Effect of gametes co-incubation time on in vitro fertilization and embryo development in buffalo species (Bubalus bubalis).

The aim of this work was to evaluate whether shortening the sperm-oocyte co-incubation time improves embryo development in buffalo species. Cumulus-oocytes complexes (n=190), recovered from slaughtered animals, were matured in vitro in TCM 199 + 10 % FCS, 0.5 g/mL FSH, 5 g/mL LH, 1 g/mL 17- estradiol and 50 M cysteamine, at 38.5°C under 5 % CO2 in humidified air for 22 hours. The mature oocytes were fertilized in vitro at 38.5°C under 5% CO2 in humidified air in Tyrode’s modified medium (TALP), in the presence of 0.01 mM heparin, 0.2 mM penicillamine and 0.1 mM hypotaurine. Frozen-thawed sperm from a tested bull was treated by the swim-up procedure and used at a final concentration of 206/ml. Presumptive zygotes were removed from TALP medium respectively at 8 h (N=94) and 20 h (N=96) post-fertilization, and cultured in SOF medium, supplemented with essential and non-essential amino acids and BSA, in a gas atmosphere of 5% CO2, 7% O2, and 88% N2 up to the blastocyst stage. On day 5 (day 0=IVF) cleavage rate was assessed and embryos were transferred into fresh SOF medium for further 2 days of culture. Embryo development, in terms of tight morulae (TM) and blastocysts (Bl), was evaluated on day 7. Data expressed in percentages were analyzed by Chi square. Although cleavage rate was significantly lower at 8 h vs 20 h of co-incubation of the gametes (48.9 % vs 63.5 % respectively; P<0.05), both the percentages of TM+Bl and of Bl, evaluated on the total inseminated oocytes, were not different (TM+Bl: 22.3 % vs 18.7 %; Bl: 18.1 % vs 12.5 %). In fact, a significantly (P<0.05) higher proportion of cleaved eggs developed into TM+Bl and Bl in the 8 h-group vs the 20 h-group (TM+Bl: 45.6 % vs 29.5 %; Bl: 37.0 % vs 19.7 %). Our results suggest that prolonging the sperm-oocyte co-incubation time results in a higher penetration and cleavage rate in buffalo without affecting post-fertilization embryo development. To our knowledge no information are available on the chronology of sperm penetration of buffalo oocytes in vitro; it is possible that an interval of time longer than 8 h is required for buffalo sperm to penetrate the oocytes in vitro. On the other hand the improved development of the cleaved oocytes in the 8 h group may indicate that extending the co-incubation time is detrimental to embryo development. This aspect may be accounted for by either a higher occurrence of polyspermia and abnormal fertilization or by the unsuitability of the fertilization medium for the zygotes, once penetration has been achieved. In conclusion shortening the co-incubation time of the gametes may improve development of cleaved oocytes in buffalo even if it results in a decrease of cleavage rate. This recent finding suggests that it will be worth to investigate the effects of intermediate co-incubation times and the interaction with other factors on buffalo in vitro embryo development.