22 RESPIRATORY ACTIVITY AND ULTRASTRUCTURAL FEATURES OF BOVINE SOMATIC NUCLEAR TRANSFER EMBRYOS

Abstract

We succeeded in determining oxygen consumption of individual bovine embryos non-invasively and quantitatively by scanning electrochemical microscopy (SECM). Recently, we have found that there is a close relationship between high oxygen consumption and developmental ability of bovine IVF embryos. However, the relationship between the respiratory activity and the quality of bovine embryos reconstructed by somatic cell nuclear transfer (SCNT) is still unclear. The aims of this study were: (1) to assess the oxygen consumption of single bovine SCNT and IVF embryos; and (2) to examine the ultrastructural features of SCNT embryos. Bovine oocytes were matured in IVMD101 medium (Research Institute for the Functional Peptides, Yamagata, Japan) and enucleated. The recipient oocytes were activated by treatment with Ca ionophore A23187 and then incubated with IVMD101 containing cycloheximide. Single donor cells (fibroblasts derived from an adult cow) were placed into the perivitelline space of the enucleated oocytes and the two cells were fused by electrofusion. The nuclear transfer embryos were cultured in IVD101 medium without bovine cumulus/granulosa cell co-culture in a humidified atomosphere of 5% CO₂/95% air at 38.5°C. Oxygen consumption rates by whole embryos were quantified individually by the SECM measuring system. Some of these embryos were prepared for observation by transmission electron microscopy. Statistical analyses were performed using one-way analysis of variance (ANOVA) and Fisher