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Semen Analysis And Stallion Fertility

Description
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.

"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."

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 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 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. We 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. "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.

    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.

    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, 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. 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.

    A common cause is ejaculation of a low number of progressively motile sperm. In such horses, however, 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.

    The goal 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.

    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.
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