Temporal leakage of Cu,Zn superoxide dismutase and loss of two low-molecular-weight forms of glutathione peroxidase-1 from buffalo (Bubalus bubalis) sperm after freezing and thawing

The postthaw motility and fertility of frozen-thawed buffalo spermatozoa are substantially low as compared with those of cattle sperm. The sperm motility and fertility have been positively correlated with the antioxidant enzyme activities of human and canine sperm. However, the extent of antioxidant enzyme loss during cryopreservation, although reported for human and cattle sperm, is still not clear for buffalo sperm. Thus, in the present study, an attempt was made to determine the activities of various antioxidant enzymes in buffalo spermatozoa cryopreserved for various durations (0, 30, and 60 days) and the mechanism of antioxidant enzyme loss, if any, during the process. Total superoxide dismutase (SOD) activity of cryopreserved sperm decreased and that of extended seminal plasma increased progressively with the increase in duration of cryopreservation indicating the possible time-dependent leakage of these enzymes from cryopreserved sperm into the extended seminal plasmas. The catalase and glutathione peroxidase (GPx) enzyme activities could not be detected in buffalo sperm but could be detected in fresh and extended seminal plasmas. Total GPx activities of extended seminal plasma decreased progressively with the increase in duration of cryopreservation. To confirm the presence of these enzymes at protein levels, specific antioxidant enzymes such as Cu,Zn SOD of 16 kDa and three molecular weight forms (57.7, 40.9, and 26.05 kDa) of GPx-1 were detected in buffalo sperm by Western blot. Furthermore, the intensities of 16-kDa Cu,Zn SOD in 60-day cryopreserved sperm and those of two low-molecular-weight forms of GPx-1 (40.9 and 26.05 kDa) in 30-day cryopreserved sperm decreased significantly (P < 0.05) as compared with those of noncryopreserved (0-day cryopreserved) sperm indicating selective and temporal leakage of only low-molecular-weight antioxidant proteins in the initial phase. However, all the mentioned GPx-1 forms disappeared in 60-day-old cryopreserved sperm. Immunocytochemistry experiment also revealed that Cu,Zn SOD proteins are distributed over the acrosomal region of noncryopreserved buffalo spermatozoa, and the fluorescence signal decreased substantially in 60-day cryopreserved sperm. Thus, the present study reported that there is temporal leakage of Cu,Zn SOD and loss of two low-molecular-weight forms of GPx-1 from the cryopreserved buffalo spermatozoa after freezing and thawing.