Ovine-specific Y-chromosome RAPD–SCAR marker for embryo sexing

An accurate, sensitive, and quick (approximately 3 h) method for determining the sex of ovine embryos was developed using polymerase chain reaction (PCR) primers derived from an ovine-specific Y-chromosome random amplified polymorphic DNA marker ( UcdO43 ). The accuracy and sensitivity of the assay were first tested using genomic DNA from 10 males and 10 females of five different sheep breeds, and then tested using serial dilutions of male-in-female DNA. The assay was 100% accurate in confirming the sex of the individuals and the ovine male-specific fragment was detected in dilutions containing as little as 10 pg of male DNA in 50 ng of female DNA. The assay was also confirmed to be specific for the ovine Y-chromosome as bovine, caprine, porcine, murine, and human DNA did not amplify. The ovine embryo sexing method is a duplex PCR system that also includes ZFY/ZFX primers. ZFY/ZFX provide an internal positive control for amplification as well as a means to confirm the results obtained with the UcdO43 primers. All embryo sexing results (36/36) from our method were in agreement with the ZFY/ZFX assay results. However, while our method requires an internal control to detect PCR failure, it has the advantages of not requiring nested PCR or restriction endonuclease digestion of the PCR product, and concerns about cross-species contamination are eliminated.