Influence of freeze rate, thaw rate and glycerol level on acrosomal retention and survival of bovine spermatozoa frozen in French straws.

A three-dimensional central composite design was used to study the effect of glycerol level, freeze rate, and thaw rate on acrosomal retention and percent motility. Glycerol levels examined were: 1, 4, 7, 10 and 13%. Average freeze rates between 1 0 and 8 0 C examined were: 6.5, 13.1, 26.3, 52.5 and 105.0 C/minute. Thaw rates studied were achieved by plunging straws into water at 5 C for 2 rain, 20 C for 1 min, 35 C for 30 sec, 50 C for 15 sec and 65 C for 7.5 seconds. Percent motility and percent intact acrosomes for one ejaculate from each of 12 bulls were recorded post-thaw at 0, 3, 6 and 9 hr of a 37 C-incubation. The multiple regression model (mean of 0-, 3-, 6and 9-hr incubation periods) described the experimental data adequately (r 2 = .83, acrosomes; r 2 = .74, motility). Glycerol level was significant (P<.01) as a quadratic component for both intact acrosomes and motility. Thaw rate was significant as a quadratic component for intact acrosomes (P<.01)and motility (P<.05). The interaction of glycerol and thaw rate was of greatest importance (P<.01). As the thaw bath temperature was increased, optimum glycerol level also increased in achieving maximum motility and acrosomal integrity. Freeze rate was relatively unimportant. Using the average freeze rate of 26.3 C/rain from 1 0 to 8 0 C, minimal cell injury was calculated to be achieved with 8.5% glycerol and a thaw water temperature of 65 C.