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DEVELOPMENT OF CLONED BOVINE EMBRYOS DERIVED FROM SERUM-STARVED AND ROSCOVITINE TREATED ADULT DONOR CELLS IN G1G2 AND BARC MEDIUM.
Gibbons, John1, Arat, Sezen1,2, Rzucidlo, S. Jacek1, Waltenburg, Rachel1, Stice, Steven1, 1 2
ABSTRACT- The objective of this study was to examine development of nuclear transfer (NT) embryos derived from cells synchonized by two different treatments, in two different embryo culture medium. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with fetal bovine serum (FBS), sodium pyruvate, penicillin/ streptomycin, rIGF-1, bFSH, and bLH. Bovine granulosa cells were isolated from ovarian follicles by ultrasound guided transvaginal aspiration and cultured in DMEM-F12 supplemented with 10% FBS, and penicillin/ streptomycin at 37 oC in 5% CO2 in air. Prior to NT, donor cells were cultured either in media with 0.5 % serum for 4 days or were exposed to 15 M Roscovitine for 24 hours. Matured oocytes labeled with DNA fluorochrome Hoechst 33342 were enucleated under UV to ensure removal of the chromatin. A single cell was inserted into the perivitelline space of the enucleated oocyte. Nuclear transfer units were fused by using a needle-type electrode in the Zimmerman's medium. After fusion, NT units were activated using a combination of calcium ionophore, cytochalasin B, and cycloheximide and cultured either in G1G2 or BARC medium for 7 days. Although the cleavage rate was higher (P<0.05) in the NT embryos constructed from donor cells treated with 0.5% serum (61.3 ± 6.0%) compared to cells treated with roscovitine (40.0 ± 6.8%), the blastocyst formation rate was not different between cells treated with 0.5% serum (18.0 ± 0.5%) or roscovitine (14.7 ± 1.1%). There were no significant differences in the rates of cleavage for embryos cultured in G1G2 or BARC (53.2 ± 8.9%, 48.1 ± 6.7%; respectively) or blastocyst formation (15.6 ± 1.4 %, 17.1 ± 0.6%; respectively). The early pregnancy rates (40-45 days) from transferred blastocysts constructed with donor cells treated with 0.5% serum (38.6 ± 8.6 %) or roscovitine (49.5 ± 7.2%) were not different, however; early pregnancy rates were lower (P<0.05) from blastocysts cultured in G1G2 (27.0 ± 3.8%) compared to BARC (61.1 ± 2.7%). These results indicate that donor cell treatment with 0.5% serum or roscovitine prior to nuclear transfer did not impact embryo development, however; embryos cultured in BARC supported early fetal development more efficiently than embryos cultured in G1G2.
KEY WORDS: bovine, cloning, embryo culture, nuclear transfer
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