Laboratory analysis:

A skin biopsy was received from the aborted fetus with hair samples from the dam, the sire and the other foal for DNA genotyping and parentage verification. Genomic DNA was extracted from the samples using an EZ-10 Spin Genomic DNA Minipreps purification kit following the manufacturer’s protocol. A total of 12 microsatellite markers (AHT4, AHT5, ASB17, ASB23, HMS6, HMS7, HTG4, VHL20, HMS3, ASB2, HTG10, HMS2) specific to Equus caballus were used in this study. All markers are included in the panel recommended by the International Society for Animal Genetics for diversity studies and parentage verification. The 12 microsatellites are amplified in one multiplex reaction using Stockmarks; horse genotyping kit (Cat. No.: PN4336407 – Applied Biosystem - USA) according to the method described by (Sargious et al., 2014). Fragment sizes of microsatellite alleles were determined using Genetic analyzer 3500 (Applied Biosystem-USA) with the aid of Liz standard. The data obtained is further analyzed using Gene Mapper V 4.1 software (Applied Biosystem, USA). The Proposed nomenclature for the 12 equine short tandem repeat loci investigated is based on the number of repeat units and is adopted from the recommendation of International Society of Forensic Genetics (ISFG) for the nomenclature of human STRs (Bozzini et al., 1996). Global standardization of molecular marker profiles and their use within animal parentage verification is currently governed by various institutes, inclusive of ISAG (https://www.isag.us/)

Table (1):