| Optimization of culture systems for in vitro fertilization of animal oocytes is achieved by supplementation with various cytokines. Previous research has shown that vascular endothelial growth factor (VEGF) can sustain the genesis, growth and development of animal gametes, and has positive effects on in vitro fertilization of animal oocytes. The present study has investigated the effects of VEGF on in vitro maturation, fertilization and subsequent early embryo development of buffalo oocytes, with the aim of optimizing the culture system for buffalo oocyte fertilization in vitro, and improving the efficiency of in vitro embryo production in buffalos. Three separate experiments were performed.In Experiment1, the effect of cumulus on the in vitro fertilization of buffalo oocytes was evaluated, and the developmental potential of zygotes with slightly deformed cytoplasm was studied. Besides, the effect of VEGF on the developmental potential of buffalo gametes was studied by preliminary test. The results showed:(â…°) the cleavage rate and blastocyst rate of oocytes fertilization without cumulus were significantly higher than that with intact cumulus (55.14%,20.38%vs.21.44%,3.68%; P<0.01),(â…±) the cleavage rate and blastocyst rate of zygotes with slightly deformed cytoplasm were higher than that in the normal zygotes, but the differences were not significant (59.96%,20.13%vs.48.59%,18.86%; P>0.05),(â…²) with10ng/mL VEGF supplementation during maturation, the cleavage rate was significantly higher than that in the control (54.36%vs.41.84%, P<0.05), and with that supplementation during fertilization, the cleavage rate was highly significantly higher than that in the control (64.28%vs.43.40%, P<0.01). The results showed that (â…°) removal of cumulus from mature buffalo oocytes was beneficial for fertilization,(â…±) zygotes with slightly deformed cytoplasm still had developmental potential, and (â…²) VEGF supplementation can improve both oocyte developmental potential and buffalo sperm fertilization ability.Experiment2investigated the effects of different concentrations of VEGF (0,1,10and100ng/mL) on in vitro maturation of buffalo oocytes. The results showed that (â…°) cumulus expansion index (CEI) in all treatment groups were higher than that in the control, but there was no significant difference in CEI among these groups (2.83,2.68,2.65vs.2.61; P>0.05),(â…±) the maturation rate in10ng/mL VEGF treatment group was significantly higher than that in the control (72.30%vs.57.30%, P<0.05), the maturation rate in1ng/mL or100ng/mL group was also higher than that in the control, but the differences were not significant (67.18%,60.58%vs.57.30%; P>0.05),(â…²) the cleavage rate in every treatment was higher than that in the control, and significant difference was observed in10ng/mL group compared to control or1ng/mL group (53.27%vs.41.60%,40.97%; P<0.05). The results proved that (â…°) VEGF has a positive effect on buffalo oocyte maturation in vitro,(â…±) it promotes their nuclear maturation, and (â…²) it could also improve their developmental potential.In Experiment3, the effects of VEGF supplementation on the fertilization ability of buffalo sperm were investigated for addition of different levels of VEGF (0,1,10and100ng/mL) to the fertilization medium. Cleavage rates in the10ng/mL and100ng/mL groups were highly significantly greater than that in the control (63.04%,56.26%vs.41.92%; P<0.01), and the cleavage rate in the1ng/mL group was also significantly higher than that in the control (55.16%vs.41.92%, P<0.05). In addition, the blastocyst rate in the10ng/mL group was significantly higher than that in the control (32.62%vs.17.00%, P<0.05).As a result of these experiments, both maturation and fertilization media were supplemented with10ng/mL VEGF in subsequent studies of its effects on embryonic development in vitro. Results showed that both the cleavage and blastocyst rates were highly significantly greater than that in the control (64.28%,27.22%vs.43.40%,19.02%; P<0.01), and the blastocyst cell number was significantly higher than the control (106.83vs.89.50, P<0.05). Thus VEGF can promote in vitro fertilization and early embryo development of buffalo oocytes, and at the same time improve the quality of the blastocysts.In summary, VEGF has positive effects on in vitro embryo production in buffalos, and can be used to optimize the culture system for buffalo in vitro fertilization;10ng/mL was found to be the optimum concentration for VEGF supplemention of both maturation and fertilization media in this study. |
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