Roanoke A. I. Laboratories, Inc. | Insemination Instructions
8535 Martin Creek Road
Roanoke, VA. 24018-5875
(540) 774-0676
Insemination Procedure Using
Frozen Stallion Semen in ½ ml French Straws
  1. Thermometer which covers range from 37° to 75°C.
  2. First water bath at 75°C (167°F)
    Bath should have volume of about ½ gallon and be at least 6" deep.
    Improvised water baths have been made from the following:
    • Crock Pots
    • Electric Coffee Pots
    • Electric Frying Pans
    • Fry Babys and Fry Daddys
    • Styrofoam Coolers and Large Styrofoam Cups
    • 1 Quart Wide Mouth Thermos Bottles
    It is usually better to heat the water hotter than 75°C and add cool water to bring the temperature down to 75°C at the moment the semen is to be thawed.
  3. Second water bath at 37°C (98.6°F)
    A 1 pint wide mouth thermos bottle is satisfactory.
  4. A STOPWATCH ! (Accuracy is imperative)
  5. A straw cutter or razor blade
  6. A pair of 8 or 10 inch hemostats or tweezers for straw retrieval
  7. Sterile inseminating (breeding) guns
  8. Sterile sleeves & lube, towels, Povidone-Iodine scrub, tail wrap
  1. Do Not expose semen straws above the frost line in the tank neck when retrieving straws for thawing. Slight warming and refreezing will damage or kill the sperm cells.
  2. Use long forceps or hemostats that have have been chilled in liquid nigtrogen to retrieve straws. Grasp the straw gently by the plug. Caution: Wear safety glasses when retrieving and thawing straws. An occasional straw may burst when thawed.
    If ordered, inseminator training straws will be shipped in the canister with the semen straws and will be red. These are used for practicing straw retrevial, thawing, and loading the inseminating gun. NEVER inseminate a mare with the contents of a training straw (the contents are non-sterile).
  1. Adjust the temperature of one water bath to 75°C and of a second bath to 37°C.
  2. Wrap mare's tail and scrub genital area with povidone-iodine scrub.
    • Rinse genital area thoroughly with water to remove any antiseptic then dry.
  3. Remove one straw of stallion semen from the canister.
    • Immeidately immerse the straw in the 75°C water bath for 6.5 to 7 seconds, timing it with a STOPWATCH. Do not turn it loose.
    • At 7 seconds, remove the straw from the 75°C bath and immerse it in the 37°C water bath for another 15 to 20 seconds.
    • Remove the straw from the second water bath and dry thoroughly with a clean towel.
  4. IDENTIFY the print on the straw! The inseminator is the last person to insure that the mare is bred to the correct stallion.
  5. Using a straw cutter or razor blade, cut off the end of the semen straw which contains the air bubble just below the plug.
    Straw Cutting...Note perpendicular, clean cut...straw cutter shown
    • Some semen straws frozen after January 1995 will have a white plug in one end and a black glass ball in the other. The black glass ball may jam in the breeding gun preventing expulsion of the semen. ALWAYS cut off the end with the black glass ball, if one is present, to open the straw.
    • If the straw should balloon or crimp at the air bubble during thawing, cut off the damaged portion so it will fit properly into the barrel of the breeding gun.
  6. Load the straw into a breeding gun.
  7. Check to make sure that the mare is still clean, and deposit the semen slowly into the body of the uterus within 2 or 3 minutes of thawing.
  1. Snip the end of the breeding gun wrapper off on the end containing the plunger rod, leaving the gun in its protective wrapper.
  2. Turn the breeding gun upside down and shake the metal plunger rod and blue straw from the barrel.
  3. Insert the open or cut end of the semen straw into the barrel of the breeding gun from the rear.
    Inserting straw into barrel of breeding gun
  4. Insert the blue straw behind the semen straw and use it to push the semen straw forward until it seats on the conical tip at the front of the barrel.
  5. Insert the metal plunger rod through the blue straw until it just makes contact with the plug in the end of the semen straw.
    Loaded breeding gun with blue straw and plunger re-inserted into barrel
    • The gun may be placed in your trousers or wrapped in a clean cotton towel, still in its protective wrap, to protect semen from temperature changes and light until you are ready to inseminate the mare.
    • The plastic plug functions like the plunger in a syringe when pushed forward by the metal rod.
  • Use only one straw for each insemination and keep inseminations at least 6 hours apart
  • Do not mix any extender with the semen or infuse the mare for 90 minutes after insemination to prevent osmotic shock to the sperm cells.
  • For the highest conception rate, inseminate as close to pre-ovulation as possible but within a window which is 6 to 12 hours pre-ovulation up to 6 hours post-ovulation. Studies have shown resorption rates are higher if the mare is only inseminated post-ovulation.
  • Frozen-thawed stallion semen has a shorter life span (about 12 hours) in the mare's reproductive tract than fresh semen, so inseminations should be timed no more than 12 hours apart if the exact time of ovulation cannot be pinpointed by palpation or ultrasound.
  • Inseminating late at night may result in higher conception rates since many mares ovulate at this time.
  • Post thaw motility of the semen cannot be determined microscopically by examining the contents directly from the straw as the sperm concentration is over 1.5 x 109/ml.
  • The proper technique for evaluation is to remove a 10 microliter aliquot of semen from the straw with a capillary tube and add to ½ ml of 37°C skim milk extender and incubate at 37°C for 10 minutes.
  • Slides, cover slips, and the microscope stage should all be warmed to 37°C. for evaluation.

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