Re-activated bull spermatozoa: motility, acrosome status and ability to penetrate cervical mucus and zona-free hamster oocytes

Abstract

Semen of four dairy bulls was inactivated by centrifugation twice through a 7% Ficoll solution. After the second centrifugation, the sperm fractions were resuspended in 14G buffer and stored for 0, 3, 6 and 9 days at room temperature. At the end of each storage period, inactivated semen was reactivated by centrifugation and the sperm plugs were resuspended to a final concentration of 400 × 10 6 sperm ml −1 in tris-based diluent supplemented with 5 mM caffeine or theophylline. Sperm motility and acrosome status were assessed during subsequent incubation. Sperm penetration into cervical mucus was also assessed. The fertilizing ability of the sperm was tested by using the zona-free hamster oocyte technique. The results showed that bovine semen inactivated by centrifugation through a 7% Ficoll solution could be stored beyond 9 days at room temperature. Satisfactory proportions of motile sperm and spermatozoa with intact acrosomes were recovered after re-activation. The inclusion of either caffeine or theophylline at a 5 mM level had a beneficial effect on the percentage of motile sperm ( P < 0.005). However, the percentage of motile sperm and spermatozoa with intact acrosomes decreased with increasing storage and incubation time ( P < 0.005). These results were confirmed by sperm penetration into cow cervical mucus. The total number of zona-free hamster oocytes to which re-activated bull sperm attached and penetrated was not significantly different from that obtained with fresh sperm (68.3% vs. 57.1%). However, increasing the storage time decreased, in general, ( P < 0.01) the total number of oocytes to which with re-activated bull sperm attached and penetrated.