Impacts of variation in experimental parameters on larval zebrafish behavior: Enhancing rigor and reproducibility in...

Larval zebrafish have become a cornerstone in neurobehavioral research. However, the reproducibility of zebrafish behavioral studies is often hindered by subtle yet significant variations in experimental parameters. This study systematically investigates how variations in experimental parameters-well geometry; area; plate opacity; genotype; holding temperature; and light intensity-influence larval zebrafish baseline activity and behavioral responses. Using automated behavioral tracking and sample sizes ranging between 73 and 192 larvae/group, we uncovered evident differences in response to changes in these conditions. When assessing geometry and area, larvae in square wells and larger well areas displayed heightened baseline and stimulus-induced behavioral responses; however, relative comparison contradicted these findings indicating smaller areas significantly exaggerated the magnitude of behavioral responses. Clear plates elicited stronger baseline activity and stimulus-driven behaviors. Wild-type fish showed higher behavioral responses than mutants. Temperatures significantly modulated baseline activity and stimulus-induced behavior, while relative comparison analysis revealed increases in stimulus-induced responses across both extremes. Lastly, intermediate light intensities elicited the strongest reactivity, indicating a non-linear relationship between illumination and visual-motor-response behaviors. Our study demonstrates that variation in experimental parameters can alter behavioral outcomes, underscoring the need for standardized zebrafish neurobehavioral assays and for accounting for methodological differences when comparing results. Developing this framework will improve the reliability and interpretability of zebrafish behavioral data.