Birth of a cloned calf derived from a vitrified hand-made cloned embryo
R. T. Tecirlioglu A , A. J. French A , I. M. Lewis B , G. Vajta C , N. A. Korfiatis A , V. J. Hall A , N. T. Ruddock A , M. A. Cooney A and A. O. Trounson AA Centre for Early Human Development, Monash Institute of Reproduction and Development, Monash University, 27–31 Wright Street, Clayton, Victoria 3168, Australia.
B Genetics Australia Co-operative Ltd, Bacchus Marsh, Victoria 3340, Australia.
C Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark.
D To whom correspondence should be addressed. email: tayfur.tecirlioglu@med.monash.edu.au
Reproduction, Fertility and Development 15(7) 361-366 https://doi.org/10.1071/RD03052
Submitted: 24 July 2003 Accepted: 1 December 2003 Published: 1 December 2003
Abstract
The hand-made cloning (HMC) technique describes a simplified nuclear transfer process without the need for micromanipulators. The technique involves manual bisection of zona-free oocytes, selection of cytoplasts by Hoechst staining and fusion of a single somatic cell and two cytoplasts. In this proof-of-principle experiment, the objective was to examine the developmental competence of HMC embryos following embryo transfer. Modifications to the original method include not selecting of matured oocytes and simultaneous fusion of cytoplasts and karyoplast. Blastocyst rates for embryos cultured in the glass oviduct system as singles (10.5%; 24/228) or in pairs (16.1%; 36/224) did not differ significantly. Fresh and vitrified–thawed blastocysts were transferred to 16 synchronised recipients (three to four embryos per recipient). Ultrasound examination on Days 35–45 showed an initial pregnancy rate of 43.8% (7/16) and a pregnancy rate >8 months of 12.5% (2/16). A male cloned calf (42 kg) derived from a vitrified HMC blastocyst was delivered by Caesarean section on Day 271. The birth and ongoing survival (15 months, 243 kg) of a healthy and apparently normal calf, combining both HMC and vitrification technologies, provides a ‘proof of principle’ of the technology and a promising alternative to traditional nuclear-transfer techniques.
Extra keywords: bovine
Acknowledgments
This research gratefully acknowledges the support of The Cooperative Research Centre (CRC) for Innovative Dairy Products. We appreciate the assistance of Ms Shara Downie and Renee Hodgson and staff (Genetics Australia Co-operative Ltd, Bacchus Marsh, Victoria, Australia) for oocyte supply and ongoing animal care, Ms Catriona Thompson, Victorian Institute of Animal Science for microsatellite analysis and Dr Garey Dawson and laboratory staff (Monash Medical Centre, Southern Health) for cytogenetic analyses.
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