Habituation with apparatus and group testing improves assessment of fish preferences.

Preference tests are commonly used to assess fish behavior and cognition in several research fields. This study aimed to investigate how fish perform in a preference test involving extended habituation to the apparatus, which was expected to reduce stress. We contrasted the choice between a sector of the apparatus with natural vegetation, expected to be the preferred stimulus, and a barren sector. Initially, we demonstrated that guppies' preference for the sector with vegetation increased after a 5-day habituation period (Experiment 1). Subsequent experiments systematically modified the testing paradigm to observe effects on the preference. Experiment 2 introduced a physical separation between sectors to facilitate discrete choices, Experiment 3 tested groups of fish, and Experiment 4 used wild guppies. Only the modification in Experiment 3 impacted preference scores: guppies tested in groups showed a higher preference for the vegetation stimulus and spent less time in the central, no-choice sector of the testing apparatus. Overall, this study supports the importance of methodological details in preference tests and highlights the benefits of extended habituation and group testing. Researchers should consider these factors when designing experiments to evaluate cognitive abilities or behavioral preferences in fish. Tailoring testing paradigms to specific research goals can improve the reliability and comparability of results, contributing to a deeper understanding of fish behavior and welfare.