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Influence of Freeze Rate, Thaw Rate and Glycerol Level on Acrosomal Retention and Survival of Bovine Spermatozoa Frozen in French Straws¹

Virginia Polytechnic Institute and State University³, Blacksburg 24061

Abstract

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 –10 and –80 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 min, 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-, 6- and 9-hr incubation periods) described the experimental data adequately (r² = .83, acrosomes; r² = .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/min from –10 to –80 C, minimal cell injury was calculated to be achieved with 8.5% glycerol and a thaw water temperature of 65 C.

¹ The authors wish to acknowledge the cooperation and support of Select Sires, Inc., Plain City, Ohio, in making this study possible. This work was also supported by the Research Grant Program of NAAB and the William M. Hackman Foundation.

² Present address: American Breeders Service, DeForest, Wisconsin, 53532.

³ Department of Dairy Science.

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