Retained Endometrial Cups
John Steiner, DVM, Dipl. ACT, of Hagyard Equine Medical Institute in Lexington, Ky., reported on a recently recognized abnormality in some mares that didn't cycle normally after either foaling or aborting.
The abnormality, Steiner said, involves retention of the endometrial cups. The endometrial cups form in a pregnant mare's uterus beginning at about the 35th day of pregnancy. They secrete pregnant mare serum gonadotrophin that is essential in helping to maintain the pregnancy at that point. At about 70-90 days of pregnancy, the cups begin to slough off, and by about 120-130 days, they normally disappear.
However, Steiner said, in 13 mares in Kentucky, it was found that the endometrial cups remained for up to 18 months. As long as they were present, the mare did not cycle normally or become pregnant. Eventually, he said, the cups disappeared and the mare was capable of becoming pregnant. In one mare, he said, the phenomonon occurred two years in a row.
Typically, he said, these mares have erratic heat cycles and might develop a 30- to 35-millimeter follicle, which does not ovulate but forms a corpus luteum that secretes progesterone.
He urged his fellow vets to be on the lookout for the syndrome. "If we are finding it (in Kentucky), then it's out there," he said.
The question of how much or how little stallion semen is required to achieve a pregnancy received detailed attention during the conference. The consensus was that while 500 million progressively motile sperm is the "gold standard," especially for artificial insemination (AI), many fertile stallions breeding fertile mares could achieve pregnancy with many fewer sperm.
Making the presentation on that topic was Steven Brinsko, DVM, PhD, Dipl. ACT, of Texas A&M University. He titled his talk, "Insemination Doses: How low can we go?"
Some 30 years ago, Brinsko said it was decided, based primarily on research work at Colorado State University, that 500 million progressively motile sperm was the recommended number for an insemination dose of fresh semen under less-than-ideal conditions.
That standard still remains, he said, even though improved techniques, extenders (which help to increase the volume of the ejaculate and provide nutrients and fertility preservation to sperm cells), and shipping containers for cooled semen have made it possible to achieve pregnancies with far lower sperm numbers. Under ideal conditions, he said, it should be possible to achieve pregnancy with no more than 100 million progressively motile sperm. However, he hastened to add, this would be true if one were involved with ideal conditions with sperm from a healthy stallion being placed in an equally reproductively healthy mare.
Research on low doses of semen is not new. Some early research on lower insemination doses was conducted years ago by the Chinese, Brinsko said.
"The Chinese reported that they had established a very successful equine artificial insemination program involving 40 stallions and thousands of mares, and they found the insemination dose could be reduced from 400 million to 100 million when diluted in powdered milk-based extenders," he said. "The other essential finding in this report was that pregnancy rates were optimized when mares were inseminated close to ovulation. Unfortunately, it appears that Western nations were slow to recognize and accept these findings and despite these results, a dose of 500 million progressively motile sperm continues to be the most commonly recommended and employed in the equine breeding industry, even under ideal conditions."
The development of the "Kenney Extender," formulated with nonfat dried milk solids glucose, leveled the playing field as far as extenders were concerned, according to Brinsko.
"The 'Kenney Extender' as it is known, revolutionized equine artificial insemination in the Western world," Brinsko said. "Once a convenient, reliable semen extender became available, the use of artificial insemination in horses increased worldwide. The effect of insemination doses could now be compared without the confounding effects of inferior extender composition."
Despite this major advance, Brinsko said, there were no controlled studies concerning lower doses for insemination. There was anecdotal evidence that insemination doses ranging from 200 million to 400 million progressively motile sperm were successful in achieving pregnancy, but these weren't backed by scientific evidence.
"Surprisingly," he told the group, "it was not until 1997 that such a study was published in a major English language journal. In that study, 75% (21/28) of mares became pregnant when bred with 300 million progressively motile sperm compared with 64% (23/36) mares becoming pregnant when bred with 500 million progressively motile sperm. Unfortunately, the authors did not investigate lower insemination doses, probably because the study was performed at a commercial breeding operation.
"However, it is clear from this study and data from more recent studies where 20 million, 50 million, or 100 million progressively motile sperm from fertile stallions did not reduce fertility, that an insemination dose of 500 million progressively motile sperm is not necessary to achieve acceptable pregnancy rates."
Brinsko told the group that today, much stallion semen is collected, cooled, and shipped. The number of progressively motile sperm shipped is based on the accepted premise that 500 million progressively motile sperm are ideal for achieving a pregnancy. The original study by Douglas Hamilton, developer of the Equitainer, indicated that there would be 50% mortality of sperm cells in the shipped semen. Thus, it was reasoned, one billion progressively motile sperm should be shipped in order to ensure that 500 million would arrive in a progressively motile state.
"Since that time," Brinsko said, "advances in transport container design and function, semen extenders, and semen dilution ratios have greatly improved sperm survival after cooling and storage; yet one billion progressively motile sperm remains the most common dose to package for shipment."
An effort was made at Texas A&M to investigate whether the total number of progressively motile sperm inseminated after being cooled and transported affected pregnancy rates. It was found, he said, that mares inseminated with 70 to 480 million progressively motile sperm had a lower per-cycle pregnancy rate than mares inseminated with 500 million progressively motile sperm.
When progressive sperm motility was less than 60%, pregnancy rate per cycle was only 48%. When the percentage of progressively motile sperm was higher than 60%, the pregnancy rate per cycle was 67%.
While Brinsko pointed out that the above was not a controlled study, he did have this to say: "Interpretation of these data suggests that when breeding with transported cooled semen, prudence dictates that a minimum of 500 million progressively motile sperm be inseminated as a starting point, unless successful testing has been conducted with lower doses. Data such as this should also provide impetus for evaluating the quality of cooled semen used to inseminate each mare.
"Before cooled semen is to be offered on a commercial basis, it is important to perform a test cool on the semen to ensure that it is suitable for this purpose," he advised. "For some stallions, even when optimal semen handling techniques are employed, insufficient numbers of their sperm cells survive the cooling and storage process to provide an adequate number of progressively motile sperm for insemination. Since sperm viability decreases with storage time and the effect varies with (each) stallion, the minimum dose of progressively motile sperm necessary to achieve satisfactory pregnancy rates for a given stallion should be tested. This would allow more efficient use of semen for stallions whose sperm motility is only slightly reduced (less than 30%) after cooling and storage, as well as indicate the need to increase the packaged dose for stallions whose semen has poor tolerance (more than 50% reduction in motility) to cooling and storage."
Brinsko then touched on another subject that engendered a bit of discussion with reproduction researcher Ed Squires, PhD, of Colorado State University.
In some cases, Brinsko said, semen shipments arrive containing two insemination doses. The question is whether both should be used at once, or whether one should be used immediately and the other stored for insemination later.
He said that both methods had been tested, but with disparate results. In one test, it appeared that a single insemination brought the best pregnancy rates and in the other test, it appeared that a second insemination dose 24 hours after the first one brought the most positive results.
Squires said he found value in using both doses immediately. He said he would rather store the semen inside the mare than in a container for later insemination.
Brinsko also addressed the issue of frozen semen, telling his listeners that post-thaw sperm motility is quite variable among stallions and even among ejaculates from the same stallion. Generally, he said, only ejaculates demonstrating more than 30% post-thaw sperm motility are selected for insemination.
The World Breeding Federation of Sport Horses, he said, is attempting to adopt uniform standards for the use of thawed frozen semen concerning recommended insemination doses.
Brinsko also addressed the matter of deep-horn insemination, pointing out that the procedure had been used by the Russians as early as the 1930s. The approach involves hysteroscopic insemination with low numbers of sperm at the uterotubal junction. Brinsko said there is something of a "resurgence" in this insemination approach.
During his summary, Brinsko said, "Although the time-tested insemination dose of 500 million progressive motile sperm has served us well over the years for both fresh and cooled semen, improvements in extender composition and mare management should allow conventional doses to be reduced to at least 100 million progressively motile sperm for fertile stallions bred to fertile mares under good management. However, when conditions are less than ideal, e.g., subfertile stallions, subfertile mares, or poor management, it would seem prudent to follow the earlier recommendation for using higher sperm numbers."
He noted that low-dose deep-horn insemination techniques can and are being used by practitioners for fresh, cooled, and frozen semen, "and it appears that the threshold dose to achieve acceptable pregnancy rates is at least 100 million progressively motile sperm even for highly fertile stallions."
Employment of these methods in the management of subfertile stallions has been met with mixed success, Brinsko said, they appear to show promise in selected cases. Reducing the insemination dose can improve the efficient use of semen, but the level of reduction that still results in acceptable fertility varies among stallions.
"Only by breeding sufficient numbers of mares with reduced sperm numbers and/or alternative insemination techniques will we know how we can go with regards to an insemination dose for any particular stallion," he stated.
Much of the information recorded in breeding records can be used diagnostically, said Charles Love, DVM, PhD, Dipl. ACT, of Texas A&M University. For example, a stallion's rate of achieving pregnancy in mares bred is highly important information. Generally speaking, he said, if a stallion achieves an 80% or above pregnancy rate in mares bred, it is considered good. Anything below that, he said, should be considered in the poor category, with the stallion's book evaluated accordingly.
Maintaining records on individual stallions takes on added importance, he said, because there is broad variance among horses. With some stallions, pregnancies might be achieved at a highly successful rate with 400 million or fewer progressively motile sperm per insemination. With others where sperm might not have the same overall good health, 700 million motile sperm might be required per insemination.
The number of mares covered during a season also can be significant, he said, with some stallions being able to service a much larger book than others.
There also are a great many factors that can affect pregnancy on the part of the mare, he said, including reproductive health, age, whether she is a maiden, has a foal at side, or is barren.
Love said foaling mares account for 50-60% of the normal stallion's book, barren mares account for 20-30%, and maiden mares account for 10-20%. Normally, he said, barren mares will have lower fertility than the other groups. The highest fertility normally is with maiden mares, with foaling mares next in fertility.
Reviewing complete records, ranging all the way from stallion fertility to a mare's reproductive health, can have a pronounced influence on making correct breeding decisions, Love said.
Juan Samper, DVM, PhD, Dipl. ACT, of Langley, British Columbia, Canada, told the group that widespread use of cooled and frozen semen around the world, plus the shuttling of Thoroughbred stallions from one continent to another, has increased the risk of disease transmission via stallions.
With artificial insemination as a tool, he said, it is not uncommon for one stallion to cover 150 mares, and in one instance, a single stallion was bred to many more mares in one season via artificial insemination. If that stallion's semen had carried disease, he said, the effect would have been dramatic.
"The high risk that the stallion poses to the mare population to which he, or his semen, is exposed, becomes a more serious threat when we realize that the stallion is an asymptomatic carrier (showing or causing no symptoms) of most reproductive diseases," Samper told the group.
Samper then gave an outline of the various diseases that can be transmitted by the stallion, ranging from contagious equine metritis to dourine.
In conclusion, he said, "To prevent the spread of any disease to susceptible populations through breeding, correct identification of infected animals as well as the implementation of appropriate managerial procedures is critical. A stallion carrier of bacterial disease should not be used for breeding through natural cover, and his semen should be treated with appropriate antibiotics prior to insemination. A stallion's semen should undergo complete evaluation, and a lab should conduct microbiological examination on his semen as well as pre- and post-ejaculation urethral swabs. Breeding hygiene should be strictly observed to avoid transmission of contaminants to mares."
John Hurtgen, DVM, PhD, Dipl. ACT, of New Freedom, Pa., told his listeners that endometritis, an inflammatory condition of the uterus, causes a substantial decline in mare fertility. The inflammatory conditions, he said, can be categorized as acute, chronic, active, subclinical, postpartum, bacterial, fungal, viral, mating-induced, persistent, and other.
"For the clinician," Hurtgen said, "it is crucial that clients understand the apparent simplicity of uterine infections in mares, but also the complexity of endometritis. If clients have an appreciation of the pathogenesis of endometritis, they will understand not only the goal of prevention and treatment efforts, but also the treatment failures that occur."
While endometritis often is not implicated when young maiden mares fail to become pregnant, the opposite is true for older mares, especially older maiden mares, he said. Mares as young as eight years can be affected by this syndrome, he said, but usually they are 12 years of age or older.
Affected mares, he said, have an elongated, closed, fibrous cervix during estrus. The uterus is usually normal in size, but it is common for affected mares to accumulate varying degrees of fluid in the uterine lumen.
Treatment can include lavage and administration of oxytocin (to induce contractions and expulsion of the fluids), he said.
The foaling mare is at risk for developing endometritis, Hurtgen said, because "foaling provides numerous challenges to the mare's reproductive tract." For example, relaxation of the vulva, vagina, and cervix might allow substantial contamination by air, debris, fecal matter, and bacteria.
Also placing mares at risk for endometritis are dystocia, or difficult birth--where damage may occur to the reproductive tract--retained placenta, and mating-induced inflammation.
Persistent endometritis can cause pregnancy failure and early embryonic death if pregnancy does occur, Hurtgen said. When that is the case, he said, "Embryo transfer is a management technique that has been invaluable as a method of obtaining foals from mares with persistent chronic endometritis, repeated early embryonic death or abortion, and non-responsive, persistent mating-induced endometritis."
Hurtgen summed up the problems involved in endometritis this way: "Endometritis is very difficult to study because of the wide array of variables associated with each affected mare. It is not prudent to assume that cases of acute, chronic, or persistent mating-induced endometris can be treated in standardized protocols. Furthermore, all of the above discussions are complicated by a lack of uniformity and precision in diagnostic methods and agreement on the interpretation of clinical findings."
Henrik Lehn-Jensen, DrMedVet, from the Royal Veterinary and Agricultural University in Copenhagen, Denmark, provided what he described as a simple remedy for what can be a complicated problem--hypersensitivity and pain in the mare's reproductive tract, particularly the vagina.
The problem, he theorized, begins with inflammation of the reproductive tract, and it spreads to the central nervous system.
Lehn-Jensen said the treatment procedure involves performing routine Caslick's surgery. He said that recovery time varied from one to six months, but that most of the mares treated were able to be bred and become pregnant.
STUDENT RESEARCH AWARDS
Awards were presented at a luncheon where Terry Blanchard, DVM, MS, Dipl. ACT, formerly of Texas A&M University and now of Hill n' Dale Farm in Kentucky, was honored as Thereogenologist of the Year (see www.TheHorse.com/emag.aspx?id=7391).
Veterinary students were invited to submit case studies on all species that were judged by a panel, with the top six being scored and invited to make presentations at the conference. Each case study was scored again when the student made the presentation during the conference. The two scores were then added together to determine a winner, with prize money being awarded.
One of the equine case studies tied for first place. It was titled, "Uterine Segmental Aplasia in a Thoroughbred Mare," and was presented by Kathy Tuhacek from the University of Pennsylvania's School of Veterinary Medicine.
The study involved a 5-year-old Thoroughbred mare that did not get pregnant following breeding. When the mare arrived at the clinic, it was thought that she had a 60- millimeter cyst in the left uterine horn. She had a history of having lost her first pregnancy between 16-60 days after ovulation.
A detailed examination revealed that a membrane had sealed off the left uterine horn from the uterus. The membrane was removed surgically and 333 days after ovulation, the mare delivered a healthy filly.
Tuhacek said to conclude her report: "This case demonstrates the value of a systematic diagnostic approach, a thorough understanding of reproductive tract embryology, and a sound working knowledge of reproductive physiology in the workup of any case of reproductive failure. It also describes the first successful surgical correction of uterine segmental aplasia (a lack of development of normal uterine tissue) in the mare."
Fourth place went to T.L. Stephens for a study on "Bilateral Seminoma in a Stallion." The case involved a 16-year-old Quarter Horse stallion that had been used successfully in a live cover breeding program. However, during the preceding year, he had been unable to impregnate mares and his testes were getting larger and larger.
The diagnosis was that the horse was suffering from testicular neoplasia, or out-of-control tissue growth of the testicles. The condition is considered rare in stallions. When it does happen, Stephens reported, it is more apt to be in just one testicle rather than in both.
In this case, the stallion was castrated. Here is the conclusion of the report: "Seminomas (cancerous growths of the testicle) may be metastatic (they can spread to other areas of the body). Life expectancy is short in the case of metastatic disease--12 to 24 months--but may be favorable in cases of benign tumors. The animal in this report was healthy 12 months past treatment--24 months after onset of clinical signs."
Fifth place went to Elizabeth James of Oregon State University for the case study of a 10-year-old mare that would not conceive. It was learned she was suffering from gonadal agenesis, or lack of ovaries. She also had underdeveloped mammary glands and small, infantile uterine horns. A whole blood sample revealed that her karyotype (complete set of chromosomes) was typical for a male horse.
James presented this conclusion: "In humans, gonadal agenesis can result from an autosomal recessive gene, in which both male and female genotypes present with female external genitalia and hypoplastic mullerian derivatives (incomplete development of fallopian tubes, uterus, cervix, and the upper portion of the vagina). This case illustrates how reproductive tract defaults to the female phenotype in the absence of gonads."
Sixth place went to Jane Merryman, also from the University of Pennsylvania. Her study, which is still ongoing, involves a 4-year-old Oldenburg mare that was presented when she failed to cycle. She was found to be suffering from a chromosomal abnormality wherein she did not have a left ovary and the right ovary had no follicles. The condition was described as XO gonadal dysgenesis.
The study is aimed at determining whether stimulation by ovarian sex steroids will cause the endometrium to become functional and harbor a pregnancy.--Les Sellnow
About the Author
Les Sellnow is a free-lance writer based near Riverton, Wyo. He specializes in articles on equine research, and operates a ranch where he raises horses and livestock. He has authored several fiction and non-fiction books, including Understanding Equine Lameness and Understanding The Young Horse, published by Eclipse Press and available at www.exclusivelyequine.com or by calling 800/582-5604.
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