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Somatic cell nuclear transfer (SCNT) allows animal cloning but remains technically challenging. This study investigated limitations to functional oocyte enucleation by actinomycin D (AD) as a means of making SCNT easier to perform. Denuding oocytes or inhibiting transcription before AD treatment revealed that the toxicity of this compound during bovine oocyte maturation is mediated by cumulus cells. Exposure of denuded oocytes to higher concentrations of AD (5-20μgmL-1 ) and stepwise reductions of the incubation period (from 14.0 to 0.25h) led to complete inhibition of parthenogenetic development. Bovine SCNT using this improved AD enucleation protocol (NT(AD)) restored cleavage rates compared with rates in the parthenogenetic and SCNT controls (P(CTL) and NT(CTL) respectively). However, NT(AD) was associated with increased caspase-3 activity in cleavage stage embryos and did not recover blastocyst rates. The removal of AD-treated oocyte spindle before reconstruction (NT(AD+SR)) improved embryo development and reduced caspase-3 activity to levels similar to those in the P(CTL) and NT(CTL) groups. Furthermore, mid-term pregnancies were achieved using NT(AD+SR) blastocysts. In conclusion, improvements in AD functional enucleation for bovine SCNT circumvents most cellular roadblocks to early embryonic development and future investigations must focus on restoring blastocyst formation.

The present study was designed to compare Day 14 bovine embryos that were produced entirely in vitro using the post-hatching development (PHD) system with in vivo-derived embryos without or with transient PHD culture from Day 7 to Day 14. Embryos on Day 14 were used for sex determination and gene expression analysis of PLAC8, KRT8, CD9, SLC2A1, SLC2A3, PGK1, HSF1, MNSOD, HSP70 and IFNT using real-time quantitative (q) polymerase chain reaction (PCR). First, Day 7 in vivo- and in vitro-produced embryos were subjected to the PHD system. A higher rate of survival was observed for in vitro embryos on Day 14. Comparing Day 14 embryos produced completely in vivo or completely in vitro revealed that the mean size of the former group was greater than that of the latter (10.29±1.83 vs 2.68±0.33mm, respectively). Expression of the HSP70 and SLC2A1 genes was down- and upregulated, respectively, in the in vitro embryos. The present study shows that in vitro embryos cultured in the PHD system are smaller than in vivo embryos and that of the 10 genes analysed, only two were differentially expressed between the two groups. These findings indicate that, owing to the poor survival rate, the PHD system is not reliable for evaluation of in vitro embryo quality.

The aim of the present study was to investigate the effects of growth and differentiation factor-9 (GDF-9) on the survival and activation of preantral follicles, as well as their subsequent progression to secondary follicles, using goat ovarian cortical culture in vitro. Pieces of ovarian cortex were cultured for 1 and 7 days in minimum essential medium (MEM) with or without different concentrations of GDF-9 (1-200 ng mL(-1)). On Day 0 and after 1 and 7 days of culture, cortical pieces were fixed for histological and transmission electron microscopy evaluation. Preantral follicles were classified according to their development stage (primordial, intermediate, primary and secondary) and on the basis of morphological features (normal or degenerated). In addition, follicular and oocyte diameters were determined before and after culture. The results showed that, compared with non-cultured cortical tissue (Day 0), the culture of ovarian tissue significantly reduced (P < 0.05) the percentage of normal follicles in all media tested, except for tissue cultured in the presence of 200 ng mL(-1) GDF-9. Furthermore, in all media tested, the percentage of primordial follicles was significantly reduced (P < 0.05), with a concomitant increase in the percentage of developing follicles. The highest percentage of secondary follicles was observed after 7 days of culture in MEM plus 200 ng mL(-1) GDF-9. At all concentrations of GDF-9 tested, follicular diameter increased significantly after 7 days of culture compared with non-cultured cortical tissue. In conclusion, the results of the present study indicate that 200 ng mL(-1) GDF-9 maintains the survival of preantral follicles and promotes activation of primordial follicles. Furthermore, GDF-9 stimulates the transition from primary to secondary follicles, maintaining ultrastructural integrity of the follicles.