The optimal combination of cooling and equilibration durations, along with the addition of melatonin, gamma-oryzanol, and canthaxanthin, for improving swamp buffalo semen cryopreservation quality

Background and Aim: The success of semen cryopreservation relies on several aspects, including breed, age, season, collection method, extender composition, cooling rate, equilibration period, freezing rate, and thawing rate. This study aimed to investigate the effects of cooling and equilibration duration, as well as the addition of antioxidants to the semen extender, on the cryopreservation of swamp buffalo semen. Materials and Methods: Semen collected from swamp buffalo bulls was subjected to four different conditions: (T1) 2-h cooling and 2-h equilibration, (T2) 1.5-h cooling and 1.5-h equilibration, (T3) 1-h cooling and 1-h equilibration, and (T4) 0.5-h cooling and 0.5-h equilibration. Spermatozoa motility was evaluated using a computer-assisted semen analyzer. Moreover, this study also investigated the effect of antioxidant supplementation during cryopreservation using tris-citrate egg yolk extenders enriched with various antioxidants: Control (Con), 1 mM melatonin (ML), 0.5 mM gamma-oryzanol (GO), 10 μM canthaxanthin (CX), 1 mM melatonin + 0.5 mM gamma-oryzanol (ML + GO), and 1 mM melatonin + 10 μM canthaxanthin (ML + CX). Results: Results showed that the (T1) 2-h cooling and 2-h equilibration and (T2) 1.5-h cooling and 1.5-h equilibration groups achieved higher progressive motility than the (T3) 1-h cooling and 1-h equilibration and (T4) 0.5-h cooling and 0.5-h equilibration groups. The ML-treated group exhibited superior progressive motility and total motility. Conclusion: The optimal approach for cryopreserving swamp buffalo bull semen involves a 1.5-h cooling period followed by a 1.5-h equilibration period, with the incorporation of ML into the semen extender. Keywords: antioxidant, cryopreservation, equilibration, semen, and swamp buffalo.