Sex determination and genetic screening of equine embryos with whole genome amplification and real-time PCR.

Sex determination and genetic screening of equine embryos can be accurately performed using a combination of whole genome amplification (WGA) and multiplex real-time PCR, allowing for informed embryo transfer decisions in horse breeding without later interventions.

Introduction and Objective

• Pre-implantation sex determination and genetic screening are important for making informed decisions before embryo transfer in equine breeding.
• These techniques help avoid the need for later interventions by identifying sex and genetic traits early.
• The study aimed to develop a rapid, reliable, and streamlined method combining whole genome amplification (WGA) and multiplex real-time PCR for sex determination and genetic screening of equine embryos.

Methodology

• Whole Genome Amplification (WGA): A rapid WGA step was introduced to amplify the tiny amounts of DNA in embryo biopsies, improving the quality and consistency of genetic material for testing.
• Multiplex Real-Time PCR: Two genetic markers were targeted:
  • ETSTY5: A Y chromosome-specific marker used for male sex determination.
  • UBC: An autosomal control gene to verify DNA presence and quality.
• Testing was performed on purified equine genomic DNA (gDNA), embryo biopsies, and cloned embryos of known sex for validation.
• The assay also included genotyping tests for two inherited diseases:
  • Hyperkalemic Periodic Paralysis (HYPP)
  • Hereditary Equine Regional Dermal Asthenia (HERDA)

Key Findings

• The sex of purified equine DNA could be accurately determined with as little as 10 picograms of gDNA and even from a single fibroblast cell.
• Direct testing of embryo biopsies without WGA resulted in inconsistent sex determination results.
• Introducing a short WGA step before PCR drastically improved accuracy, yielding tight allelic discrimination and 100% accurate diagnostic calls.
• This reliable method worked effectively even for cloned embryos where sex is already known, demonstrating robustness.
• The WGA product was also useful for targeted genotyping of the two disease-associated alleles, enabling pre-implantation genetic diagnosis.

Implications and Applications

• The combined WGA and multiplex real-time PCR workflow offers a practical, reliable method for early sex determination and genetic disease screening in equine embryos.
• It supports improved decision-making in in vitro produced embryo and embryo transfer procedures.
• This pipeline reduces the need for invasive or later-stage interventions by providing crucial genetic information prior to implantation.
• It has the potential to enhance breeding management in the equine industry by ensuring desired sex ratios and reducing the transmission of genetic diseases.