Viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis

Abstract

The equine embryo possesses a capsule that is considered essential for its survival. We assessed viability after breaching the capsule of early (Day 6) and expanded (Day 7 and 8) equine blastocysts by micromanipulation. The capsule was penetrated using a Piezo drill, and trophoblast biopsy samples were obtained for genetic analysis. Pregnancy rates for Day-6 embryos, which had intact zonae pellucidae at the time of recovery, were 3/3 for those biopsied immediately after recovery and 2/3 for those biopsied after being shipped overnight under warm (w28 8C) conditions. The pregnancy rates for encapsulated Day-7 expanded blastocysts were 5/6 for those biopsied immediately and 5/6 for those biopsied after being shipped overnight warm. Two of four encapsulated Day-8 blastocysts, 790 and 1350 mm in diameter, established normal pregnancies after biopsy. Nine mares were allowed to maintain pregnancy, and they gave birth to nine normal foals. Biopsied cells from eight embryos that produced foals were subjected to whole-genome amplification. Sex was successfully determined from amplified DNA in 8/8 embryos. Identification of disease-causing mutations matched in the analyses of 6/6 samples for the sodium channel, voltage-gated, type IV, alpha subunit (SCN4A) gene and in 6/7 samples for the peptidylprolyl isomerase B (PPIB) gene, in embryo-foal pairs. Thus, the capsule of the equine embryo can be breached without impairing viability. Further work is needed to determine whether this breach is transient or permanent. These findings are relevant to the understanding of equine embryo development and to the establishment of methods for micromanipulation and embryo cryopreservation in this species.

Assisted reproduction techniques in horses – clinical application by different programs around the world

“…Additionally, PGD has also been used to detect specific mutations associated with genetic disorders (Choi et al 2010, Choi et al 2015 or genes associated to specific phenotypes as, for example, coat color (Guignot et al 2015). PGD has been successfully incorporated to a commercial embryo transfer program, to select the female embryos produced from Polo Argentino horses (Herrera et al 2014).…”

Cryopreservation of Equine Embryos: Current State‐of‐the‐Art

“…(2011). In an earlier study to establish techniques for embryo biopsy for pre‐implantation genetic diagnosis, the authors noted that embryo puncture and collapse did not compromise viability (Choi et al. 2010).…”

“…The most recent approach to improving the freezability of expanded equine embryos was arrived at serendipitously by Choi et al (2011). In an earlier study to establish techniques for embryo biopsy for pre-implantation genetic diagnosis, the authors noted that embryo puncture and collapse did not compromise viability (Choi et al 2010). In a follow-up study, they used a piezo drill to similarly penetrate the capsule and trophoblast, and then deliberately aspirated blastocoele fluid prior to vitrification; moreover, with one set of CPAs they achieved promising pregnancy rates (5 ⁄ 7: 71%) for embryos up to 650 lm (Choi et al 2011).…”

“…Embryo cryopreservation would also allow ‘temporary genetic banking’, which could be advantageous to the owners of young mares from interesting bloodlines that have still to prove themselves in competition; embryos could be frozen when the mare was young and fertile and transferred at a later date only if the mare proved its worth. A more recent development for which cryopreservation is a desirable adjunct is pre‐implantation genetic diagnosis, during which a small number of cells are recovered from ET‐stage embryos to test for specific heritable diseases (Choi et al. 2010); after the biopsy procedure, the embryos are ideally stored until the test results are available so that only embryos free of the genetic defect are transferred.…”

OMICS for the Identification of Biomarkers for Oocyte Competence, with Special Reference to the Mare as a Prospective Model for Human Reproductive Medicine

“…To the best of our knowledge no studies have been reported to date on genomic analysis in the equine oocyte. A recent paper in the horse by Choi et al, (2010) reports the identification of disease-causing mutations in trophoblastic biopsies from equine in vivo-recovered pre-implantation embryos. These authors demonstrated for the first time the correct identification, by embryo biopsy and whole genome amplification, of sex and genotype at the causative mutation sites for two disease-linked genes (SCN4A and PPIB).…”