145 EFFECT OF RELAXIN ON FERTILIZING ABILITY OF BUFFALO SPERM

Abstract

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 × 10⁶ mL^–1 concentration and incubated in TALP medium in the absence of capacitating agents (negative control), in the presence of 10 μg mL^–1 of heparin (positive control) and 100 ng mL^–1 of relaxin for 2 h. Following incubation, sperm were exposed for 15 min to 60 mg mL^–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 10 μg mL^–1 of heparin and 100 ng mL^–1 of relaxin. Twenty hours after IVF, presumptive zygotes were denuded and cultured in SOF for 24 h, when cleavage rate was evaluated and confirmed by fixation with absolute ethanol overnight and staining with 2.5 μg mL^–1 of Hoechst 33342 after zona removal by pronase (2 mg mL^–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 2 h of incubation, 100 ng mL^–1 of relaxin significantly (P < 0.05) increased the percentages of live AR sperm (P < 0.05) compared with the negative control (31.3 ± 2.2 and 25.8 ± 2.8, respectively), with intermediate results in the positive control (27.0 ± 2.2). Motility was significantly improved (P < 0.05) when sperm were exposed to 100 ng mL^–1 of relaxin compared with both the negative and positive control (73.7 ± 2.4, 60.0 ± 4.1, and 60.0 ± 7.1, respectively). A significant (P < 0.05) improvement of cleavage rate was recorded both in the positive control (71.5 ± 4.8) and in the group treated with 100 ng mL^–1 of relaxin (70.7 ± 0.5) compared with negative control (52.1 ± 1.5). In conclusion, these preliminary results indicate that relaxin at the concentration of 100 ng mL^–1 improves sperm motility, capacitation, and the IVF capability of buffalo sperm.