A study on methods for preimplantation genetic diagnosis on equine embryos

Abstract

Determination of embryonic sex or the presence of alleles associated with genetic conditions is commonly applied in equine reproduction. To date, two methods for preimplantation genetic diagnosis (PGD) have been described for equine embryos: trophoblast cell biopsy (TCB) or blastocele fluid aspiration (BFA). While TCB has been described in both in vivo- (IVV) and in vitro-produced (IVP) embryos, BFA has been reported only for IVV embryos. Alternative methods for PGD, including analysis of cell-free DNA (CFD) in the medium where IVP has been cultured, are commonly used in humans, but not for equine embryos. In the current study, three experiments were performed to determine the agreement among methods for PGD on both IVV and IVP equine embryos. In Experiment 1, IVV (n = 10) and IVP (n = 13) embryos were subjected to BFA, cultured for 24 hours, subjected to TCB, and then cultured for additional 24 hours. No detrimental effect on embryonic diameter was observed for either embryo group (IVV: before BFA: 740 ± 618 µm vs. after TCB: 718 ± 526 µm; IVP: before BFA: 174 ± 8 µm vs. after TCB: 176 ± 10 µm; P > 0.05). In Experiment 2, the agreement among BFA, TCB, and the whole embryo (Whole) was studied by detecting the genes SRY (Y chromosome) and AR (X chromosome) using PCR. For IVV embryos (n = 14),there was a 100% agreement (14/14 embryos) between Whole and TCB for both SRY and AR, while a 64% agreement (9/14 embryos) between Whole and BFA, or between BFA and TCB. On the other hand, for IVP embryos (n = 13), there was a 75% agreement (10/13 embryos) between Whole and TCB for both SRY and AR, while a 31% agreement (4/13 embryos) between Whole and BFA, and a 46% agreement (6/13 embryos) between BFA and TCB. In Experiment 3, CFD was performed in IVP embryos (n = 10), to determine embryonic sex by means of whole genome amplification (WGA) followed by detection of the genes SRY (Y chromosome) and AMEL (X and Y chromosome) using PCR. For this, WGA was performed in both Whole, CFD samples, and Control medium (negative control). Overall, CFD samples yielded results for SRY and AMEL in 6/10 embryos (60%); the sex determined in CFD samples corresponded to the same results for the Whole group. None of the Control samples amplified any DNA. In conclusion, equine embryos can be subjected to 2 biopsy procedures without apparent detrimental effects on embryonic size. For IVV, but not IVP embryos, BFA can be considered a potential alternative to TCB for PGD. Finally, CFD can be further explored as a non-invasive method for PGD in IVP equine embryos.