The aim of this work was to evaluate the effect of relaxin, known to improve fertility parameters of frozen-thawed sperm in other species (Miah et al. 2006 J. Reprod. Dev. 52, 773-779; Miah et al. 2007 Anim. Sci. J. 78, 495-502), on buffalo sperm motility, capacitation, and fertilizing capability. Frozen-thawed sperm from 2 bulls (4 replicates each) were separated by Percoll, diluted to a 20×10(6)mL(-1) concentration and incubated in TALP medium in the absence of capacitating agents (negative control), in the presence of 10μgmL(-1) of heparin (positive control) and 100ngmL(-1) of relaxin for 2h. Following incubation, sperm were exposed for 15min to 60mgmL(-1) of lysophosphatidylcholine, a fusogenic agent known to induce the acrosome reaction only on capacitated sperm. To evaluate acrosome-reacted (AR) live sperm, cells were fixed and stained with Trypan blue-Giemsa (Kovacs and Foote 1992 Biotech. Histochem. 67, 119-124) and evaluated (800 sperm counted/group). Sperm motility was examined by a phase contrast microscope, whereas the fertilizing capability was evaluated by heterologous IVF. Abattoir-derived bovine oocytes (n=258, 86 per group) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355) with buffalo sperm in the absence of capacitating agents and in the presence of 10μgmL(-1) of heparin and 100ngmL(-1) of relaxin. Twenty hours after IVF, presumptive zygotes were denuded and cultured in SOF for 24h, when cleavage rate was evaluated and confirmed by fixation with absolute ethanol overnight and staining with 2.5μgmL(-1) of Hoechst 33342 after zona removal by pronase (2mgmL(-1)) digestion. The differences in the percentages of AR sperm and cleavage among groups were analysed by a chi square test and those in sperm motility by Student's t-test. Acrosomal loss was observed in 10.8% of the sperm after thawing, which may indicate freezing-induced capacitation, and, hence, this value was detracted from the percentages of AR recorded following incubation. After 2h of incubation, 100ngmL(-1) of relaxin significantly (P |
