Rapid sex determination of chick embryos using the polymerase chain reaction1

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Abstract
The sex of early chicken embryos was determined by DNA dot‐blot hybridization and the polymerase chain reaction (PCR). An oligonucleotide probe, specific to the XhoI family of repetitive DNA of the W chromosome, was hybridized to crude and purified preparations of DNA obtained from blood or cells from embryos and detected using chemiluminescence. Complete agreement was observed in sexing 100 embryos using the dot‐blot assay and visual inspection of embryonic gonadal tissue. Although the dot‐blot system was useful for determining the sex of embryos prior to and throughout incubation, the time required for hybridization and detection did not allow for the immediate manipulation of embryos. Therefore, a PCR assay was developed using primers specific to the XhoI repetitive element and rapid thermocycling with an air thermocycler. A female‐specific amplification product was observed using DNA from as few as two cells in the PCR. Furthermore, female DNA was detected in mixtures of male and female DNA at levels equivalent to one female cell in 5000 male cells. The high level of sensitivity permitted the sexing of unincubated embryos using a small biopsy of cells sampled in ovo. In general, the entire procedure, including sample preparation, the PCR, and electrophoresis could be completed within 2.5 hours thereby saving a considerable amount of time over techniques requiring hybridization. The ability to rapidly determine the sex of the unincubated chick embryo will facilitate studies on gonadal differentiation, the examination of sex differences during development, and assist current efforts to manipulate the avian genome using germline chimeras.