The Application Of Hand-Made Nuclear Transfer On The Somatic Cloning In Cattle

Based on bovine oocytes, this study means to explore a more suitable system for demecolcine-assisted enuleation on cattle. Following that, did some work on hand-made nuclear transfer (hand-made cloning) on bovine, such as zona-free nuclear transfer and WOWs culture system etc. The purpose of this study is to set up a suitable system of hand-made cloning for our laboratory and complete the basic work for the simplification of nuclear transfer and reduction on the cost of it.1. The demecolcine was used to induce the nucleus extrusions on bovine oocytes in this study. And some factors effecting the formation of nuclear extrusion were assessed, including oocytes with or without the first polar body, different concentration and duration of demecolcine-treatment. The result demonstrated: (1) treated with 0.5μg/mL demecolcine, oocytes induced for 1 h (76.00%) gained higher rate of extrusion than those for 0.5 h (61.18%),1.5 h (64.10%) and 2 h (63.46%). (2) And in 1 h demecolcine-treatment, 0.4μg/mL and 0.5μg/mL demecolcine group obtained the higher rate of extrusion (75.24% and 77.31% respectively) compared to the 0.3μg/mL (46.54%) and 0.6μg/mL group (60.66%). (3) And there was no significant difference on the extrusion rate between oocytes with (83.24%) or without (77.54%) first polar body. To sum up, bovine oocytes induced with 0.4μg/mL demecolcine for 1h can gain the higher rate of extrusion on bovine oocytes.2. Manipulated hand-made nuclear transfer and traditional nuclear transfer on bovine oocytes. (1) compared the technical procedures and time needed in these two methods; (2) compared the rates of enucleation in blind aspiration and demecolcine-assisted enucleation. (3) compared the developmental rate of reconstructed embryos in traditional nuclear transfer and hand-made nuclear transfer. And the results demonstrated that: the time needed in hand-made nuclear transfer (263～360 min) could be at least 60 min shorter than that in traditional one (345～450 min); as for the accuracy of enucleation, the demecolcine-assisted enucleation could gain higher accuracy(98.57%)rather than blind aspiration (63.21%). However, as for the overall rate of enucleation, the demecolcine-assisted enucleation still obtained the higher rate (45.48%) than blind aspiration (33.00%), although there was no significant difference between them. In addition, the rate of cleavage and blastocysts were still higher in hand-made nuclear transfer(78.79%和39.51%), but no significant difference compared with traditional one(72.00%和27.78%).