Semen Evaluation

Breeding horses today often involves the use of artificial insemination (AI) with fresh, cooled, or frozen semen (except for Thoroughbreds). But whether you are using AI or live cover, the main goal is to get a viable sperm to fertilize a mature, healthy ovum (also known as an oocyte or egg). Therefore, it is important to evaluate and understand the sperm-producing capabilities of a stallion and understanding the quality of his semen.

Terry Blanchard, DVM, MS, Dipl ACT, of Texas A&M University, says that in general, if a stallion has good-quality fresh semen, that semen has a good chance of surviving the cooling process. Figuring prominently into the equation of semen survivability is the human handling factor during collection and insemination. The handling of semen at every level can be improved through education and experience, but that won’t affect the initial quality of the semen a particular stallion produces.

Thus, says Blanchard, it is highly important to evaluate a stallion’s semen well in advance of collecting and shipping it. If it is not of the best quality, there might be some steps that can be taken to improve the survivability of spermatozoa, but determining what those steps should be will come in the wake of semen evaluation.

Anatomy and Physiology

Before beginning a discussion on evaluation, we must know what is being evaluated. The point of discussion here is a specialized cell called a spermatozoon ("spermatozoon" is singular, while "spermatozoa" or "sperm" is plural). One of the most succinct descriptions of a spermatozoon -- how the cell is structured and how it functions -- is provided in the handbook Cooled and Frozen Stallion Semen published in 1999 by Colorado State University, with E.J. Squires, DVM, PhD, as the chief author. Much of the information that follows relative to spermatozoal structure and function is based on information in that publication.

First of all, since spermatozoa have only one function, they possess special characteristics to carry out that function, but do not have the same capabilities as other cells. For example, because they must travel through the mare’s reproductive tract, spermatozoa have movement capability. However, they have little ability to repair themselves if something should adversely affect the cellular structure.

Each spermatozoon is compartmentalized into a head (containing an acrosome for penetrating the egg’s membrane as well as the nucleus), neck, middle piece, principal piece, and end piece. The tail consists of the neck, middle piece, principal piece, and the end piece. If something deleterious should happen to any one of these pieces, the spermatozoon could be rendered incapable of fertilizing the egg.

Covering these structures -- head, neck, middle piece, principal piece, and end piece -- is the plasma membrane, which is composed of many lipids and proteins.

Damage to any part of the plasma membrane can compromise the ability of the spermatozoon to perform its function. Thus, we again emphasize the importance of proper handling, as the plasma membrane is very sensitive to collection, extending, shipping, and cooling procedures.

Now, for a breakdown, piece by piece, of a spermatozoon.

Head -- The head of a stallion spermatozoon is broad and relatively flat. It includes the nucleus, containing the DNA or genetic material, and the acrosome, containing enzymes. The acrosomal membrane is designed to fuse with the plasma membrane directly over it (this is known as vesiculation) and release the acrosomal enzymes that help the spermatozoon penetrate the ovum and its vestments. This vesiculation is known as the acrosomal reaction. The plasma membrane overlying the acrosome is capable of lipid reorganization required for acrosomal reaction (more on this later). Proteins of the head’s plasma membrane allow the spermatozoon to bind to receptors on the ovum’s zona pellucida (coating on the outside of the oocyte) and later to the ovum’s plasma membrane during fertilization.

Neck -- The neck connects the head to the middle piece. It has a relatively fragile "ball and socket joint." Separation of the head from the middle piece often occurs due to damage to this structure.

Middle Piece -- The middle piece is described by Squires in the handbook as "the powerhouse of the spermatozoon." It contains the mitochondria, cellular compartments which convert energy sources such as glucose into adenosine triphosphate (ATP). The ATP transfers energy to other cell components called organelles to power all cell processes. The middle piece also contains a series of fibers that continue through the length of the tail and are responsible for motility. This is what enables the spermatozoon to "swim" through the mare’s reproductive tract.

Principal Piece -- The fibers from the middle piece continue into the principal piece, surrounded by a protein sheath. Spermatozoal motion occurs as these fibers slide back and forth, producing tail movement. This is an important part of the spermatozoon’s "swimming" action or motility.

End Piece -- The tail fibers end in this section of the spermatozoon, and here the plasma membrane tapers to an end.

At the end of the above descriptions of various parts of a spermatozoon, Squires offers this conclusion:

"To fertilize an ovum, a spermatozoon must have all of these spermatozoal compartments intact and functioning properly. A spermatozoon can be sterile for numerous reasons: a) acrosomal loss or damage; b) plasma membrane damage; c) poor mitochondria function; d) loss of motility, etc.

"A spermatozoon exhibiting acrosomal damage after handling, cooling, or freeze-thawing will be just as sterile as the spermatozoon that is immotile (unable to swim). Therefore, breeders must take precautions to maintain all of the compartments of spermatozoa to maximize potential fertility of a seminal sample."

Again, the point is made that proper handling of a seminal sample at all stages is necessary if pregnancy is to result from a breeding via artificial insemination, especially if the breeding is done with cooled or frozen semen (cooling and freezing always  compromise spermatozoon fertility to some degree).

But, as stated earlier, before it can be handled properly, the technicians must know what they are dealing with. For an explanation of semen evaluation, we turn to Blanchard, who has written and spoken extensively on the subject.

In 1996, Blanchard prepared a draft for the American Association of Equine Practitioners (AAEP) on "Recommendations for Breeding with Cooled Transported Equine Semen." One of Blanchard’s recommendations was that "each stallion for which breeding with cooled transported semen is planned should be tested prior to the breeding season to ensure the spermatozoa survive the cooling process."

The examination and evaluation of semen, Blanchard pointed out, can include everything from merely examining the spermatozoa with a microscope to doing highly sophisticated tests, as well as testing its sensitivety to cooling and freezing.

He also emphasizes the need for proper handling of the semen after it is collected to prevent damage to fragile sperm: "Immediately after its collection, semen should be quickly transported to a laboratory while minimizing physical trauma, exposure to light, cold shock, or excessive heat.

To enhance its reliability, the semen evaluation should be performed in a thorough, methodical manner by an experienced person in an adequately equipped laboratory.

"All materials that come in contact with semen (including the seminal extender) should be pre-warmed to body temperature in an incubator."

An important first step, Blanchard says, is to remove the gel, along with any other debris, from the semen. The gel-free portion of the semen is what contains most of the spermatozoa.

Once that is done, Blanchard says, the gel-free semen should immediately be poured into a graduated cylinder to measure volume. "Though volume itself is seldom important to fertility," he says, "it is used to calculate the total number of sperm in an ejaculate. Consequently, accurately measuring volume is essential."

It is also essential to accurately measure sperm concentration, Blanchard tells us, because "the total sperm number in an ejaculate is derived by multiplying sperm concentration and gel-free semen volume. An imprecise estimate of sperm concentration produces a correspondingly inaccurate calculated sperm number in an ejaculate."

Available today is sophisticated equipment that can help the technician determine quickly and accurately the number of spermatozoa present in a given seminal sample, provided the above preceding steps are handled correctly. Utilized for this function are spectrophotometers that are pre-calibrated and dedicated specifically for counting equine spermatozoa. Several types of spectrophotometers are available.

Blanchard says recent investigations at Texas A&M revealed that substantial variations exist among many of the commercial systems for counting sperm.

"Total sperm number, calculated as the product of sperm concentration and semen volume, is one of the more important measurements used in estimating a stallion’s fertility," Blanchard says.

According to Blanchard, the total number of sperm in an ejaculate is influenced by a number of factors, including season, frequency of ejaculation, age, testicular size, the efficiency with which the testes produce sperm, the size of extragonadal (duct system outside the testes) sperm reserves, and various forms of reproductive disease.

Stallion ejaculates, according to Blanchard, typically will contain from three to 20 billion sperm.

"When the stallion is young and on a frequent breeding schedule, sperm numbers are usually at the lower end of this range," Blanchard explains. "Sperm numbers are usually at the upper end of this range when a stallion is older and on an infrequent breeding schedule. Fertile stallions should be expected to have at least four billion total sperm in the first ejaculate and two billion total sperm in the second ejaculate after one week of sexual rest."

The Look and Movement

Now we come to two other essentials that figure prominently into an evaluation of sperm -- motility and morphology. Motility refers to the motion capability of spermatozoa, and morphology refers to the structure of the spermatozoa.

While there are some sophisticated laboratory instruments available for these determinations, most veterinarians and technicians at breeding farms will use a microscope to examine semen samples for determining motility and morphology.

"Subjective analysis of sperm motility by visual estimation, using a microscope equipped with phase-contrast optics and a warming stage, is acceptable when personnel are experienced in analyzing sperm motility," says Blanchard.

The motility examination is divided into three parts, according to Blanchard:

Gross or total sperm motility -- The percentage of sperm exhibiting motility of any form.

Progressive sperm motility -- The per-centage of sperm that are moving in a rapid linear manner.

Sperm velocity -- The movement that can be arbitrarily scored on a scale of 0 to 4.

"Progressive sperm motility generally is considered the most credible gauge of sperm motion to predict the fertilizing capacity of a semen sample," says Blanchard.

"The accuracy and repeatability of the sperm motility evaluation," he adds, "can be improved markedly by diluting the semen in an appropriate extender before analysis. Warmed nonfat dry skim milk-glucose extender works well."

The morphologic study is also carried out with a microscope with some of the sperm affixed to a slide. At least 100 to 200 spermatozoa should be evaluated for morphological defects, Blanchard notes.

The type of defects can sometimes indicate where they might have originated.

One classification for morphologic abnormalities is as follows:

Primary -- Considered to be associated with spermatogenesis and are therefore of testicular origin.

Secondary -- Abnormalities created in the excurrent duct system; commonly associated with prolonged storage in the epididymides (duct system).

Tertiary -- Abnormalities developed as a result of improper semen collection or handling procedures.

Just how long the spermatozoa will remain viable when cooled will vary from stallion to stallion. Generally speaking, however, the longer the spermatozoa remain in a cooled state, the more its fertilizing capability will be compromised.

Blanchard puts it this way: "Fertility trials using cooled-stored equine semen have yielded pregnancy rates varying from 0% to greater than 70% per cycle, emphasizing that there is great variability among stallions in spermatozoal ability to maintain fertilizing capacity during cooling and storage.

"Breeding with equine semen cooled for 48 hours at 5°C (41°F) usually reduces pregnancy rates to approximately half of that achieved with fresh semen. Since pregnancy rates per cycle achieved by breeding with semen cooled for 24 hours at 5°C (41°F) average 60-70%, one should expect near-normal pregnancy rates when semen is used for breeding after short-term storage (in the 24-hour range), provided semen quality is good following this cooling period."

Problem Stallions

The evaluation process previously described above is considered the normal approach and would be all that is needed for many stallions. However, some fertility problems in stallions are subtle, and more sophisticated evaluation techniques are required.

Blanchard and his colleague Dickson Varner, DVM, MS, Dipl. ACT, have described them in detail. Here is a capsule look:

Karyotype Analysis -- This test will occasionally yield useful information re-garding a genetic basis for subfertility and infertility. It involves an in-depth study of chromosomes.

Chemical Analysis of Seminal Plasma -- This involves a close study of seminal plasma that is included in the ejaculate. It has been found that high concentrations of seminal plasma can adversely affect sperm motility during cooling and storage. It has been found that some stallions produce seminal plasma that is toxic to spermatozoa.

Electron Microscope Analysis of Sperm -- As the title implies, this involves examining sperm under an electron microscope to evaluate structures invisible with a light microscope.

Sperm Chromatin Structure Assay -- This test is designed to evaluate the integrity of sperm chromatin. The test is designed to assess the health of DNA in the nucleus by examining it with a flow cytometer.

Antisperm Antibody Test -- This test is designed to search for antibodies that can interfere with the fertilizing capacity of sperm.

Acrosomal Test -- This test determines the ability of sperm to undergo the acrosome reaction. This involves addition of sperm to special media that contains substances capable of inducing the reaction. Over a set time, the percentage of sperm with acrosomes that react is compared to that in a similarly prepared sample from a highly fertile stallion.

The rate of success in achieving pregnancy is on the rise because of the understanding of what is normal in stallion reproduction, and how to test sperm for abnormalities. Researchers are working to learn more about how sperm change as they pass through the mare’s reproductive system, how to safely collect, store, and ship semen, and what can be done to promote fertility in problem stallions. The end result will be to get more mares pregnant, whether through AI, natural cover, or other means.

About the Author

Les Sellnow

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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