A modified protocol for the extraction and analysis of cortisol in individual zebrafish, Danio rerio, embryo, and larva samples has been developed and evaluated. Recovery efficiency of the method was high, specifically calculated at 93.8% ± 6.5%. Dilution tests showed high parallelism, while increasing the number of individuals used in each extraction sample resulted in a linear, although slightly underestimated, increase of cortisol yield. Results of cortisol content from 0, 3, and 5 days postfertilization (dpf) fish using the proposed protocol were within the range of most published studies analyzing cortisol in pooled samples of 10-30 individuals. Moreover, 5 dpf larvae had significantly higher cortisol levels than embryos, a pattern commonly observed in literature. Finally, application of an osmotic stress in 5 dpf larvae led to a statistically significant increase in cortisol content.
Keywords: cortisol extraction; embryos; larvae; protocol; zebrafish.