Evaluation of Centrifuged Egg Yolk Extender with L-Methionine for Improving Cryosurvival of Kankrej Bull Spermatozoa

The present study was undertaken to evaluate the effect of a centrifuged Tris-fructose egg yolk glycerol (TFEYG) extender supplemented with L-methionine on post-dilution and post-thaw semen quality, oxidative stress, and enzymatic activity in Kankrej bull semen. Eighteen ejaculates collected from three healthy, sexually mature Kankrej bulls were processed using three extender formulations: conventional TFEYG (Group I), centrifuged TFEYG (Group II), and centrifuged TFEYG supplemented with 2.5 mM L-methionine (Group III). Semen quality was assessed in terms of individual sperm motility, viability, abnormalities, hypo-osmotic swelling test (HOST)-reacted sperm, malondialdehyde (MDA), superoxide dismutase (SOD), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) at the post-dilution and post-thaw stages. Cryopreservation reduced semen quality in all treatment groups; however, semen extended with centrifuged TFEYG, particularly when supplemented with L-methionine, retained significantly (p<0.05) higher sperm motility, viability, and HOST‑reacted sperm, along with significantly lower sperm abnormalities than the conventional extender. The most favourable postthaw semen characteristics were consistently observed in Group III. Although MDA, SOD, and ALT values were not significantly affected by the treatments, post‑thaw AST activity was significantly lower in the L‑methionine‑supplemented group, indicating reduced membrane damage, while the group also showed lower MDA and higher SOD values after thawing. The results suggest that removal of deleterious egg yolk components through centrifugation, together with the antioxidant action of L‑methionine, improved the ability of spermatozoa to withstand cryopreservation. Supplementation of a centrifuged TFEYG extender with 2.5 mM L‑methionine can therefore be considered a useful approach for improving the post‑thaw quality of Kankrej bull semen.