Effects of Repeated Partial Thaw and Refreeze on Post-Thaw Parameters of Stallion Semen Cryopreserved in Cryovials

The current study evaluated post-thaw semen parameters of stallion semen cryopreserved in cryovials and subjected to multiple partial thaw-refreeze cycles. Five fertile stallions were collected twice, and ejaculates were analyzed for concentration, percent membrane integrity, motility, morphology, and sperm chromatin structure (SCSA). Semen processed with freezing extender from each ejaculate was cryopreserved in both 1.2-mL cryovials and 0.5-mL straws. Cryovials were subjected to eight subsequent partial thaw-refreeze cycles. Cryovials were warmed for approximately 30 seconds; then, a sample of cryopreserved semen was removed with a 16-gauge needle, and the cryovial was immediately refrozen in liquid nitrogen. A piece of 0.5-mL straw cut under liquid nitrogen from the same stallion and ejaculate was thawed alongside each cryovial to serve as a control. Thawed samples were analyzed for percent membrane integrity, motility, and SCSA. Post-thaw parameters of motility and membrane integrity were analyzed by one-way or two-way analysis of variance with repeated measures when appropriate. The SCSA data were analyzed using a mixed regression model. Post-thaw motility and percentage of intact sperm were significantly lower when sperm was cryopreserved in cryovials compared to straws. However, these parameters may remain adequate for use in assisted reproductive techniques (ARTs) such as intracytoplasmic sperm injection through all cryovial thaws. Additionally, DNA denaturability was not affected by semen packaging method and was only affected by thaw number, increasing at post-thaws 5 and 6. This technique may offer a unique approach for cryopreservation and utilization of stallion sperm for ARTs in the future.