Use of a non-contact, infrared laser for zona drilling of mouse embryos: assessment of immediate effects on blastomere viability.

To compare the effects of using a non-contact infrared laser with acid Tyrode's for zona drilling, mouse embryos were used to examine both immediate effects on blastomere viability, using fluorescent markers, and development to the blastocyst stage in vitro. Preliminary experiments with isolated blastomeres established that the safe working distance for the lowest setting of the laser (pulse duration 5 ms) was 8 micro m. At the 2-cell stage, when it was possible to drill through the zona while maintaining this safe distance from adjacent blastomeres, no immediate damage was detected. At the 8-cell stage, blastomeres are generally <8 micro m away from the outer surface of the zona and often touch its inner surface. Hence it was not possible to drill the zona while maintaining a safe distance and as a consequence almost all embryos had at least one, and often more, damaged blastomeres. Acid Tyrode's drilling also frequently lysed an adjacent blastomere at this stage and after 24 h in culture cavitation was delayed compared with laser-drilled embryos. Cell numbers at the late morula/early blastocyst stage, analysed by differential labelling of trophectoderm and inner cell mass nuclei, were significantly reduced following zona drilling. With the laser-drilled embryos, this was in proportion to the immediate effects on blastomere viability, whereas with acid Tyrode's, the reduction was greater, indicating an adverse effect on cleavage rate. It is concluded that use of a non-contact laser, at an appropriate pulse duration and distance from adjacent blastomeres, avoids blastomere lysis and adverse effects on development caused by acid Tyrode's zona-drilling.