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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.