31 IN VITRO SURVIVAL AND PREGNANCY OUTCOME OF ZONA-FREE TRANSGENIC GOAT CLONED EMBRYOS AFTER OVIDUCTAL TRANSFER TO FEMALE RECIPIENTS ON DAY 1 OF DEVELOPMENT

Abstract

Survival to term following embryo transfer is usually low after cloning by somatic cell nuclear transfer (SCNT). The aims of this study were to evaluate the effects of the zona pellucida (presence or absence) and the cell nucleus transfer method (cell fusion or cell injection) on the in vitro survival and pregnancy outcome of Day-1 goat cloned embryos transferred into the oviduct of recipient females. In vitro-matured goat oocytes from slaughterhouse ovaries were polar body selected, with a group of oocytes subjected to enzymatic zona pellucida removal. Zona-free (ZF) and zona-intact (ZI) oocytes were enucleated by micromanipulation procedures (Oback and Wells 2003 Cloning Stem Cells 5, 3–12; Keefer et al. 2000 Biol. Reprod. 66, 199–203). Somatic nucleus donor cells from 3 transgenic females for mammary gland expressing human lysozyme (Maga et al. 2003 Trans. Res. 12, 485–496) were either fused (CF) with enucleated ZI and ZF oocytes or injected (CI) into enucleated ZI oocytes, with the assessment of fusion or injection survival rates performed after 60 min. Two direct-current (DC) pulses were used to induce fusion in the ZI group (2 kV cm^–1 each for 10 µs) and in the ZF group (1.0 kV cm^–1 for 20 µs). Embryo reconstruction using ZI oocytes was done by micromanipulation. Zona-free (CF) or ZI (CF or CI) reconstructed cloned embryos were chemically activated in ionomycin/DMAP, followed by in vitro culture for 12 h prior to the surgical embryo transfer into the oviducts of synchronous female recipients (Day 1). Pregnancy diagnosis was carried out on Day 30 of gestation by ultrasonography. Survival after cell fusion, cell injection, and embryo transfer were compared by the χ² test, for P < 0.05. After 16 replications, 1047 in vitro-matured oocytes obtained from 208 does were used for embryo reconstruction (Table 1). In vitro survival was higher in ZI oocytes that were injected with somatic cells than in ZI oocytes or ZF subjected to cell fusion. Pregnancy rates were similar between groups, irrespective of the cell nucleus transfer method or the presence or not of the zona pellucida, but the overall efficiency (fetal survival/recipients) was higher in the ZI-CF group. Currently, 2 ongoing pregnancies carrying 3 cloned concepti from ZI oocytes fused to somatic cells are in late gestation (>110 days). In conclusion, the cell injection method promoted higher survival and, consequently, better efficiency than cell fusion for the reconstruction of goat cloned embryos. However, the zona removal did not affect subsequent in vivo embryo development, as the transfer of zona-free embryos into the oviducts of synchronous recipients resulted in similar pregnancy rates than with zona-intact embryos.

Table 1.  In vitro survival and pregnancy outcome of goat cloned embryos after embryo reconstruction and transfer to female recipients on Day-1 of development