Mitochondrion appears to be the main source of ROS, which plays an important role on sperm physiology. However, an imbalance between ROS and antioxidant mechanisms (i.e., oxidative stress) is lethal to the spermatozoa. The aim of this study was to evaluate the effect of inhibiting mitochondrial activity on sperm functional status in rams. Ejaculates were collected from four adult rams, divided in four aliquots and treated as follows: 0 (control), 5, 10 and 20 µM of CCCP (carbonyl cyanide meta-chlorophenyl hydrazine), uncoupler of oxidative phosphorylation. Samples were incubated for 30 min (37°C) and evaluated for computerized analysis of motility (CASA), integrity of plasma and acrosomal membranes (nigrosin-eosin and fast green-bengal rose, respectively), mitochondrial activity (3'3 diaminobenzidine) and lipid peroxidation (TBARS). Despite no changes on overall motility, the pattern of sperm movement was influenced by the treatment, as observed by the decrease on VAP (average pathway velocity), VSL (strait-line velocity), VCL (curvilinear velocity) and Linearity (VSL/VCL). A higher percentage of spermatozoa with high mitochondrial activity was observed in the control samples when compared to those incubated with 5 and 10 µM (58.75±1.49^a, 44.75±4.34^b and 42±3.39, respectively). However, a higher percentage of cells with impaired mitochondrial activity was found in the control group when compared to the CCCP treated groups (control:9.25±1.10^a, 5µM: 3.25±1.25^b, 10µM: 1.75±0.25^b, 20µM:  1.66±0.66^b). Results indicate that other pathway such as glycolysis may be the main responsible for sperm motility. However, inhibition of mitochondrial activity modified the pattern of sperm kinetics. In contrast, CCCP provided a decrease on the number of cells with low mitochondrial activity. Since previous studies indicate that the partial impairment of mitochondrial activity is extremely deleterious to the spermatozoa, the inhibition of mitochondrial activity with CCCP may be an alternative to diminish the effects of oxidative stress in these cells.

Acknowledgements: The authors want to thank FAPESP for financial support (process 2014/02576-0)