Flow cytometry approach combined with intracellular antioxidant enzymes evaluation, data suggest that the most significant impact on sperm cell during cryopreservation is actually when spermatozoa is exposed to temperatures lower than 5 , probably during fast freezing period, compromising membranes (plasma, acrosomal and mitochondrial) and DNA integrity. This quality is not transitory and nonreversible, as it cannot be recovered after 2 hs ofCastro et al. Journal of Animal Science and Biotechnology (2016) 7:Page 5 ofFig. 2 (See legend on next page.)Castro et al. Journal of Animal Science and Biotechnology (2016) 7:Page 6 of(See figure on previous page.) Fig. 2 Evaluation of sperm profile separated by treatment group. a DNA damage (SCSAm); b High mitochondrial potential (HMP); c medium mitochondrial potential (MMP); d intact membrane and intact acrosome (IMIA); e intact membrane and damaged acrosome (IMDA); f damaged membrane and intact acrosome (DMIA); g damaged membrane and damaged acrosome (DMDA); h damaged membrane (DM); i damaged acrosome (DA). ab Different letters indicate differences between treatmentsincubation. Interestingly, antioxidant enzymes cannot repair PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28506461 the damage caused by cryopreservation, since their activity is unchanged during the process. Probably they act as a buffer system only to maintain the intracellular homeostasis from physiological sperm metabolic processes.Cryopreservation effects on plasma membrane and acrosome integrityThe stress on plasma membrane during sperm cooling may occur due to changes in the asymmetry of the phospholipid bilayer and the altered functional state of the membrane. Lipids and proteins in a fluid state, solidifies into gel, producing a rigid and fragile structure, more sensitive to injuries [21]. Furthermore, sperm cells are exposed to a hyperosmotic environment that induces an influx of water and ions across the membrane, leading to cell dehydration [22]. Our study shows that a progressive loss of integrity occurred in both acrosome and plasma membranes during cryopreservation. Interestingly, no differences were found when we compare fresh sperm and those evaluated after the cooling rate (5 ). Evaluations of MGCD516 web cooled group were performed after equilibrium period (Fig. 1), when sperm cells had already been exposed to the toxic effect of the cryoprotectant. Sperm damage was observed only when cells were exposed to freezing temperatures, indicating that intracellular crystal formation is probably more deleterious to the spermatozoa than the solution effect and cell dehydration. This could justify why sperm from other domestic species do not tolerate well cryopreservation processes. Susceptibility to cold temperatures and differences among species concerning sperm survival seems to be linked to the ratio of unsaturated and saturated fatty acid content [23], specially related to the proportion of cholesterol in the plasma membrane [24].Sperm membrane injuries were more evident in the plasma membrane, with a decrease of 56 on the percentage of intact cells in contrast to the 33 decrease observed for acrosome integrity after cryopreservation. Damaged acrosome in cryopreserved sperm could be related to an event known as cryocapacitation, when alterations on membrane fluidity externalize inner phospholipids [2, 25] inducing premature acrosome reaction. This capacitation-like changes would, in turn, reduce sperm lifespan [26].Cryopreservation effects on mitochondrial membrane potentialWe ob.