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Melatonin, the main hormone secreted by the pineal gland, plays many roles in reproduction. In ram spermatozoa, melatonin administration increases plasminogen activator activity (Tsantarliotou MP et al. 2007 Theriogenology 69, 458-465), known to be involved in sperm capacitation and acrosome reaction (Taitzoglou IA et al. 1996 Mol. Androl. 8, 187-197). The aim of this study was to evaluate the efficiency of melatonin to induce buffalo in vitro sperm capacitation, indirectly assessed by estimating the capability of spermatozoa to acrosome-react. Frozen-thawed semen from 4 different bulls was pooled and treated by swim-up in order to select only the motile population. Spermatozoa (n = 829) were assessed immediately after swim- up separation, to evaluate the incidence of acrosomal loss in non-treated cells (time 0). The remaining spermatozoa were incubated in the absence of capacitating agents (negative …