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The Study of Telophase Enucleation Method in YanBian Yellow Cattle Somatic Cell Nuclear Transfer

Author: LinTao
Tutor: FangNanZuo
School: Yanbian University
Course: Animal Genetic Breeding and Reproduction
Keywords: Telephase enucleation Oocyte Activation Injection Nuclear transfer Yanbian cattle
CLC: Q813
Type: Master's thesis
Year: 2009
Downloads: 9
Quote: 1
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Abstract


[Purpose] In general the enucleation of oocytes at the second metaphase stage(MII) were used as recipient cytoplasts for nuclear transfer, MII recipient cytoplasts enucleated with excitable fluorochromes and ultraviolet light .in order to improve the enucleation rate, Telophase II enucleation methods have been used for somatic cell nuclear transfer, it can improve enucleation rate and development of embryo, without using ultraviolet light and DNA stain, less cytoplasm were removed.[Methods] Oocytes were matured and exposed in 7% ethanol for 5min then returned to the incubator for 2-3h to allow for extrusion of the second polar body, enucleation subsequently, the first and the second polar bodies(pb1 and pb2) closely attached to cytoplasm were extruded, simultaneously, donor cells from an adult yanbian cattle injected by subzonal injection and intracytoplasmic injection, then electro-fusion and cultured in vitro, the control groups were MIL[Result] Oocytes matured for 28h activated showed higher rate of pb2 extrusion, only 35% of oocytes showed angle less 30°between pb1 and pb2,we found 100% of oocytes showed adjoin of the position of two polar bodies and chromatin when angles of Pb1 and pb2 less 30°by staining of DNA with Hoechst33342.The high enucleate rate was obtained by removed a small amount of cytoplasm surrounding pb1 and pb2 at Telophase II enucleation ; Enucleation by removing pb1 and pb2 and a small amount of cytoplasm (20% and 10%) were significantly higher enucleation rate than control, a larger amount of cytoplasm(30%) surrounding pb1 were removed at metaphase II stage, the cleavage rate(P<0.05) was improved in telephase II group, but blastocyst formation were not significantly. The telephase recipient oocytes received donor cells by subzonal injection supporting blastocyst formation, however, the directly cytoplast injection did not. Cleavage rate and proportion of blastocyst were not significantly different when activated oocytes at 24h ,26h ,28h and 30h of in vitor maturation(IVM).[Conclusion] Our results indicated that only 35% of oocytes showed that angle less 30°between pb1 and pb2, but100% of oocytes showed adjoin of the position of two polar bodies and chromatin when angles of Pb1 and pb2 less 30°. High enucleate rate was obtained by removed a small amount of cytoplasm surrounding pb1 and pb2 at Telophase II enucleation. Subzonal injection was superior to cytoplast injection in reconstruction of bovine embryos with telephase recipient oocytes; the effect of reconstructed embryo development were not significantly different in test of activated oocytes at IVM 24h, 26h, 28h and IVM 30h.

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