2010's Top Equine Reproduction Studies (AAEP 2010)
The science of equine reproduction enjoyed many significant advancements in 2010, and these were discussed during the popular Kester News Hour session of the American Association of Equine Practitioners convention, held Dec. 6-10 in Baltimore, Md. 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, presented the top published stories in this area during the convention.
In a study Macpherson termed "attention-getting," researchers inserted tiny colored fluorescent beads into mares' oviducts, then performed uterine lavage one and two days later, to assess whether the oviducts were patent (open, or allowing the beads to pass along the oviduct to the uterus). Beads were only recovered from 40% of mares, suggesting blockage of one or both oviducts was present in the other 60% of mares (which averaged 8 years of age compared to 4 years for the patent mares).
"While this isn't a first line of defense for the barren mare, this novel approach could provide answers for mares with long-standing, unexplained infertility," Macpherson comented. "Similar studies have also shown that oviductal flushing can sometimes resolve obstruction and result in future pregnancy."
- Arnold,C.E. & Love,C.C. Laparoscopic evaluation of oviductal patency in the standing mare. Animal Reproduction Science 121, 86 (2010).
"Work from several investigators in the past five years has suggested that clinical diagnosis of a uterine infection can vary depending on the organism involved," said Macpherson. In other words, different bacteria cause different levels of inflammation in the uterus (endometritis), causing veterinarians to diagnose them differently. She described an Animal Reproduction Science (ARS) study in which researchers reported that Streptococcus zooepidemicus bacteria caused significantly more inflammation than Escherichia coli in susceptible mares (those that tend to get uterine inflammation/infection more easily than others).
Also, resistant mares (those that do not get uterine infections as easily) did not have any bacteria recovered from their uteri, meaning they were able to clear the infection on their own. However, their susceptible counterparts yielded Strep after infection with that bacterium, but generally did not have E. coli cultured even after infection with that bacterium.
Researchers in two more studies (in Journal of Equine Veterinary Science, JEVS, and ARS) also evaluated uterine infections; both reinforced the findings of more positive culture results with Streptococcus compared to E. coli. Also, endometrial biopsy samples were found to be better for cytology and culture testing than guarded swab samples.
"All uterine infections are not created equally," said Macpherson. "E. coli infections may be more insidious in nature and require more aggressive diagnostic procedures to detect."
In a related study, researchers from Colorado State University validated a polymerase chain reaction (PCR) test for detecting bacteria in the uterus. This is a "highly sensitive tool" that identified bacteria in 33% of mares with endometritis, whereas standard culture identified bacteria in only 22% of cases.
"PCR has great potential as an adjunct tool for mares with a long-standing history of infertility," Macpherson commented. Also, she added, "Preliminary data from a PCR test used to detect fungal DNA from uterine samples shows great promise for difficult-to-detect fungal organisms."
- Eaton,S.L., Raz,T., Chirino-Trejo,M., Bergermann,J. & Card,C. Comparison of endometrial inflammation following intrauterine inoculation with genital strains of Streptococcus equi subsp. zooepidemicus or Escherichia coli in the mare. Animal Reproduction Science 121, 101-102 (2010).
- Nielsen,J.M.l. et al. Diagnosis of Endometritis in the Mare Based on Bacteriological and Cytological Examinations of the Endometrium: Comparison of Results Obtained by Swabs and Biopsies. Journal of Equine Veterinary Science 30, 27-30 (2010).
- Burleson,M.D., LeBlanc,M., Riddle,W.T. & Hendricks,K.E.M. Endometrial microbial isolates are associated with different ultrasonographic and endometrial cytology findings in Thoroughbred mares. Animal Reproduction Science 121, 103 (2010). AAEP 2010
- Ferris,R.A., Veir,J.K., Lappin,M.R. & McCue,P.M. Development and clinical application of a broad range 16S quantitative PCR assay for detection of bacteria in the uterus of the mare. Animal Reproduction Science 121, 98-100 (2010).
One group of researchers researched treating placentitis (placental infection/inflammation, a leading cause of foal loss). Veterinarians treated mares with clinical signs of induced placentitis using a combination of trimethoprim sulfamethoxazole, pentoxifylline, and altrenogest at standard doses. Treatment continued from the onset of clinical signs until foals were born or aborted.
According to Macpherson, 83% of the treated mares delivered live foals compared to zero control mares. Ten of 12 live foals of treated mares had no bacteria in their bloodstreams, whereas all of the aborted foals had bacteria in at least one of the following locations: Blood, stomach contents, and/or chest fluids.
"The workers attributed the rapid and aggressive treatment, within 96 hours of infection, to the successful delivery of live foals in treated mares," said Macpherson.
Another group of researchers tested several drugs against induced placentitis, using antibiotics alone (such as trimethoprim sulfamethoxazole) or in combination with immunomodulators (in this case, immune system boosters) such as the corticosteroid dexamethasone, aspirin, and progestins (synthetic hormones similar to progesterone). Treatment was initiated with onset of clinical signs (within 48 hours of infection), and two-thirds of the mares delivered viable foals regardless of which treatment they received.
"These findings beg the question of whether drug choice is the determining factor in successful treatment of bacterial placentitis, or (if it is) early detection of disease and rapid initiation of treatment," Macpherson said. She also noted that treated mares delivered about 10 days earlier than healthy mares, suggesting that accelerated foal maturation occurred with the disease.
Lastly, she discussed an Australian study in ARS which mares were classified as being at "high-risk" of placentitis if they had less than a 50% live foal rate within the last three years. High-risk mares were monitored with ultrasound and visual observation starting at five months of gestation, and treated with antibiotics and altrenogest if they showed any signs of placentitis. More persistent cases also got anti-inflammatory medications.
With this treatment protocol, more than 80% of high-risk mares delivered live foals, compared to only about 25% before the study.
"Monitor mares at risk for premature delivery to ensure prompt diagnosis," recommended Macpherson. "Initiate treatment early, treat aggressively and for the duration of pregnancy. To ensure continued reproductive health for the mare, she should also be treated in the postpartum period--clean those mares up!"
Another mare management study (in Theriogenology) involved evaluating the effects of Regu-Mate (altrenogest, a progesterone hormone product). "Many of us use Regu-Mate, liberally at times, for support of pregnancy," Macpherson said.
Researchers found no difference in pregnancy rates with Regu-Mate, but they did find that older mares (which had smaller embryos at all time points) that were given the product had significantly larger embryos from Days 30-45 of gestation compared to older mares that didn't get Regu-Mate. Also, some mares then produced more equine chorionic gonadotropin hormone (which also helps support pregnancy).
"Early treatment (no later than Days 5-6) of pregnant mares, particularly aged ones, with altrenogest may have a positive effect on embryo and placental development," Macpherson noted.
- Bailey,C.S. et al. Treatment efficacy of trimethoprim sulfamethoxazole, pentoxifylline and altrenogest in experimentally induced equine placentitis. Theriogenology 74, 402-412 (2010).
- Christiansen, D.L. et al. Evidenced-based medicine approach to develop efficacious therapies for late-gestation mares presenting with uterine infections using an experimentally-induced placentitis model. Animal Reproduction Science 121, 345-346 (2010).
- Carrick,J.B., Begg,A.P., Perkins,N.R., O'Meira,A.O. & O'Meara,D.O. Ultrasonographic monitoring and treatment of pregnant mares at risk for placentitis. Animal Reproduction Science 121, 331-333 (2010).
- Willmann,C., Schuler,G.,Hoffman,B.,Parvizi,N., Aurich,C. Effects of age and altrenogest treatment on conceptus development and secretion of LH, progesterone and eCG in early-pregnant mares. Theriogenology, 2011 Feb;75(3):421-8.
Macpherson also reported that an injectable, sustained-release deslorelin product (for managing estrus in mares, using the same primary ingredient as the product Ovuplant, which has been off the market for some time) gained Food and Drug Administration approval in the week before the convention, and was expected to be available by January 18, 2011. The product, called SucroMate, is being distributed by Bioniche Life Sciences.
Broodmares and Financial Return
In the financial arena, Macpherson reported that the general economic downturn has not spared the Florida horse production industry, which saw a "staggering drop in Thoroughbred production" of nearly a 30% reduction in foal crops from 2009 to 2010.
"I am guessing that few of us in this room are prepared to give up breeding mares, racing horses, or being equine veterinarians," she commented. "Instead, we need to make better decisions about what we do and how we do it. A first start is evaluating our breeding practices."
In that vein, Macpherson described a recent production data study in Equine Veterinary Journal (EVJ) where researchers evaluated mare value and productivity, with an eye toward helping breeders maximize returns and mare productivity.
Most mares' foaling dates tend to drift later in the year with subsequent seasons, often resulting in a missed or barren season, she noted. Of the 1,176 mares in the study, 63% failed to produce a registered foal every season within the seven-year study period. For those mares, the average time to a barren season was 3.4 years.
Factors including increasing mare age, foaling after April 1, requiring multiple breedings in a season, and producing fewer foals in previous years were associated with reduced foal production during the study. Additionally, the researchers found that a mare must produce a live foal in at least six out of seven years to recoup the breeder's investment in her, and that higher-value mares yield better financial gains.
"Mares are long-term investments," concluded the study authors. "Improving our understanding of mare, stallion, and management factors that affect the likelihood of producing a live foal is critical to ensuring a positive financial return."
Also, Macpherson reported that breeding earlier in the year yielded higher pregnancy rates, and that early breeding was especially important for maiden and barren mares. Foal heat breeding was also effective in reproductively healthy mares in one study, but not in another.
On the stallion side of things, some stallions were found to be more fertile than others in one study, and reinforcement breeding was again found to improve pregnancy rates when used in a natural cover program. Later covers in the day were associated with decreased pregnancy rates, as was the presence of neutrophils (a type of immune cell) in the dismount sample (possibly indicating uterine inflammation in the mare). Tranquilizers given to mares also had a detrimental effect (whether that was because mares were not ready for breeding or because of a pharmacologic effect of the tranquilizer drug was not clear).
"The majority of factors associated with variation of fertility in this study, both positive and negative, were attributable to the mare," said Macpherson.
- Bosh,K.A., Powell,D., Shelton,B. & Zent,W. Reproductive performance measures among Thoroughbred mares in central Kentucky, during the 2004 mating season. Equine Veterinary Journal 41, 883-888 (2009).
- Bosh,K.A., Powell,D., Neibergs,J.S., Shelton,B. & Zent,W. Impact of reproductive efficiency over time and mare financial value on economic returns among Thoroughbred mares in central Kentucky. Equine Veterinary Journal 41, 889-894 (2009).
- McDowell, K.J., Little,T.V. & Baker,C.B. Models to predict reproductive success in horses. Animal Reproduction Science 121, 126-127 (2010).
- Blanchard,T.L. et al. Some Factors Associated With Fertility of Thoroughbred Stallions. Journal of Equine Veterinary Science 30, 407-418 (2010). http://www.j-evs.com/article/S0737-0806(10)00225-X/abstract
Stallion Vocalizations for Teasing?
Could farm managers tease mares to assess estrus by simply playing tape recordings of stallion vocalizations? The results of a recent study confirm the effectiveness of this practice, which could be used by some breeding farms.
Researchers on an ARS study played taped stallion vocalizations to broodmares and were thereby able to identify estrous behavior. The effectiveness of this practice was improved when the mares were also presented with stallion scent, with a successful estrus detection rate of over 80%.
"This tactic might provide a less threatening environment for mares, which could be especially important for maiden or timid mares or on small farms with no stallions," Macpherson added. "Further, if physical finding of the reproductive tract were combined with behavioral responses, as is typical in practice, one would expect the sensitivity of this tool to be even better."
- Janson,K.M. & Mcdonnell,S.M. Estrus detection in mares using contextually congruent stallion vocalization playback with and without stallion scent. Animal Reproduction Science 121, 123-125 (2010).
Stallion semen is generally centrifuged, or spun at high speeds, during processing for extending/shipment so sperm are concentrated. However, this procedure can crush some sperm depending on the technique used, so researchers often evaluate techniques to reduce this sperm loss, such as by using a cushion medium in the tube.
Macpherson discussed one study in which researchers modified cushioned centrifugation procedures to use less cushion medium (1 mL instead of 3.5) and larger sperm samples (3 billion instead of 1), and found that sperm recovery rates were still higher than 90% regardless of cushion volume or sperm number. Sperm characteristics were also unaffected even after cooling for 24 hours.
"More sperm and less cushion means less tubes to process, which can increase efficiency and reduce costs," explained Macpherson.
Another study she was "particularly excited about" looked at increasing the concentrations of sperm in cooled, shipped semen from 25 million sperm per milliliter of shipped semen to 250 million sperm/mL. No negative impact on sperm characteristics was noted with this protocol.
"So we can ship more sperm, perhaps even up to 20 billion sperm," Macpherson noted. "This could be useful for providing more sperm for traditional artificial insemination (AI) or for low-dose AI. The one qualifier for both of these studies is that these protocol changes have not been tested for fertility."
- Bliss,S.B. et al. Effect of sperm number and cushion volume during centrifugation on the quality and recovery rate of stallion sperm. Animal Reproduction Science 121, 220-221 (2010).
- Voge,J.L. et al. Cooled storage of semen at high or low sperm concentrations: effects on sperm motility, membrane integrity, and chromatin quality. Animal Reproduction Science 121, 210-211 (2010).
Contagious Equine Metritis
Macpherson also discussed a study of contagious equine metritis (CEM), a highly contagious bacterial infection that causes acute endometritis in mares and subsequently impacts fertility. The disease caused significant consternation recently in the U.S. breeding industry (see "CEM Investigation: 23 Positive Stallions"). For this study, researchers evaluated whether mixing antibiotics with semen extender would prevent transmission of the bacterium that causes CEM, Taylorella equigenitalis, from infected stallions to mares. They found that growth of the bacterium was "significantly inhibited" by the antibiotics and that mares bred with this semen did not become ill, whereas all mares bred with raw (not extended), infected semen developed CEM.
"The risk of transmission of CEM can be significantly reduced by mixing semen with extenders containing antibiotics," summarized Macpherson.
- Klein,C. et al. Antibiotic-containing semen extender reduces the risk of transmission of CEM. Animal Reproduction Science 121, 222-223 (2010).
"Exciting advances in embryo biopsy techniques for diagnosing genetic diseases were revealed in 2010," said Macpherson. In humans, she added, this technique is commonly used to screen for genetic disease before implanting embryos created by in vitro fertilization.
Texas A&M University researchers have found good success with biopsying equine embryos that were 6-8 days old, then transferring the embryos into mares or freezing them for later use. Fresh, shipped embryos still yielded better than 75% pregnancy rates.
Diseases detected from the biopsied cells included hyperkalemic periodic paralysis (HYPP) and hereditary equine regional dermal asthenia, and embryo sex was also determined.
"This laboratory is working hard to perfect gene amplification procedures to allow for diagnosis of additional diseases," Macpherson said. (For more information, see "Assisted Reproduction in Horses: Practical Usage (AAEP 2010).")
Another study, this one from the University of Kentucky, also looked at vitrification (freezing) of biopsied embryos.
"In this groundbreaking study, transfer of frozen/thawed embryos subjected to biopsy resulted in a 75% pregnancy rate at day 14," reported Macpherson. "Three live foals were born from these biopsied embryos, including the first reported live foal following these procedures. Successful embryo biopsy for pre-implantation genetic diagnosis of equine diseases may revolutionize the way we manage genetic diseases in horses."
- Choi,Y.H. et al. Viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis. Reproduction REP-10 (2010).
- Troedssen,M.H.T. et al. Transfer success of biopsied and vitrified equine embryos. Animal Reproduction Science 121, 295-296 (2010).
- Seidel,G.E., Cullingford,E.L., Stokes,J.E., Carnevale,E.M. & McCue,P.M. Pregnancy rates after vitrification, warming and transfer of equine embryos. Animal Reproduction Science 121, 299-300 (2010).
- Brosnahan,M.M.,Brooks,S.A.,Antczak,D.F. Equine clinical genomics: A clinician's primer. Equine Veterinary Journal 42, 658-670 (2010).
- Alvarenga,M.A., Melo,C.M., Magalhaes,L.C.O. & Papa,F.O. A new method to concentrate equine sperm. Animal Reproduction Science 121, 186-187 (2010).
- Brinkerhoff,J.M. et al. Influence of mare age, pre-breeding mare status, breeding method, and stallion on first cycle pregnancy rates on a large commercial breeding farm. Animal Reproduction Science 121, 159 (2010).
- Diaw M. et al. Characteristics of endometrial culture and biopsy samples taken immediately postpartum from normal mares compared with those from mares with induced placentitis. Anim Reprod. Sci. 121S, 369-370 (2010).
- Esteller-Vico,A., Liu,I.K., Brosnan,R.J. & Steffey,E.P. Uterine vascular elastosis and its effect on uterine blood flow in cyclic mares. Animal Reproduction Science 121, 87-88 (2010).
- Hayden,S.S. et al. Low-dose deep-horn insemination: a comparison of transrectally guided and hysteroscopic techniques. Animal Reproduction Science 121, 155 (2010).
- Kotoyori,Y. et al. Transrectal 3-dimensional ultrasound examination of the equine fetus during the first half of gestation. Animal Reproduction Science 121, 327-328 (2010).
- Ryan,P. et al. Use of bioluminescence imaging technology and lux-modified bacteria to determine pathogen progression during uterine infections in the pregnant mare and rate of clearance post partum. Animal Reproduction Science 121, 341-342 (2010).
- Schnobrich,M.R., Riddle,W.T., LeBlanc,M. & Stromberg,A.J. The effect of manual twin elimination on live foal rate in Thoroughbred mares. Animal Reproduction Science 121, 324-325 (2010).
- Sheerin,P.C., Howard,C.E., LeBlanc,M. & Stromberg,A.J. Effects of operator, treatment and mare age on the live foal rate of mares after manual twin reduction. Animal Reproduction Science 121, 312-313 (2010).
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.
POLL: University Equine Hospitals