This study investigated the beneficial effects of relaxin on cryotolerance of buffalo spermatozoa and reproductive hormones during low breeding season. Collected semen was diluted in five aliquots with relaxin addition (0.25 mg/mL, 0.50 mg/mL, 0.75 mg/mL, 1 mg/mL, and control). After gentle dilution (37 degrees C), cooling (4 degrees C, 2 h), equilibration (4 degrees C, 4 h), and packaging (straws, polyvinyl French, 0.5 mL), frozen (cell freezer), and thawed (37 degrees C, 30 s) for analysis. Blood samples were collected at different time intervals i.e.,-60,-30 and 0 min (pre -dose) and 30, 60, 90, 120 and 150 min (post-dose) from a jugular vein. This study manifest that adding relaxin (1 mg/ mL) in freezing medium ameliorates sperm motility, functionality (%), and seminal plasma total antioxidant capacity (TAC, mu M/L) than control during low breeding season. Furthermore, we found that relaxin supplementation at 1 mg/mL significantly improves seminal plasma ATP concentrations (nmol/million) than control, 0.25 mg/mL, and 0.50 mg/mL, and fertility (control, and 0.75 mg/mL). Further, relaxin injection significantly improves plasma T, LH and IGF-1 levels (150 and 120 min vs.-60, and -30), and FSH, KP, and GnRH concentrations (150 min vs.-60), during low breeding season. Taken together, this study revealed that relaxin ameliorates motility, functionality, and fertility of buffalo spermatozoa. Moreover, relaxin injection (1 mg/mL) improves essential reproductive hormones levels in buffalo signifying its importance in the field of reproductive physiology. Further studies are required to determine the exact mechanism of action of relaxin in enhancing semen quality, fertility and reproductive hormones.
