Stallion owners must determine if their stallions have what it takes physically to make a good breeding prospect, including good semen quality, before the start of the breeding season. In his presentation at the sixth annual Equine School at the Alltech Symposium, Peter Sheerin, DVM, Dipl. ACT, a theriogenologist at Rood and Riddle Equine Hospital in Lexington, Ky., asked the audience: “What is good semen quality?”

“One definition of good semen quality is an ejaculate that contains a high population of normal sperm cells that provides a normal rate of fertilization (80-90%),” he said. However, he noted that pregnancy rates don’t indicate fertility rates because of the high occurrence of early embryonic loss in the mare.

To tell whether you have a stellar stallion with quality semen you need a veterinarian to perform a breeding soundness examination. The veterinarian not only will examine the stallion’s genitalia, but he will perform a series of tests to determine semen motility, morphology, total sperm numbers, and ratio of live to dead sperm. The typical breeding soundness exam stops at this point, with the tests after this point being performed in a more elaborate workup which is typically done in difficult fertility problem cases. These include testing for  integrity of the membrane and acrosome, quality of the acrosome reaction and of the DNA, mitochondrial function, and the ability of the sperm to impregnate the oocyte.

While it might sound good to know all of these details, many times the cost of these tests--and the limited number of facilities that can perform these tests--prevent stallion owners from having them done unless there is a known problem with fertility of a particular stallion, said Sheerin.

Motility--the ability of the sperm to move in a forward motion--is examined in raw and extended semen, said Sheerin. He said that although semen can be evaluated visually by a technician with the use of a light microscope, the new technology of computer-assisted semen analysis (CASA) is more accurate. Sheerin pointed out that the relationship between motility and fertility has been found to be poor; however, the longevity of motility might indicate fertility. One study found that semen motile for longer than 20 hours was considered capable of producing a normal pregnancy rate. He also said this is true only for fresh semen and might not be true for frozen or shipped-cooled semen.

The morphology (semen structure) can be examined using bright field, phase contrast, differential interference contrast (DIC), or electron microscopy. Primary abnormalities generally occur during spermatogenesis (formation of sperm), while secondary abnormalities happen after spermiogenesis (when immature spermatids become spermatozoa), said Sheerin. Some studies have shown that a high number of abnormalities results in low fertility, while other studies have been unable to make this correlation.

To measure total sperm numbers, calipers are used to measure the length, width, and height of each testicle to estimate testicular volume. Ultrasound can also be used for these measurements. Daily sperm output (DSO) is estimated, and this number is used to estimate the number of mares that a stallion should be able to breed, according to Sheerin.

In addition, the concentration of raw semen is determined with a modified spectrophotometer or by diluting the semen and doing a manual count. Total sperm number is a product of the gel free volume of the ejaculate and the concentration.

Advanced Testing
Advanced tests can be used to evaluate the plasma membrane, acrosome, DNA, and mitochondria, said Sheerin. “Sperm cells with intact plasma membranes are considered viable and thus potentially capable of fertilization,” he continued. With a variety of staining methods available, it has been found that cells with damaged plasma membranes will one color, while live cells will stain another (the colors produced depend on the stain used).
In addition, functionality can be tested by placing sperm in a hypo-osmotic solution and observing swelling of the cells, which is evidenced by coiling of the tail. This is considered normal.

“In humans, the hypo-osmotic swelling test has positively correlated with <I>in vitro<I> and <I>in vivo<I> fertilization as well as sperm penetration assays,” he said. ”The relationship to <I>in vivo<I> fertility has not been closely examined in the horse.”

The acrosome is a membrane-bound compartment at the head of a sperm that digests the outer surface of the egg and allows the sperm to inject its DNA. An intact acrosome is necessary for sperm to bind to the zona pellucida (a translucent, elastic, non-cellular layer surrounding the ovum). Acrosome-intact cells will stain a distinct color (the color depends on the stain used) over the acrosomal cap.

Once sperm reach certain secretions in the mare’s reproductive tract, surface changes of the acrosome begin that will eventually allow the sperm to fertilize the ovum. This is called capacitation. This process can only be evaluated indirectly by incubating sperm in a TALP-TEST buffer and assessing the ability of the sperm to acrosome-react in the presence of progesterone.

Another test involves filtering out sperm that have acrosomes that are damaged, or sperm that have begun capacitation. It was found that first cycle pregnancy rates in nine stallions using frozen semen were higher in the stallions that had a larger percentage of sperm pass through the filtration column.

Another test can evaluate the mitochondrial activity of the sperm. Mitochondria (cells responsible for producing energy and for cell respiration) that have high membrane potential are motile and stain orange. Low potential mitochondria are non-motile and stain green using the fluorescent stain called JC-1.

For the oocyte to be penetrated, the sperm must be motile with functional receptor proteins. In addition, they must be able to undergo an acrosome reaction and bind to the plasma membrane of the oocyte. Penetration assays can evaluate these factors.
Solving Semen Quality Problems
In those breeds using live cover only, little can be done to improve fertility unless the stallion is suffering from an illness or injury that can be treated.  However, if cooled or frozen semen is used, modifications in the process and in the spermatozoa’s environment might improve fertility.

Decreased motility might be a result of too much seminal plasma in the ejaculate. Centrifugation of the ejaculate can remove some of the excess seminal plasma. In addition, motility might be affected by the composition of the extender, said Sheerin. Improper handling of the semen might also be a factor. Semen must be gradually cooled to avoid cold shock. Gradual cooling, and the use of lipids or lipoproteins in the cryopreservation media, can prevent damage to the sperms’ plasma membranes. The use of Concanavalin A can improve motility and the percentage of cells with intact acrosomes, said Sheerin.

It is important to note that the semen from different stallions will react differently to the freezing process. Therefore, the centrifugation media and cryopreservation media can be altered for each stallion to maximize the sperm’s fertility.

As sperm are being formed, there can be a variety of insults that can cause low fertility or infertility.

“Morphological abnormalities can occur during spermatogenesis, sperm transit, disease, trauma, or can be created artifactually,” Sheerin said. “Testicular degeneration, various toxins, illness, high temperatures, and trauma are associated with morphological abnormalities. Increased scrotal temperature due to trauma or long-standing fevers can cause morphological changes in spermatozoa. Within four days of insult, an increased incidence of detached heads will be seen. If the cause of the increased scrotal temperature is identified and treated, sperm morphology will eventually return to normal provided the duration of the insult did not affect the testicular parenchyma.”

Testicular degeneration, steroid use, and progesterone use can reduce sperm concentration or total sperm numbers.

A variety of studies have been done to try and improve semen fertility. The use of hormones has not been shown to be beneficial. More studies need to be done on the role of nutritional supplements and on changing ration composition as well as adding vitamins and minerals to the diet. It is known that an obese stallion is less fertile than a stallion of proper weight. This is due to the fat insulating the testicles causing them to be maintained at a higher than normal temperature.

About the Author

Sarah Evers Conrad

Sarah Evers Conrad has a bachelor’s of arts in journalism and equine science from Western Kentucky University. As a lifelong horse lover and equestrian, Conrad started her career at The Horse: Your Guide to Equine Health Care magazine. She has also worked for the United States Equestrian Federation as the managing editor of Equestrian magazine and director of e-communications and served as content manager/travel writer for a Caribbean travel agency. When she isn’t freelancing, Conrad spends her free time enjoying her family, reading, practicing photography, traveling, crocheting, and being around animals in her Lexington, Kentucky, home.

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