Behavioral test batteries in zebrafish: methodological variability, experimental design, and implications for...

The zebrafish (Danio rerio) is a widely used model organism for studying neurobehavioral processes and neuropsychiatric disorders. Numerous protocols in adult zebrafish assess anxiety-like, locomotor, social and cognitive responses. The increasing experimental complexity and experimentation throughput has led many studies to combine multiple assays into behavioral test batteries. However, the methodological implications of such multi-test designs remain insufficiently understood. Here, we examined current practices in zebrafish behavioral batteries through an initial laboratory survey followed by a systematic literature analysis. Responses from 24 active zebrafish behavioral neuroscience laboratories were used to characterize experimental workflows and guide a targeted PubMed literature search. We further analyzed 357 studies employing the novel tank test (NTT) to assess the prevalence of multi-assay designs, identify commonly co-reported behavioral paradigms, and evaluate methodological variability. Our analyses confirm the central role of the NTT, frequently combined with the light-dark, open field, social interaction, and Y-maze tasks, and indicate that multi-assay experimental designs are common in the literature. However, methodological descriptions also often lacked sufficient clarity on whether assays were conducted sequentially in the same animals (as test batteries) or performed in independent cohorts. These findings highlight an important methodological gap in the field, and emphasize the need for greater clarity and consistency in the design and reporting of zebrafish behavioral batteries to improve the interpretability and comparability of multi-domain behavioral phenotyping.