Quality assessment of goat oocytes and effects on goat cloning efficiency after in vivo or in vitro maturation

Oocyte quality plays a key role in nuclear reprogramming after cloning by somatic cell nuclear transfer (SCNT), with the oocyte source having a significant effect on the developmental outcome. Although the use of in vivomatured oocytes for goat cloning appears to be more efficient, despite the variation in maturation, the use of in vitromatured oocytes provides more homogeneous and synchronous cytoplasts for cloning. The refinement of evaluation criteria for oocyte quality may improve in vitro and in vivo survival after cloning. The aim of this study was to evaluate the effect of the ooplasm morphologic quality, chromatin configuration and polar body/MII nearness between in vivo- and in vitro-matured oocytes on the in vitro survival and pregnancy outcome after goat cloning by SCNT, following our established procedures (Martins et al., 2016, Cellular Reprogramming, in press). In vivomatured or immature oocytes were obtained from 65 and 94 FSH-stimulated does by aspiration of pre-ovulatory follicles from abattoir-derived ovaries (in vivo group) or by laparoscopic ovum pick up (in vitro group). Immature oocytes were in vitro-matured for 22 h. Following polar body selection, MII oocytes from both maturation groups were individually classified during the enucleation procedure regarding ooplasm quality as good, regular, or poor, chromatin configuration as normal or abnormal, and polar body/MII nearness as far or near. After reconstruction using transgenic goat fibroblast cells, and following embryo activation (Day = 0) and a 12-h long in vitro-culture, cloned embryos from both maturation groups were surgically transferred to the oviduct of synchronous recipients (13 embryos/recipient). Pregnancy diagnosis was performed by ultrasonography on Day 23. Data on maturation, ooplasm quality, chromatin configuration, polar body/MII nearness, survival upon reconstruction and pregnancy rates were compared between groups by ANOVA or chi-square tests (P < 0.05). After 10 (in vivo) and 15 (in vitro) replications, maturation rate was higher for in vitro- (996/1682, 59.2%) than in vivo-matured (580/1526, 35.7%) oocytes, with no differences observed between groups for the mean number of MII oocytes per doe (10,6 vs. 8,9), for oocytes evaluated as good (23.7 ± 2.8 vs. 14.8 ± 2.8%), medium (38.1 ± 2.4 vs. 37.0 ± 2.4%) or poor (38.2 ± 4.1 vs. 48.2 ± 4.1%), with chromatin abnormalities (98/858, 11.4% vs. 53/562, 8,9%), or for the in vitro survival following embryo reconstruction (243/562, 90.4% vs. 797/858, 92.9%), respectively. Although the near polar body/MII rate was higher in in vivo- (243/501, 47.2%) than in vitro-matured (92/724, 12.8%) oocytes, pregnancy rates were higher in the in vitro (14/41, 34.1%, for 539 transferred embryos) than the in vivo group (4/29, 13.7%, for 379 transferred embryos). Under our conditions, a higher goat cloning efficiency was obtained using in vitromatured oocytes.