Seasonal Variation of DNA Integrity Assessed by Comet Assay in Buffalo (Bubalus bubalis)

In buffalo, seasonal differences of sperm fertility might be related to the occurrence of DNA damage. The occurrence of DNA strand breakage damage was evaluated all year round using the comet assay. Semen of buffaloes A and B was collected each month from December to November. Ejaculates were assessed using a computer sperm analyzer. Spermatozoa (106/ml) embedded in low melting point agarose on a microscope slide were lysed, DNA was unwound and electrophoresis carried out (pH 11). Four replicates were performed for each sample. DNA was visualised after staining with ethidium bromide, 200 sperm cells per slide were scored for the presence and absence of comets (DNA-damaged cells). The percentage differences among months and subjects were assessed by ANOVA. Comet percentages were higher in June (A = 40 ± 2%; B = 20 ± 4%) and July (A = 9 ± 1%; B = 44 ± 2%) than in April (A = 5 ± 1%; B = 8 ± 1%) and September (A = 2 ± 1%; B = 1 ± 2%). Observed values were significantly different (p < 0.01) in June (buffalo A) and July (buffalo B) compared with the overall trend. Our data suggest an increase of DNA damage in the summer period (despite an observed increase in blood testosterone values), with a slight time shift between subjects. It is concluded that seasonality may exert a biological effect on the regulation of the apoptotic process at the testicular level, which has negative consequences on sperm fertility. This preliminary study addresses concerns that seasonal variations observed in buffalo reproductive function are related to spermatozoa integrity (Supported by MIUR COFIN).