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Effect of cryopreservation on fusion efficiency and in vitro development into blastocysts of bovine cell lines used in somatic cell cloning

Published online by Cambridge University Press:  03 January 2006

O. Hayes
Affiliation:
Division of Animal Biotechnology, Centre for Genetic Engineering and Biotechnology, PO Box 6162, Havana 10600, Cuba.
LL. Rodríguez
Affiliation:
Department of Animal Sciences, Faculty of Veterinary Medicine, University of Concepción, Chillá;n, Chile.
A. González
Affiliation:
Division of Animal Biotechnology, Centre for Genetic Engineering and Biotechnology, PO Box 6162, Havana 10600, Cuba.
V. Falcón
Affiliation:
Division of Chemistry and Physics, Centre for Genetic Engineering and Biotechnology, PO Box 6162, Havana 10600, Cuba.
A. Aguilar
Affiliation:
Division of Animal Biotechnology, Centre for Genetic Engineering and Biotechnology, PO Box 6162, Havana 10600, Cuba.
F.O. Castro
Affiliation:
Department of Animal Sciences, Faculty of Veterinary Medicine, University of Concepción, Chillá;n, Chile.

Abstract

The outcome of the process of cloning by nuclear transfer depends on multiple factors that affect its efficiency. Donor cells should be carefully selected for their use in somatic nuclear transfer, and the protocols used for keeping frozen cell banks are of cardinal importance. Here we studied the effect of two protocols for freezing donor cells on fusion rate and development into blastocysts. Our hypothesis is that freezing affects cell membranes in a way that interferes with the fusion process upon cloning but without hampering normal cell development in vitro. We found that freezing cell lines without controlling the cooling rate gives lower yields in the fusion step and in the final development into blastocysts, compared with cells frozen with a controlled cooling rate of approximately 1°C/min. Transmission electron microscopy of the cells subjected to different freezing procedures showed major damage to the cells frozen with a non-controlled protocol. We conclude that freezing of donor cells for cloning is a critical step in the procedure and should be monitored carefully using a method that allows for a step-wise, controlled cooling rate.

Type
Research Article
Copyright
2005 Cambridge University Press

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