Ascorbic acid and biotin modulate heat shock protein mRNA expression after thawing in rat sperm

The successful freezing of rat spermatozoa and the development of innovative cryoprotectant formulations are of great importance in the field of reproductive biotechnology. In the present study, 4 mg/mL Ascorbic acid and 20 nM Biotin were added to cryopreservation medium containing 8% lactose-monohydrate, 23% egg yolk, and 10% tris-aminomethane to investigate the freezability of rat sperm. After cryopreservation, motility, viable spermatozoon ratio, plasma membrane integrity, abnormal acrosome ratio, and mRNA levels of certain heat shock proteins that play a critical role in the sperm cryopreservation process were evaluated. After thawing, the highest motility rate was found in the ascorbic acid group (17.50 ± 3.27), which was significantly higher compared with the control group (10.13 ± 1.16) (p < 0.05). Likewise, the highest viability rate was found in the ascorbic acid group (41.75 ± 3.69), while it was found to be significantly higher compared with the control group (19.63 ± 1.44) (p < 0.05). When the cells were evaluated in terms of plasma membrane integrity and abnormal acrosome rate, no statistical difference was found between the groups (p > 0.05). The expression levels of heat shock protein-related genes were determined using the RT-qPCR technique. Addition of biotin and ascorbic acid significantly reduced Hsp40, Hsp60, and Hsp70 mRNA levels compared with the control group (p < 0.05). In conclusion, based on the evaluated parameters, it was revealed that the addition of the antioxidant Ascorbic acid and biotin to rat sperm extender provides effective protection against cryodamage during the cryopreservation process. These findings provide a valuable foundation for research into the development of next-generation mediums for sperm cryopreservation from a molecular biotechnology perspective.