364 AMPLIFICATION AND APPLICATION OF THE HMG BOX OF BOVINE SRY GENE FOR SEX DETERMINATION

Abstract

A fast and reliable method for bovine sexing has been developed through amplification of the bovine high motility group (HMG) box of the sex-determining region of the Y chromosome gene (SRY). We describe a similar PCR-based method, which involves amplification of the HMG-box sequence of the bovine SRY gene with optimized PCR parameters and apply it to bovine sex-determination. Oligonucleotide primers were designed according to the conserved bovine SRY HMG box sequence motif (Gen-Bank Accession NM-011564); the forward primer was 52-CACGCATCTTACTTCCTCCCCTTTTAAAAG-32 and the reverse primer was 52-AGACGCCTTTGTTGAGCGAGAGTAAGGAAG-32. In agarose gel electrophoresis, a normal bull showed 1 SRY band, and a normal cow showed no SRY band. After optimization, the PCR procedure for sex determination was applied to 14 embryo biopsies. The biopsied embryos were transferred into 14 recipient cows on the same day (Day 7 of the estrus cycle) that the embryos were collected and sex of the calf was confirmed after parturition. Nine calves were born and anatomical sex corresponded to the sex determined by PCR in all cases (100% accuracy). Thus, this study showed for the first time that the present method can be applied in bovine breeding programs to facilitate manipulation of the sex ratio of offspring and also allows a quick diagnosis for the XY-bovine offspring by amplification of the HMG box of the bovine SRY gene. This method also differs from that of Thomsen and Poulsen working with pigs and Mara in which the sex of in vitro-produced ovine embroys was determined by duplex PCR.