FACTORS AFFECTING CELL VIABILITY DURING BISECTION OF BOVINE EMBRYOS

The aim of this study was to investigate the effects of temperature, c ytochalasin B, sucrose, cell number and developmental stage of embryos on cell loss and cell lysis during embryo splitting. Day-7 morulae an d blastocysts were bisected using a metal blade. In Experiment 1, spli tting of embryos in control medium (PBS+10% fetal calf serum) was comp ared with splitting in the presence of 7.5 mu l/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 viabil ity 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 n o effect of cell number (P=0.85), the influence of the developmental s tage can be contributed to morphological changes rather than to increa se of cells associated with this change. In Experiment 1, the cytochal asin B-treated embryos contained a higher percentage of viable cells t han the control embryos after removal of the developmental stage effec t (P<0.01). In Experiment 2, no effect on sucrose could be observed. I n Experiment 3, the combined use of sucrose and cytochalasin B tended to increase the proportion of cells surviving bisection, but this diff erence was not significant. In Experiment 1, there was a correlation b etween viable cells and temperature during splitting (r=0.42, P=0.05; temperature range 8.1 degrees C to 15.6 degrees 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.