Assessing Chronodisruption Distress in Goldfish: The Importance of Multimodal Approaches

SIMPLE SUMMARY: The disruption of circadian rhythms is considered a potential source of distress, but the extent of its consequences is under study. This research investigated whether the disruption of the light/dark and feeding/fasting cycles can trigger stress and anxiety-like behavior in fish. For this purpose, we first optimized behavioral tests to measure anxiety-like states in goldfish. Second, we studied anxiety and stress responses in two groups of goldfish exposed to either continuous light or randomly scheduled meals for two months. Both conditions led to anxiety-like behavior, evidenced by increased thigmotaxis in the open field with object approach task. Fish exposed to constant light also showed higher locomotor activity, suggesting greater energy expenditure. Additionally, chronodisruption led to increased cortisol levels throughout the experiment, with evidence of a possible axis fatigue due to chronic stress in fish under continuous light. Altogether, these findings support that these chronodisruptive conditions cause distress in the animals, and they should be considered for welfare improvement. The correlation analyses suggested that cortisol, thigmotaxis, and scototaxis are not dependent on each other, while there is a high influence of individual behavioral traits. Thus, the need for combining multiple parameters when assessing discomfort in fish is emphasized. ABSTRACT: Chronodisruption caused by factors such as light at night and mistimed meals has been linked to numerous physiological alterations in vertebrates and may be an anxiogenic factor affecting welfare. This study aims to investigate whether chronodisruption causes measurable changes in the anxiety responses of goldfish under two conditions: randomly scheduled feeding (RF) and continuous light (LL). Anxiety-like behavior was assessed in the open field with object approach and black/white preference tests, which had been validated using diazepam. An increased thigmotaxis response and decreased object exploration under both chronodisruption protocols indicated anxiety states. Furthermore, locomotor activity was increased in LL fish. The black/white preference test discriminated anxiolysis induced by diazepam but was unable to detect anxiety caused by chronodisruption. Plasma cortisol increased in both RF and LL fish throughout the experiment, confirming that both conditions caused stress. The LL fish also showed an apparently desensitized hypothalamus–pituitary–interrenal HPI axis, with a decrease in pomc and crf expression. Individual analysis found no correlation between anxiety-like behavior and stress axis activation nor between scototaxis and thigmotaxis responses. However, individual differences in sensitivity to each test were detected. Altogether, these results highlight circadian disruption as a stressor for fish and endorse a multiple variable approach for reliably assessing animal discomfort.