Development of embryo transfer system of bovine embryos sexed with PCR

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Our objective was to test the practical use of direct transfer of frozen-thawed bovine embryos sexed with PCR method using bovine Y chromosome specific DNA probes. First, to develop bovine Y-specific DNA probes, cloning of bovine sex determining region of Y gene (bSRY), which is supposed to induce male differentiation of gonad during bovine embryogenesis was designed. As a result, using cloning techniques, such as RT-PCR with human SRY sequence as primers and total RNA extracted from bovine testes, and 5'RACE,two different sequences of bSRYcDNA were determined, but both had the evolutionary-conserved HMG box sequence. Therefore, the difference of the sequence may be attributed to the circular structure of bSRY mRNA,as has been described in mouse Sry. It was moreover found that the primers derived from bSRYcDNA were useful for sexing of bovine embryos with PCR method. Next, the transfer of frozen-thawed bovine embryos sexed with PCR method was directed. When the embryos biopsied were frozen using a freezing medium containing PVP as a cryoprotectant, the survival rate of embryos after thawing was very high and after transfer of those embryos, we could produce efficiently youngs with predicted sex.