Ultra-rapid freezing in spheres yields a higher cryoresistance than in straws but remains inferior to conventional slow freezing of stallion sperm

This study evaluated the cryoresistance of stallion sperm frozen by ultra-rapid (UR) methods using microspheres and straws or by the conventionally-slow (CS) method. Sixteen ejaculates from four stallions were each divided into three aliquots according to the freezing method: UR freezing in 30-μL spheres (UR-Spheres) by direct immersion in liquid nitrogen (LN2); UR freezing in 0.25-mL straws (UR-Straws) by direct horizontal submersion in LN2; and CS freezing in LN2 vapors. Ultra-rapid freezing medium included 100 mM trehalose +1 % BSA, and the CS freezing medium contained 5 % dimethylformamide. Conventional-slow freezing yielded higher sperm cryoresistance than both UR freezing methods (P < 0.05). UR-Spheres produced higher values of sperm kinematic, viability, and acrosomal integrity than UR-Straws (P < 0.05). Sperm head area and perimeter in both UR freezing groups increased compared to fresh and CS frozen samples (P < 0.05). In conclusion, the UR-Spheres method produced greater cryoresistance of stallion semen than UR-Straw method.