Impact of Non-physiological Incubation Temperatures on Spermatological and Functional of Thawed Buffalo Spermatozoa

Cryopreservation is a widely accepted technique for preserving male gametes; however, post-thaw incubation conditions may substantially affect sperm quality and function. This study aimed to investigate the influence of different post-thaw incubation temperatures on the spermatological and functional parameters of buffalo bull spermatozoa. Fifteen semen straws from the same bull were thawed at 37°C for 30 seconds, pooled to eliminate inter-straw variation, and equally divided into three groups: control (37°C), cold shock (4°C), and thermal stress (45°C). All samples were incubated for 30 minutes before evaluation. Sperm motility and kinematics were analyzed using a computer-assisted sperm analysis system (CASA). Viability was assessed with eosin-nigrosin staining, plasma membrane integrity with the hypoosmotic swelling test, acrosome integrity via SpermBlue®️ staining, and chromatin condensation using Toluidine Blue staining. Incubation temperature had a statistically significant effect on all examined parameters (P<0.05). Both the cold shock and thermal stress groups exhibited a significant decrease in motility and progressive motility, kinematic parameters (VCL, VSL, VAP), viability, and membrane integrity when compared to the control group (P<0.05 to P<0.001). Chromatin decondensation levels were significantly higher in both groups compared to the control, and acrosome integrity was significantly compromised. Furthermore, thermal stress induced a significantly decrease in progressive motility, chromatin integrity, and acrosomal structure compared to cold shock (P<0.001). In conclusion, post-thaw exposure to non-physiological temperatures was observed to negatively affect buffalo sperm quality, highlighting the importance of thermal regulation in post-thaw handling during assisted reproduction procedures. Further studies are needed to evaluate the effects on live fertility outcomes.