39 CHROMOSOME ABNORMALITIES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED BY “HANDMADE CLONING”
A.S. Jakobsen A , G. Vajta B , P.D. Thomsen A , H. Callesen B and T. Greve AA Department of Agricultural Science, Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Denmark
B Department of Breeding and Genetics, Danish Institute of Agricultural Sciences, 8830 Tjele, Denmark. Email: asj@kvl.dk
Reproduction, Fertility and Development 17(2) 169-169 https://doi.org/10.1071/RDv17n2Ab39
Submitted: 1 August 2004 Accepted: 1 October 2004 Published: 1 January 2005
Abstract
Embryos produced by nuclear transfer using somatic cells as donors exhibit a lower developmental competence compared to in vivo developed and IVM/IVF/IVC embryos. The so-called handmade somatic cell nuclear transfer (HSCNT or handmade cloning) that was recently presented (Vajta et al. 2003 Biol. Reprod. 68, 571–578) has, however, improved the reconstructed embryo and subsequent blastocyst rate. Further, the high total cell number (n = 216) and the high ratio of cells allocated to the inner cell mass (35%) as well as the initial pregnancy rate of 48% on Day 28 following nonsurgical transfer of HSCNT embryos indicated a high average quality. But still the pregnancy loss of HSCNT embryos was high and resulted in a birth rate of 8%. The aim of this study was to estimate the chromosomal variation in HSCNT embryos using fluorescent in situ hybridization (FISH) and to evaluate this as an additional parameter of embryo quality. Nuclei from 49 Day 7 HSCNT embryos from five independent trials were isolated by hypotonic treatment and fixed. Then the nuclei were hybridized with differentially labelled cJAB8 and p33E39 probes that hybridize specifically to the centromeric region of chromosome 6 and 7 (Viuff et al. 2000 Biol. Reprod. 63, 1143–1148). A total of 6715 nuclei were analyzed for chromosomal abnormalities, and the percentage of nuclei with false negative scores represented 1.8%. Only 4.1% of the embryos (2 of 49) had a completely normal diploid composition, while the remaining 47 embryos exhibited different types of mixoploidy. Of the 47 mixoploid embryos, 87% contained more than one type of chromosomal variation with diploid/triploid/tetraploid being the most frequent constitution (43% of the 47 mixoploid embryos). No pure polyploid embryos were observed. The percentages of HSCNT embryos in the groups of 0%, 1–25%, and 26–100% polyploid cells were 4.1%, 67.5%, and 28.6%, respectively. This is significantly higher than the corresponding figures produced and analyzed by comparable methodology of bovine IVM/IVF/IVC embryos (Viuff et al. 1999 Biol. Reprod. 60, 1273–1278). However, 71.4% of the HSCNT embryos contained less than 25% polyploid cells; this low level may not have compromised the developmental competence of these embryos.
