Rapid, visual and highly sensitive sexing of bovine embryos by recombinase polymerase amplification with CFI staining

Early bovine embryo sexing both increases the number of offspring of the desired sex, and reduces the subsequent costs of processing unwanted offspring of the opposite sex. The need for cattle of different sexes varies from industry to industry, and a range of tools have been set up to meet this need, but most are energy- and time-consuming, hence it is important to establish a fast and convenient method for bovine embryo determination. Herein, we established a recombinase polymerase amplification (RPA) method combined with CFI dye (RPA-CFI) for sexing of bovine embryos. The assay is highly sensitive, specific, rapid and simple; it can be carried out in only 5 min at 37 °C in a metal bath, and results are visualised using a fluorescent colorimeter. Highly specific male-female common and male-specific primers were designed based on the 1399 bp repeating unit of bovine 1.715 satellite DNA and the male-specific S4 repeating sequence, respectively. The limit of detection (LOD) of RPA-CFI with male-female common primers was 1 pg/μL, and the LOD with male-specific primers was 2 pg/μL. RPA-CFI could determine the sex of bovine embryos from only two cells. This is the first report using RPA-CFI for sex determination of bovine embryos. The assay could be applied to other economically important animals to improve efficiency in livestock industries. Additionally, the assay could relieve pressure on food demand due to human population growth, and contribute to economic development of global stockbreeding.