Housing and husbandry factors affecting zebrafish novel tank test responses: a global multi-laboratory study
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.
Authors
- Allan V. Kalueff (ORCID: https://orcid.org/0000-0002-7525-1950)
- Tatiana O. Kolesnikova (ORCID: https://orcid.org/0000-0002-5561-8583)
- Stefani M Portela (ORCID: https://orcid.org/0000-0002-6254-4068)
- Jamie Corcoran (ORCID: https://orcid.org/0009-0001-7880-475X)
- Júlia Robert de Sousa Teixeira
- João V Borba
- Maurizio Porfiri (ORCID: https://orcid.org/0000-0002-1480-3539)
- Leonardo M. Bastos (ORCID: https://orcid.org/0000-0001-8538-6405)
- Matthew O. Parker (ORCID: https://orcid.org/0000-0002-7172-5231)
- Amaury Farías-Cea (ORCID: https://orcid.org/0000-0001-5468-4936)
- Alexander V. Zhdanov (ORCID: https://orcid.org/0000-0001-5787-2035)
- Bárbara D. Fontana (ORCID: https://orcid.org/0000-0003-2832-400X)
- David S. Galstyan (ORCID: https://orcid.org/0000-0001-6220-3485)
- Caroline H. Brennan (ORCID: https://orcid.org/0000-0002-4169-4083)
- Adele Leggieri (ORCID: https://orcid.org/0000-0003-0449-9665)
- Bárbara Dutra Petersen (ORCID: https://orcid.org/0000-0003-3781-0077)
- Patricio Iturriaga‐Vásquez (ORCID: https://orcid.org/0000-0003-0553-1014)
- Ana Carolina Luchiari (ORCID: https://orcid.org/0000-0003-3294-7859)
- Ângelo Piato (ORCID: https://orcid.org/0000-0001-5109-7306)
- Ryan Y. Wong (ORCID: https://orcid.org/0000-0003-0236-672X)
- Justin W. Kenney (ORCID: https://orcid.org/0000-0001-8790-5184)
- Luiz Moutinho (ORCID: https://orcid.org/0000-0001-9074-4449)
- Sergey L. Khatsko (ORCID: https://orcid.org/0000-0001-5921-6680)
- Eduardo Ronconi Dondossola (ORCID: https://orcid.org/0000-0002-5494-7369)
- Maureen L. Petrunich-Rutherford (ORCID: https://orcid.org/0000-0002-5142-0457)
- Denis B. Rosemberg (ORCID: https://orcid.org/0000-0003-2538-8761)
- Catia A Salazar
- Violeta Medan (ORCID: https://orcid.org/0000-0002-3612-0659)
- Emily Read (ORCID: https://orcid.org/0000-0002-0376-2989)
- Matheus Gallas‐Lopes (ORCID: https://orcid.org/0000-0001-5375-2335)
- Stefani Altenhofen (ORCID: https://orcid.org/0000-0001-7229-6494)
- Eduardo Pacheco Rico (ORCID: https://orcid.org/0000-0003-0019-328X)
- Carla Denise Bonan (ORCID: https://orcid.org/0000-0001-9715-6244)
- Robert Hindges (ORCID: https://orcid.org/0000-0003-0095-7913)
- Karissa Barthelson (ORCID: https://orcid.org/0000-0002-4693-8833)
- Murilo S. de Abreu (ORCID: https://orcid.org/0000-0001-5562-0715)
- Courtney Hillman (ORCID: https://orcid.org/0000-0003-4787-7400)
- Eduardo Rivera García
- Tamara G. Amstislavskaya (ORCID: https://orcid.org/0000-0002-2656-8687)
- Ana M. Valentim (ORCID: https://orcid.org/0000-0003-1428-0691)
- Michael Lardelli (ORCID: https://orcid.org/0000-0002-4289-444X)
- М. А. Gorbunova (ORCID: https://orcid.org/0000-0002-3196-0544)
- Luis M Martinez-Duran
- Thailana Stahlhofer-Buss
- Ewan Gerken (ORCID: https://orcid.org/0000-0002-5654-8556)
- Cássio M. Resmim
- Xian Wang (ORCID: https://orcid.org/0000-0003-0041-7589)
- Guilherme Lodetti
- Giulia Lombardelli
- Austin Cooper
- Maxim A. Kleshchev
- Evgeny V. Nekhoroshev
Institutions
- Universidad de La Frontera (CL)
- University of Nebraska at Omaha (US)
- Universidade Federal do Rio Grande do Sul (BR)
- Ural Federal University (RU)
- Universidade Federal de Ciências da Saúde de Porto Alegre (BR)
- Queen Mary University of London (GB)
- Flinders University (AU)
- St Petersburg University (RU)
- King's College London (GB)
- Wayne State University (US)
- University of Surrey (GB)
- Western Caspian University (AZ)
- Universidade Federal de Santa Maria (BR)
- Universidade Federal do Rio Grande do Norte (BR)
- Universidade do Sul de Santa Catarina (BR)
- Institute of Physiology and Basic Medicine (RU)
- Sirius University of Science and Technology (RU)
- Indiana University Northwest (US)
- Adelaide Institute for Sleep Health (AU)
- Clinical Research Consortium (US)
- i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto (PT)
- Pontifícia Universidade Católica do Rio Grande do Sul (BR)
- Fundación Ciencias Exactas y Naturales (AR)
- The University of Adelaide (AU)
- New York University (US)
- Xi’an Jiaotong-Liverpool University (CN)
Publication Details
- Journal
- Lab Animal
- Published
- 2025-05-26
- DOI
- https://doi.org/10.1038/s41684-025-01548-x
- Citations
- 13
- Primary Topic
- Zebrafish Biomedical Research Applications
- Type
- article
- Field-Weighted Citation Impact
- 14.97
Funders
- National Science Foundation
- National Centre for the Replacement, Refinement and Reduction of Animals in Research
- Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
- Conselho Nacional de Desenvolvimento Científico e Tecnológico
- Saint Petersburg State University
- Xi’an Jiaotong-Liverpool University
- Dementia Australia
- Medical Research Council Centre for Neurodevelopmental Disorders
- National Institutes of Health
- Defence Science and Technology Laboratory
- Medical Research Council
- Fundação para a Ciência e a Tecnologia
- Agencia Nacional de Promoción Científica y Tecnológica
- Division of Civil, Mechanical and Manufacturing Innovation