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Title: CELL DONOR INFLUENCES SUCCESS OF PRODUCING CATTLE BY SOMATIC CELL NUCLEAR TRANSFER

Author
item Powell, Anne
item Talbot, Neil
item WELLS, K - REVIVICOR, INC, VA
item KERR, D - UNIV OF VERMONT
item PURSEL, V - USDA, ARS, ANRI, BGL
item Wall, Robert

Submitted to: Biology of Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/3/2004
Publication Date: 7/1/2004
Citation: Powell, A.M., Talbot, N.C., Wells, K.D., Kerr, D.E., Pursel, V.G., Wall, R.J. 2004. Cell donor influences success of producing cattle by somatic cell nuclear transfer. Biology of Reproduction. 71(1):210-6.

Interpretive Summary: To try to identify some of the parameters that limit the success rate of bovine somatic cell nuclear transfer (cloning), nuclear donor fibroblasts from 6 Jersey fetuses we culture under a number of different conditions. The resulting healthy cloned embryos were transferred to foster mothers. Varying the kind of culture media, amount of serum in the media, or the amount of oxygen to which the donor cells were exposed had an influence on the proportion of embryos that reached the blastocyst stage of development. But the best condition differed depending on the fetus from which the cells came. There we not enough embryo transfers performed to detect a difference in the various culture conditions on pregnancy rate or calves born. However, it was clear that the fetuses from which the donor cell was harvested made a difference in the overall success rate of nuclear transfer. All of the 8 live calves came from 2 of the 6 fetuses tested. The pregnancy rate of cows receiving embryos produced by those 2 'good' fetuses was twice as high as pregnancy rates of the other recipients and may be a useful parameter for distinguishing between 'good' and 'bad' donor fetuses.

Technical Abstract: To assess sources of variation in nuclear transfer efficiency, bovine fetal fibroblasts (BFF), harvested from 6 Jersey fetuses were cultured under various conditions. After transfection, frozen-thawed lung or biceps femoris muscle BFF donor cells were selected in MEM or DMEM media, with 5 or 20% O2 or 0.5 or 10% serum and fused to enucleated oocytes. Couplets were activated with ionomycin and 6-dimethylaminopurine. Of 4007 fused couplets generated, 80% fused yielding 927 (23%) blastocysts of which 650 were transferred to 330 recipients. Fusion rate was not influence by any parameter, source fetus influenced proportion couplets developing to blastocysts (P = 0.004) and often interacted with other parameters tested. Overall, 30 d non-return rate was 51%, but 82% of pregnancies were lost by 8 weeks. Cells from 2 of the 6 fetuses produced embryos that maintained pregnancies to term, resulting in 8 live calves. Pregnancy rates of recipients carrying embryo from the 2 productive donor fetuses, on day 56, was at least double that of other recipients and may provide a fitness indicator of BFF cell sources for nuclear transfer. We conclude that a significant component in determining the success of somatic cell nuclear transfer success is the source of the nuclear donor cells.