The dating game - Older stallions require thorough evaluations to gague potency

You have seen the signs little by little, and you are pretty sure that it is about time. The older stallion that you have been managing has had a poor season, and pregnancy rates are unacceptable. You are consulted as to whether or not he should be retired from the breeding shed. Sometimes the answer is easy if the stallion has no libido, very low sperm numbers or such an arthritis problem that mounting is becoming painful. Other times there are only slight problems and no readily apparent answers to explain the drop in conception. The decision to retire a stallion often becomes as much about economics and emotion as it is about science and statistics. Yet, for veterinarians, attention to good, complete semen analysis and the use of some newer tests will allow you to recommend either an early retirement for a particular stallion or a return engagement for next year's breeding season.

When evaluating the fertility of an older stallion, a complete analysis of the stallion, his book of mares and the entire breeding facility is warranted because there are so many places where the reproductive process can break down. Many older stallions slowly fall from the public eye and become less desirable breeders and are replaced by younger, more recognizable, trendier stallions. Additionally, there is the unfounded belief, especially among the racing community, that older stallions produce weaker, poorer-quality offspring. Consequently older stallions do not have their pick of the best broodmares and are often servicing older, poorer quality mares.

Dr. Ernest Baily of the Maxwell Gluck Research Center at the University of Kentucky refutes this belief.

"From an understanding of molecular genetics, there's no reason to expect age of the stallion to have any effect on the performance of the offspring," he says. "There is no basis to say that a horse's genetic contribution will improve or decline with age."

Still, this combination of older mare and older stallion combines for lower reproductive efficiency. One simple way to boost the production of an older stallion is to ensure mating to the best possible mares, preferably younger, proven producers. Older stallions also might become more particular in their preferences and habits. Some stallions will breed better at certain times of the day. Some older stallions prefer mares of a particular color or any number of other idiosyncrasies. Attention to these small details can help improve fertility in these stallions.

Fitness test

A complete physical examination of the older stallion should be the next step in this evaluation process. Medical problems unrelated to the reproductive system can have a significant effect on breeding ability. Dental care and nutritional status should be evaluated. Older horses need additional vitamins and minerals because their digestive systems are not as efficient as they once were. Stallions use tremendous energy and body reserves during the course of a breeding season, and older stallions are more affected by this daily strain. Older stallions should be neither too heavy nor too thin as both extremes will reduce fertility. Many supplements are available claiming to improve fertility, including vitamin E, chromatin and others. More research must be done before the exact relationship is confirmed between many of these products and the possibility of improved fertility. A good-quality diet with a balanced supplement is the best overall nutritional support for older stallions.

The musculoskeletal system of the older stallion also deserves special attention. Osteoarthritis and muscle, ligament and tendon problems all can affect a stallion's willingness and ability to tease and mount. A stallion that has strong libido, mounts (but cannot ejaculate) might be experiencing back pain or hock soreness or any number of other conditions that can manifest themselves as breeding difficulties. A thorough examination done before and after the breeding season will identify any potential problems. The use of glucosamine, chondroitin, various forms of hyaluronic acid and other products can make an older stallion much more comfortable, which translates into better performance in the shed.

The cardiovascular and respiratory system should also be examined. Many older stallions suffer from some form of decreased heart function, and the stress of breeding can be potentially deadly to these horses. Anemia, from many causes, can result in lowered libido and lack of interest in breeding as well.

Finally, an examination of the reproductive tract should be done. The stallion's testicles should be measured. Ultrasound provides the most easy and accurate means of obtaining testicular size. This information then is used to correlate how much semen that stallion is producing as compared to how much semen he should be producing, according to Dr. Dixon Varner of the College of Veterinary Medicine at Texas A&M University. Daily semen output is compared to testicular size, and this information allows veterinarians and breeding managers to follow a stallion's reproductive ability. Evaluation of the testicles also can reveal inflammation—suggesting an inflammatory process, tumors or atrophy. Testicular degeneration leading to atrophy is perhaps the most common cause of reduced fertility in older stallions. As the testicles degenerate, there are fewer and fewer seminiferous tubules producing sperm, and an overall reduction occurs in the quantity and quality of the sperm. The testicles themselves become soft, then smaller and finally firm. Though aging is the most-common cause of testicular degeneration, it also can be caused by an increase in heat in the testicles (trauma, infection, systemic viruses), adverse reactions to some antibiotics, exposure to toxins, chemicals and other factors.

Material strength

If an evaluation of the breeding practices, the quality of broodmares, their preparation and handling, and a complete physical examination of the stallion has not yielded any reasons for poor breeding efficiency, then the next step is a careful semen analysis. Semen should be evaluated for gross appearance and should be milky white to turbid without clots and without smell. Any pink or red color in the ejaculate indicates hemospermia and definitely will correlate to decreased fertility. Yellow color and a dilute sample can indicate urine in the ejaculate and also correlates to breeding problems.

This is a problem that usually can be corrected at the time of collection though natural service (in breeds that prohibit artificial insemination) remains a concern with stallions that tend to urinate prior to ejaculation, so more work-up might be required with these horses. Volume of the ejaculate should be recorded exactly. Average volume is from 60 ml to 120 ml, with a gel-free fraction of 50 ml to 100 ml. There is tremendous variability between stallions however and consistency is more important than specific numbers.

Motility should be evaluated immediately upon collection using a pre-warmed slide. This is still a subjective evaluation, and the average stallion will show around 80-percent motility with 60 percent being progressively motile.

All stallions will show some percentage of dead sperm, but this number should be watched. If a stallion shows a decrease in the number of motile sperm or an increase in the number of dead sperm, then a problem is developing and should be attended to. The sperm should be evaluated as to longevity as well. Check the sperm at five-minute intervals [while on a warm (body temperature) slide] for 30 minutes and you should see consistent motility percentages. A rapid reduction in motility signals poor ability of the sperm to exist long enough to reach and penetrate the egg and points toward poor fertility.

Sperm pH should also be measured. Normal values are between 7.0 and 7.7. Abnormal pH and the presence of white blood cells can indicate the presence of an infection. Sperm sample slides should be stained with a nigrosin-eosin stain and evaluated for morphology. The gross appearance of the individual sperm should be noted, and abnormalities such as double heads or tails, abnormal mid-pieces and other irregularities should be recorded.

Sperm concentration is a very concrete number that can provide tremendous information to the veterinarian and breeder, but it sometimes can be used incorrectly as a measure of breeding ability. It is felt that a sperm concentration of 500X10(6) provides maximum pregnancy rates. But there are stallions that remain effective despite counts much lower and stallions whose count is above average but whose pregnancy rate is below average. These are the older stallions that cause concern for veterinarians asked for an evaluation and recommendation as to future breeding ability. There are additional tests that can identify damage to sperm and problems on a deeper, cellular level. Dr. Varner says the sperm chromatin assay is a very valuable test for evaluation of an older stallion.

"Sperm quality is dependent on the amount of damage to sperm DNA or DNA fragmentation," Varner says.

Technological alterations

The double helix structure of DNA begins to breakdown in some people and animals as they age. The degree of fragmentation of sperm DNA correlates very highly in humans, with the inability of sperm to initiate a birth regardless of the technology used to fertilize the egg such as artificial insemination (AI), in vitro fertilization (IVF) or intra-cytoplasmic sperm injection (ICSI). Additionally, sperm with fragmented DNA can fertilize an egg, but embryo development stops at an early stage, and early pregnancy loss commonly occurs. Statistics from some older stallions confirm this as mares will become pregnant, but consistent embryo loss will occur at or around the same time of gestation. This concept of DNA fragmentation now is clearing up many cases of previously unexplained infertility. DNA fragmentation has little to do with the para- meters measured in a standard semen evaluation and is a totally independent variable. Varner suggests that a sperm chromatin assay be performed on all older stallions and on any stallion with a fertility question. This test currently is being done at a number of veterinary schools and private labs. A semen sample is collected and shipped on dry ice to the lab. The sample is thawed and treated by stress exposure to a low-pH solution. The sample then is labeled with an orange dye that binds exclusively to the broken or fragmented DNA. A flow cytometer then views the sample and uses a special wavelength of light to measure the amount of orange (fragmented DNA) verses green (intact DNA), and a computer plot yields a DNA fragmentation index (DFI). Stallions that show normal breeding behavior and acceptable semen analysis but that have a poor DFI are likely "over the reproductive hill." Very little in the way of technological help can be given to these stallions, and their breeding careers are likely over.

Varner advises veterinarians to discuss with clients the possibility of freezing sperm from these and other older stallions.

"Technology is advancing rapidly and it is very likely that in the not-too-distant future science will be able to utilize methods to repair DNA and to use frozen sperm itself in methods of high-tech regeneration and pregnancy production," Varner says.

Currently, many breed associations do not allow such technology, but should science advance and current rules be changed, then many older stallions would be able to contribute their DNA to far future generations.

Whit Byers, a partner in Select Breeders Southwest, a company that uses mobile reproductive laboratories to provide breeding and reproductive services to the equine industry, is more concerned with what can be done for some older stallions in the here and now. Byers says there are very few stallions that can't have their productivity improved and very few that are truly too old to reproduce. He agrees with Varner that a sperm chromatin assay is an extremely important part of the work-up of an older stallion, but if that assay shows a low DFI, then he recommends centrifugation of semen samples from stallions with low motility or low sperm counts. Centrifugation at 300 G to 350 G for 10 minutes to 12 minutes and then re-suspension of the pellet in extender produces a highly concentrated low volume of semen. Byers next recommends using a more focused breeding delivery approach for insemination. Some veterinarians use a flexible infusion pipette and attempt to inseminate as close as possible to the end of the horn near the breedable follicle. Byers takes this "deep-horn insemination" procedure one better and believes that using a video endoscope further improves the breeding odds. He uses a 3/10-cc insemination dose of concentrated semen deposited at the uterine-tubal junction.

"The use of highly concentrated semen applied at precisely the right place" has produced good results in some stallions that were previously problem breeders and whose reproductive careers were almost over, Byers says.

Breeds that do not allow artificial insemination or technical manipulation cannot use this procedure because of its invasive nature. Therefore, fewer older Thoroughbred stallions continue to breed compared to older Quarterhorse, Arab and other breed stallions. But more new tests continue to be developed that will help identify specific sperm-related problems and solutions surely will follow close behind what will benefit all breeds. Genetic variability always will ensure that some stallions, for any number of reasons, are born infertile or lose their reproductive ability as they age, and some stallions just keep on going strong. Veterinarians often are asked to evaluate these older stallions, and good sperm analysis and knowledge of these new tests and procedures will allow more complete and accurate recommendations.

Dr. Marcella, a 1983 graduate of Cornell University's veterinary college, was a professor of comparative medicine at the University of Virginia. His interests include muscle problems in sport horses, rehabilitation and other performance issues.