Cytogenetic analysis of diploidy in cloned bovine embryos using an improved air-dry karyotyping method.

Of the few published studies on the cytogenetic analyses of bovine nuclear transferred (NT) embryos, results differ between air-dry and fluorescent in situ hybridization (FISH) procedures. A modified air-dry procedure is reported in this study that provides more metaphase plates for analysis. Day 5 and Day 7 bovine NT embryos were cultured in colcemid-containing CR1aa for 10-12 or 16-18 h, then treated in hypotonic sodium citrate for 3-5 min. The standard procedure of 5h in colcemid and 15-20 min in hypotonic solution was the control. A much higher (P<0.01) percent of mitotic nuclei was observed in the experimental groups. The 33 and 41% mitotic nuclei were obtained from 10 to 12 h and 16 to 18 h-colcemid-treated Day 5 embryos, respectively, which was higher (P<0.001) than the control (15%). The mitotic nuclei in Day 7 NT embryos were 24% in 10-12 h- and 28% in 16-18 h-colcemid-treated groups, which also was higher (P<0.05) than the control (10%). The majority of analyzable embryos were diploid. Analyses of mixoploid embryos showed on average that 70% of the cells were diploid. Day 5 mixoploid embryos contained numerically higher polyploid cells than Day 7 embryos, although statistically there were no differences. We concluded that the modified air-dry method provided a larger source of mitotic nuclei for chromosome analyses of cloned bovine embryos.