| In vitro fertilization (IVF) is a wildly-used technology both in basic and applied sciences. The technology has played a central role in researching the mechanisms involved in fertilization and early development, treating human infertility, and enhancing of the productivity of farm animals, especially for the dairy and beef cattle, but its relatively low efficiency and sub-optimal embryonic and fetal survival have become a limitation of its wide applications in beef and dairy cattle industries. This study was, therefore, conducted with the objective of devising improved methods of in vitro production of bovine embryos. The main results were as follow:1 Oocytes and embryos cultured in vitro generated more ROS (Reactive oxygen species) than in vivo due to the high oxygen concentration in the incubator, extra ROS was detrimental to cells, which was considered to be one of the main reasons of the low efficiency of IVP.β-mercaptoethonal (β-ME) is a low molecular weight thiol compound. It was hypothesized that the addition ofβ-ME in embryo culture media can increase the synthesis of intracellular GSH, and subsequently decrease the damage of ROS to cells. In the first experiment, oocytes and embryos were culture in vitro in the presence ofβ-ME, and its effects on oocyte maturation, fertilization and embryo development were assessed.(1) The maturation rate and cleavage rate of in vitro oocytes were not significantly increased (P>0.05) when supplemented with 0μM (Control)、50μM and 100μM ofβ-ME in maturation media, however, it showed an improved maturation rate of oocytes as the concentration ofβ-ME increased.(2) Supplementation with 50μM and 100μMβ-ME in maturation and embryo culture media significantly (P<0.05) increased the developmental speed of IVF-derived embryos, however, no significant (P>0.05) difference were observed in the final blastocyst rate(8d).Thus, addition ofβ-ME increased some maturation rate of COC’s without affecting the cleavage rate and the blastocyst development, however, improved the developmental speed of embryos.2 In the second experiment, the effects of different methods of purifying and capacitating bovine sperm, the duration required for sperm and oocyte co-incubation and different breeds on the results of IVF were assessed. The results are as follow:(1) Sperms from the same bull were either purified by Percoll method and then used to fertilize oocytes in IVF-TALP medium or purified by swim-up and then used to fertilize oocytes in IVF-BO medium in order to compare the two procedures for the ability of spermatozoa to fertilize oocytes in vitro. It was found that sperm processed with Percoll and IVF-TALP significantly (P<0.05) increased the cleavage rate, however, no significant (P>0.05) effects were observed in terms of blastocyst rate.(2) Two dairy cattle (Holstein) and two breeds of beef cattle’s (Simmental and Limousine) sperm were used to fertilize in vitro maturated oocytes(local yellow cattle), the results showed that the cleavage rate of presumptive zygotes fertilized by Holstein were significantly (P<0.05) higher than Simmental and Limousin, however, there was no significant difference (P>0.05) among these breeds in terms of blastocyst rate.(3) In this experiment, sperm and oocytes for IVF were co-incubated for 6h,9h and 12h respectively to determine the appropriate duration of co-incubation for successful IVF. The results showed that incubation for 9h and 12h significantly (P<0.05) increased the cleavage rate when compared with 6h, there was no significant (P>0.05) difference in blastocyst rate among the three groups.In conclusion, using Percoll and TALP-IVF medium system for bovine sperm purification and capacitation can improve in vitro fertilization of bovine oocytes; co-incubation for 9h is sufficient enough for successful IVF and using sperm from dairy cattle can increase the cleavage rate of bovine oocytes fertilized in vitro. |
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