Impact of Cryopreservation on Caspase-3 Gene Expression, Cell Viability, and Sperm Motility in Bull Testicular Tissue and Epididymal Sperm

This study investigated the effects of cryopreservation of testicular tissue and epididymal sperm in bulls on spermatological parameters, cell viability, and caspase-3 gene expression, a marker of apoptosis. Epididymal sperm and testicular tissue pieces obtained from testes collected from bulls (n: 15) were cryopreserved and stored in liquid nitrogen (-196°C). Epididymal sperm were frozen in liquid nitrogen vapor using the straw method, while testicular tissue pieces were frozen in cryotubes using a slow freezing protocol with Dimethylsulfoxide (DMSO) and Ethylene Glycol (EG) as cryoprotectants. Fresh semen exhibited significantly higher motility, progressive motility, kinematic parameters, and viability values compared to those after thawing (P < 0.05). Higher cell viability was achieved in DMSO-cryopreserved testicular tissues (53.72 ± 6.36) compared to EG-cryopreserved testicular tissues (41.83 ± 7.22) (P < 0.05). Caspase-3 gene expression was determined to be 42.66 ± 98.26-fold increased in DMSO-cryopreserved tissues and 65.05 ± 106.03 fold increased in EG-cryopreserved tissues after thawing. In epididymal sperm, gene expression levels increased by an average of 12.22 ± 34.60 fold compared to fresh samples. These findings suggest that cryopreservation affects cellular integrity and triggers apoptotic pathways and repair mechanisms, highlighting the need to optimize protocols to reduce cryo-induced damage and increase post-thaw viability.