Abstract

The aim of the present study was to optimize the conventional method of sperm freezing in liquid nitrogen (LN<sub>2</sub>) vapour for successful cryopreservation of Wallachian ram sperm, the genetic resources of the Czech Republic. Sperm in straws were frozen using the conventional freezing method via a static exposure of sperm doses to LN<sub>2</sub> vapour, or by four different modified freezing methods. Under modified freezing, straws were frozen by a discontinuous, time-dependent decremental change in the distance between the straws and the surface of LN<sub>2</sub>. The viability of sperm was evaluated by flow cytometry after sperm equilibration, and immediately after thawing. Besides the observed inter-sire and daily variation, the obtained results suggest the methodological weakness of the conventional freezing method via the static exposure of sperm doses to LN<sub>2</sub> vapour. With the use of the optimized freezing procedure, all parameters of thawed sperm were significantly (P < 0.05) improved in comparison with the conventional method: percentage of thawed sperm viability increased up to 48.3%, percentage of sperm with plasma membrane damage after thawing decreased to 6.58%, percentage of sperm with acrosome damage decreased to 24.4%, and percentage of sperm with deteriorated mitochondrial activity decreased to 6.28%. In conclusion, our results suggest that an optimized freezing procedure should be routinely used instead of the conventional method to cryopreserve Wallachian ram sperm.

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