Reliability of sex determination in ovine embryos using amelogenin gene (AMEL)

Sex specific sequence variability of the amelogenin gene has been used for sex determination in the family of Bovidae. In our study, suitability and reliability of the amelogenin gene for ovine sex determination in embryos was studied. The specificity of the method was previously demonstrated by testing 579 blood samples from several Spanish sheep breeds (161 males and 198 females). No amplification failures and very high agreement between genotypic and phenotypic sex was found (578/579), in contrast to humans, where errors in sex determination has been reported because of mutations in AMELX or AMELY genes. However, one female animal showed a male genotypic sex, being the most plausible explanation that a recombination event has happened during the meiosis. In our study only 0.17% (1/579) of the samples tested has been misdiagnosed using the amelogenin gene. Finally, 1-10 cells from each of 67 compact morulae were aspirated through the zona pellucida, and genotyped for sex determination. The efficiency in sex determination was 95 and 98% when more than two and more than three cells were sampled, respectively. The total time required for the genetic test, was less than 4h. These results confirm that the amelogenin gene can be used for rapid sex determination in ovine embryos, with a high efficiency and accuracy (100%) when three or more cells are sampled, allowing transferring sexed fresh embryos in MOET programmes. To our knowledge, this was the first time that sex determination using the amelogenin gene was performed in ovine embryos.