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Zebrafish (Danio rerio) has in recent years become widely used and has become the second most common lab species in Sweden. This species is most commonly used in biomedical, behavioural, and neuroscientific research due to its cost efficiency, high reproductive rate, and physiological similarities to humans. This thesis aimed to provide evidence-based insights into the effects of stocking density, tank size, social isolation, and enrichment on stress physiology, behaviour, and neurochemistry.

Three comprehensive experiments were conducted. First, three stocking densities, 1, 3, and 6 fish/L, were compared with or without enrichment. A significant increase was found in aggression and cortisol levels at the lowest density, suggesting a density between 3 and 6 fish/L as optimal. The second study investigated the interaction between two densities (3 and 6 fish/L), two standard tank sizes (6 and 9.5 L), and a novel medical-grade silicone enrichment. Enrichment increased aggression and exploratory behaviour, while the higher density elevated cortisol. Tank size effects were not significant. The third study investigated the effect of long-term (2 months) and short-term (24 hours) social isolation, with or without visual contact with conspecifics, on behaviour, feeding pattern, and brain monoaminergic signalling in male and female zebrafish. Social isolation did not affect the brain monoaminergic system, possibly indicating a relief from social stress created in the housing groups. Males had a lower stress-coping ability, and visual contact with conspecifics had no detectable effect.

The findings of this thesis indicate the difficulties of evaluating the connection between stress biology and welfare in zebrafish, as it varies and is not a one-size-fits-all analysis. Instead, environmental, social, and biological factors interact in non-linear ways to shape behavioural and neuroendocrine outcomes. Additionally, this thesis reports the importance of including both sexes as a critical biological variable. Overall, this work emphasises the need for standardised, context-specific rearing towards improving welfare and scientific validity in zebrafish research.

The refinement of housing conditions for zebrafish (Danio rerio) is essential for fish welfare. This study explored how environmental enrichment, stocking density and tank volume influenced behaviour, stress physiology and neurochemical profiles in adult zebrafish, housed in groups. A novel, medical grade silicone enrichment was used across two tank volumes (6 L and 9.5 L) and two densities (3 and 6 fish/L). Aggression was scored over 7 weeks, followed by behavioural profiling in the zebrafish multivariate concentric square field™ (zMCSF) test. Cortisol levels were measured in the water and in whole-body samples following an acute stress. Finally, brain monoamines and metabolites were analysed. Enrichment significantly increased aggression and promoted risk-taking behaviour. Higher stocking density resulted in lowered aggression but higher cortisol levels. Brain monoaminergic differences were influenced by sex and enrichment-density interactions. Tank volume had no significant effects. These findings highlight the complex interplay between rearing conditions and welfare, underscoring the need for context-specific enrichment strategies in laboratory setups.

In the lab, social isolation of zebrafish is a common procedure to allow individual recognition. Even though neurobiological and behavioural consequences of social isolation in zebrafish have been described, how these effects may differ between sexes remains insufficiently characterized. This study evaluated the effects of short-term (24 h) and long-term (2 months) social isolation, with and without visual contact, on behaviour, feeding, and brain monoaminergic signalling in male and female zebrafish. We did not observe any effects of social isolation on brain monoaminergic functions, suggesting that social isolation is a relatively mild stressor. Isolation may, in fact, provide a relief from the social stress experienced by group-reared fish. However, the most obvious result was that male and female fish differed in their response to social isolation, males being less able to cope. The results also show that visual contact between isolated fish is insufficient to modify the effects of social isolation.