The aim of this study was to describe an optimal sonication procedure for sperm cells. Therefore, we used several parameters such as damaged spermatozoa rate (%), mitochondrial activity rate (%), levels of lipid peroxidation, and total antioxidant potential. Ejaculates were collected from rams (n = 3) and were divided into aliquots and 3-, 6-, and 10-s duration times; 1, 3, 5, and 8 repetitive application groups were established. In the groups with 3-, 6- and 10-s duration times, with the increasing number of repeated applications, damaged spermatozoa rates increased (P < 0.05) while mitochondrial activity rates decreased (P < 0.05). In relation with sonication duration time, total antioxidant potential levels increased (P < 0.05) in single-application groups compared to those in control groups and gradually decreased as the repetitions increased. The most effective results were obtained in the group with 8 repetitions and 10-s duration based on damaged spermatozoa rate and mitochondrial activity rate.