelsang, M.; Sigler, D.; Hinrichs, K. Intrauterine Temperature of Mares Under Different Management Conditions. Journal of Equine Veterinary Science 2009; 29(5):344-345.
Semen centrifugation product In a series of studies scientists investigated the use of a single-layer silica colloid centrifugation technique (available in Europe) for selecting optimal populations of sperm prior to breeding or assisted reproductive techniques. The single-layer technique was compared to more traditional double-layer centrifugation protocols used for sperm selection. The overall quality of sperm recovered after single-layer colloid centrifugation was better than that of unprocessed sperm. Also, all measures of sperm quality were better using the single-layer gel product on sperm that had been cooled, stored at room temperature, or frozen and later thawed.
"The most telling thing was that the quality of sperm was maintained even after 24 hours of storage at room temperature," Macpherson commented. "This alone has the possibility of revolutionizing the way we store and transport semen and could make our jobs logistically that much simpler."
The researchers also showed the technique could be applied to a large-volume ejaculate using a readily available benchtop centrifuge and centrifugation supplies.
However, she noted, sperm yield tended to be lower with this method and, thus, it might be best reserved for use with poor-quality semen and/or with deep-horn artificial insemination.
Morrell, J.M.; Johannisson, A.; Dalin, A.M.; Rodriguez-Martinez, H. Morphology and Chromatin Integrity of Stallion Spermatozoa Prepared by Density Gradient and Single Layer Centrifugation Through Silica Colloids. Reproduction of Domestic Animals 2009; 44(3):512-517.
Morrell, J.M.; Johannisson, A.; Strutz ,H.; Dalin, A.M.; Rodriguez-Martinez, H. Colloidal Centrifugation of Stallion Semen: Changes in Sperm Motility, Velocity, and Chromatin Integrity during Storage. Journal of Equine Veterinary Science 2009; 29(1):24-32.
Pregnancy location The location of a pregnancy (i.e., whether it is in the left or right uterine horn) clearly influences the location of the next pregnancy. A study published in Theriogenology found that in nearly 1,400 Thoroughbred mares, 79% of pregnancies occurred in the uterine horn that did not house the fetus in the previous season. The number of pregnancies in the previously gravid horn (the one where the previous fetus implanted) increased with mare age and time between foaling and conception. Lastly, Macpherson reported that there was a higher incidence of early embryonic death (17% vs. 5%) when the embryo implanted in the previously gravid horn.
"Particularly in older mares, we may want to skip a cycle between foaling and breeding or even consider skipping a year," she commented. "Alternatively, embryo collection and transfer to a surrogate mare may be a good option for early breeding in these mares."
Davies-Morel, M.C.G.; Newcombe, J.R.; Hinchliffe, J. The relationship between consecutive pregnancies in Thoroughbred mares. Does the location of one pregnancy affect the location of the next, is this affected by mare age and foal heat to conception interval or related to pregnancy success. Theriogenology 2009; 71(7):1072-1078.
Semen types, time of ovulation, and embryo size Breeding with natural cover vs. frozen/thawed semen didn't seem to impact embryonic vesicle size in a Theriogenology study, but breeding timing (i.e., just before or just after ovulation) did. At Days 12-19 of pregnancy, vesicles in mares bred post-ovulation were smaller than in those bred pre-ovulation.
"You might see smaller vesicles during early pregnancy examinations with post-ovulation matings, and this might not be significant to embryo health, but just due to the breeding method," said Macpherson. These embryos ultimately "catch up" in size. "Also, when harvesting embryos for transfer after post-ovulation breeding (i.e., artificial insemination with frozen-thawed semen), it's probably worthwhile to wait until Day 7.5 or 8 for embryo flushes to improve chances for embryo recovery."
Cuervo-Arango, J.; Aguilar, J.; Newcombe, J.R. Effect of type of semen, time of insemination relative to ovulation and embryo transfer on early equine embryonic vesicle growth as determined by ultrasound. Theriogenology 2009; 71(8):1267-1275.
Cloned foal health While the survival rate of somatic cell transfer-produced foals (clones) is higher than that of cloned calves, these foals still tend to be high-risk pregnancies and high-maintenance foals, reported Macpherson. Researchers on a study of 14 cloned foals produced at Texas A&M (published in JAVMA) found the following:
- 14 live foals were born from 54 transferred embryos. Two were born mature, but early, while one was born late and small.
- Some mares had significant edema (fluid swelling) in the combined thickness of the uterus and placenta.
- All foals received oxygen therapy, seven for more than 12 hours post-foaling.
- All foals received good-quality colostrum or hyperimmune plasma within 12 hours.
- Seven foals had an abnormal umbilicus.
- Eight foals had flexural deformities of the limbs.
- Two died, one from pneumonia and one that was weak at birth and had seizures (died during anesthesia for bladder exploratory surgery).
"These are clearly high-risk pregnancies, and the recommendation from Texas A&M is to transfer these mares to a referral facility at 300 days of gestation so the intensive care the foals may need is available," reported Macpherson.
Johnson, A.K.; Clark-Price, S.; Choi, Y.; Hartman, D.L.; Hinrichs, K. Physical and Clinicopathologic findings in foals produced by somatic cell transfer - 14 cases (2005-2008). Journal of the American Veterinary Medical Association (in press).
Sperm hyperactivation Hyperactivity of sperm, characterized by disorganized, hyperactive movement patterns when viewed under a microscope, is necessary for equine sperm to be able to penetrate and fertilize an egg. Cornell University researchers have recently made some "groundbreaking" discoveries in regards to sperm capacitation (the series of changes on the surface of the sperm that allow the first step of the fertilization process) and hyperactivity, reported Macpherson. One was a current study that used procaine to hyperactivate sperm.
Macpherson quoted Dr. Sylvia Bedford, the lead investigator for this study, as describing the result of sperm treated with procaine as "sperm on drugs." This protocol combining induced capacitation and hyperactivation yielded more than 60% success rates with in vitro fertilization (IVF) over several estrous cycles (normally IVF success rates for horses are quite poor).
"This may change what we do with assisted reproductive technologies in the future and gives us hope for using IVF--it's less expensive and involved than other techniques," Macpherson commented. "Getting the yield rates up may mean we can actually use it."
McPartlin, L.A.; Suarez, S.S.; Czaya, C.A.; Hinrichs, K.; Bedford-Guaus, S.J. Hyperactivation of Stallion Sperm Is Required for Successful In Vitro Fertilization of Equine Oocytes. Biology of Reproduction 2009; 81(1):199-206.
About the Author
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.