FACTORS AFFECTING THE EFFICIENCY OF NUCLEAR TRANSPLANTATION IN THE RABBIT EMBRYO

Procedures to improve nuclear transplantation efficiency in the rabbit were evaluated. We report the influence of recipient oocyte age on the different steps of nuclear transplantation. The effect of multiple pulses and the influence of manipulation medium and cytochalasin B in the post-fusion/activation medium on activation and development were studied. Recently ovulated oocytes were enucleated at a higher rate (60%) than aged oocytes (3%, p < 0.005); they also fused at a higher rate (85% vs. 26%, p < 0.001). Activation was low with freshly ovulated oocytes compared to aged oocytes (3% vs. 37%, respectively; p < 0.005), but was increased by using multiple pulses (85% vs. 68%, p < 0.05). Multiple pulses also improved development to blastocysts (48% vs. 5%, p < 0.001). Incubation of oocytes in a bicarbonate-buffered medium with 10% fetal calf serum for manipulation also enhanced rates of activation (100% vs. 89%, p < 0.05) and development of oocytes to blastocysts (77% vs. 26%, p < 0.001). Furthermore, 7.5 μg/ml cytochalasin B in the post-fusion/activation medium increased activation rates (78% vs. 50%, p < 0.05) and development to blastocysts of manipulated embyros (46% vs. 11%, p < 0.001). When the above modifications were applied, 10% (23/230) of the total nuclear transplant embryos (8-16-cell-stage donor nuclei) or 21% (23/110) of those transferred to recipients developed to offspring, rates similar to the development of nonmanipulated control embryos (10%, 4/41, p > 0.1).