Methionine, Cysteine and Butylated Hydroxytoluene Enhance Cryosurvival of Ram Semen on Post-Thaw and Post-Incubation Time Points

Abstract

Abstract Despite there have been many experiments conducted about antioxidants, the best sole or combination use of antioxidants to include as a standard ingredient to freezing extenders yet to be found. This study conducted to investigate the different doses of methionine (2.5 and 5mM), cysteine (1 and 2mM) and butylated hydroxytoluene (BHT) (1 and 2mM) for ram semen cryopreservation on post-thaw and post-incubation (6 h) time points over spermatological parameters. Semen samples collected from Kivircik rams via electro-ejaculator in breeding season. After essential spermatological evaluations, appropriate samples were pooled then split into 7 equal aliquots to create study groups (antioxidant free control, 2.5mM methionine, 5mM methionine, 1mM cysteine, 2mM cysteine, 1mM BHT and 2mM BHT). Semen samples put into French straws (0.25mL) and freezing procedure (two-step) conducted via a programmable gamete freezer. At both time points; motility, HOST, PSA-FITC and TUNEL assays were conducted to discover the impacts of cryopreservation and incubation process over sperm cells. Antioxidant supplemented groups yielded better results comparing to control groups in terms of various spermatological parameters not only at post-thaw time point but after incubation for 6 h of time. The study demonstrated that supplementing sperm freezing extenders with abovesaid antioxidants may create new approaches to cryopreservation procedures and through increasing success rate of freezing, fertility results may increase to better results in near future.