Effect of co-culture with theca interna on nuclear maturation of horse oocytes with low meiotic competence, and subsequent fusion and activation rates after nuclear transfer.

We conducted this study to examine whether or not co-culture with theca cells improves the maturation rate of horse oocytes with compact cumuli and to evaluate the cytoplasmic competence of oocytes after maturation by assessing fusion, activation and cleavage rates after nuclear transfer. We collected oocytes by scraping follicles from slaughterhouse-derived ovaries and classified them as having an expanded or a compact cumulus. Expanded oocytes were matured in M199 supplemented with 10% FBS and 5 microU/ml FSH for 24 h: compact oocytes were cultured in the same medium, or they were co-cultured in the same medium with theca interna explants, for 24 or 42 h. Oocytes were held with or without 10 microg/ml cytochalasin B, before washing and micromanipulation. and they were fused with donor fibroblasts by electrical pulse. Fused oocytes were activated with Ca ionophore/cycloheximide, cultured for 5 days, and stained with Hoechst to assess nuclear development. We considered oocytes with an enlarged nucleus, or having cleavage with multiple nuclei, to be activated. There was no significant difference in overall maturation rate between compact oocytes cultured with theca and compact controls. When these two groups were combined, there was a significant increase in the proportion of oocytes in MII between 24 and 42 h (P < 0.05). Expanded oocytes had a significantly higher rate of maturation than did compact oocytes (64% versus 25-30%; P < 0.001). There were no significant differences in rates of successful enucleation, fusion, activation or cleavage between compact control and compact + theca oocytes, nor between compact and expanded oocytes; however, expanded oocytes treated with cytochalasin B had a significantly higher survival rate after enucleation than did untreated expanded oocytes (P < 0.05). Three embryos developed from recombined oocytes, with maximum cleavage to 10 cells. The results of this study indicate that co-culture with theca cells does not increase either nuclear or cytoplasmic maturation of compact oocytes. Cytochalasin B is helpful in increasing survival of horse oocytes during enucleation. In vitro matured equine oocytes have the potential to develop into embryos after nuclear transfer; this is the first full report of production of cloned embryos in this species.