Preimplantation genetic testing in horses: biopsy of Piezo-ICSI embryos for sex, coat color, and disease alleles

The introduction of ovum pick-up, Intracytoplasmic Sperm Injection (ICSI) and in vitro embryo production in equine reproduction offers significant advantages, including overcoming infertility, high blastocyst yields, embryo cryopreservation, and preimplantation genetic testing (PGT) by collecting few cells extruded spontaneously during embryo growth using a mouth pipette. PGT enables embryo selection before transfer and is particularly useful in breeds with a preference for specific offspring genders, coat color, and detecting genetic diseases. Here, we present the first large-scale validation of a non-invasive biopsy method for equine ICSI-derived blastocysts, confirming its safety and reliability, preserving embryo integrity. Biopsied cells enabled successful PGT for sex determination, Tobiano white spotting and clinical DNA screening for disorders such as Warmblood Fragile Foal Syndrome Type 1 (WFFST1) and Glycogen Branching Enzyme Deficiency (GBED), using PCR-based assays validated by Sanger sequencing. Our results showed a 92.76 % success rate in sex determination among 746 ICSI blastocysts biopsied, with an overall mean of 56.21 % male and 43.79 % female, and no significant differences among breeds. Tobiano genotypes were identified in over 90.91 % of 22 biopsies analyzed from embryos of heterozygous or homozygous horses. Additionally, 17 of 19 embryos from a WFFST1 carrier stallion were successfully diagnosed, with 29.5 % identified as heterozygous. Finally, 19 of 20 embryos from a GBED carrier stallion were identified, with 21.05 % confirmed as heterozygous. This study demonstrates the reliability of non-invasive PGT of OPU-ICSI embryos as a tool for informed embryo breeding programs, controlling genetic diseases incidence, and contributing to future advancements in equine assisted reproduction.