Unfamiliar conspecifics increase freezing and shift movement towards immobility in zebrafish, most clearly in males

Familiar companions can buffer stress in zebrafish (Danio rerio), but whether an unfamiliar shoal is itself a stressor, and whether the sexes respond alike, is unclear. We exposed adult zebrafish to a familiar or an unfamiliar shoal for at least 75 min, video-tracked their behaviour over the last 60 min and then measured whole-body cortisol. In males, an unfamiliar shoal increased freezing (+14.5 percentage points), reduced swim bouts and shifted movement towards a frozen state (8% to 49% of frames). In females, the changes were smaller and not significant. With the sexes pooled, whole-body cortisol was 1.6-fold higher after exposure to the unfamiliar shoal. Sex differences were only found for swim bouts and movement states, not for freezing or cortisol. We conclude that an unfamiliar shoal is a likely stressor for zebrafish, with behavioural effects clearest in males. Thus, who a zebrafish shares its tank with is not a neutral background, and the familiarity of conspecifics should be considered whenever fish are placed in new social settings in the lab.