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Updated: Aug 28, 2025

A Standardized Protocol for Preference Testing to Assess Fish Welfare
Published on: February 22, 2020
Female zebrafish (Danio rerio) demonstrate stronger preference for established shoals over newly-formed shoals in the
Andrew J Velkey1, Caroline H Koon1, Isabel A Danstrom1
1Neuroscience Program, Christopher Newport University, Newport News, VA, United States of America.
Abstract:
Zebrafish (Danio rerio) share a considerable amount of biological similarity with mammals, including identical or homologous gene expression pathways, neurotransmitters, hormones, and cellular receptors. Zebrafish also display complex social behaviors like shoaling and schooling, making them an attractive model for investigating normal social behavior as well as exploring impaired social function conditions such as autism spectrum disorders. Newly-formed and established shoals exhibit distinct behavior patterns and inter-member interactions that can convey the group's social stability. We used a three-chamber open-swim preference test to determine whether individual zebrafish show a preference for an established shoal over a newly-formed shoal. Results indicated that both sexes maintained greater proximity to arena zones nearest to the established shoal stimulus. In addition, we report the novel application of Shannon entropy to discover sex differences in systematicity of responses not revealed by unit-based measurements; male subjects spent more time investigating between the two shoals than female subjects. This novel technique using established versus newly-formed shoals can be used in future studies testing transgenics and pharmacological treatments that mimic autism spectrum disorder and other disorders that affect social interaction.

