Domestic cat embryos generated without zona pellucida are capable of developing in vitro but exhibit abnormal gene expression and a decreased implantation rate

Abstract

The removal of the zona pellucida has been used to improve the in vitro development of domestic cat embryos generated by IVF and SCNT. However, the in vivo development of domestic cat embryos generated without the zona pellucida has not been evaluated. The objective of this study was to evaluate the effects of zona pellucida removal on the in vitro and in vivo development of domestic cat embryos generated by IVF. For this purpose, two experimental groups were created: 1) domestic cat embryos cultured in vitro (Zona-intact group, ZI) and 2) domestic cat embryos cultured in vitro without the zona pellucida (Zona-free group, ZF). Domestic cat embryos were generated by IVF and cultured in vitro for 8 days. In the ZF group, the zona pellucida was removed after IVF, and embryos were cultured using the well of the well system (WOW). Cleavage, morula and blastocyst rates were evaluated in both groups. The diameter and total cell number of blastocysts were assessed. Relative expression of pluripotency (OCT4, SOX2 and NANOG), differentiation (CDX2 and GATA6) and apoptotic markers (BAX and BCL2) was evaluated in blastocysts. Finally, to evaluate in vivo development, embryos at days 5, 6 and 7 of development were transferred into recipient domestic cats, and ultrasonography was performed to evaluate implantation. No differences were observed in the cleavage, morula or blastocyst rates between embryos from the ZI and ZF groups. The diameter (mean ± SD) of blastocysts from the ZF group was greater (253.4 ± 83.3 μm) than that from the ZI group (210.5 ± 78.5 μm). No differences were observed in the relative expression of OCT4, CDX2 or GATA6. However, the relative expression of SOX2 and NANOG was significantly reduced in ZF blastocysts compared to ZI blastocysts. Furthermore, the relative expression of BAX was higher in ZF blastocysts than in ZI blastocysts. Finally, four pregnancies were confirmed after the transfer of ZI embryos (n = 110). However, no pregnancies were observed after the transfer of ZF embryos at the morula or blastocyst stage (n = 56). In conclusion, domestic cat embryos cultured without the zona pellucida were able to develop in vitro until the blastocyst stage. However, the removal of the zona pellucida negatively affected the gene expression of pluripotency and apoptosis markers, and ZF embryos were unable to implant. This might indicate that the removal of the zona pellucida is detrimental for the implantation and in vivo development of domestic cat embryos.

Keywords: Embryo culture; Embryo transfer; Felid embryos; Gene expression; In vitro fertilization.