Not all mice are alike: Mixed-strain housing alters social behaviour

•Effects of mixed-strain and same-strain housing on mouse welfare were investigated.•Anxiety, stereotypies, and stress hormones did not differ between groups.•However, changes in social behaviours and activity rhythm were found.•This might point towards mild impacts on welfare under co-housing conditions.•Thus, when housed together strains need to be carefully selected for compatibility. The use of millions of mice in scientific studies worldwide emphasises the continuing need for a reduction of sample sizes, however, not at the expense of scientific validity. Split-plot designs have been suggested to enhance statistical power while allowing a reduction of animal numbers in comparison to traditional experimental designs. Recently, a promising approach of a split-plot design has been implemented and proven useful using mixed-strain housing of at least three different mouse strains. However, the impact of co-housing different strains of mice in one cage on animal welfare has still to be defined. This study aimed at comparing the effects of mixed-strain and same-strain housing of female C57BL/6J and DBA/2N mice on welfare and behaviour in two experimental phases. In a first phase, mice were housed in either mixed- or same-strain pairs. Home cage behaviour, activity rhythm, body weight, and faecal corticosterone metabolites were assessed. Furthermore, tests for anxiety-like and exploratory behaviour as well as spatial learning were performed. In a second phase, sociability was investigated in newly formed mixed-strain quartets. Mixed-strain housing did not induce alterations in anxiety, locomotion, learning, stereotypic behaviour, and stress hormone levels. However, changes in social behaviours and activity rhythm were observed. Increased agonistic and decreased socio-positive behaviours might point towards mild impacts on welfare in C57BL/6J mice under co-housing conditions. Altogether, scientific research may greatly benefit from co-housing mice of different strains within the same cages (e.g. for the realisation of a split-plot design), provided that strains are carefully selected for compatibility.