119 Single closed-tube qPCR assay with dual-labelled probes for improved sexing of equine embryos

In addition to fulfilling many breeders’ curiosity, equine embryonic sex determination can have a profound commercial impact. However, currently described assays suffer from suboptimal diagnosis rates (50–94%) and accuracies (84–90%), with sensitivity (65–80%) being the main problem. In addition, while pregnancy results of in vivo-produced embryos following invasive biopsy equal those of nonbiopsied embryos, the effect on in vitro-produced embryos is unknown. We aimed to develop an improved sex assay that can be directly performed on embryo biopsies of 5–20 cells in a closed-tube format, avoiding potential sample loss or contamination. A multiplex qPCR assay with dual-labelled probes was designed to allow simultaneous generation of both male-specific and control fragments in a single reaction. To improve sensitivity, multicopy genes were chosen to be specifically amplified, i.e. 8 copies of Y-chromosomal ETSTY5 as male-specific and 4 autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA. At the optimal annealing temperature, the assay was 100% sex specific with a high sensitivity (down to 10 pg DNA). The assay was applied on 58 biopsies. The biopsy procedure involved puncture of Grade 1 or 2 in vitro-produced blastocysts cultured for 8–13 days, with a bevelled biopsy micropipette and suction of 5–20 trophectoderm cells at the contralateral side. The biopsy, hatching embryonic cells (when present), and 35 biopsied embryos were tubed in 2 µL PBS-1%PVP and stored at −80°C. Other biopsied embryos were vitrified before transfer (n = 23). The assay reaction mix (8 µL: 3.7 µL H₂O, 1 µL 10× Key Buffer, 5 µM of each primer, 40 mM dNTP’s, 0.5 U tempase, 7 µM UBC and 3 µM ETSTY5 TaqMan probes) was either directly added to the biopsy-containing tube (n = 44) or added to 2 µL (1 ng/µL) of DNA following whole-genome amplification of the biopsy (REPLI-g SC kit; n = 14). qPCR cycling parameters were 95°C (14′ 40″), 49 cycles of 95°C (20″) and 68°C (40″). The assay success rate on non-preamplified and preamplified equine embryo biopsies did not differ significantly (89% and 64%, respectively; P > 0.05; Fisher’s exact test). Exactly half of the embryos were found to be fillies and colts. The results were validated in 30 embryos with the same assay on a second biopsy, hatching cells, or the rest of the embryo (n = 28), or following transfer and ultrasound-based fetal sex determination around 120 days post-gestation (n = 2) with 100% accuracy. Of 6 biopsied Day 9 embryos transferred last season, 5 resulted in an initial pregnancy, 1 was lost early in gestation, and 1 was lost in the last trimester. Nonbiopsied Day 9, Grade 1 or 2 blastocysts produced in vitro and transferred over the same period resulted in 55% initial pregnancy (n = 29) and 21% early pregnancy loss (n = 14). In conclusion, we report a reliable, fast, and cost-effective method for sex determination of equine embryos. Initial results do not reveal an effect of invasive, micromanipulation-based embryo biopsy on the viability of in vitro-produced embryos after transfer.