Abstract

The objective of this study was to compare the development to the morula and blastocyst stages, after either cycloheximide (CHX) or ethanol (ETOH) activation, in somatic nuclear transfer (NT) goat embryos derived from 2 sources of oocytes. In vivo- and in vitro-matured oocytes were obtained from FSH-stimulated goats (Native, Saanen, and Native-Saanen crossbred goats). Gonadotropin treatment was performed with a modified program of a previous report (Reggio et al. 2001 Biol. Reprod. 64, 849-856). In vivo-matured oocytes were flushed from the oviduct of donor goats by exposing the reproductive tract via a small ventral laparotomy incision. In vitro-matured oocytes were aspirated and cultured in maturation medium (M199 + 10% FCS, 10 µg mL^-1 FSH, 10 µg mL^-1 LH, and 1 µg mL^-1 17²-estradiol) for 22 h, at 38.5°C in 5% CO₂ and air. Donor cells were prepared from ear skin fibroblasts of a female goat (Native breed). Cells, at passage 3-9, starved by culturing in 0.5% FCS for 4-8 days, were used for NT. Matured oocytes were enucleated, and cell-cytoplast couplets (n = 162 in vivo-, and n = 190 in vitro-matured oocyte groups, respectively) were fused by applying 2 DC pulses of 2.2 kV cm^-1 for 30 µs. One to 2 h after fusion, fused embryos were either incubated in 10 µg mL^-1 cycloheximide plus 5 µg mL^-1 cytochalasin B for 5 h (CHX treatment) or in 7% ethanol for 5 min followed by a 4-h incubation in 2 mM 6-dimethylaminopurine plus 5 µg mL^-1 cytochalasin B (ETOH treatment). NT embryos were then cultured in B₂ medium supplemented with 5% FCS and Vero cells for 9 days. At the end of the culture period, the NT embryos were fixed and stained with Hoechst 33342 (Begin et al. 2003 Theriogenology 59, 1839-1850). The numbers of nuclei were counted under ultraviolet light. Fusion, cleavage, and development rates were compared using chi-square test or Fisher's exact test. For the in vivo-matured oocyte group, there were no significant differences in fusion rates (78.1% vs. 68.7%), cleavage rates (87.7% vs. 87.0%, based on the numbers of embryos fused) between the CHX and ETOH treatment groups, respectively (P > 0.05). However, the development rates to morula and blastocyst stages of NT embryos derived from either in vivo- or in vitro-matured oocytes were significantly higher in the ETOH group than in the CHX group (in vivo: 15.2% vs. 0%, and in vitro: 7.1% vs. 0%, for ETOH and CHX groups, respectively; P < 0.05). For the in vitro-matured oocyte group, no significant differences were found between the CHX and ETOH groups in fusion rates (78.6% vs. 83.6%; P > 0.05), cleavage rates (80.5% vs. 83.9%: P > 0.05, based on the numbers of embryos fused). NT embryos from the CHX treatment group derived from in vivo- or in vitro-matured oocytes did not develop beyond the 16-cell stage. These results demonstrate that activation with CHX plus cytochalasin B treatment affects the development to the blastocyst stage of cloned goat embryos whether derived from in vivo- or in vitro-matured oocytes.