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This Article Full Text Full Text (PDF) All Versions of this Article: 68/2/571    most recent biolreprod.102.008771v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vajta, G. Articles by Callesen, H. Search for Related Content PubMed PubMed Citation Articles by Vajta, G. Articles by Callesen, H. Agricola Articles by Vajta, G. Articles by Callesen, H.

Handmade Somatic Cell Cloning in Cattle: Analysis of Factors Contributing to High Efficiency In Vitro¹

^a Section of Reproductive Biology, ^b Department of Animal Breeding and Genetics, and Section for Metabolism, Growth and Lactation, Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark ^c Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia ^d Genetics Australia Co-operative Ltd., Bacchus Marsh, Victoria 3340, Australia ^e Department of Anatomy and Physiology ^f Section of Reproduction, Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Denmark

Widespread application of somatic cell cloning has been hampered by biological and technical problems, which include complicated and time-consuming procedures requiring skilled labor. Recently, zona-free techniques have been published with limited or no requirement for micromanipulators. The purpose of the present work was to optimize certain steps of the micromanipulator-free (i.e., handmade) procedure, to analyze the morphology of the developing blastocysts, and to explain factors involved in the high efficiencies observed. Optimization of the procedure included selection of the appropriate medium for enucleation, orientation of pairs at fusion, timing of fusion, and culture conditions. As a result of these improved steps, in vitro efficiency as measured by blastocysts per reconstructed embryo and blastocysts per working hour was among the highest described so far. The cattle serum used in our experiments was superior to other protein sources for in vitro embryo development. One possible explanation of this effect is the considerable mitogenic activity of the cattle serum compared with that of commercially available fetal calf serum. Morphological analysis of blastocysts by inverted microscopy, inner cell mass-trophoblast differential staining, and transmission electron microscopy revealed high average quality. A high initial pregnancy rate was achieved after the transfer of single blastocysts derived by aggregation of two nuclear transfer embryos into recipients. The improved handmade somatic cell nuclear transfer method may become a useful technology as a simple, inexpensive, and efficient alternative to traditional somatic cell nuclear transfer.

¹ This work was supported by grant 53-00-0294 from the Danish Agricultural and Veterinary Research Council and grant FREM98-8 from the Danish Directorate for Development.

² Correspondence: Gábor Vajta, Department of Animal Breeding and Genetics, Danish Institute of Agricultural Sciences, Research Centre Foulum, DK-8830 Tjele, Denmark. FAX: 45 89 99 13 00; gabor.vajta{at}agrsci.dk

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