Induction of bovine sperm capacitation by TEST-yolk semen extender

Ejaculated bull semen was diluted 1:10 in the TEST-yolk buffer, cooled slowly to 4 degrees C, and stored for up to 48 h. Aliquots were taken at 0, 4, 8, 16, 24, and 48 h and washed once or three times in bovine serum albumin-saline and the sperm pellets resuspended in this saline. Fertilization of zona-free hamster oocytes was used to assess sperm capacitation. Motility differed between samples washed once or three times (53.7 vs. 21.7%). Motility was highest at 4 h storage but did not differ between 16, 24, or 48 h of storage. More sperm without intact acrosomes were found at 4 h than at 0 h, but the percentage did not change further until after 24 h. Penetration of oocytes was not different between sperm washed once or three times (28.5 vs. 26.9%). No penetration occurred at 0 h, and highest penetration rates occurred at 4 and 8 h of storage (32.1 and 33.4%). Penetration rates at 16, 24, and 48 h were not different (25.3, 25.2, 22.5%). In conclusion, storage of bull sperm in TEST-yolk buffer for 4 to 48 h resulted in capacitation. Even though capacitation was induced by 4 h, at least 71% of the sperm population had not undergone an acrosome reaction by 48 h of storage. This may explain why penetrability was maintained over this period.