A rapid method for sexing the bovine embryo

This study aimed to detect the sex of bovine embryo by adding ethidium bromide (EB) or 5 μl of CuSO4 at different concentrations (0.5 M, 1 M, 1.5 M, 2 M, 3 M and 5 M) to the product of loop-mediated isothermal amplification (LAMP) reaction. The result of these additions was a colour change and a precipitate. This allows detection with the naked eye without the use of electrophoresis or a turbidity meter. The in vitro produced bovine embryos were divided into one to eight pieces using a microblade attached to a micromanipulator. The cell number in each piece was counted before sexing. Sexing of DNA samples extracted from one to five biopsies cells was performed by LAMP. After biopsy, the remaining part of the embryos was used to confirm the sex by polymerase chain reaction (PCR). The time for the whole procedure was about 45 min. LAMP is a novel DNA amplification method which amplifies a target sequence specifically under isothermal condition. We used this method to examine 58 bovine embryos and the accuracy of sex prediction was 100% when the blastomeres dissociated from a morula exceeds three. This study showed that the present method can be applied in bovine breeding programs to facilitate manipulation of the sex ratio of offspring. The aim of this study is to develop an improved embryo sexing technique based on the LAMP reaction. This new method is economic and more suitable for field application without turbidity meter or electrophoresis.