Effect of Different Equilibration Times and Freezing Rates on Bull Spermatozoa Cryotolerance

Sperm cryopreservation is a complex process consisting of several steps, the details of which are not yet universally agreed upon. The objective of this study was to evaluate the effects of different equilibration times and freezing rates on post-thaw resilience and functional integrity. Collected semen from five Holstein bulls (30 samples in total) were split into 4 aliquots to produce frozen straws after 2 different equilibration times (2 h and 4 h) and freezing rates (2-phase and 3-phase). After thawing, the sperm characteristics were measured by flow cytometry. A linear model procedure was used to determine the effects of bull, equilibration time, freezing rate and interaction between them, on sperm variables. The evaluated sperm characteristics included spermatozoa with intact plasma and acrosomal membrane (PMAI), PMAI with high mitochondrial membrane potential (HMMP), spermatozoa with plasma membrane damage (PMD), and spermatozoa with acrosome damage (AD) were analysed immediately, 1 h and 2 h after thawing. In conclusion, the extension of the equilibration time from to 4 h in combination with 2-phase freezing rate led to a significant improvement in all measured parameters of frozen-thawed spermatozoa (P < 0.01).