Ajor oxidative damages. Although there is no difference between cryopreservation stages, interesting correlations were observed between antioxidants enzymes activities and sperm functional profile. The GPX activity was positively correlated with medium mitochondrial potential (MMP) and negatively correlated with the percentage of sperm showing intact membrane and acrosome (IMIA) in all steps of cryopreservation process, which support the idea that this enzymatic machinery is responsible for the control of the ROS formed during aerobic respiration. Nichi et al. [18] demonstrated that GPX activity was higher in Bos taurus when compared to Bos indicus bulls. The increased GPX activity could be related to higher level of ROS in Bos taurus bulls in response to the heat stress. Similarly to our study, GPX activity also correlated negatively with membrane integrity of avian cryopreserved sperm [52]. Based on these findings and our results, we suggest that intracellular sperm GPX could be a marker of sperm injury in stressing situations, such as cryopreservation and heat stress, associated with loss of membrane integrity and increased number of cells with impaired mitochondrial PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28506461 membrane potential. Then, we can assume that GPX intracellular protection is not enough to protect cryopreservation damage. Correlations between SOD activity, mitochondrial membrane potential and membranes integrities indicate that this enzyme acts as a buffer to maintain the oxireduction and cellular homeostasis equilibrium during cryopreservation. In the NIK333 chemical information scenario of fresh semen (physiological condition), SOD correlates positively with HMP. High mitochondrial membrane potential seems to favor ROS production, specially by complex III of mitochondrial electron transport chain [53]. In fact, 1? of the oxygen consumed in the oxidative phosphorylation is converted into superoxide anion [54]. In the cooling condition, SOD is maintained as a metabolism buffer enzyme, correlating negatively with MMP and positively with IMIA. On the other hand, in the thawed group, when the damage is established, the correlation between SOD and IMIA inverses (positive to negative) indicating that its role as buffering is no longer enough to maintaincellular integrity. Our results highlight the contribution of the antioxidant enzymes to support sperm metabolism. However, during the cryopreservation process such enzymes are probably not sufficient to avoid oxidative cryodamages.Conclusion In conclusion, cryopreservation process can damage sperm cell in different compartments such as membranes (plasma and acrosome), mitochondria and even chromatin damages, without recovery after 2 hs of incubation. During this process, the critical moment is when sperm are subjected to freezing temperatures. In addition, our study indicates that intracellular antioxidant machinery of sperm cell (SOD and GPX enzymes) is not enough to control cryodamage.Competing interests The authors declare that they have no competing interests. Authors’ contributions LSC, TRSH and MN designed the experimental study, LSC, TRSH and CMM performed the animal management and analyzed sperm. LSC, TRSH and MN performed enzymatic activities assays, LSC and MN drafted the manuscript, whereas MEOAA completed critical revision and approval of the article. JAV and VHB contributed with laboratory equipment and infrastructure. All authors read and approved the final manuscript. Acknowledgments This study was supported by S Paulo Research Foun.
