Dairy bull sperm subpopulation behaviour in frozen-thawed semen across breed, temperature, and thawing time.

Optimising dairy cattle reproduction with assisted reproductive technologies, such as artificial insemination, requires standardised semen handling and analysis. This study evaluated the kinematic structure of the spermatozoan subpopulation in frozen-thawed Holstein and Jersey bull semen doses under different thawing protocols. We used frozen semen doses taken from four bulls of each breed. Nine semen doses were collected from each animal, for a total of 72 cryopreserved doses. Straw thawing was performed at three temperatures (35 °C, 37 °C, and 40 °C) and three times (30 s, 40 s, and 45 s). Sperm kinematic patterns were evaluated using a CASA-mot system (ISAS®v1). Sperm kinematic variables for each breed, temperature, and thawing time identified four subpopulations. The analysis revealed an effect of breed and semen thawing protocols (P < 0.05) on sperm subpopulation distribution, sperm movement, and swimming patterns. Subpopulation analyses based on semen assessment are needed to further interpret the relevance and effect on fertility.