Veterinary Andrology and Reproductive Techniques

PREPARATION OF SPERMS

Semen samples

• The frozen semen samples of bulls maintained at the Semen Banks can be used for carrying out this test.
• On the day of use, the semen straws should be thawed at 37^oC for 30 seconds in a water bath and used for capacitation.

Medium for sperm handling

• The mBWW medium used for handling of oocytes can be used as medium for handling spermatozoa.
• The capacitation medium is prepared by adding bovine serum albumin at a concentration of 35 mg/ml of mBWW medium.

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Swim-up technique

• Swim-up technique should be adopted to improve the quality of semen to be used for sperm penetration bioassay.
• Four mini straws (0.25 ml) are thawed, to which 5 ml of mBWW medium was added to remove the egg yolk and seminal plasma by centrifugation.
• The supernatant is discarded and the pellet is resuspended in 0.5 ml of medium.
• 100 to 200 ml of sperm suspension is placed in three centrifuge tubes, to which two ml of fresh medium is carefully layered.
• The tubes are then placed in an air incubator at 37oC for one hour in slanting position for the motile spermatozoa to swim-up.
• At the end of one hour, the supernatant is collected and centrifuged.
• After centrifugation, the supernatant is discarded and the sperm pellet is again resuspended in fresh medium.

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Capacitation of spermatozoa

• After swim-up, the sperm suspension is capacitated in 0.5 mM concentration of calcium ionophore A23158 (CaI) for one minute and centrifuged to remove CaI.
• The supernatant is discarded and the pellet is resuspended in capacitation medium.
• The sperm concentration is adjusted to 10⁷ million spermatozoa per ml and kept in an air incubator at 37oC for one hour.
• The individual sperm motility is estimated and observing head to head association of two to three spermatozoa can confirm capacitation.

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