Cooled Semen Preparation

Procedure if centrifugation not required - Raw ejaculate > 80 X 10^6/ml.

  1. Warm (37 - 39°C) a small tube of extender to evaluate semen motility; usually about 0.5 ml of extender.
  2. Warm (37 - 39°C) sufficient extender to dilute semen, this could range from 20 - 300 ml.
  3. Collect semen
  4. Save aliquote to evaluate sperm concentration and small amount to extender to evaluate motility
  5. Dilute remaining semen 3:1 (extender:semen)
    1. extender should be one suitable for cooling semen
    2. always be sure antibiotics are in the extender
  6. Determine the concentration of sperm and evaluate motiltiy
  7. Dilute semen further to 50 million sperm/ml if possible (50 x 10^6 sperm/ml)
  8. Place 1 billion sperm in storage vessel (plastic tube, syringe, or whirlpak)
  9. Cool semen slowly over at least 2 hr to 5°C in the refrigerator (beaker with 200 ml water + tube of extended semen), use Equitainer, Clipper or similar device as directed by the manufacturer.
  10. Fill a cooled semen transport data sheet. This sheet should contain information about the ejaculate volume, motility, sperm morphology, extender, antibiotic type, amount of sperm and volume of semen per insemination dose. (Example of UW Cooled Semen Transport Sheet, word, pdf).
  11. Preliminary trials should always be done to ensure sperm from a particular stallion will survive the cooling and storage process. You may need to try several extenders if a stallion's semen does not cool well in the extender you first choose to test.

Centrifugation required - Raw ejaculate < 80 X 10^6/ml.

  1. 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.
  2. Make IMV centrifugation buffer or other clear buffer solution as per package and store in refrigerator. Prior to collection, warm (37 - 39°C) centrifugation buffer, 20 ml, in 50 cc centrifuge tubes. You will be adding 20 - 25 ml of semen to each tube so make sufficient tubes to centrifuge all semen collected. Usually this is no more than 3 or 4 tubes of the centrifugation buffer.
  3. Warm 100 to 200 ml of cooling extender to room temperature.
  4. Warm (37 - 39°C) a small tube of cooling extender to evaluate semen motility; usually about 0.5 ml of extender.
  5. Collect semen
  6. Save aliquote to evaluate sperm concentration and small amount to extender to evaluate motility
  7. Put 20 – 25 ml of semen (gel-free) into the 50 ml centrifuge tubes with the 20 ml warm centrifugation buffer and mix by inversion several times.
  8. 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.
  9. Centrifuge the semen, buffer and cushion at 2000 RPM for 20 minutes (approx. 1000 x g).
  10. 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. 
  11. 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.
  12. Determine total number of sperm to be extended for cooling (volume of sperm pellet X concentration of sperm in pellet).
  13. Dilute semen with room temperature cooling extender to 50 million sperm/ml
    1. ml extender to add to sperm pellet = [(number of sperm in pellet, expressed in millions or X 10^6) / (50 X 10^6 sperm/ml)]- volume of pellet in ml
    2. example; [3575 X 10^6 sperm / 50 X 10^6 sperm/ml] - .75 ml in pellet = 71.5 -.75 = 70.75 ml of extender needs to be added.
  14. Place 1 billion sperm in storage vessel (plastic tube, syringe, or whirlpak)
    1. this is an insemination dose for cooled semen
    2. if you diluted semen using the procedure described above, this will be 20 ml of the extended semen.
  15. Cool semen slowly over at least 2 hr to 5°C in the refrigerator (beaker with 200 ml water + tube of extended semen), use Equitainer, Clipper or similar device as directed by the manufacturer.
  16. Fill a cooled semen transport data sheet. This sheet should contain information about the ejaculate volume, motility, sperm morphology, extender, antibiotic type, amount of sperm and volume of semen per insemination dose. (Example of UW Cooled Semen Transport Sheet, word, pdf).
  17. Preliminary trials should always be done to ensure sperm from a particular stallion will survive the cooling and storage process. You may need to try several extenders if a stallion's semen does not cool well in the extender you first choose to test.