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Osmotic Pressure and pH Effects on Survival of Frozen Bovine Spermatozoa

Department of Animal Science, Cornell University, Ithaca, New York

ABSTRACT

Semen from ten bulls was extended in egg yolk extenders buffered with live concentrations of Tris adjusted to four pH levels, and containing two levels of glycerol, to study their interactions during semen freezing. Spermatozoa in these 40 media (5x4x2) were equilibrated for 1.5 and 6.0 hr, frozen at 0.8 and 3.0 C/minute in the critical range, and stored seven days at -196 C. Duplicate estimates of the percentage of motile spermatozoa made immediately after thawing (6,400 observations) indicated that the freezing rate and equilibration time had little effect on survival. The percentages of motile spermatozoa for the 6.4 and 8.8% glycerol levels were 30 and 28, for the 0.15, 0.20, 0.25, 0.30, and 0.35 M Tris-buffered yolk extenders 37, 40, 37, 28, and 5; and for the pH's of 6.0, 6.5, 7.0, and 7.5, 25, 29, 31, and 31, respectively. These effects were statistically significant (P < .005). Several main factors interacted in opposing ways, thus reducing their overall differences. The highest percentage of motile spermatozoa was observed in the extender adjusted to pH 6.50 containing 0.20 M Tris, 6.4% glycerol and 1% fructose; motility averaged over the two equilibration periods was 43% upon thawing and 33% after an additional storage period of 24 hr at 5 C. This extender without glycerol was slightly hyper-osmotic to sperm, as evidenced by a freezing point depression of 0.62 C.

¹ Present address: Department of Agriculture, University of Ibadan, Nigeria.