Stallion Semen Freezing Protocol Using IMV Extender

Or Any Extender Requiring Cooling Prior to Freezing

(Adapted by John Parrish, January 2006)

Media to Prepare (or thaw)

  1. IMV Freezing extender as per package instruction but without glycerol.
    1. Put 100 ml double distilled water in a bottle
    2. Pour contents of package in bottle and dissolve
    3. Wash media back in foil package a couple of time.
    4. Pour 96 ml of the extender in a graduated cylinder, add 4 ml of egg yolk, mix and return to the bottle. Egg yolk may need to be certified if semen is to be exported.
    5. This can be stored in aliquots frozen (10 ml is a good size) and thawed for use.
  2. Mix the IMV freezing extender 1:1 with glycerol.
    1. For this make 10 ml glycerol + 10 ml freezing extender and freeze 1 ml aliquots.
    2. Each day thaw as many aliquots as you will need.  Usually this is only 1 or 2 per ejaculate collected. Place the thawed glycerol extender in a cold room (5°C, 40°F).
  3. Place 2 - 3 ml of cushion solution in a tube and warm to room temperature. The cushion solution may be either IMV's (Eqcellsire Solution B), Minitubes Cushion solution, or 90% percol.
  4. Make IMV centrifugation buffer as per package and store in refrigerator.
  5. When thawing, warm some IMV insemination buffer. Alternatively you may use INRA 96, or any milk based extender used for cooled semen such as Minitube's Equi-pro or Eazi-Mixin.

Freezing Procedure

  1. Place 20 ml of centrifugation buffer in a 50 ml centrifuge tubes and place in water bath at 37 – 39°C. You will be adding 20 - 25 ml semen to the tube so put sufficient tubes in for the normal size ejaculate of the stallion you are collecting.
  2. Put 0.5 ml aliquots of cushion solution in 12 X 55 tubes and place in water bath.
  3. Put 10 – 20 ml of IMV freezing extender in water bath to thaw or warm up.
  4. Go and collect semen.
  5. Put 20 – 25 ml of semen (gel-free) into the 50 ml centrifuge tubes with the warm centrifugation buffer and mix by inversion several times.  Save some raw semen for motility and concentration determination.
  6. Using a Pasteur pipette, put 0.5 ml cushion solution below the semen and centrifugation buffer to make a cushion for sperm to be pelleted on top.
  7. Centrifuge the semen, buffer and cushion at 1750 – 2000 RPM for 20 minutes (approx. 1000 x g).
  8. Remove the freezing buffer from the water bath and place on the counter to cool to room temperature.
  9. Remove supernatant and cushion solution if still visible as a clear layer at the bottom. The supernatant is usually removed with an aspirator. The cushion solution is removed with a pipet. Carefully transfer the sperm pellet to a small tube. 
  10. Remove 10 µl of sperm and mix with 90 µl of centrifugation buffer in a small tube. Determine the concentration of this sample and multiply by 10 to get the concentration of the sperm pellet.
  11. Dilute semen to 250 X 10^6 sperm/ml with the freezing extender without glycerol.  Put the tubes containing this semen in 100 ml room temperature water and cool to 5°C over 2 hours (place in a cold room).
  12. Once cooled, to each ml of extended semen add 80 µl of the cold 50% glycerol solution and mix quickly by inversion (this yield 4% glycerol in the extended semen).
  13. Fill and seal straws in cold room.
  14. Freeze semen 3 cm above liquid nitrogen for at least 15 minutes before plunging into liquid nitrogen.

Thawing Procedure

  1. Put 10 ml of insemination buffer into a tube and warm to 37 – 39°C.
  2. Thaw at least 7 straws in 37 – 39°C water for 1 minute. This yields an insemination dose of approx. 800 x 10^6 sperm.
  3. Dry straws carefully.  For each straw, cut one end, place straw above tube of insemination buffer and cut the other end of the straw to allow contents to flow into the tube.  Blow out contents of straw into tube to make sure all semen goes into the tube.  Repeat procedure for all straws.
  4. You may inseminate the total volume leaving a small amount to evaluate or you may examine the semen microscopically after 10 – 15 minutes in the water bath.
  5. A caffeine coated slide will allow you to determine maximal motility from your sample if you so desire and will not require the 10 – 15 minute incubation.  Remember to always use heated slides and coverslips, and a heated stage on the microscope.