In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovi...

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Bibliographic Details
Published in:Genetics and molecular research 2010-01, Vol.9 (1), p.295-302
Main Authors: Gerger, R.P.C, Ribeiro, E.S, Forell, F, Bertolini, L.R, Rodrigues, J.L, Ambrósio, C.E, Miglino, M.A, Mezzalira, A, Bertolini, M
Format: Article
Language:English
Subjects:
animal breeding
Animals
Cattle
Cell Aggregation
cell culture
Cell Culture Techniques - methods
Cells, Cultured
Cloning, Organism - methods
embryo (animal)
Embryo, Mammalian - cytology
Embryo, Mammalian - embryology
molecular cloning
Research Embryo Creation - methods
Skin - cytology
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Summary:The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.
ISSN:1676-5680
1676-5680
DOI:10.4238/vol9-1gmr690