Housing and Husbandry Factors Affecting Zebrafish (Danio rerio) 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 and global collaborative studies offer a unique solution. This study is the first global collaboration using the zebrafish (Danio rerio) novel tank test, a 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 females exhibit more anxiety-like behavior than males, underscoring sex as a critical variable. Housing conditions, including higher stocking densities and specific feed types, influenced anxiety levels. Optimal conditions (5 fish/L) and nutritionally rich feeds (e.g., rotifers), mitigated anxiety-like behaviors. Environmental stressors, like noise and transportation, significantly impacted behavior. We recommend standardizing protocols to account for sex differences, optimal stocking densities, nutritionally rich feeds, and minimizing stressors to improve zebrafish behavioral study reliability.

Keywords: Anxiety; Data reproducibility; Experimental standardization; Novel tank test; Sex differences; Zebrafish.

Fig. 1:

Summary of the different fields of scientific research that have publications using the zebrafish novel tank behavioral assay. This demonstrates the wide and varied use of the assay regardless of the field of study. Data obtained from the PubMed search described previously.

Fig. 2.

(A) Approximate locations of the different laboratories involved in the data acquisition. An interactive version of this map can be found: https://osf.io/4chwt/?view_only=024aa4208a83420c8fa38e2e0c64943a (B) Distribution of quantitative parameters across laboratories. (C) Differences on main variables comparing each laboratory to the average of the data. Data were represented as mean ± SEM and analyzed by ANOVA followed by Dunnett’s post-hoc test. Asterisks indicate statistical differences compared to the average (*p < 0.05, **p < 0.01, ***p < 0.001 and **** p < 0.0001).

Fig. 3.

Forest plots illustrating the effect size of A) distance traveled, B) entries to the top zone and C) time in the top zone for various laboratories when comparing females to males. Positive effect size represents higher values for females, and a negative effect size represents higher values for males. Each row represents a different lab, with the overall estimate shown at the bottom calculated using a random effects method. The size of each square is proportional to the weights of each effect size of each laboratory and the arrows indicate the confidence intervals of each lab effect size.

Fig. 4.

Interaction between rotifer feed (1 = yes, 0 = no) and time (min 1–5 in test) for time in top (s) (A). Interaction between transport prior to testing (1 = yes, 0 = no) and time (min 1–5 in test) for time in top (s) (B). Sixteen laboratories performed the test in a separate room and two laboratories fed rotifers.