Housing and husbandry factors affecting zebrafish novel tank test responses: a global multi-laboratory study

Abstract

The reproducibility crisis in bioscience, characterized by inconsistent study results, impedes our understanding of biological processes. Global collaborative studies offer a unique solution to this problem. Here, we present a global collaboration using the zebrafish (Danio rerio) novel tank test, a popular behavioral assay for anxiety-like responses. We analyzed data from 20 laboratories worldwide, focusing on housing conditions and experimental setups. Our study included 488 adult zebrafish, tested for 5 min, focusing on a variety of variables. Key findings show that female zebrafish exhibit more anxiety-like behavior than males, highlighting sex as a critical variable. Housing conditions, including higher stocking densities and specific feed types, also influenced anxiety levels. Optimal conditions (5 fish/L) and nutritionally rich feeds (for example, rotifers) mitigated anxiety-like behaviors. Environmental stressors, such as noise and transportation, significantly impacted behavior. We recommend standardizing testing protocols to account for sex differences, optimal stocking densities, nutritionally rich feeds and minimizing stressors to improve the reliability of zebrafish behavioral studies.

Fig. 1. Comparative analysis of quantitative parameters across laboratories.

a, The distribution of quantitative parameters across laboratories. b, Differences in main variables comparing each laboratory with the average of the data. Data are represented as mean ± s.e.m. and analyzed by ANOVA followed by Dunnett’s post-hoc test. Asterisks indicate statistical differences compared with the average (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).

Fig. 2. Forest plots of sex differences in behavioral metrics across laboratories.

a–c, Forest plots illustrating the effect size of distance traveled (a), entries to the top zone (b) and time in the top zone (c) for various laboratories when comparing females and males. A positive effect size indicates higher values for females, while a negative effect size indicates higher values for males. Each row represents a different laboratory, with the overall estimate shown at the bottom (green diamonds), calculated using a random-effects method. The size of each circle is proportional to the weights of each effect size of each laboratory, and the arrows indicate the CIs of each laboratory effect size.

Fig. 3. Effects of rotifer feeding and transport on time spent in the top of the tank.

a, The interaction between rotifer feed (1 = yes, 0 = no) and time (minutes 1–5 in the test) for time spent in the top of the tank (s). Fish that were fed rotifers (1) showed a more rapid increase in time spent in the top compared with those that were not (0) throughout the test period. b, The interaction between transport before testing (1 = yes, 0 = no) and time (minutes 1–5 in the test) for time spent in the top of the tank (s). Fish tested in the same room where they were housed (0) spent more time in the top overall. However, those transported to a separate testing room (1) showed a gradual increase in time spent in the top, finishing the testing period with a similar amount of time in the top as those tested in their home tank room (0). Sixteen laboratories performed the test in a separate room, and two laboratories fed rotifers.