A study on methods for preimplantation genetic testing (PGT) on in vivo- and in vitro-produced equine embryos, with emphasis on embryonic sex determination

• Published in: Theriogenology 2024-10, Vol.227, p.41-48
• Main Authors: Ramírez-Agámez, Luisa, Castaneda, Caitlin, Hernández-Avilés, Camilo, Grahn, Robert A., Raudsepp, Terje, Love, Charles C.
• Format: Article
• Language: English
• Subjects: androgen receptors; animal reproduction; Animals; biopsy; Blastocoele fluid; Cell-free DNA; Cell-Free Nucleic Acids; DNA; Embryo biopsy; Embryo Culture Techniques - veterinary; Embryo, Mammalian; Equine embryo; Female; Fertilization in Vitro - veterinary; genes; Genetic Testing - methods; Genetic Testing - veterinary; horses; Horses - embryology; Male; PGT; Preimplantation Diagnosis - methods; Preimplantation Diagnosis - veterinary; sex determination; Sex Determination Analysis - methods; Sex Determination Analysis - veterinary; trophoblast; Trophoblast cells; X chromosome; Y chromosome
• ISSN: 0093-691X; 1879-3231; 1879-3231
• DOI: 10.1016/j.theriogenology.2024.07.009

Two methods for preimplantation genetic testing (PGT) have been described for equine embryos: trophoblast cell biopsy (TCB) or blastocoele fluid aspiration (BFA). While TCB is widely applied for both in vivo- and in vitro-produced embryos, BFA has been mostly utilized for in vivo-produced embryos. Alternative methods for PGT, including analysis of cell-free DNA (CFD) in the medium where in vitro-produced embryos are cultured, have been reported in humans but not for equine embryos. In Experiment 1, in vivo- (n = 10) and in vitro-produced (n = 13) equine embryos were subjected to BFA, cultured for 24 h, then subjected to TCB, and cultured for additional 24 h. No detrimental effect on embryonic diameter or re-expansion rates was observed for either embryo group (P > 0.05). In Experiment 2, the concordance (i.e., agreement on detecting the same embryonic sex using two techniques) among BFA, TCB, and the whole embryo (Whole) was studied by detecting the sex-determining region Y (SRY) or testis-specific y-encoded protein 1 (TSPY) (Y-chromosome), and androgen receptor (AR; X-chromosome) genes using PCR. Overall, a higher concordance for detecting embryonic sex was observed among techniques for in vivo-produced embryos (67–100 %; n = 14 embryos) than for in vitro-produced embryos (31–92 %; n = 13 embryos). The concordance between sample types increased when utilizing TSPY (77–100 %) instead of SRY (31–100 %) as target gene. In Experiment 3, CFD analysis was performed on in vitro-produced embryos to determine embryonic sex via PCR (SRY [Y-chromosome] and amelogenin – AMEL [X- and Y-chromosomes]). Overall, CFD was detected in all medium samples, and the concordance between CFD sample and the whole embryo was 60 % when utilizing SRY and AMEL genes. In conclusion, equine embryos can be subjected to two biopsy procedures (24 h apart) without apparent detrimental effects on embryonic size. For in vivo-, but not for in vitro-produced equine embryos, BFA can be considered a potential alternative to TCB for PGT. Finally, CFD can be further explored as a non-invasive method for PGT in in vitro produced equine embryos. •Equine in vivo- and in vitro-produced embryos were subjected to two biopsies.•An apparent negative effect on embryonic diameter was not observed.•TSPY was more efficient than SRY as a target gene for sex determination of equine embryos.•Blastocele fluid aspiration yielded similar results to trophoblast cells for in vivo embryos.•Cell-free DNA can be isolated