Recombinant heat shock protein HSPA1A enhances the cryosurvival of frozen-thawed or vitrified-warmed dog epididymal spermatozoa

HSPA1A is a heat shock protein belonging to the HSP70 family that acts during cryopreservation by mitigating oxidative stress, stabilizing denatured proteins, preventing their aggregation, and maintaining proteostasis. This study evaluated the effect of recombinant HSPA1A protein on the cryosurvival of dog epididymal spermatozoa subjected to slow-freezing (SF) and kinetic vitrification (VIT). Twenty adult dogs were orchiectomized, and epididymal sperm from the left testes were cryopreserved with SF, while the right ones were with VIT. Four treatments were established according to the addition of 10 µg/ml (+HSPA1A) or 0 µg/ml (-Control) of recombinant bovine HSPA1A, produced using Escherichia coli Rosetta 2 (DE3): pET15b-HSPA1A strain and IMAC purified. The different cryopreservation treatments evaluated were SF+HSPA1A, SF-Control, VIT+HSPA1A, and VIT-Control (n = 20 samples per treatment). Samples from SF+HSPA1A and SF-Control treatments were frozen in 0.25 ml straws by exposing them to liquid nitrogen (LN₂) vapors. For VIT, samples were vitrified by direct immersion of 30 µl drops into LN₂ and stored in cryotubes. Results showed that SF+HSPA1A and SF-Control yielded significantly higher (P < 0.05) progressive motility, velocities, linearity and straightness, and beat cross frequency compared to VIT+HSPA1A and VIT-Control. Furthermore, sperm treated with HSPA1A (both SF and VIT) exhibited significantly improved (P < 0.05) motility, viability and DNA integrity compared to their respective controls. In conclusion, the addition of recombinant HSPA1A to both slow-freezing and vitrification media enhanced the post-cryopreserved quality of dog epididymal spermatozoa, demonstrating its protective effect on motility, viability, and integrity of acrosome and DNA.