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The aim of the present study was to optimize the conventional method of sperm freezing in liquid nitrogen (LN 2 ) 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 2  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 2 . 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 2  vapour. With the use of the optimized freezing procedure, all parameters of thawed sperm were significantly (P &amp;lt; 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.

Optimizing the conventional method of sperm freezing in liquid nitrogen vapour for Wallachian sheep conservation program