Impact of Non-physiological Incubation Temperatures on Spermatological and Functional of Thawed Buffalo Spermatozoa
Abstract
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.
Keywords
Buffalo spermatozoa, Cold shock, Post-thaw incubation, Sperm quality, Thermal stress
Ethical Statement
This study is not subject to the permission of HADYEK in accordance with the “Regulation on Working Procedures and Principles of Animal Experiments Ethics Committees” 8 (k). The data, information, and documents presented in this article were obtained within the framework of academic and ethical rules.
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