Osmolarity and composition of cell culture media affect further development and survival in zebrafish embryos

Abstract

With the aim of carrying out chimaerism and somatic cell-midblastula transition (MBT) embryos co-culture experiments in freshwater fish species, we evaluated the effect of osmolarity and composition of two media commonly used in cell fish culture on MBT zebrafish embryos and their further development and survival. To this end, wild zebrafish dechorionated embryos in midblastula stage were cultured for 6 days (Experiment 1: 189 embryos) or 1 h (Experiment 2: 150 embryos) in three different media: Hanks' 10% (H-10), 35 mOsm; Hanks' 100% (CH), 315 mOsm; and L-15 with serum (L-15: 315 mOsm). High osmolarity affected the survival rate (6 days: L-15: 45.1% v. CH: 72.34% v. H-10: 100%, P , 0.05; after 6 days: 0% both in L-15 and CH) and slowed their developmental timing. Embryos showed tail deformation (curly) as well as body paralysis at 48 h when they showed tail movements at 28 h. Differences in tail deformation were observed between high-osmolarity groups (CH: 85.10% v. L-15: 98.04%; P , 0.05). In Experiment 2, no effects on survival rate were observed. Teratogenic effects were only observed in L-15 (L-15: 12.98% v. CH: 0%; P , 0.05). Loss of motility was not detected in any group at 48 h. Optimum osmolar condition for cultured cells and also embryonic cells is around 315 mOsm and so, during chimaerism experiments (usually practised at MBT stage), present results indicate that midblastula embryos can acceptably bear the effects caused by 315 mOsm (CH) for 1 h, even though this involves a certain delay in developmental timing.

Reduction in activity by noxious chemical stimulation is ameliorated by immersion in analgesic drugs in zebrafish

“…However, citric acid at a concentration of only 5% significantly altered the osmolarity of the tank water, which represents an almost 10-fold change. This value could be potentially damaging; however, a study by Perez-Camps and Garcia-Ximenez (2008) reported that the survival rate was 100% when zebrafish larvae were kept for 48 h at 315 mOsm and that larvae kept for 1 h at the same osmolarity had a survival rate of 100% after 6 days. Thus, further studies are needed to clarify the impact of osmolarity both in isolation and combined with pH.…”

EMBRYOLOGICAL AND MICROMANIPULATION TECHNIQUES IN ZEBRAFISH (Danio rerio) AND PACIFIC OYSTER (Crassostrea gigas)

“…Moreover, the fact of keeping the embryos in HBSS only during micromanipulation and transferring them to H10 medium for incubation (with the consequent sharp osmolarity change), did not prevent embryos continuing their development without any delay (Pérez-Camps and García-Ximénez, 2008). So, these results are of interest to take into account in future chimaerism works.…”

“…The other drop was the handling drop, which means the place where the chimaerism was performed and it was composed by HC medium (Pérez-Camps and García-Ximénez, 2008). The number of injected cells per recipient embryo ranged from 50 to 100 cells and they were deposited into the animal pole as was described by Lin et al (1992).…”

“…In a previous work, we tested that the survival and further development were not affected by the culture of embryos at mid blastula transition state (MBT) in an isosmolar medium (300 mOsm) for 1 hour (Pérez-Camps and García-Ximénez, 2008) and, no references related to these manipulation particularities in chimaerism were found in the reviewed literature. So, the aim of this study was to assess donor cell viability and chimaeric embryo survival when different manipulation media osmolarities are used in chimaerism assays.…”

Epigenetic reprogramming of somatic cells by nuclear transplant in zebrafish

“…However, it was proved that MBT embryos could support high osmolarities (300 mOSm) for 1h without further damage in the development (Pérez-Camps and García-Ximénez, 2008). In addition, Francisco-Simao and Cardona-Costa et al (2009) reported that the low osmolarity commonly used in chimaerism experiments does not affect the viability of the transplanted cells, but that it affects embryonic survival.…”