Effects of different temperature treatments applied to deep stored bull semen on post-thaw cold shocked spermatozoa

Various treatments were applied to frozen semen during storage in containers with different nitrogen level and the effects of temperature changes due to these treatments on the resistance of spermatozoa against post-thaw cold shock were studied. Straws with frozen bull semen were placed into two identical field containers. One of these containers was the low nitrogen level container with 3 cm nitrogen level that is 2 cm above the lower end of straws and the other one was the high nitrogen level container with 17 cm nitrogen level that is 2 cm above the upper end of straws. Canister No. 1 in both containers served as control without any manipulation. Canister No. 2 were raised up to the neck of the container and kept at this level for 10 s. Canister No. 3 were taken out to such a position that their base was at the entrance of the container and kept at this level for 20 s. These manipulations were repeated 100 times for canisters No. 2 and 3, in both containers. Motility, defected acrosome, and hypo-osmotic swelling test were carried out at post-thaw (37 degrees C for 30 s) and after cold shock in a cold water bath (5-6 degrees C for 30 s). Post-thaw cold-shock was observed to cause significant damage to motility, membrane integrity, and acrosome integrity of spermatozoa. Low nitrogen level was not capable of protecting viability and morphological structures of spermatozoa. In container with low nitrogen level, raising the canister from the container and keeping it at this level for 20 s made the spermatozoa more sensitive to cold shock. In conclusion, container nitrogen level and canister manipulations had negative effects on both shocked and unshocked spermatozoa. Also the results provide evidence that not only canisters misuse but also low nitrogen level made spermatozoa more sensitive to post-thaw cold shock.