Amplification of Ultra-Trace DNA from Early Sheep Embryos Based on qPCR: Establishing a Gender Identification System

The ability to identify and control the sex of embryos holds significant commercial implications for livestock production. Due to the high costs and instability of detection methods, it is necessary to establish a time-saving, effective, simple, and reliable method to identify the sex of mammalian embryos. This study designs primer probes based on the <i>SRY</i> gene sequence in the sex-determining region of the Y chromosome of sheep and combines qPCR to amplify both the <i>SRY</i> gene and the internal reference gene <i>GAPDH</i> as a control. By adjusting and optimizing the detection system with ultra-trace of genomic DNA (gDNA) from sheep blood and embryos, we sexed sheep embryos cultured in vitro. The results show that male embryos exhibit specific amplification bands for both the <i>GAPDH</i> and <i>SRY</i> genes, while female embryos only have amplification bands for the <i>GAPDH</i> gene. The sensitivity of the sheep embryo sex identification system established in this study can be as low as 5.64 pg of DNA, with an accuracy rate of 100% for embryo sex identification.