Abstract

BACKGROUND-AIM Domestic cat embryos cultured without zona pellucida have a reduced implantation capacity after embryo transfer at the blastocyst stage. The objective of this study was to evaluate the expression of trophectoderm markers in domestic cat blastocysts cultured without zona pellucida. METHODS Two experimental groups were done: 1) Domestic cat embryos cultured until the blastocyst stage (Zona intact group, ZI). 2) Domestic cat embryos cultured until the blastocyst stage without zona pellucida (Zona free group, ZF). Ovaries were collected from domestic cats subjected to ovariohysterectomy. Cumulus-oocyte complexes (COCs) were matured in vitro in supplemented Medium-199, in 5% CO2, at 38.5°C for 26 hours. For IVF, 20-30 COCs were co-cultured with 1.5-2.5 x 106 spermatozoa/mL in supplemented TALP medium, in 5% CO2, at 38.5°C for 24 hours. After IVF, the zona pellucida was removed by incubation in 2 mg/mL pronase for 4 minutes. ZF-zygotes were cultured in microwells using the well of the well system (WOW). In both groups, IVC was done in supplemented SOF, in 5% CO2, 5% O2 and 90% N2, at 38.5°C, for seven days. The cleavage, morula and blastocyst rates were estimated. The relative expression of the trophectoderm markers CDX2, YAP1, TEAD4 and E-cadherin and the apoptosis marker CASP3 was evaluated in the blastocysts by RT-qPCR using the ΔΔ-Ct method. SDHA was used as internal control. The Wilcoxon test was used to evaluate differences (P< 0.05). RESULTS No differences were observed in the in vitro development between the ZI and ZF groups. Cleavage rate: ZI = 310/641 (48.4%); ZF = 216/375 (57.6%). Morula rate: ZI = 180/310 (58.1%); ZF = 124/216 (57.4%). Blastocyst rate: ZI = 81/310 (26.1%); ZF = 55/216 (25.5%). No statistical differences were found in the relative expression of CDX2, YAP1, TEAD4 and CASP3 between blastocysts from the ZI and ZF groups. The relative expression of E-cadherin was higher in ZF-blastocysts than in ZI-blastocysts. This higher expression might be needed to enhance cellular adhesion and compaction of blastomeres in the ZF-embryos. CONCLUSIONS In conclusion, the culture without zona pellucida did not affect the in vitro development of domestic cat embryos and their expression of trophectoderm markers. ANID FONDECYT 3200352