Interactions Between Feeding Protocols, Larval Density, and Methylene Blue During Zebrafish Larval Development

Conducting longitudinal experiments on zebrafish disease models has the potential to deepen understanding of disease progression. Longitudinal experiments require rearing of zebrafish embryos/larvae in the laboratory for extended time periods to enable use of laboratory equipment for serial observation. We observed that in our laboratory, wild-type larvae did not survive past 14 days. As we were unable to identify a study that compared different factors that could affect survival of larvae raised in the laboratory, we aimed to define the most relevant parameters that impact larval survival and growth. We found that prolonged access to food had the greatest beneficial effect on survival, regardless of rearing density or rearing media. However, rearing density did impact the activity of 2.5-week-old larvae. Methylene blue is a frequent additive to zebrafish embryo-rearing medium. Methylene blue can impact cellular health, and methylene blue mitigated the decrease in motility of densely grown larvae. Methylene blue also promoted growth: larvae raised in methylene blue were significantly larger at 1 month than their counterparts raised without methylene blue. These data highlight factors that promote survival and growth of larval zebrafish raised in the laboratory for longitudinal studies.