Adding the mitochondrial peptide humanin to the freezing solution helped buffalo sperm survive the freeze‑thaw process better – they moved more, stayed alive longer, and showed less damage from oxidative stress.

Semen cryopreservation results in deleterious effects on spermatozoa, including lipid peroxidation and a reduction in the total antioxidant components of seminal plasma. The ultimate outcome of these changes is a reduction in post-thaw semen quality. A mitochondrial derived peptide, humanin, a potent cytoprotective and antioxidant agent was used in the present study. To evaluate the efficacy of a mitochondrial-derived peptide, humanin to improve the post-thaw quality of buffalo spermatozoa. A total of 18 ejaculates from three Murrah buffalo bulls (n=6 each) were collected. Each ejaculate was divided into four aliquots. The first aliquot was diluted with standard EYTG dilutor (Group I, control), whereas the other three aliquots were diluted with EYTG supplemented with 2 µM (Group II), 5 µM (Group III) and 10 µM humanin (Group IV), respectively. Semen was evaluated for physico-morphological and functional attributes such as progressive motility, viability, abnormality, acrosome integrity, plasmamembrane integrity of fresh samples, pre-freeze and post-thaw stages. Oxidative stress parameters [lipid peroxidation (LPO) and total antioxidant capacity (TAC)] were also measured at the pre-freeze and post-thaw stages. Humanin supplementation resulted in significantly higher (p < 0.05) post-thaw motility in all treatment groups and, higher (p < 0.05) viability in Groups III and IV in comparison to the control at the post-thaw stage. Spermatozoa with intact acrosome and plasma membrane were higher (p < 0.05) in Groups III and IV as compared to Groups I and II. The LPO levels at the post-thaw stage were found to be lower (p < 0.05) in all treatment groups versus the control group, whereas, higher (p≤0.05) TAC values were recorded in Groups III and IV in comparison to the control and Group II. Humanin supplementation in the extender improved the freezabilty of buffalo spermatozoa.