Highly Efficient In Vitro Production of Bovine Blastocyst in Cell-Free Sequential Synthetic Oviductal Fluid vs. TCM199 Vero Cell Co-Culture System

Background: The aim of this study was to establish a cell-free sequential culture system that can support high levels of in vitro embryo development and blastocyst formation from bovine zygotes. To this end, this investigation was carried out to evaluate the effects of glucose, serum and EDTA on bovine zygote in vitro development. Materials and Methods: Bovine presumptive zygotes were derived from oocytes matured, and fertilized in vitro and cultured in synthetic oviductal fluid sequential medium in a two-steps manner; SOF 1 for the first 3 days and SOF 2 for the second 5-6 days of in vitro embryo development. In order to evaluate the effect of different modifications of the basic medium on embryo development, glucose was added to the second phase (SOF A), serum was added to the first phase (SOF C) and EDTA alone (SOF D) or in combination with serum (SOF E) was added into the first phase of in vitro embryo culture. The results of each composition were compared with each other and with the results of embryo development in TCM199 vero cell co-culture system. Results: Glucose addition to the second phase of embryo culture, improved the developmental competency; however, the differences were not significant. Serum addition to the first phase of embryo culture, significantly improved the developmental competency of embryos beyond the cleavage stage, compared to all the treatment and TCM199 co-culture groups. EDTA supplementation of culture medium, either alone or in combination with serum, significantly inhibits the embryo development beyond the morula stage. Conclusion: The results indicated that culture of bovine presumptive zygotes in two steps cell-free culture system, can support embryo development, and addition of serum throughout the culture and glucose to the second step significantly increased overall developmental competency compared to TCM199 co-culture system.