Three Non-Invasive Tests Reveal Anxiety-like Responses During Food Anticipation in Rainbow Trout

Anxiety-like behavior in fish is commonly assessed using non-invasive behavioral paradigms such as the Light/Dark preference, Novel Tank, and Open Field tests. In this study, we validated these three assays in rainbow trout (Oncorhynchus mykiss), a species of commercial relevance, to characterize their anxiety-related responses. To explore behavioral changes associated with feeding anticipation and satiety, we implemented a feeding schedule consisting of two daily meals and conducted behavioral tests at specific times before and after feeding. Trout exhibited clear patterns of scototaxis, geotaxis, and thigmotaxis, consistent with anxiety-like behavior described in other teleosts. Our results showed a significant increase in anxiety-like responses before feeding, coinciding with food anticipatory activity observed prior to expected feeding schedules, which diminished after food intake, as evidenced by each test individually. Moreover, multivariate analysis combining parameters from all three tests improved discrimination between anxious and relaxed fish. The behavioral states before and after feeding resembled anxiety-like and anxiolytic conditions reported in other species, supporting that food anticipatory activity reflects an anxious state in rainbow trout as well. These findings endorse using a multi-test behavioral battery to assess anxiety-like states and provide a framework for studying neurobiological mechanisms of emotional regulation related to feeding in teleosts.