Semen Analysis

A stallion produces untold billions of sperm during a lifetime, and in a normal ejaculate, he can send four billion of them on a race to fertilize a single egg. Only one sperm can succeed. Unfortunately, there are times when none of them succeed. The result is an open mare, something that can cost the mare owner time, money, and patience.

There are multitudes of reasons why sperm fail to fertilize an egg. In some cases, the problems that exist can be solved. In other instances, they can't, and the stallion is retired from the breeding shed.

While not every troublesome condition can be solved for the stallion, the stallion manager at least has at his or her disposal a method for determining whether the horse is fertile--semen analysis.

Before getting into the topic of semen analysis, we should understand what it is we are discussing.

First, anatomy. The normal stallion has two testes. Their prime job is to produce spermatozoa and testosterone. At birth, each testis normally will weigh between five and 10 grams. It will remain at that weight and size during the first 10 months of life. A slight growth occurs between 11 and 16 months of age, and rapid development of both testes starts at about 18 months of age. In some cases, the testes don't reach final maturation until the stallion is 12 to 13 years old.

Each testis is covered by a thick lining of connective tissue called the tunica albuginea. Fused to the outer surface of this capsule is the thin visceral vaginal tunic. Supporting strands of connective tissue extend from the tunica albuginea to divide the testis into lobules.

The non-capsular part of the testis is called the parenchyma. It consists of seminiferous tubules (capable of producing and conveying semen) and interstitial tissue situated between the seminiferous tubules. Located within the seminiferous tubules are Sertoli's cells. They are involved with the production of spermatozoa. Located in the interstitial tissue between the seminiferous tubules are Leydig's cells. They are involved in the production of testosterone, which causes the stallion to be sexually aggressive.

During the first 18 to 24 months after birth, the testes do not produce sperm and the colt is infertile. At about 18 months of age, the testes grow and develop rapidly. Several months later, they gradually begin to produce sperm.

In the adult stallion, billions of spermatozoa are produced daily in the convoluted seminiferous tubules. This figures out to be at the rate of about 70,000 per second.

Although there is this prolific production of sperm on a second-by-second basis, the development and maturation of each individual spermatozoa require between 54 and 57 days. During this time frame, a great many things can go awry and result in the sperm's being unable to carry out the fertilization role.

The process of sperm development is called spermatogenesis. It is basically divided into three phases. Phase one is known as spermatocytogenesis. This is the initial differentiation and subsequent division of the germ cells known as spermatogonia to increase their number.

The second step is meiosis--the process where genetic rearrangement occurs between homologous chromosomes and where spermatogonia are reduced in chromosome number by division to form spermatids. The final step in the process is referred to as spermiogenesis, where spermatids differentiate into mature spermatids.

Once spermatids are released from the seminiferous epithelium into the lumen of the seminiferous tubule, they are referred to as spermatozoa.

Each of the above three phases requires 18 to 19 days.

Once spermatozoa are released within the seminiferous tubule, they travel through a series of ducts to the epididymis, which is lightly attached to the upper surface of each testis. It is here that spermatozoa complete the maturation process and are stored.

The epididymis is divided into three sections--head (caput), body (corpus), and tail (cauda). It is while traveling through the epididymis, from head to tail, that the spermatozoa reach maturity and acquire the ability to fertilize an egg. The migration through the epididymis takes approximately eight days. Once the migration and maturation process is completed, the spermatozoa are stored in the tail of the epididymis.

During breeding season, the spermatozoa are released through ejaculation. When the stallion is not being used in the breeding shed, he will, nevertheless, produce sperm. When the epididymis is filled to overflowing, the excess sperm are voided in the urine.

The minimum number of spermatozoa produced within a 24-hour time period is known as daily sperm production. This will vary among stallions and is strongly influenced by testicular size. Daily sperm output refers to the number of spermatozoa that can be collected per 24-hour period from a stallion and is determined by collecting the stallion daily for seven days.

The number of spermatozoa produced varies from stallion to stallion. Contrary to some long-held beliefs, frequent ejaculations do not stimulate a more rapid production of spermatozoa. In other words, whether a stallion is collected daily or every other day for a week will not have an effect on the number of spermatozoa produced during that week. The stallion, quite simply, has an individual productive capability and the number of times he is collected or used in live cover has no bearing on sperm production.

Thus, a strong case is made for ongoing semen analysis during a breeding season.

It also is significant to note that the entire process involved in the development and maturation of spermatozoa takes approximately 65 days. If something negative should occur during that process, it could have harmful effects on the spermatozoa being produced, but might not harm those that are in storage in the epididymis. Thus, if a stallion suffers an injury to the testes, the full implications might not be manifested until two months down the line.

Again, a strong argument in favor of semen analysis.

Before getting into that, however, let's take a quick look at the route the spermatozoa travel from epididymis to the female's vagina. The reason for doing so is that much can happen to the spermatozoa during this trip.

The Reproductive Trail

From the tail of the epididymis, the spermatozoa, during ejaculation, are delivered to the deferent duct, which is a continuation of the epididymal duct. As the deferent duct approaches the pelvic urethra, it widens into a structure termed the ampulla of the deferent duct. The ampulla is about 18 millimeters in diameter as compared to four to five millimeters for the deferent duct. During ejaculation, spermatozoa are moved through the ducts and into the urethra by muscular contractions.

Along the route through the ducts that the spermatozoa will travel are the two vesicular glands, which are elongated, hollow pouches about 15 to 20 centimeters long and five centimeters in diameter. Fluid secreted by the vesicular glands contributes a major portion of the seminal plasma in an ejaculate.

Next along the route is the prostrate gland. The secretion of the prostate gland is thin and watery and likely helps cleanse the urethra during ejaculation. The secretion also contributes to seminal plasma.

Next are the bulbourethral glands. Their secretions also contribute to seminal plasma.

As the sperm complete their journey through the deferent ducts, fortified along the way with seminal plasma, they ultimately arrive at the urethra. The urethra is a long mucous tube that extends from the bladder to the free end of the penis.

At this point, the ejaculate is called semen. This is the term used to describe the whitish fluid of the male reproductive tract consisting of spermatozoa suspended in the secretions of the accessory glands.

Semen Analysis And Stallion Fertility

Terry S. Blanchard, DVM, MS, Dipl. ACT, and Dickson Varner, DVM, MS, Dipl. ACT, of Texas A&M University, recently collaborated on a series of papers involving stallion fertility. Facts contained in those papers provide much of the information that follows.

The two researchers point out that stallions typically are classified as satisfactory, questionable, or unsatisfactory prospective breeders. A stallion with a satisfactory rating should be able to achieve a season pregnancy rate of 75%, they say.

A minimum 75% seasonal pregnancy rate assumes that the stallion breeds a standard book of mares during a typical breeding season of 135 to 150 days. For Thoroughbreds in the United States, this normally means 40 to 47 mares, and for other breeds that permit artificial insemination, it means 120 to 135 mares.

The Society for Theriogenology has established guidelines for classifying stallions as to breeding potential. For a satisfactory rating, the Society requires that light-breed stallions ejaculate at least four billion total sperm in the first ejaculate after a period (typically one week) of sexual rest. The second ejaculate, collected about an hour after the first, should contain at least two billion total sperm.

Blanchard and Varner offer some firm guidelines for semen analysis:

"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 semen analysis data should be recorded on a breeding soundness examination form.

"All materials that come in contact with the semen (including the seminal extender) should be prewarmed to body temperature (37-38° C) in an incubator.

"The gel-free semen should immediately be poured into a graduated cylinder to measure volume. Though volume itself is seldom important to fertility, it is used to calculate the total sperm number in an ejaculate."

Blanchard and Varner say that 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. Total number of sperm in an ejaculate is expected to change from season to season during the year, and number can be affected by numerous other factors, including frequency of ejaculation, age, testicular size, size of extragonadal sperm reserves, and various forms of reproductive disease.

"Total number of sperm in stallion ejaculates typically ranges from three billion to 20 billion. When the stallion is young and on a frequent breeding schedule, sperm numbers are usually at the lower end of this range; sperm numbers are usually at the upper end of this range when the stallion is older and on an infrequent breeding schedule."

It is also important at this point to learn the pH of the gel-free semen. Abnormally high semen pH can be associated with inflammatory lesions of the internal male genital tract.

Once the sperm count has been made and the pH level determined, it is time to take a look at sperm motility. Many researchers strongly believe that there is a definite relationship between sperm motility and fertilizing capability. Yet, the correlation at this point is not considered to be an absolute.

Assessment of sperm motility includes gross or total sperm motility (the percentage of sperm exhibiting motility of any form) and progressive sperm motility (the percentage of sperm moving in a rapid linear manner).

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

Also assessed during a semen analysis is the longevity of the sperm motility. However, say the Texas A&M researchers, the relationship of longevity of sperm motility and fertilization capacity is controversial.

A microscopic examination of the sperm for good health is next on the agenda during a semen analysis. Blanchard and Varner recommend that at least 100 to 200 sperm be evaluated for evidence of morphological defects. Abnormalities in sperm morphology traditionally have been classified as primary, secondary, or tertiary.

The classifications are broken down as follows:

Primary--Associated with a defect in spermatogenesis and are therefore of testicular origin.

Secondary--Defects that are created in the excurrent duct system.

Tertiary--Defects that develop in vitro as a result of improper semen collection or handling procedures.

In addition, there are some sperm defects where the cause is unknown.

While a study of sperm morphology can provide clues concerning fertility, it is not absolute. Blanchard and Varner note that, "The value of sperm morphology studies in predicting the fertility of a stallion is met with a degree of skepticism since some stallions can have many sperm morphological abnormalities and still achieve good pregnancy rates when bred with good management. Conversely, some stallions exhibit decreased fertility even though the percentage of morphologically normal sperm in their ejaculates is high.

"A recent study investigating effects of sperm morphological defects on fertility in 64 stallions found the percentage of abnormal heads, midpieces, and proximal droplets significantly adversely affected fertility--the higher the percentage of these defects, the lower the fertility."

The good news is that it does not appear that the abnormal sperm in an ejaculate have a negative effect on normal sperm. Thus, it could be reasoned, the number of normal sperm in an ejaculate is what really matters.

Blanchard and Varner say that a minimum of one billion "morphologically normal progressively motile" sperm are recommended to be present in the second ejaculate of stallions with one week of sexual rest.

"Conversely," they add, "some investigators believe that many of the important sperm morphological abnormalities that adversely affect fertility also show up as non-motile or non-progressive motile sperm; therefore, they believe determinations of breeding soundness should be based on the number of progressively motile sperm in ejaculates."

The semen analysis approach described above normally gives a fairly accurate look at the potential of a stallion's fertility. However, there are cases where the basic semen analysis doesn't yield specific results, especially for the stallion having fertility problems.

When that is the case, modern-day laboratory techniques take semen analysis to another, higher level. Here are some of the sophisticated tests that can be performed:

Karyotype Analysis--This analysis involves a study of the chromosomes. Karyotyping involves arranging computerized images, drawings, or photomicrographs of chromosomes. Karyotyping permits visual scrutiny of chromosomes for numeric or structural changes that could affect reproductive performance. It has been estimated that 2% of stallions have chromosomal defects. Blanchard and Varner say that in a study population of subfertile stallions, 18 of 36 subfertile or infertile stallions had abnormal karyotypes.

Chemical Analysis of Seminal Plasma--High concentrations of seminal plasma adversely affect sperm motility during cooling and storage. There can be another problem. Some stallions are suspected of producing seminal plasma that is toxic to spermatozoa. Thus, the fluid that is to provide an assist to the spermatozoa sent on its way during ejaculation could instead carry a death sentence.

Transmission Electron Microscopic Analysis of Sperm--This involves evaluating sperm morphology with a very high-powered microscope. Normally, using a phase-contrast or light microscope at X1,000 magnification is sufficient for most breeding soundness examinations. However, in certain cases where a high incidence of a particular abnormality shows up, it might be necessary to use a microscope with more power. Used in those instances are scanning electron or transmission electron microscopic techniques that typically provide magnifications of X4,000 to X60,000. Although expensive, these two methods offer high-resolution detail and permit closer examination of spermatozoal morphology.

Sperm Chromatin Structure Assay--The sperm chromatin structure assay is a procedure that has been developed to evaluate the structural integrity of sperm chromatin. This assay defines abnormal chromatin structure as a heat- or acid-induced susceptibility to DNA denaturation (destruction of the substance). Lower fertility occurs when greater sperm denaturation is found. Blanchard and Varner see this as a good tool in predicting fertility: "The potential of the sperm chromatin structure assay for predicting fertility in stallions has recently been evaluated with encouraging results. Some subfertile stallions with otherwise normal semen quality had a high percentage of sperm with unstable sperm cell DNA. The sperm chromatin structure assay may have potential for identifying some stallions as subfertile when routine breeding soundness examination fails to do so."

Antisperm Antibody Tests--These tests are aimed at determining if the stallion's body is producing antibodies that can attack and kill sperm. Normally, that does not happen because there are tight barriers in the seminiferous tubules that are aimed at preventing the Sertoli's cells from coming into contact with blood. If there is a commingling of blood and maturing germ cells (including sperm) in the testis, the body would begin producing antibodies. Equine antisperm antibody tests are being developed to assess the potential role of these antibodies in the infertility of both stallions and mares.

Hormonal Assays--Reproductive malfunction due to hormonal abnormalities has not been firmly established in stallions, except for those given testosterone, anabolic steroids, or altrenogest, all of which have a negative effect on spermatogenesis. However, abnormal concentrations of reproductive hormones commonly point to dysfunction in infertile or subfertile stallions. A hormonal assay is designed to detect which components in the reproductive system might be contributing to abnormal reproductive function.

Success Parameters

Earlier, we pointed out that a stallion, given a classification as a "satisfactory prospective breeder," is one which is able to achieve a season pregnancy rate of 75%.

There are two other classifications--unsatisfactory and questionable.

An unsatisfactory classification, in the wake of a breeding soundness examination, including semen analysis, would go to the stallion which

1. Does not have two normal testes.

2. Harbors a venereal pathogen or is ejaculating semen containing blood, purulent material, or urine.

3. Exhibits abnormal mating behavior or ejaculatory dysfunction.

4. Is severely deficient in two or more categories of the semen analysis.

If a stallion is borderline in two or more categories, say Blanchard and Varner, it should be classified as a questionable prospective breeder, "meaning the clinician questions the stallion's ability to achieve satisfactory pregnancy rates (per cycle or season) when bred to a normal book of mares under routine management procedures."

Neither of these classifications makes the statement that the stallion is sterile. The Texas A&M researchers recommend that stallions in these two classifications be re-examined at intervals--usually 60 days. This would be especially true for stallions classified as questionable and would be especially important for young stallions which have recently retired from a performance career. These stallions might have small testes and ejaculate fewer than normal sperm. However, in many instances, their fertility improves as they adjust to new surroundings and mature sexually.

In some cases, stallions which do not pass a breeding soundness examination with flying colors can be given assistance. There is evidence that a good many such stallions exist in breeding sheds around the country. Blanchard and Varner cite one study of 1,044 stallions where 36% failed breeding soundness examinations.

A common cause is ejaculation of a low number of progressively motile sperm. In such horses, however, the researchers state, satisfactory pregnancy rates can be achieved by limiting the book size to fewer mares and by intensifying mare management by utilizing hormones to control ovulation time in the mares being bred (see related article on page 42).

The goal, they point out, should be to breed each mare one time 24 to 48 hours before ovulation. Reducing the number of services required to breed the same number of mares increases the number of sperm per ejaculate, which will optimize pregnancy rates.

The researchers recommend that if a stallion is to stand at stud despite failing a breeding soundness examination, semen should be collected once a day until daily sperm output is established. Knowing the number of normal, motile sperm ejaculated daily enables the booking of an appropriate number of mares for such stallions.

As all horsemen know, breeding can be a precarious business. Fortunately, modern science has given us tools to at least make predictions about a stallion's fertility before a breeding season is lost.

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 or by calling 800/582-5604.

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