Sex diagnosis of equine preimplantation embryos using the polymerase chain reaction.

A rapid and reliable method for sex determination of preimplantation-stage equine embryos has not been available. The aim of the present study was to find an enzyme which would distinguish sexes in the horse by finding a polymorphic restriction site between the ZFY and ZFX homologues amplified by the polymerase chain reaction (PCR). Altogether, 38 different restriction enzymes were tested using female and male DNA extracted from blood. The primers used for amplification were selected from conserved sequences between human ZFY and ZFX genes and mouse Zfy-1 and Zfy-2 genes. Nine enzymes cut the PCR product of approximately 450 basepairs, but only Bsm I yielded different banding patterns in female and male DNA. All blood samples were correctly diagnosed. To test the method on embryonic cells, 17 horse demi-embryos were obtained from expanding blastocysts 220 to 950 mum in diameter. Demi-embryos were further cut into 3 to 7 parallel samples which were analyzed individually to test the repeatability of the method. Eight of the original embryos were diagnosed as females and 9 as males. No misidentifications were observed within the embryonic samples, suggesting that this sexing method is highly reliable. This study provides a rapid and accurate method to sex horse embryos.