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Effects of Thawing Rate and Cold Post-Thaw Temperatures on Bovine Semen Packaged in Glass Ampules¹ ,^,2

Washington State University⁴, Pullman 99163

Abstract

Experiment I was designed to test the effect of thaw rate on semen packaged in glass ampules. One ejaculate from each of 10 Holstein bulls was diluted in egg yolk-citrate-glycerol, packaged in 1.0 ml glass ampules and frozen in liquid nitrogen vapor. Water bath thaws of 5 G for 10 min, 20 C for 4 min, 35 C for 4 min, 50 C for 75 sec, 65 C for 48 sec, 80 C for 3 7 sec and 100 C for 20 sec were compared. Comparisons were based on percentage of motile spermatozoa and percentage intact acrosomes. Initial estimates of motility and percentage intact acrosomes were made immediately following thaw (0 hr) and again at 4 and 8 hr of incubation at 37 C. Overall means (averages of 0, 4 and 8 hr for all ejaculates) for percentage intact acrosomes were 34.5, 24.2, 32.4, 46.3, 47.6, 50.3 and 52.2 for the 5 C, 20 C, 35 C, 50 C, 65 C, 80 C and 100 C thaws, respectively. Significant damage to spermatozoa (P<.01) resulted following thaws of 20 C while thaws of 35 C did not differ from 5 C thaws. For motility and percentage intact acrosomes there was a significant (P<.01) ejaculate X thaw rate interaction. However, all ejaculates responded favorably to thaws above 35 C. Overall means for percentage of motile spermatozoa (all ejaculates at 0, 4 and 8 hr) were greater (P<.01) following thaws above 35 C when compared to the 5 C and 20 C thaws.

Experiment II was designed to compare the effect of sudden post-thaw temperature reduction on semen thawed at various rates. Thaws of 5 C for 10 min, 35 C for 4 min, 50 C for 75 sec and 100 C for 20 sec were used. Post-thaw treatments of -20 C for 40 sec, 1 C for 4.5 min, 37 C for 40 sec and 37 C for 4.5 min were used. When compared to the 5 C thaw, semen thawed at 35 C, 50 C and 100 C had significant acrosomal damage (P<.01) when exposed to the —20 C or the 1 C post-thaw treatments. Semen thawed at 50 C and 100 C resulted in higher (P<.01) percentage of motile spermatozoa and percentage intact acrosomes when followed by exposure to 37 C post-thaw treatments than semen thawed at 5 C and 35 C. With the exception of semen thawed at 5 C, the —20 C post-thaw treatment resulted in greater acrosomal damage (P<.01) than did the 1 C post-thaw treatment. Semen thawed at 100 C resulted in higher (P<.01) percentage intact acrosomes than semen thawed at 35 C or 50 C regardless of the post-thaw treatment.

¹ Scientific Paper No. 4608. College of Agriculture Research Center, Washington State University, Pullman. Project No. 0237.

² Supported in part by All West Breeders/Select Sires, Burlington, WA 98233.

³ Present address: Animal Reproduction Laboratory, Department of Physiology and Biophysics, Colorado State University, Fort Collins, CO 80521.

⁴ Department of Animal Sciences.