VGO 511: Veterinary Andrology and Reproductive Techniques (1+1)

Preparation of sperms, sperm - oocyte interaction and scroring

PREPARATION OF SEMEN

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.

Sperm - Oocyte Co-incubation

• After one hour of capacitation, 100 ml of capacitated sperm drops is placed in a sterile disposable petridish to which 15-20 zona-free oocytes are added.
• Mineral oil should be layered over the sperm drop to avoid possible loss by evaporation.
• The petridishes are then incubated in air incubator at 37oC for three hours to allow sperm-oocyte coincubation.
• At the end of coincubation, the oocyte-sperm complexes are washed several times with handling medium to remove loosely attached spermatozoa.
• The oocytes are then subjected to either fluorescent staining or aceto-orcein staining to assess sperm penetration.
• Spermatozoa with decondensed heads and associated tails are considered to have penetrated the oocyte

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Scoring

• The fertilizing ability of frozen spermatozoa of different bulls is assessed by fertilization percentage and fertilization index.

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