Effect of different equilibration periods pre-cryopreservation on post-thaw sperm motility in Nguni and Boran bulls

Abstract

Compared to natural selection, the use of artificial insemination (AI) and other reproductive technologies rapidly increase the rate of genetic change in any population. In order to achieve success with AI, the semen used to inseminate cows must be of the highest possible quality. When semen is frozen, generally only about 50% of the spermatozoa survive the cryopreservation process. Thus, any factors possibly affecting the survival of spermatozoa through the numerous freezing-thawing steps should be studied, in order to identify the optimal conditions for the survival of spermatozoa. The discovery of protective agents within egg yolk and glycerol was a major milestone in sperm cryopreservation. These agents protect bovine spermatozoa during cooling and freezing procedures and result in increased survival rates. Cryopreservation of spermatozoa has become the most common technique for the preservation of male fertility of genetically superior sires even after their death. Using cryopreserved sperm to artificially inseminate females has become standard practice in commercial dairy cattle herds and the application of this reproductive management tool is also expanding to beef herds worldwide. The use of glycerol as a cryoprotectant for bovine spermatozoa is credited as the reason for the success in bovine semen cryopreservation. The purpose of this research was to quantify the effects of different cooling periods, as well as different glycerol equilibration periods on the post-thaw motility percentages and recovery fractions of semen collected from Boran and Nguni bulls. The research was subdivided into two experiments. In each experiment different cooling and glycerol equilibration times were researched. The first experiment involved shorter cooling times (30, 60, 120 and 240 minutes) with each cooling time followed by several longer equilibration times (4, 5, 6, 7 and 8 h). In the second experiment the cooling and equilibration times from the first experiment were reversed. This resulted in longer cooling times (4, 5, 6, 7 and 8 h) with each cooling time having shorter glycerol equilibration times (30, 60, 120 and 240 minutes). An egg yolk-Tris two-step extender was used in both the experiments. The general trend for the glycerol equilibration periods studied in Experiment 1 was that the resulting overall average post-thaw motility percentage and average recovery fraction increased with longer periods. There was a breed difference when comparing the average post-thaw motility percentages after 4, 5, 6 and 8 h (p<0.05), while the average post-thaw motility percentages also tended to differ after 7 h of equilibration. The general trend observed for equilibration periods used in Experiment 2 was that the average post-thaw motility percentage increased as glycerol equilibration period increased up to 120 minutes, but after 240 minutes of glycerol equilibration, there was a slight decline. The differences in average post-thaw motility percentage after the respective glycerol equilibration periods were not statistically significant. The results of each experiment were used to create a matrix that can be used in practice. The matrix using results from Experiment 1 demonstrated that a cooling period glycerol equilibration period combination of 240 minutes and 7 h resulted in the highest (not significantly different from most other combinations) average post-thaw motility rates. The matrix formed from the results of Experiment 2 demonstrated that an 8 h cooling period combined with a 60 minute glycerol equilibration period yielded the highest (not significantly different from most other combinations), average post-thaw motility percentage.