Semen Collection from the Stallion and Handling for Artificial Insemination

Semen from stallions is usually collected with an artificial vagina with the stallion mounted on a mare in heat or a dummy mount. Freshly collected semen can then be evaluated and used for artificial insemination of mares.

To start a semen collection/artificial insemination program, minimum requirements include:

  • A breeding shed, optimally with a phantom mare or dummy mount
  • An artificial vagina and related supplies
  • Laboratory equipment, such as a microscope to evaluate sperm motility, a sperm counting machine to determine sperm concentration, an incubator to keep supplies warm, and miscellaneous utensils, such as microscope slides, pipettes and disposable plastic cups.

    Most stallions can be easily trained for semen collection with an artificial vagina provided both the handler and semen collection technician are experienced. The libido or sexual drive of the stallion and breeding experience of the stallion are also important factors to consider when training a stallion for semen collection.

    Artificial insemination programs with fresh semen provide excellent results as long as the semen quality is acceptable and the semen is handled appropriately after collection. It is also very important to breed the mare at the right time during her cycle; therefore, mares should be teased until they come in heat and then optimally evaluated by palpation and/or ultrasound to ascertain good breeding timing in relation to ovulation.

    The main advantage of performing artificial insemination with fresh semen is that one ejaculate can be split among several mares that are ready to be bred, therefore increasing the book of mares for a particular stallion in a given season. Performing artificial insemination also avoids the inherent risks of handling stallions and mares for natural cover, or breeding mares that do not stand well for breeding during heat.

    The main disadvantage of performing artificial insemination is the requirement for specialized equipment and training. If semen is not handled appropriately and timing of insemination is not accurate, pregnancy rates may be sub-optimal in artificial insemination programs.

  • Semen from stallions is most commonly collected with the aid of an artificial vagina while mounting a mare in heat, an ovariectomized mare or a dummy mount. There are several types of artificial vaginas, and the most commonly used in the United States are the Missouri, Colorado State University and the Japanese or Nishikawa. Other methods of semen collection are available, including collection of semen with the stallion standing on the ground or manual stimulation instead of an artificial vagina. These are good alternative methods in some cases, particularly with special needs stallions, but have not been used widely for routine semen collection and require special training.
  • The choice of artificial vagina to be used depends on the preference and experience of the semen collection technician. For example, the Missouri model is easy to handle and wash and most stallions respond well with this type of vagina. Additionally, the length of this vagina fits with the length of the penis of most stallions avoiding the heat shock of the sperm that will result if semen is exposed to the inside water jacket of the vagina. The design also eliminates the possibility of water contamination of the semen sample.

    Preparing the Artificial Vagina

  • Prior to semen collection the inside of the vagina should be clean and dry.
  • A sterile whirl-pack bag or other type of receptacle (i.e. baby bottle or baby bottle liner) is secured to the distal end of the artificial vagina to collect the semen during ejaculation.
            
  • The inside of the artificial vagina is lubricated with a non-spermicidal water-soluble sterile lubricant (i.e. KY Jelly is not acceptable because it contains a bactericidal substance that can affect sperm viability). The lubricant is spread by inserting the operator's arm into the vagina protected by a clean shoulder-length palpation sleeve. The vagina should only be lubricated to about two-thirds of its length to avoid excessive contamination of the semen sample with lubricant.
  • The water jacket of the vagina is then filled with hot tap water at around 50 to 55 degrees Celcius. A non-breakable thermometer can be used to measure the temperature inside the opening of the vagina, but be sure to remove the thermometer prior to semen collection! A final temperature of 42 to 48 C is appropriate for most stallions. Many stallions do not appear to get enough stimulation to ejaculate at lower temperatures. Temperatures higher than 50 C may burn the penis of the stallion resulting in a long-term negative experience.
  • Once the artificial vagina is at the right temperature, the outer leather shell is secured and the pressure of the inside of the vagina is adjusted by releasing water according to the size of the penis and pressure preferences of the stallion to be collected. Some stallions require more pressure than others for adequate stimulation and only by working with a stallion in a regular basis one can learn his individual preferences. As a general rule, one should be able to fit their arm comfortably inside the lumen of the vagina and make a fist with the hand.

    Cleaning and Care of the Artificial Vagina

  • The artificial vaginal should be thoroughly cleaned after semen collection to avoid both cross contamination between stallions and contamination of the semen sample. The exact cleaning steps vary among farms and laboratories.
            
  • Most people first clean the rubber liner with tap water only. Then the rubber liner is submersed in an alcohol bath (70 percent isopropyl alcohol) for about 20 minutes for disinfection and is then hung in a clean cabinet for drying. Most detergents will leave spermicidal residues even after thorough rinsing, so should be avoided altogether. Rubber products are particularly vulnerable to harboring spermicidal residues. Spermicidal agents can have varying degrees of damage to sperm. Some kill sperm immediately, some slowly. So you may not detect damage even under a microscope before semen is inseminated but damaged sperm may not yield expected pregnancy rates.

    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.

    Semen Preparation

    Once the semen has been collected and appropriately evaluated, it is ready for insemination of mares. Although not always necessary, adding an extender for insemination will optimize survival of sperm during the time needed to prepare the mare for breeding and will reduce contamination of the mare's uterus at breeding. This is especially important in mares susceptible to developing uterine infections after breeding. For immediate insemination, adding an equal volume of extender than that of semen (1:1 dilution ratio) is usually sufficient and does not require any special calculations.

    As long as enough progressively motile sperm are present, the extended ejaculate is simply split between the number of mares to be bred, or the whole ejaculate is used to breed a single mare. The minimum insemination dose necessary to keep from compromising fertility rates is 500 million progressively motile sperm per mare. However, as much sperm as is available should be used for insemination.

    Mare Preparation

    Once it is ascertained that a mare (or group of mares) is ready for breeding, the mare's tail is wrapped and the perineal area around the vulva is thoroughly scrubbed and generously rinsed three times. Povidone iodine scrubs can be used for this purpose, but all residues should be thoroughly rinsed with water to avoid introducing this disinfectant into the uterus while inserting the pipette for insemination. After thorough scrubbing and rinsing, the perineal area is dried with clean paper towels. The mare is ready for insemination.

    Insemination Procedure

    In preparation for artificial insemination, the corresponding volume of extended semen is drawn into one or more syringes (with no rubber plunger) connected to a disposable long plastic pipette.

    The veterinarian or manager performing the procedure should wear a clean plastic sleeve and a sterile glove on the hand that will be used to introduce the pipette into the uterus. The gloved hand may be lubricated with a water soluble sterile jelly to facilitate the procedure. Then, the pipette is cupped within this hand, and directed through the vulvar lips, vestibule, into the vagina, and then inserted through the cervix, while the opposite hand remains outside the mare to push the syringe plunger and inject the semen through the pipette. For successful insemination, the semen must be deposited inside the uterus.

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