An Improved Enucleation Method of Bovine Somatic Cell Nuclear Transfer

The aim of this work was to improve the rate of conventionally blind enucleation for bovine somatic cell nuclear transfer. The cross section of a 0.5 ml Eppendorf tube was attached with a sheet of 400 mesh/inch²-cell screen after the bottom of the Eppendorf tube had been cut, and put into a 1 mL Eppendorf tube. In experiment 1, the oocytes in the metaphase ? stage were placed on the membrane in the Eppendorf tube, and centrifuged at 1,000, 2,000, or 3,000 r/min for 10 min, respectively. The oocytes were stained with Hoechst 33342 and then the relative position of the first polar body to the chromosomes, and the efficiency of enucleation were evaluated. In experiment 2, enucleated oocytes were fused with granulosa cells, following centrifugation and enucleation, and the potential development of the reconstituted embryos was estimated. The results indicated that the rate of enucleation in oocytes after centrifugation at 2,000 r/min for 10 min was 86.6% with an angle less then 20° between the first polar body and chromosomes. The rate of enucleation in cells spun at 2,000 r/min was higher than that of controls (87.4% vs. 64.4%, P < 0.05). Furthermore, centrifugation of recipient oocytes did not have a detrimental effect on the development of reconstituted embryos following nuclear transfer. In conclusion, centrifugation assisted enucleation may significantly improve the rate of bovine oocyte enucleation.