Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

Comparison of two approaches to nuclear transfer in the bovine: hand-made cloning with modifications and the conventional nuclear transfer technique

R. Tayfur Tecirlioglu A D E , Melissa A. Cooney A D , Ian M. Lewis B , Natasha A. Korfiatis A D , Renee Hodgson B D , Nancy T. Ruddock A D , Gábor Vajta C , Shara Downie B D , Alan O. Trounson A , Michael K. Holland A D and Andrew J. French A D

A Centre for Early Human Development, Monash Institute of Medical Research, Monash University, Clayton, Victoria 3800, Australia.

B Genetics Australia Co-operative Ltd, PO Box 195, Bacchus Marsh, Victoria 3340, Australia.

C Danish Institute of Agricultural Sciences, Postboks 50, 8830 Tjele, Denmark.

D Cooperative Research Centre for Innovative Dairy Products (CRC-IDP), Melbourne, Victoria 3000, Australia.

E Corresponding author. Email: tayfur.tecirlioglu@med.monash.edu.au

Reproduction, Fertility and Development 17(5) 573-585 https://doi.org/10.1071/RD04122
Submitted: 11 October 2004  Accepted: 5 April 2005   Published: 23 May 2005

Abstract

The aim of the present study was to compare the in vitro and in vivo developmental competence of hand-made cloning (HMC) embryos with the conventional nuclear transfer (NT) method using five somatic cell lines and in vitro-fertilised (IVF; control) embryos. Modifications to the HMC procedure included fusion efficiency optimisation, effect of cytoplasmic volume and cloned embryo aggregation. The developmental competence of blastocysts from each of the treatment groups and cell lines used was assessed following transfer to 345 recipients. Vitrification was also used to enable management of recipient resources and to assess the susceptibility of membranes to cryopreservation following zona removal. Increasing cytoplasmic volume to 150% or aggregating two embryos improved the blastocyst development rate and increased the total cell number. Although HMC embryo transfers established a significantly higher pregnancy rate on Day 30 than fresh IVF or NT embryo transfers, the overall outcome in terms of cloned live births derived from either fresh or vitrified/thawed HMC or NT embryo transfers across the five cell lines did not differ. The birth and continued survival of clones produced with HMC technology with equivalent efficiency to NT shows that it can be used as an alternative method for the generation of cloned offspring in the bovine.

Extra keyword: aggregation, cytoplasmic volume, nuclear transfer, vitrification, zona free.


Acknowledgments

The authors gratefully acknowledge the support of the CRC-IDP. The authors appreciate the assistance of Genetics Australia Co-operative (Bacchus Marsh, Victoria, Australia) for oocyte supply and ongoing animal care, Ms Catriona Thompson (Victorian Institute of Animal Science, Attwood, Victoria, Australia) for microsatellite analyses, Dr Mark M. Williamson (Victorian Institute of Animal Science, Attwood, Victoria, Australia) for post-mortem examinations, and Dr Garey Dawson and laboratory staff (Southern Cross Pathology Australia, Monash Medical Centre, Southern Health, Clayton, Victoria, Australia) for cytogenetic analyses.


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