Social housing conditions, hierarchical status and testing order affect behavioral test outcomes of male C57BL6/J mice

Male mice are frequently used for behavioral neuroscience research, but outcomes of behavioral tests are often variable across studies, contributing to poor reproducibility. Social housing conditions, social hierarchical status and within-cage order of testing are factors that likely influence behavioral outcomes, but it is unknown to what extent and if these factors interact. For this purpose, behavior of socially and individually housed male C57BL6/J mice was studied upon subjection to the open field test, the elevated plus-maze test and the three-chamber social test. In socially housed animals, effects of social hierarchical status and within-cage testing order were evaluated. We show that the differences in behavior outcomes between individually and socially housed mice depend on the social hierarchical status and the test order of the socially housed mice. Careful consideration of these factors in the design, analysis and interpretation of behavioral experiments with socially housed mice can lead to more precise results and more reliable research outcomes.