53 MODIFIED AIR-DRY METHOD REVEALS THAT DIPLOID CELLS ARE PREDOMINANT IN CLONED BOVINE EMBRYOS

Abstract

Chromosomal analysis is important for evaluating embryo quality. Reports on the cytogenetic analyses of bovine nuclear transfer (NT) embryos, however, have varied according to methods used (Mohamed NMS et al. 2000 J. Vet. Med. Sci. 62, 339–342; Arat S et al. 2002 Biol. Reprod. 66, 1768–1774; Booth PJ et al. 2003 Biol. Reprod. 68, 922–928). An improved air-dry method was developed in this study to specifically investigate the chromosomal composition of bovine NT embryos. NT embryos were obtained by transferring cumulus cells into enucleated oocytes, the fused embryos were then co-cultured in CR1aa with bovine cumulus cells. Day 5 embryos were cultured in the presence of 0.01μg mL⁻¹ colcemid for 18 h (classical method is 5 h) and then in 1% sodium citrate for 3 to 5 min (classical method is 15 to 30 min), and fixed on slides, total of 55 embryos were analyzed. The categories for the chromosome composition were classified as follows: diploid (2n), triploid (3n), tetraploid (4n), mixoploid (including 2n/3n, 2n/4n, and 2n/3n/4n). Any embryo that did not show an interpretable metaphase spread due to gross overspreading or clumped chromosomes was not classified. The mitotic index (number of metaphase plates/total cell number of analyzed embryo) was 41.1% (700/1701), which is significantly higher (P < 0.001) than that obtained by the classical method (11.9%, 50/419). Of 700 metaphase plates observed, 608 (86.9%) were diploid. Triploidy and tetraploidy were 49 (7%) and 42 (6%), respectively. There were 72.7% (40/55) of the embryos diploid, and 27.3% (15/55) polyploid or mixploid, which included 3n (1/15), 4n (1/15), 2n/3n (5/15), 2n/4n (3/15), and 2n/3n/4n (4/15). Of the 15 non-diploid embryos, 64% of the total cells were diploid. The results of this study indicate that diploidy is the predominant state in bovine Day 5 NT embryos.