Stallion Semen Tests: Which to Use and What Do They Mean

Because breeding is a business, having a veterinarian evaluate your stallion's semen to determine his fertility (or lack thereof) is a key component of a breeding soundness examination. But semen evaluations are not always black and white, and there are many types of tests your veterinarian can perform. So at the 2013 Society for Theriogenology Conference, held Aug. 7-10 in Louisville, Ky., Dickson Varner, DVM, Dipl. ACT, professor and Pin Oak Stud Chair of Stallion Reproductive Studies at Texas A&M University, walked attendees through the tests he would use to evaluate semen.
Although most of these tests haven't changed in recent years, Varner said they were worth reviewing, as "there are a lot of tests we currently perform that we might not be doing correctly, or the people we're overseeing at the farms aren't doing correctly."
The following are what he listed as important semen tests:
Sperm numbers Although this is a traditional breeding exam test, Varner said it's one of the hardest to get right. "Determination of total sperm number (semen volume times concentration) in an ejaculate would seem to be a relatively simple procedure, but accurate measurement can be fraught with error," he said. “This is due to inaccuracies in measurement of both semen volume and sperm concentration.”
Varner recommended determining volume based on weight, as 1 milliliter is equivalent to 1 gram. He also indicated that the best way to measure sperm concentration in the commercial setting is with a device called a NucleoCounter.
Part of calculating an accurate sperm count, he explained, is determining daily sperm output for a particular stallion, because analysis of a single ejaculate has limited value. He said that, ideally, the practitioner would like to calculate the number of sperm the stallion produces on a daily basis, called the daily sperm output.
"It's important to know this because we can also measure the testicles and determine, based on the size of the testicles (testicular volume), how much sperm the stallion should be producing on a daily basis," Varner explained. "We can then determine how much sperm the stallion is actually producing on a daily basis and determine if there's a disconnect. In aging stallions, for instance, a decline in daily sperm output frequently precedes declines in testicular size or semen quality.”
Sperm concentration The current standard tool for estimating sperm concentration accurately is the NucleoCounter. "In my view, the NucleoCounter has revolutionized stallion reproduction to the extent that ultrasound has revolutionized mare reproduction," Varner said.
Semen volume Varner said veterinarians and breeding managers don't often do a good job of measuring sperm volume, and suggested that the most accurate method for doing so is to measure volume, based on specimen weight (as described above).
Sperm motility Measuring sperm motility, or its movement/swimming action, is a fundamental test, Varner said. The only drawback he mentioned was that scores—from 0 (stationary) to 4 (fast)—among practitioners are subjective and can vary in accuracy. "This can be minimized when a high-quality microscope with phase contract optics and a warming stage is used," he explained. He also noted that computer-assisted sperm analysis systems can provide a more objective means of measuring sperm motility.
Varner also cautioned that sperm motility is susceptible to environmental conditions such as excessive heat or cold, lubricants, light, disinfectants, etc., "so it is necessary to protect the semen from injurious agents or conditions prior to analysis."
Sperm morphology Varner believes that the most effective way to evaluate sperm morphology (structure) is with a differential interference contrast (DIC) microscope. The question then becomes, what is abnormal sperm morphology?
"If you have one defect by itself, in all likelihood that defect isn't going to be associated with reduced fertility," Varner explained. "Usually, subfertility or infertility is associated with a combination of defects. If there are enough normal sperm in an ejaculate, you sometimes do not have to worry about presence of abnormal sperm. Such structural defects are called compensable defects, as they can be neutralized by morphologically normal sperm when present in sufficient numbers."
For example, if your stallion has 17% morphologically normal sperm and a breeding dose is calculated to be two billion sperm, more than 300 million morphologically normal sperm will still be present in the breeding dose. This could be sufficient for normal fertility.
Varner reviewed specific abnormal sperm structures veterinarians commonly see:

• Detached heads "When you see these it's beautiful, because you can generally solve the problem and be a hero," Varner said. He explained that while detached heads are problematic for a given ejaculate, they generally don't lend a poor prognosis overall; rather, they represent stagnation of sperm in the epididymides and ductus deferens (the ducts that store and transport sperm). Veterinarians can typically correct this issue with a frequent semen-collection schedule, sometimes in conjunction with medications that promote muscular contraction of these ducts.
• Proximal droplets Varner said he generally considers sperm with these remnants of germ cell cytoplasm attached to be functionally normal. He cited one study by Texas A&M researchers whereby sperm with distal or proximal droplets had better viability over time than morphologically normal sperm. Varner cautioned that sometimes, however, proximal droplets can indicate early stages of testicular dysfunction in aging stallions.
• Abnormal midpieces Varner considers these sperm to be problematic. "Anytime you have disruptions to the mitochondrial helix, those sperm have reduced longevity and are, in all likelihood, less capable of fertilization," he said.
• Coiled tails These defects can be problematic and represent a degree of testicular dysfunction, even when in relative low prevalence (5-6%).
• Premature germ cells (PGCs) In low prevalence, these abnormalities are not a “kiss of death” as was previously thought, Varner said. Secretariat, for instance, was initially believed to be infertile when a semen collection revealed PGCs, but the stallion went on to have a very successful breeding career. "If PGCs are present in higher percentages and persist, then it's an indicator of testicular dysfunction," Varner said. "But don't be alarmed if you see 1, 2, 3% (PGCs) in an ejaculate."

Varner cited an influential stallion sperm morphology study led by Charles Love, DVM, PhD, Dipl. ACT, in 2000: Every time a stallion at one Central Kentucky Thoroughbred breeding farm ejaculated into a mare, Love collected the dismount sample, put it in buffered formal saline, and looked at its morphology. He determined for each sample whether the mare got pregnant, and he did this for hundreds of mares. "That particular study showed that the most significant predictors of fertility were normal sperm (positive effects on fertility), and abnormal heads, detached heads, abnormal midpieces, coiled tails, and PGCs (negative effects on fertility)," Varner summarized. "Droplets did not have predictive value in that study."
Testicular size Although not directly part of a semen evaluation, Varner mentioned that testicular size is a good indicator of how many sperm a stallion should be producing over a period of time or on a daily basis. Using ultrasound and tranquilization, practitioners can measure testicle length, width, and height to estimate a stallion's semen volume.
Sperm chromatin quality "If I had to pick only three things to evaluate a stallion's fertility, I would measure testicle size, look at sperm morphology, and evaluate sperm chromatin," Varner said. Sperm chromatin is the DNA bound by proteins called protamines, which help protect sperm during transport to, and within, the mare reproductive tract, as well as promote decondensation of sperm at the time of fertilization.
Using the sperm chromatin structure assay (SCSA), developed by scientist Don Evenson, PhD, veterinarians can expose the sperm to acid and evaluate their susceptibility to DNA denaturation (the destruction of the usual nature of a substance). Varner indicated they sometimes also use a TUNEL assay in conjunction to the SCSA for more critical analysis of sperm DNA. "The assays do measure different things, even though they're both measuring parts of DNA," he said. He has also used the Comet assay for determining DNA fragmentation in sperm but added that the test's downside "is that results can be skewed when looking at fresh versus frozen semen."
Acrosomal integrity and function The sperm acrosome fits over the head of the sperm like a bathing cap and develops pores to release enzymes that are necessary for fertilization. Varner says his laboratory performs an acrosomal responsiveness assay on some stallions to test the sperm acrosome's ability to react when challenged with calcium ionophore, A23187, a potent inducer of the acrosome reaction. "If you have a semen sample that looks very good based on traditional tests, but you can't explain why mares are not getting pregnant, I'd perform an acrosomal responsiveness assay, especially in Thoroughbred stallions," Varner suggested.
He added that genomics researchers at Texas A&M recently showed that there is a specific problematic gene on Chromosome 13 that is upregulated (has a heightened cellular response) in all of the stallions they have studied with this problem. Thus, there is likely a genetic predisposition for this form of acrosomal dysfunction.
In summary, Varner advised being as complete as possible when performing a breeding soundness examination on a stallion. A battery of laboratory tests will be more likely to help predict stallion fertility than just a couple tests. Accurate measurements are also important to the examination outcome.