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Semen Collection from the Stallion and Handling for Artificial Insemination

By: Dr. Sylvia Bedford-Guaus

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All utensils that come into contact with semen should be kept in an incubator at 98.6 degrees Fahrenheit (37 C). Slides and coverslips may be kept on top of a warming stage also set at 98.6 F (37 C). A sudden changes in temperature will result in decreased viability of spermatozoa and may jeopardize the breeding program. However, sperm also die at temperatures above 98.6 F, and most incubators are set well above that. For this reason, semen should not be placed in an incubator or on top of a slide warming stage after collection, and the temperature of the incubator should always be checked when removing a supply item that will contact the semen. It is better to have the supplies that come into contact with sperm a little too cool than a little too hot.

Likewise, all utensils should be clean and dry prior to contact with the sperm sample. Water and residues from detergents or disinfectants are spermicidal. For this reason, the use of disposable utensils is highly recommended. All should be evaluated to be sure they are not toxic to sperm.
Process semen samples in a laboratory kept at a constant room temperature of approximately 72 to 75 F (23 to 25 C). The semen sample should be handled carefully. Avoid pouring into the bottom of a graduated cylinder; instead, pour carefully down the side of the cylinder. Additionally, avoid exposure to cold drafts or direct sunlight. The key to success is to be thorough but fast in processing and extending the semen sample, whether for packaging for shipment or for immediate insemination on the farm.

Again, once the semen sample is processed do not place it back into the incubator or in warm water bath. Instead, let it cool slowly to room temperature until used for artificial insemination.

Rubber plungers on traditional-type syringes are sometimes coated with a substance that may be toxic to sperm. Non-spermicidal all-plastic syringes (without rubber plungers) should be used for handling semen and for insemination.

Semen Extenders

Semen extenders are used to dilute semen providing a source of energy for sperm cells, protecting spermatozoa against changes in temperature and or pH, and to avoid excessive bacterial contamination of the sample. In general terms, extenders EXTEND the life of the semen sample.

The extender most commonly used for both artificial insemination and transported-cooled semen is a milk based extender that was developed by Dr. Kenney at the University of Pennsylvania in 1975. This extender contains non-fat dried skim milk (i.e. Sanalac® or store brand), glucose and an antibiotic. The components are mixed in sterile water.

Originally, the antibiotics added to the extender were penicillin and gentamicin. More recent research has shown that other antibiotics may be more suitable since they are less toxic to sperm over storage time. Presently, the antibiotics most commonly used for semen extenders are ticarcillin, amikacin, a combination of penicillin and amikacin, or polymyxin B.

Making your own extender requires adequate training in laboratory skills, an accurate laboratory scale and a trustworthy source of sterile de-ionized water, which may be purchased although it is expensive. Otherwise, the pre-measured components of the extender and pre-measured water may be obtained commercially from different companies selling supplies for equine semen collection and artificial insemination.

On-Farm Semen Evaluation

Optimally, the volume, concentration of sperm and percentage of progressively motile sperm should be evaluated for every ejaculate. Therefore, every breeding operation should have a laboratory equipped with an incubator, disposable semen containers (graduated cups or cylinders), a microscope and some means of evaluating sperm.

The following are suggested steps for evaluation of the semen sample:

  • Appearance of the sample. This mostly refers to color. Semen is normally whitish to grayish in color and creamy to translucent depending on the concentration of sperm. For example, a yellow sample may indicate contamination with urine, and a pink or brown-tinged sample may indicate contamination with blood, both of which will kill the sperm. A transparent sample may indicate that only seminal fluid, and no sperm, was ejaculated. Any clumps, shreds, or other debris should be noted.

  • Volume. This is measured by placing the semen in an accurate graduated cylinder or cup. The volume is only important as a means to calculate the total number of sperm in the sample and has no direct bearing on the quality of the ejaculate.

  • Concentration. The concentration of sperm in the sample can be measured by means of a hemocytometer or commercially available machines specially designed to count stallion spermatozoa. Concentration is given on millions of sperm per milliliter of semen. The concentration of sperm is meaningless on its own in regards to semen quality, and is only important in calculating the total number of sperm.

  • Total number of sperm. The total number of sperm can be calculated by multiplying the number of sperm per milliliter (or concentration) times the volume of the sample. The total number of sperm is traditionally expressed in billions.

  • Percentage of motile sperm. The percentage of motile sperm is evaluated by placing a drop of semen on a warm microscope slide and a warm cover slip and observing it under the microscope. Two types of sperm cell motility are usually estimated. The total motility is the percent of sperm that are just moving, in any form or direction. The progressive motility includes only the percent of sperm moving in a straight line and is the most important.

  • Total number of progressively motile sperm. A breeding dose with fresh-extended semen should contain a MINIMUM of 500 million progressively motile spermatozoa. Once the total number of sperm has been calculated and the percentage of progressively motile sperm has been evaluated, one may calculate the total number of progressively motile sperm. This will allow us to know how many mares can be bred with a given ejaculate.

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