Study of Methods for Early Fetal Sex Identification Using Cell‐Free Fetal DNA in the Peripheral Blood of Pregnant Cows

Livestock sex control is one of the core bioengineering technologies for improving quality and efficiency in modern animal husbandry, holding profound practical significance for sex-limited livestock production systems. Precise identification of fetal sex at the early gestational stage constitutes a key prerequisite for achieving targeted sex regulation. The discovery of cell-free fetal DNA (cffDNA) in the peripheral blood of pregnant cows has paved an important technical avenue for establishing a non-invasive and high-precision diagnostic system for early fetal sex identification. In this study, plasma and serum samples collected from pregnant cows were used as experimental materials, and three protocols-phenol-chloroform extraction, heat-based extraction, and a commercial kit specifically designed for isolating cffDNA from plasma/serum-were employed for cffDNA purification and isolation. Y-chromosome-specific genes (either the TSPY or SRY gene) were selected as molecular markers, and optimised detection systems were established by integrating polymerase chain reaction (PCR), real-time quantitative PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP) techniques. A systematic comparison was conducted to evaluate the efficacy and accuracy of different cffDNA extraction methods combined with various amplification technologies for fetal sex identification in both early and late stages of gestation. The actual calving outcomes were used as the standard for validation. The results demonstrated that the quality of cffDNA templates extracted by the commercial kit method was significantly superior to that obtained by the heat-based and phenol-chloroform methods, with the corresponding sex identification accuracy reaching the highest level. Notably, the LAMP technique exhibited unique advantages in detecting fetal sex in extremely early gestational samples (at 1-2 months of pregnancy). Characterised by its simplicity of operation, rapid reaction kinetics, and elimination of the need for sophisticated instrumentation, LAMP is particularly well-suited for on-site large-scale rapid primary screening of fetal sex in livestock farms. It enables the efficient exclusion of male foetuses within a short timeframe, thereby substantially improving the efficiency of breeding selection. Based on the aforementioned findings, this study proposes a combined detection model of "LAMP-based primary screening plus PCR-based confirmation", which can effectively balance detection efficiency and identification accuracy. The research outcomes provide empirical data and methodological references for constructing a non-invasive, early-stage, and high-precision technical system for fetal sex identification in dairy cows. This holds great value for promoting the implementation of precise early reproductive management in dairy farms and enhancing the economic benefits of the livestock industry.