Dilution and cryopreservation procedure
Each pooled semen sample was divided into three aliquots and subjected to cryopreservation. Before freezing the three aliquots were diluted with the basic extender containing 5 mM of NAC, 200 U/mL of SOD and no antioxidant (control) at the ratio of 1:2. Semen samples were frozen in accordance with the procedure adapted from Tselutin et al.[43]. Diluted samples were stored for 15 min at 8°C, then dimethylacetamide (DMA) was added to a final concentration of 6% and after 3 min of equilibration the solution was pipetted and plunged drop-by-drop directly into the liquid nitrogen. After 3 months of storage frozen semen was thawed in water bath at 60°C for 6 s. Fifteen semen collections and freezing procedures were performed