Effects of housing density on anxiety-like behavior of zebrafish in the plus maze with ramp

Population density in experimental animals is a crucial factor in maintaining the wellbeing of the organisms. Inadequate housing conditions can compromise the validity and reliability of research results, making comparisons between studies difficult. In sociable species such as zebrafish (Danio rerio), which are housed in groups, overcrowding or undercrowding represents a variable that needs to be considered. In this study, we evaluated the effects of housing at different densities for different exposure times on the anxiety response measured in the Plus Maze with Ramp test in zebrafish. The subjects (144) were divided into three large groups according to the housing time (1, 7, and 30 days). Each group was divided into six subgroups based on the density of the fish (0.25, 0.5, 1, 2, 4, 6 fish/liter, n = 8) and housed in a 4-liter aquarium. After the housing conditions, each animal was tested individually in the PMR. Time and housing density altered the exploratory behavior of zebrafish. Increased housing time reduced the time spent in the ramp arms, with groups kept for 30 days spending less time in this compartment. Density increased the time spent in the flat arms in groups with 2 and 6 fish/liter and, conversely, reduced the exploration of the ramp arms. Isolation, on the other hand, increased the exploration of the ramp arms, indicating an anxiolytic effect. In this study, we demonstrate that housing conditions can act as low-intensity chronic stressors that alter anxiety-like behavior in zebrafish when tested in the PMR protocol.