Protective effect of epidermal growth factor on cryopreservation of dromedary camel epididymal spermatozoa: Evidence from in vitro and in silico studies

Epidermal growth factor (EGF) plays a crucial role in maintaining male reproductive capacity in mammals, however, its protective effects on cryopreserved dromedary camel epididymal spermatozoa have not been thoroughly investigated. This study aims to investigate the potential protective role of EGF on cryopreserved camel epididymal spermatozoa, supported by evidence from a molecular docking study. We assessed sperm motility, kinematics parameters, oxidative stress, ultrastructural changes, apoptosis, and molecular docking markers in camel epididymal spermatozoa following cryopreservation. Camel epididymal spermatozoa (n = 30 pairs of testes) were collected from local slaughterhouses. The epididymal spermatozoa were diluted with a freezing medium (SHOTOR extender) supplemented with different concentrations of EGF; 0 (EGF0), 50 (EGF50), 100 (EGF100), 200 (EGF200), and 400 (EGF400) ng/mL in SHOTOR extender and cryopreserved using a standard protocol. All EGF groups showed significant improvements in sperm progressive motility, viability, and sperm membrane function after equilibration at 5 °C for 24 hours. Regarding frozen-thawed samples, sperm progressive motility and some kinematic parameters (DAP, VSL, VCL and AHL) were significantly higher in the EFG400 group compared to the other groups (P < 0.01). A significant increase in the percentage of live/acrosome-intact sperm was observed, accompanied by a significant decrease in malondialdehyde levels in all EGF groups (P < 0.05). Both the EGF200 and EGF400 groups showed significantly higher sperm viability and significantly lower percentages of apoptotic and necrotic sperm compared to the other groups (P < 0.05). EGF supplementation preserved the ultrastructural integrity and cryotolerance of epididymal camel spermatozoa. The docking analysis indicated that EGF exhibited higher binding affinity with apoptosis sperm markers, including caspase-3 and bcl-2-associated X (Bax) proteins, with binding energies of -502.0 and -621.0 kcal/mol, respectively. In conclusion, the addition of EGF to SHOTOR extender was found to have beneficial effects on sperm motility, kinematics parameters, sperm viability, acrosome integrity, sperm ultrastructural features, and reduced oxidative stress and apoptosis-like changes in cryopreserved epididymal camel spermatozoa.