1995 Fiscal Year Final Research Report Summary
Study of cloning embryos in bovine using parthenogenetic cells
| Project/Area Number |
05806041
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Applied veterinary science
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| Research Institution | United Graduate School of Veterinary Science, Yamaguchi University |
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Principal Investigator |
SUZUKI Tatsuyuki United Graduate School of Veterinary Science, Yamaguchi University Professor, 大学院・連合獣医学研究科, 教授 (00216409)
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| Project Period (FY) |
1993 – 1995
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| Keywords | Aggragation / Parthenogenetic / IVF / Chimera / Cloning |
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| Research Abstract |
Oocytes were aspirated from the ovaries collected from abatoir and matured for 30-32h at 38.5C under 5% CO2 in air. To induce parthenogenetic activation, these matured oocytes were suspended in culture media containing 7% ethanol for 10mins followed by treatment using cytochalasin D (5h) to suppress the second polar body extrusion, so producing diploid parthenogenes. The oocytes were then washed and cultured in vitro on feeder layrs of bovine cumulus cells for further development. The normal fertilized embryos were obtained by in virtro maturation, fertilization and culture procedures. The follicular oocytes were matured for 20-24h at 38.5C under 5%CO2 in air. These matured oocytes were then fertilized by in vitro capacitated sperm and followed by cultured in vitro. Aggregation chimeras were produced by methods : (1) injection of 2 blastomeres obtained from 16 cell stage fertilized embryo into 4-cell parthenogenetic cell ; 2/16F->4/4P, (2) injection of 4 balstomeres obtained from 16 cell stage fertilized embryo into 4-cell parthenogenetic cell ; 4/16->4/4P.(3) injection of fertilized demi-embryo (8-cell stage) into parthenogenetic demi-embryo (8cell-stage) ; 4/8F->4/8P, (4) aggregation of whole fertilized embryo and parthenogenetic cell ; 8/8F<->8/8P.The developmental rate of aggregated embryos produced by aggragation of whole embryos to morulae and balstocysts were significantly higher (P<.05) than injection of 2/16F-> 4/4p, 4/16F-> 4/4P and 4/8F -> 4/8P.By method (2), the identical twin male calves were delivered on day 267 from 1 recipient in which XY chromosome plates were detected in each calf. From method (4), single male calf which XX and XY chromosome plates were detected was delivered on day 261.
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