Parentage testing of Day 10 equine embryos by amplified PCR analysis of microsatellites.

The research conducted DNA-based parentage testing of horse embryos 10 days post ovulation and accurately identified the true sire for all embryos, using microsatellite-based PCR amplification.

Introduction

The study revolves around the process of parentage identification of day 10 equine embryos. The focus here is not only on the identification of the mother but also of the three possible fathers for these embryos. The method utilized here involves a sophisticated DNA analysis technique and is particularly important in breeding contexts where understanding lineage can be pivotal in driving decisions.

Materials and Methods

• A total of 21 embryos were collected non-surgically on the 10th day after ovulation from known female horses (mares).
• DNA of each embryo was further extracted and typed using radioactive Polymerase Chain Reaction (PCR) amplification process. This application targeted ten specific sequences in the extracted DNA referred to as microsatellites.
• These amplification processes made use of satellites HMS 1, 2, 5, 6, 7, and 8 as well as HTG 3, 4, 6, and10 as per previous studies by Guérin and Marklund respectively.
• The 21 mares and the three potential sires were also genotyped using DNA extracted from blood samples which was further amplified by PCR.

Procedure and Results

• The PCR products forming the embryos and the parent DNA were subjected to electrophoresis and autoradiography for visualization.
• The alleles (specific versions of a gene) of the embryo were matched with those of the females to identify the maternal alleles.
• The paternal alleles were consequently singled out and a search was carried out in the three potential sires’ genotype (genetic makeup) to identify which sire was carrying the paternal alleles.
• In this process, it was identified that the stallions exhibiting specific alleles corresponding to the embryos were considered compatible with the embryos for the particular microsatellite, hence determining the true sire.
• Through this method, the accurate sire was successfully identified for all the 21 embryos collected for the study.

This study highlights the contribution of PCR amplification and the study of microsatellites to accurate sire identification in equines, showcasing the applicability of such profiling in animal breeding, lineage tracing, and potentially resolving disputes around parentage.