Sex segregation affects exploratory and social behaviors of zebrafish according to controlled housing conditions

•Little is known about the influence of sex distribution and presence of different cues in fish behavior.•We analyze the exploratory and social behavior of different sex distribution and housing conditions.•Segregated females and males are more active than mixed animals and the females are more anxious.•Fish visualizing and smelling the opposite sex are more active and less anxious.•The presence of structural enrichment lowers the activity and anxiety.•These variables should be considered to improve the reproducibility of experiments. Zebrafish has become an animal model in research and articles have established ideal conditions for their maintenance. However, little is known regarding the influence of gender and other cues on zebrafish behavior. Thus, here we analyzed the exploratory and social behavior of different sexes (male and female, mixed or segregated) under different housing conditions with various types of stimuli (visual or/and chemical cues and structural environmental enrichment). Segregated females and males were more active than mixed individuals and females were more anxious. Fish that visualized and smelled the opposite sex presented higher activity and were less anxious than individuals that only smelled or visualized the opposite sex. Fish segregated by sex while being allowed to visualize and smell the opposite sex with the presence of structural environmental enrichment exhibited lower activity and anxiety-like behavior than fish without structural environmental enrichment. Thus, we emphasized that these variables should be taken into account in housing conditions and should be detailed for better replicability and reproducibility of experiments performed with zebrafish.