Single closed-tube quantitative real-time PCR assay with dual-labelled probes for improved sex determination of equine embryos.

This research article discusses the development of a highly sensitive equine sex determination assay that allows for simultaneous detection of male-specific and control fragments in a single reaction. The techniques used and results were validated on in vitro-produced embryos.

Research Background and Purpose

• The ability to determine the sex of equine embryos is not only interesting for breeders but can also have significant commercial implications. However, the applications available for this purpose have been ineffective, mostly due to low sensitivity.
• While the effects of needle aspiration biopsy on in vivo-flushed embryos have been studied before, the impacts on in vitro-produced embryos were unknown.
• The main goal of the research was to design an assay that could determine the sex of an equine embryo effectively and exactly and evaluate the effect of a needle aspiration biopsy on the viability of in vitro-produced embryos.

Methodology

• A multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was used. It was designed to allow the creation of male-specific and control fragments simultaneously, in a single closed tube, reducing the chance of contamination or sample loss.
• To enhance sensitivity, multicopy and polymeric genes were chosen for amplification. Eight copies of Y-chromosomal ETSTY5 acted as male-specific amplifiers, while four autosomal UBC monomers acted as control fragment.
• The assay’s specificity was improved by the use of equine-specific ETSTY5 and dual-labelled probes.
• In testing the assay, equine male and female genomic DNA samples were used. The method demonstrated 100% accuracy and >95% qPCR efficiency down to 10 pg of DNA.
• The researchers used the optimized assay to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies using needle aspiration biopsy and herniating cells.

Results and Conclusion

• Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Result validation was performed on a secondary sample from the biopsied embryo (n = 18) or through ultrasound-based sex determination of the foetus (n = 7).
• The needle aspiration biopsy procedure did not impair the initial pregnancy rate or early pregnancy losses compared to non-biopsied embryos.
• In conclusion, the team developed a reliable, efficient, and cost-effective method for equine sex determination that was validated for in vitro-produced embryos. Biopsy did not compromise the embryo’s viability. The assay and biopsy strategy could have applications for other uses in the future.