THE POTENTIAL PRODUCTION OF RECONSTRUCTED EMBRYO OF LOCAL GOAT THROUGH INTRACYTOPLASMIC DIRECT NUCLEAR INJECTION USING CUMULUS DONOR CELL



Ciptadi G.(1*), S.B. Sumitro(2), A. Boediono(3), M. S. Djati(4), T. Susilawati(5), Budi Siswato(6)

(1) Fac. Of Animal Husbandry, Brawijaya University, Malang
(2) Fac. MlPA-Biology, Brawijaya University, Malang
(3) Fac of Veterinary Sci., IPB Bogor
(4) Fac. MlPA-Biology, Brawijaya University, Malang
(5) Fac. MlPA-Biology, Brawijaya University, Malang
(6) Fac. Of Medicine /RSUD Syaiful Anwar, Brawijaya University, Malang
(*) Corresponding Author

Abstract


The aim of this research is to develop methods for producing reconstructed
embryo of local goat after intra cytoplasmic direct nuclear injection with cumulus donor cells into enucleated M-ll oocyte recipient. Reconstructed embryo was performed by injection of cumulus donor cell intracytoplasmic. Donor cells were cultured in standard protocol until confluence with 10 % FBS supplementation. Cells culture done using CO1 incubator 5 “/0 38 “C with maximum humidity, cultured in 2 — 3 passages and then it’s used as donor cells after trypsinized with 0.1 % trypsin-EDTA. Enucleation was done by blind enucleation method of l0 — 25 % cytoplasmic near second polar body of M-II oocyte recipient. Reconstructed cells were activated chemically by Ethanol + 6- DMAP and (‘n ionophore A-23187 + 6—DMAP. Variable observed are cleavage rate and embryo development compared with paithenogenetic activation as a control. Results showed that rcconstructctl embryo still very low success to reach morula stage ( 1.2 — 1.7 %) in in \-itro development culture system using TCM 199. The cleavage rate of this reconstructed embryos are 12.41 % (ethanol + 6-DMAP) and l4.58 % (Ca ionophore A 23187). Viability of donor cell is about 78.30 %, selected for their medium and small size of population that probably in G0/G1 phases (4 — 6 um). It was concluded that intra cytoplasmic direct nuclear injection could be use as method of reconstructed embryo production of local goat. lt is necessarily to done further research on analyze of cellular damage after enucleation and injection


Keywords


Recmrstrucled Embryo, Intracytoplasmic, Enucleation, Cumulus Cells, Activation.

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