2.2. Extender Preparation and Semen Processing and Evaluation

The pooled semen samples were diluted in a 1 : 2 basic extender [41] containing 20 mM Tris, 57 mM citric acid anhydrous, 5 mM fructose, 22 mM glucose, and 4.6 mM streptomycin along with 10 mL egg yolk and completed to 100 mL with distilled water with pH 6.8–7, and osmotic pressure was kept at pH 6.9, 300 mOsm. The diluted pooled semen samples were divided equally into 7 fractions (1 fraction/treatment). The diluted semen samples were then supplemented with 0 (control with diluent only without additives), 0.5, 1, and 2 mM glutathione (GSH; cat. no. Y0000517, Merck KGaA, Darmstadt, Germany) and 1, 5, and 10 mM taurine (cat. no. T0625, Merck KGaA, Darmstadt, Germany). The pH was adjusted to 6.8–7 with a dilution factor of 1 : 2 (semen: prepared extenders), and the diluted semen was kept at 5°C. Then, changes in semen quality were evaluated after 24, 48, and 72 h of chilling. Sperm motility, viability (% of live spermatozoa), acrosome reaction, fructose and glutamic pyruvic transaminase (GPT) concentrations, and antioxidant enzymes activity (catalase (CAT), superoxide dismutase (SOD), GSH, and malondialdehyde (MDA)) as oxidative biomarkers were assessed. Relative transcriptomic levels of SOD and CAT genes were also evaluated. All the evaluations were repeated ten times throughout the trial (ten weeks).