Drug compounding, the ethics of treating cryptorchids, and sexually transmitted diseases were the hot topics discussed by equine practitioners at the Reproduction Forum, led by Steven Brinsko, DVM, PhD, Dipl. ACT, associate professor of theriogenology at Texas A&M University, and Mats Troedsson, DVM, PhD, Dipl. ACT, of the University of Florida.
Cryptorchids are colts with at least one testicle that hasn't descended into the scrotum; this descent usually occurs by 10 days after birth.
Forum leaders raised the question of ethics in treating cryptorchid horses--is treatment doing the horse any good? Are veterinarians propagating the cryptorchidism horses by treating the condition? Or are the owner's finances the only benefit of treating these horses?
The heritability of cryptorchidism in horses is unknown, although studies in other species suggest the condition is highly heritable, and practitioners are aware that certain breeds (e.g., Percherons, Welsh ponies) are more susceptible.
Forum leaders questioned whether it is correct to treat this condition, given that it appears to be passed on to the stallion's progeny. Additionally, testicles retained in the abdominal cavity are incapable of normal spermatogenesis due to the elevated temperature and more prone to neoplasia (cancer).
Contagious Equine Metritis
In October 2006, two stallions tested positive for contagious equine metritis (CEM) during a breeding soundness test, the first U.S. cases since 1998. They had bred test mares in 2004 during the importation process and were certified to be clean. Following exposure to those mares, they were bred. So how did they contract it, and how did they pass quarantine? Are there more positives like these in the country?
Attendees discussed the current importation testing procedure and considered possible solutions, including the use of polymerase chain reaction (PCR) tests.
One forum attendee cited a study performed in Iceland, where horses have never been exposed to CEM. Researchers performed PCR tests on stallions and found 30% were positive for CEM, raising the possibility that there are pathogenic and non-pathogenic strains of the virus.
Because of these high positive PCR results on unexposed horses, PCR testing was considered too sensitive to use as the sole test for use in imported horses.
Equine Viral Arteritis
Attendees also discussed the outbreak in Quarter Horses of equine viral arteritis (EVA) in New Mexico and Utah in 2006. It was noted that since the outbreak, many stallions and pregnant mares have been vaccinated, potentially signaling a shift in the way horse owners and veterinarians approach the disease.
Although many people vaccinated against the disease, very few precautions were taken at shows and events. It is possible for a stallion to pick EVA up via the respiratory route, and then shed the virus for the rest of his life. Forum attendees suggested stallion advertising should start declaring, "guaranteed tested and vaccinated for EVA," as mares can be protected via vaccine.
It was suggested that the disease could be eliminated if all colts and serologically negative stallions were vaccinated.
Surviving Uterine Torsion
All types of horses can get colic, but in the pregnant mare, the painful signs of colic might be caused by something other than a gastrointestinal problem. The mass of the foal and placenta causes a lot of crowding in the mare's body, and uterine torsion--a twisted uterus--can be the result. But there is good news; a recent study found that at less than 320 days of gestation, chances are good that both mare and foal will survive uterine torsion with proper treatment. And there is a good chance the mare will be sound for future breeding after recovery.
Kristin Chaney, DVM, a resident at Michigan State University, described the retrospective study that evaluated records of 63 mares with uterine torsion treated at four referral hospitals from 1985-2005--it found that 53 of 63 mares (85%) survived. "Two factors significantly affected survival: stage of gestation and heart rate at admission," reported Chaney.
At more than 320 days of gestation, only 17 of 26 mares (65%) survived; she explained that at that stage, increased fetal size and weight complicate correction techniques. At less than 320 days of gestation, 36 out of 37 (97%) of mares survived (one mare was euthanatized after a catastrophic accident during recovery).
The study also found mares that survived had considerably lower heart rates than mares that did not survive (59 beats per minute compared to 74 beats per minute). Although the data were not statistically examined, subjectively, it didn't appear that heart rate correlated with the degree of torsion or pain, commented Chaney.
She said future mare reproductive soundness after successful uterine torsion correction is good.
Superovulation and Embryo Transfer
Embryo transfer is expensive, and its success rates are lower than those of some other methods of equine reproduction. "Commercially, single embryo recovery attempts have a 50% recovery rate, and there is a 50-65% pregnancy rate per transferred embryo," said Tal Raz, DVM, a graduate student in theriogenology at the University of Saskatchewan's Western College of Veterinary Medicine. "Economically, there is a need to increase the success rate with embryo transfer."
Raz presented a study on superovulating embryo donor mares. Superovulation means inducing the mare to ovulate several eggs instead of one at a time, so that more eggs can be fertilized and more embryos can be recovered. Ideally, this increases the pregnancy rate. It might also have a place in other assisted reproductive techniques such as oocyte transfer and gamete intrafallopian transfer (combining eggs and sperm outside the body, then placing them in the fallopian tubes to achieve fertilization), he said. Currently, single-embryo recovery attempts are more common.
In this study, equine follicle-stimulating hormone (eFSH) was used to superovulate 12 donor mares (four to 17 years old), and 37 young, fertile recipients were evaluated. The donors were untreated on the first cycle (which served as the control cycle), were treated with eFSH for the second cycle, and were injected with human chorionic gonadotropin (hCG) on the last day of treatment. They were bred via artificial insemination, and recipients' estrous cycles were synchronized as needed.
Raz reported that eFSH significantly stimulated follicular development, but the number of ovulations per cycle (2.3 in treated mares) was not significantly different from controls (1.5). "This was an unexpected finding," he commented. He noted that during eFSH-treated cycles, mares had more follicles larger than 30 mm per cycle, but only 70% of those ovulated compared to 100% of follicles larger than 30 mm in control cycles.
However, eFSH did significantly increase embryo recovery rate (1.3 embryos per cycle versus 0.5 in untreated cycles). Mean embryo age was significantly lower in eFSH-treated cycles, as additional ovulations occurred 24-48 hours later during those cycles.
Embryos from eFSH-treated cycles were of poorer quality than control embryos. This is also seen in superovulating cattle, and it might be a reason why post-transfer recipient pregnancy rates were lower when embryos were obtained and transferred from eFSH-treated donor mares (33% compared to 67% of controls). However, this difference was not statistically significant, possibly because of the small number of mares in the study.
Raz concluded that more study of eFSH with more mares is needed to clarify its effects in different situations. Age and genetic makeup of donor mares might affect its success, as could estrous synchronization protocols and embryo handling techniques. Also, observing the "coasting" period now recommended by Bioniche, the company that produces eFSH, might give different results than those found in this study. In this protocol, mares receive their last eFSH treatment when the majority of follicles are around 35 mm in size, then 36 hours elapse before an injection of hCG is given to induce ovulation.
In summary, under the experimental conditions Raz reported, eFSH increased the number of preovulatory follicles in treated mares, did not affect ovulation rate, increased embryo recovery rate, and did not increase pregnancy rates.
Hormones to Treat Cryptorchids
James Brendemuehl, DVM, PhD, Dipl. ACT, assistant professor of veterinary clinical medicine at the University of Illinois-Urbana, discussed a study using hCG to influence testosterone production and testicular descent in 16 Thoroughbred colts.
During his presentation, Brendemuehl explained that hCG is "routinely used in prepubertal boys to facilitate testicular descent of inguinally retained testes. The goals of this study were to compare the endocrine (hormone) response of prepubertal colts with descended or cryptorchid testicles to repeated hCG administration, and to determine whether hCG treatment influences testicular descent."
Six of the 16 colts had both testes descended, while 10 had only one testicle descended (two had the testicle retained in the abdomen, while the retained testicle was just above the inguinal ring in the other eight colts). All 16 colts, which were 180-240 days old, received 2,500 IU hCG via intramuscular injection twice weekly for four weeks.
While testosterone was undetectable in all colts before treatment, all responded with a significant increase in testosterone production. Brendemuehl noted that this response was comparable to that of adults. The cryptorchid colts had lower levels of testosterone (78.5 pg/mL) than colts with both descended testicles (132.5 pg/ml), but this number was not significant, possibly due to the small number of colts in the study. Testicular volume was slightly higher after treatment, but this was also not statistically significant.
Half of the colts with testes retained above the inguinal ring had those testes descend after treatment.
"While it is possible that delayed descent could and does occur spontaneously in certain individuals, such as colts with enlarged inguinal rings, this has not been supported by well-documented case reports," commented Brendemuehl. "It is therefore unlikely that testicular descent would have occurred in 50% of the treated group without hCG administration.
"Additional studies are necessary to determine if early hormone intervention in prepubertal cryptorchid colts minimizes Sertoli cell degeneration (which has been shown in cryptorchid boys) and improves subsequent fertility," he concluded. "The genetic implications of cryptorchidism in horses are poorly characterized and highly controversial."
Reinforcing Reinforcement Breeding
It's common practice at some breeding farms to collect the drippings from a stallion's penis after he breeds and dismounts a mare, evaluate the sample to confirm that ejaculation did occur, then place the sample in the mare. Reinforcement breeding, as the last step of that procedure is called, was found to increase pregnancy rates by up to 11.7% in a recent study.
Terry Blanchard, DVM, MS, Dipl. ACT, resident veterinarian at a Central Kentucky Thoroughbred farm, evaluated records from all 2005 matings at the farm. These described 2,171 estrous cycles from 1,406 mares that were bred to one of 13 stallions. Initially in the breeding season, reinforcement samples were infused into the uteri immediately after cover in those mares returning on second (or later) cycles of the season. Early results indicated a dramatic advantage to using the procedure with two stallions, so reinforcements were used on all of their subsequent matings.
"The take-home message here is that we had an average per-cycle pregnancy rate increase of 12% in eight of our 13 stallions," Blanchard said. "The effectiveness of reinforcement was influenced by the number of sperm recovered in the dismount sample that was infused into the uterus."
Blanchard described the following possible reasons why reinforcement breeding might improve pregnancy rates for some covers:
- A higher total number of sperm is deposited in the mare.
- Extender added to the dismount sample might protect sperm to improve their viability within the uterus, particularly if the uterine environment is a "hostile" one with fluid accumulation, urine pooling, or endometritis (uterine inflammation).
- Antimicrobials in the extender might help control bacteria introduced at mating and reduce endometritis.
- He also offered the following suggestions for those performing or considering reinforcement techniques:
- Practice safety first for stallion, mare, and personnel.
- Attempt to collect as much of the dismount sample as possible; sperm number affected success rates in this study.
- Use a disposable wide-mouth collection cup.
- Add prewarmed extender as soon as possible to protect sperm against cold and osmotic shock.
- Pour the sample through a filter to remove debris and dirt.
- Use disposable supplies and clean the working area thoroughly between samples (hands, counters, working areas, and supply-holding areas).
Breeding Older Maiden Mares
Success when breeding older maiden mares depends on the management of several factors that contribute to the retention of excessive fluid in the uterus, including insufficient lymph drainage, a uterus hanging over the pelvic brim, and a tight cervix, according to Jonathan Pycock, B. Vet. Med, PhD, DESM, MRCVS, of Equine Reproductive Services in North Yorkshire, England.
"Unless your therapy program addresses all these problems, it's going to end up not doing quite the job it ought to do," Pycock said. "If you're going to come across one of these very susceptible maiden mares, it's important to pull all the stops out the first time you inseminate her."
Semen causes an inflammatory reaction in the uterus, prompting the production of fluid to clear the uterus of the semen. However, in some mares, this fluid does not clear the uterus. The resulting inflammation decreases the chances of embryo survival. According to Pycock, it's normal to see fluid in the uterus up to 12 hours following breeding, but some mares retain fluid beyond 24 hours. These mares require special planning during the breeding process, breeding, and after breeding to have the best chance of pregnancy success.
Pycock suggested that care be taken when scheduling the breeding of these mares. Using ovulation induction agents can help practitioners to more accurately estimate when the mare will ovulate. Timing is important, as endometrial edema scores (amount of fluid in the uterus) decrease 24 to 36 hours before ovulation. It is ideal to breed these mares between one and three days before ovulation as long as they are naturally covered or inseminated with fresh semen. If permitted by the breed registration, fresh semen insemination is preferable to natural covering to reduce the challenge to the uterus.
Scanning the mare for fluid often prior to breeding can help practitioners know if fluid is present, in which case Pycock suggests using oxytocin to clear the uterus for breeding. According to Pycock, it is easier to breed older maiden mares on their second ovulation of the year, as the ovulation date is easier to estimate. Pycock recommended the administration of oxytocin every six hours post-breeding, or every four hours in certain cases, alternating between short- and long-acting formulas. Pycock also suggested uterine flushing, broad-spectrum antibiotics, and dilating the cervix.
Low-Tech Uterine Torsion Correction
Uterine torsion can be corrected using ropes, a plank of wood, and a burly volunteer, according to Laura M. Riggs, DVM, a clinical instructor of large animal surgery at the University of Georgia's College of Veterinary Medicine.
Riggs uses these tools to help roll around a mare's twisted uterus while the organ is held steady from the outside. Riggs says she tries this option before attempting surgery to correct the twist (without removing the foal). The causes of uterine torsion are largely unknown, although vigorous rolling and trauma are suspected to cause a portion of cases.
"We use this unless we have a reason not to, and proceed with surgery," she said.
The veterinarian using this method first determines the direction of the twist--whether counterclockwise or clockwise. The mare is then anesthetized and placed on her side (in recumbency). The veterinarian places a plank of wood that's at least eight feet long on the mare's belly at the flank (vertically between the tuber coxae and 18th rib). The largest person available stands or sits on the center of the plank on the mare's belly while other people roll the mare in the direction of the torsion using rope leg hobbles. The volunteer's weight holds the uterus still. Riggs says she re-examines the recumbent mare manually to ensure the twist was corrected, and if not, she repeats the procedure.
Riggs estimates that the average case can be resolved in 10 to 45 minutes using the roll method. She knows of one case that required five rolls to correct, but she suggests that if the uterus hasn't returned to its normal position after two or three rolls, surgery should be considered.
Riggs said that 60-70% of mares survive uterine torsion, along with 30-70% of their foals. The type and duration of the torsion seems more clearly correlated with survival than the method used to correct it.
Ulcers In Broodmares
While the rigors of training, showing, stall confinement, and travel have previously been shown to increase a horse's risk for gastric ulcers, a new study suggests broodmares at pasture--with none of these risk factors--are likely to have ulcers.
Conducted by Sarah S. le Jeune, DVM, Dipl. ACVS, Dipl. ECVS, staff veterinarian at the University of California, Davis, the study showed 44 of 62 (70.9%) Thoroughbred broodmares scoped had gastric ulcers.
These mares had not been transported for at least two weeks prior to scoping, and all lived on the same farm under similar management.
"We know (or we thought) that pastured horses would have perfect mucosa," le Jeune said, noting the rate of horses affected by ulcers in this study is comparable to those in active race training.
While the overall ulcer rate in the broodmares was higher than expected, the mean grade of ulcer was 3.3 (on a scale of 1 to 5, one being least severe), which is considered mild. Only two mares with poor body condition scores had ulcers in the glandular region of the stomach (an area that is well-protected against acid damage).
The researchers found no strong correlation between the prevalence of ulcers and any of the study parameters, which included age, pregnancy, race earnings, last race start date, size of herd, medical history (including use of non-steroidal anti-inflammatory drugs), number of live foals, number of years the mare was bred, and type of feed (whether alfalfa or grass hay).
Le Jeune said that one study hypothesis was that mares in a later stage of pregnancy would be more likely to have ulcers in the squamous portion (the nonglandular region of the horse's stomach, located in the inner one-third of the stomach and covered by "tender" tissue with minimal protection against acid injury) of their stomachs due to the intrabdominal pressure exerted by the foal pushing stomach acid into this portion.
Researchers discovered that this hypothesis was incorrect--mares scoped at a late stage of pregnancy and after foaling showed no change in stomach health.
Inducing Ovulation With reLH
Kory Niswender, DVM, MS, Dipl. ACT, of Reata Equine Hospital in Weatherford, Texas, discussed a study to determine an effective dose of recombinant equine luteinizing hormone (reLH) and compare its effectiveness to the currently used hCG in inducing ovuation.
Theoretically hCG and reLH should induce similar responses in the mare, since hCG mimics the activity of luteinizing hormone and reLH is an engineered form of luteinizing hormone. (Luteinizing hormone hastens the maturation, and it induces ovulation of the dominant ovarian follicle.)
There are some concerns with hCG and GnRH use, Niswender said: "Mares become refractory to hCG over time (it is recognized as a foreign molecule and they develop antibodies to it, so it becomes less effective after multiple uses). And there have been downregulation problems in the past when GnRH implants were used (some mares had delayed return to estrus if they did not get pregnant on that cycle)."
In contrast, he reported that no antibodies were found to reLH in the initial safety study of this project, and one mare ovulated within 48 hours on each of six treated cycles, so the hormone's effectiveness should not decrease with multiple uses.
During the question and answer session, Niswender noted that he had not seen any deleterious effects of reLH on pregnancy rates, and that the medication can be obtained from Bioniche Animal Health for approximately $14 per dose.
Uterine Inflammatory Response
One big challenge in successful equine reproduction is uterine inflammation. Increased inflammation can destroy spermatozoa before they have a chance to fertilize the egg, and it can create a hostile environment that kills an embryo.
Temporary inflammation is a natural result of placing semen in the mare, especially frozen semen, but the cause is not fully understood. Nor is it clear whether bacterial contamination plays a significant role. To better understand this phenomenon, researchers at the University of Saskatchewan's Western College of Veterinary Medicine in Canada studied uterine inflammation caused by frozen spermatozoa, bacteria, or both. Vet student Barbara Hunter presented the study results.
"The mild or acute uterine inflammatory response that follows breeding is normal and necessary to clear the uterus of debris, bacteria, and excess spermatozoa," she explained. "Uterine inflammation should be completely resolved within 48 hours of breeding. Persistent acute post-breeding uterine inflammation is a leading cause of subfertility in mares, and it has been reported to cause lower pregnancy rates in mares bred with frozen semen."
Bacterial contamination introduced during insemination might also contribute.
"Sperm and bacteria are often found together in the uterus--is there an additive or synergistic relationship between bacteria and sperm that increases inflammation?" she asked the audience.
To answer this question, researchers placed 13 young, reproductively healthy mares in three treatment groups for study. They induced ovulation, then placed either frozen semen, cultures of Streptococcus equi zooepidemicus bacteria (the most common bacterium isolated from mares with endometritis), or both in their uteri. They evaluated a total of 20 mare cycles with the treatments, monitored the mares with ultrasound, and collected uterine fluid samples at 72 hours to assess bacteria levels, inflammation, percentage of neutrophils (a type of inflammatory cell), and total number of neutrophils.
Hunter reported there were no significant differences in percentage or number of inflammatory cells between any groups. The group receiving only bacteria had a significant accumulation of fluid (1 mm) compared to the other two groups.
"It is possible that the presence of spermatozoa in the frozen semen and frozen semen/bacteria groups increased uterine clearance compared with the bacteria group through the stimulation of prostaglandin (hormone that arises from uterine inflammation) secretion, cytokine production, or another mechanism modulating uterine tissue like contractility, fluid secretion, or drainage," Hunter commented.
It's also possible synergistic/additive inflammatory effects of bacteria might occur earlier than 72 hours post-insemination and have been cleared, she noted.
In short, "No synergistic, additive effects of bacteria and frozen semen together were found in this study," Hunter concluded. "Further research is needed to determine if there is an acute difference in 24 hours (although if this does not affect pregnancy rates, it might not be clinically relevant), and if there is a difference between normal and susceptible mares.
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