Efficiency of embryo biopsy and PCR for sex determination in a commercial equine embryo transfer program in Argentina

Abstract

In some horse breeds, such as Argentinian Polo Horses, limiting pregnancies to the desired foal sex has both economic and welfare implications. Biopsy of equine embryos to assess embryonic sex by polymerase chain reaction (PCR) on DNA from sampled cells has been reported under research settings and in a limited number of commercial programs. We previously reported the results of embryo biopsy and PCR of biopsied trophoblast cells for sexing in our commercial equine embryo transfer program. In that study, including one season and results on biopsy of over 450 embryos, there was no significant difference in pregnancy rate between biopsied and non-biopsied embryos (72% vs 73%) or for biopsied embryos recovered at the center (73%) compared with those shipped overnight (72%). Initially, sampled cells were probed with both a genomic (confirmatory) DNA probe and a Y-specific probe, however a sizable proportion (14%) of biopsies did not provide a sex diagnosis (no signal for either probe). We later found that determination only of presence of Y DNA was equally or more effective than using the genomic DNA control; it appeared that the division of the DNA isolated from the biopsy into two aliquots (for analysis of genomic and Y-specific DNA) reduced the accuracy.Overall, the accuracy (correct foal sex) was 95% for embryos diagnosed as male and 78% for embryos diagnosed as female. In the present breeding season (2021-22), we used only Y-DNA assessment. At the time of writing, we have biopsied and assessed over 300 embryos. Embryos were recovered at the Center or shipped overnight to the Center. Biopsy was performed via micromanipulation, and the biopsied cells were evaluated for Y-chromosome specific DNA via PCR. Only embryos without a Y-DNA signal were transferred. Pregnancy rates after transfer of biopsied embryos are currently 124/163 (76%). Sexing of fetuses from transferred embryos via ultrasonographic evaluation at 60-70 days gestation is ongoing and at the time of writing, 48 fetuses have been evaluated. The proportion of female fetuses resulting from embryos with a “female” biopsy/PCR diagnosis (no Y-DNA signal) appears to have varied through the season, being 80% for the first 20 fetuses evaluated and 68% for the next 28 fetuses evaluated. We are currently examining the system to determine possible reasons for the reduction in the accuracy of the PCR diagnosis, and updated results will be presented at the time of the symposium.