The Effects of Various Levels of Sodium Citrate, Glycerol, and Equilibration Time on Survival of Bovine Spermatozoa after Storage at −79° C

Abstract

Summary Experiments were designed to determine the optimal combination for per cent sodium citrate, per cent glycerol (by volume), and equilibration hours which would allow maximum survival of motile spermatozoa after being frozen for 5 days by use of dry ice. A sodium citrate–yolk glycerol diluent was used which contained approximately 24% yolk in the final dilution. A dilution rate of 20million spermatozoa per milliliter of diluted semen was used. A three-dimensional central composite experimental design was used, which is adapted to estimating the optimal level of the factors studied. The estimated optima were: sodium citrate, 2.9%; glycerol, 7.6%; equilibration time, 14.9 hours. As equilibration time was decreased, per cent survival decreased only slowly. An interaction between sodium citrate and glycerol was noted. Although semen from bulls was different, the pattern of response to treatments was the same with only absolute magnitude of response varying. An eosin-aniline blue live-dead stain was not satisfactory for estimating live spermatozoa when used on freshly thawed semen samples.