The effects of washing and protein supplementation on the acrosome reaction of ram spermatozoa in vitro

Ram spermatozoa were incubated for 12 h at a concentration of 1-5 × 106 ml-1 in a modified BWW medium with TRIS (pH 8.0 in air, 308 mOsm) and a variety of pre-treatments were examined. These included washing in hypertonic (360 mOsm) medium by centrifugation and also supplementing with purified bovine serum albumin (BSA), with fatty acid-depleted BSA and with 5% v/v heat-inactivated sheep serum. Motility was assessed by phase contrast microscopy, and the incidence of acrosome reactions among live spermatozoa was estimated from nigrosin-eosin live-dead smears. All treatments showed a steady decline in sperm motility with time, and progressive increases in the proportion of live, acrosome-reacted spermatozoa (% ARS). However, there were no significant differences between washing regimes except for the treatment with hypertonic medium., where % ARS was elevated significantly after 9 and 12 h incubation. No differences were seen in % ARS between the various protein supplementations, although serum promoted significantly better survival