The influence of diluents and processing time after ejaculation on the revival of deep-frozen bull spermatozoa

Abstract

For highest revival rates in experiments on the deep-freezing of bull spermatozoa to – 79° C., a period of incubation of the undiluted semen at 30° C. did not eliminate the need for overnight equilibration at 5° C. The addition of 1·25% fructose or arabinose to the diluent improved revival rates of non-equilibrated spermatozoa but these rates still did not equal those of equilibrated spermatozoa.Glycerol apparently penetrated spermatozoa rapidly and spermatozoa survived in glycerol solutions as high as 2·0m if 0·1m buffered citrate was also present. Omission of the citrate solutions caused immediate death of the spermatozoa.A one-ninth replication of a 37 factorial experiment showed that, by all scoring criteria, 40° C. was superior to 0° C. as a thawing bath temperature. A diluent containing 8% (v/v) glycerol was superior to either 4% or 6% if activity of spermatozoa after thawing was the scoring criterion, but there was no difference in the percentage of spermatozoa stained by congo-red-nigrosin when frozen in diluents containing these levels of glycerol.In the range of tonicity 80, 100 and 120% (0·9% NaCl = 100% tonicity), using the diluent composed of a mixture of fructose and citrate, there were more motile spermatozoa than in buffered citrate or fructose alone. Increasing tonicity had an adverse effect on revival rates of spermatozoa in the buffered citrate diluent. Spermatozoa stored chilled at 5· C. for periods up to 36 hr. prior to freezing survived best in the mixture of fructose and citrate.The investigation of the equilibration phenomenon showed that an improvement in survival after deep-freezing occurred if diluted semen was stored for up to 12 hr. after dilution, but this was not dependent on the presence of glycerol in the diluent during this ageing process.