Factors affecting cell viability during bisection of bovine embryos

Abstract

The aim of this study was to investigate the effects of temperature, cytochalasin B, sucrose, cell number and developmental stage of embryos on cell loss and cell lysis during embryo splitting. Day-7 morulae and blastocysts were bisected using a metal blade. In Experiment 1, splitting of embryos in control medium (PBS + 10% fetal calf serum) was compared with splitting in the presence of 7.5 μl/ml cytochalasin B. In Experiment 2, the control medium was compared with medium supplemented with 200 mM sucrose. In Experiment 3, the control medium was compared with medium supplemented with sucrose and cytochalasin B. Cell viability was measured by staining nuclei of embryos with Hoechst 33258 and propidium iodide. Cells with nuclei exhibiting pink fluorescence were considered lysed, while blue fluorescence was considered an indication of viable cells. Cells disaggregated during splitting were classified as extruded cells. An effect of the developmental stage was observed in the pooled data from the control groups of the 3 experiments, with a higher proportion of viable cells in bisected morulae compared with bisected blastocysts (77.6 vs 70.0%; P = 0.003). However, as there was no effect of cell number (P = 0.85), the influence of the developmental stage can be contributed to morphological changes rather than to increase of cells associated with this change. In Experiment 1, the cytochalasin B-treated embryos contained a higher percentage of viable cells than the control embryos after removal of the developmental stage effect (P < 0.01). In Experiment 2, no effect on sucrose could be observed. In Experiment 3, the combined use of sucrose and cytochalasin B tended to increase the proportion of cells surviving bisection, but this difference was not significant. In Experiment 1, there was a correlation between viable cells and temperature during splitting (r = 0.42, P = 0.05; temperature range 8.1 °C to 15.6 °C). No correlation was found in any other group in any of the experiments, nor in the pooled data from the control groups in the 3 experiments.