Micromanipulation medium osmolarity compromises zebrafish (Danio rerio) embryo and cell survival in chimaerism experiments

In zebrafish chimaerism experiments, the cell injection can involve intra-embryonic cell lyses by osmolar effects. Moreover, the donor cells can be injured during manipulation due to osmolar changes into the transplant pipette. Therefore, the present study aimed to assess the effects of manipulation medium osmolarity on embryonic survival and donor cell viability. In Experiment I, 0.1 mu l to 0.15 mu l approximately of an isosmolar solution (300 mOsm) was injected into recipient embryos, which were kept at 300 (El) or 30 mOsm (E2). Survival at day 1 was significantly higher in the E2 group than in E1 (E1: 68% vs E2: 81%, p < 0.05), but after 5 days embryo survival in the El group was slightly higher. In Experiment II, donor cells from zebrafish embryos were exposed (or not) to a possible osmolarity change (inner pipette medium: 300 mOsm vs external medium: 30 or 300 mOsm) using two different micropipette outer diameters, 40-50 and 60-70 mu m. Cell mechanical damage was detected in the 40-50 mu m pipette (p < 0.05), but not by the handling medium osmolarity. Results recommend the use of a 300 mOsm manipulation medium and bore-sized pipettes adjusted as closely as possible to the donor cell size.