Development of embryo transfer system of bovine embryos sexed with PCR

本研究旨在探讨用牛Y染色体特异性DNA探针PCR法鉴定牛胚胎性别后直接移植的实用性。首先，为了开发牛Y特异性DNA探针，设计克隆了牛Y基因的性别决定区（bSRY），该基因被认为在牛胚胎发育过程中诱导性腺的雄性分化。结果，利用克隆技术，如以人SRY序列为引物，从牛睾丸中提取总RNA的RT-PCR和5 'RACE，确定了两个不同的bSRYcDNA序列，但都具有进化保守的HMG盒序列。因此，序列的差异可能归因于bSRY mRNA的环状结构，如小鼠Sry中所述。根据bSRYcDNA序列设计的引物可用于牛胚胎性别鉴定。然后，对PCR法性别鉴定的牛胚胎进行冻融移植。当使用含有PVP作为冷冻保护剂的冷冻培养基冷冻活检的胚胎时，解冻后胚胎的存活率非常高，并且在移植这些胚胎后，我们可以有效地生产出具有预测性别的年轻胚胎。

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.