Hidden Husbandry Bias: Improving Animal Welfare and Experimental Reproducibility in Zebrafish Research

Abstract

A biomedical laboratory encountered variability in the behavioural, endocrine, and molecular results of its adult zebrafish, although it used the same experimental methods. This case investigates how daily routine husbandry introduced previously unrecognised experimental bias and demonstrates the value of standardised animal care and management practices in improving animal welfare and enabling research to be replicated. The study conducted by an interdisciplinary team produced behavioural, endocrine, and molecular results that were unpredictable and inconsistent, despite using identical experimental methods, standardised analytical procedures, and the same strain of zebrafish. Since the previous study found that the inconsistency was not due to technical or methodological errors, the team went further to determine whether everyday husbandry methods were the source of experimental bias. The investigation focused on four common husbandry-related variables: alterations in the light period, repeated tank relocation, frequent handling, and variability among animal caregivers. A controlled experimental approach combined behavioural assessments with assays of whole-body cortisol, brain catecholamine concentrations, and stress-related gene expression to evaluate their biological influence. Overall, the outcomes showed that routine husbandry practices significantly affected behavioural responses, endocrine activity, neurochemical profiles, and molecular parameters, indicating that seemingly minor management variations can alter physiological baseline conditions before experiments even begin.