239 CAPACITATION OF BUFFALO SPERMATOZOA IN VITRO

Abstract

The efficiency of in vitro embryo production (IVEP) in buffalo is hampered by the poor cleavage rate. The quality of the frozen semen may affect fertilization efficiency, due to damages of the male gamete that occur following cryopreservation. However, it is not possible to rule out that the process of capacitation, required by spermatozoa to acquire the fertilizing ability, is impaired in the IVF system. Although several agents have been proven to induce sperm capacitation in vitro, heparin is still the most efficient method in most of the domestic species. The aim of the study was to evaluate the efficiency of buffalo estrus serum (BES) and follicular fluid (FF) to induce buffalo sperm capacitation in vitro, indirectly assessed by estimating the capability of spermatozoa to acrosome react. Frozen semen from a bull previously tested for IVF, thawed at 37°C for 40 s in water, was treated by swim-up in order to select only the motile population. Spermatozoa (n = 1546) were assessed immediately after swim-up separation, to evaluate the incidence of acrosomal loss in nontreated cells (time 0). The remaining spermatozoa were incubated in the presence of 0.01 mM heparin (control; n = 3531), 20% BES (n = 2442) and 20% FF (n = 1419), the latter recovered from a pool of dominant follicles, for 1, 2, and 3 hours. Sperm was then exposed for 15 min to 60 μg mL⁻¹ of lysophosphatidylcholine, an agent known to induce acrosome reaction only on capacitated spermatozoa. Trypan blue was used to differentiate live from dead spermatozoa, and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The proportion of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. Differences between groups were analyzed by χ². No dead spermatozoa were found in all groups. Acrosomal loss was observed in only 15.3% of the sperm population at time 0; it may be accounted for either by damages preceding cell death or by freezing-induced capacitation. No differences were found between incubation times within each treatment group. Interestingly, sperm treatment with both BES and FF resulted in a significantly higher incidence of acrosome reaction compared with heparin (84.3, 94.5 vs. 50.1%, respectively; P < 0.001), the capacitating agent currently used in the IVF system, and, in particular, FF showed the highest percentage of acrosome reaction at all incubation times, even when compared with BES (P < 0.01). It is likely that factors derived by BES and FF, present in the oviduct environment around fertilization, play a critical role in processing the male gamete in vivo. These preliminary results show the possibility of significantly improving the efficiency of sperm capacitation in vitro in buffalo species with BES and FF and strongly suggest investigating the effects of these factors also on the fertilizing capability of buffalo spermatozoa.

The authors thank to Dr. O. Paciello for his technical assistance.