Effects of membrane piercing and the type of pronuclear injection fluid on development of in vitro-produced bovine embryos

We studied the effects of mechanical damage of plasma and pronuclear membranes on the development of in vitro-produced bovine zygotes. The effects of the type of injection fluid and the presence of DNA in the zygotes were also studied. In the first experiment, either the plasma membrane or both the plasma and pronuclear membranes of zygotes were pierced with a capillary filled with DNA-buffer. Additionally, pronuclear microinjections with either MilliQ-water or buffer were performed. In the second experiment, pronuclear microinjections with buffer containing either none or 2 μg/ml of DNA were performed. Development of cleaved embryos to compact morulae and blastocysts at Day 7 was monitored. Results of Experiment 1 indicate that membrane piercing does not decrease development of cleaved embryos as compared with that of the controls (30.8, 28.8 and 29.9% compact morulae and blastocysts for controls, plasma membrane and pronuclear membrane pierced groups, respectively). Pronuclear microinjections decreased development significantly as compared with that of the controls, but no differences were observed between the effects of water and buffer (29.9, 18.4 and 15.5% compact morulae and blastocyst, respectively). Results of Experiment 2 showed that inclusion of DNA into the injection buffer decreased development even more drastically (36.7, 27.5 and 14.5% compact morulae and blastocyst in the control, buffer-injected and DNA-injected groups, respectively).