Dietary casein source modulates water intake and anxiety- and cognition-related behaviors in C57BL/6J male mice

INTRODUCTION: Semi-purified diets are commonly used in animal studies to improve experimental reproducibility. However, the composition of these diets often differs, particularly in ingredient sources such as casein, and the influence of these compositional differences on physiological and behavioral phenotypes remains unclear. Therefore, this study aimed to compare commonly used semi-purified diets and to test whether ingredient-level variation influences physiological and behavioral outcomes in mice. METHODS: Male C57BL/6J mice were fed semi-purified diets based on the AIN-93G or an OpenStandard diet (OSD), the latter containing inulin. Each diet was formulated using either lactic acid casein or mineral acid casein and administered for 114 days. Feed intake, water intake, body weight, tissue weight, and various behavioral tests were assessed. RESULTS: Mineral-acid casein was associated with higher total water intake than lactic-acid casein across diet formulations. In contrast, lactic-acid casein was associated with a higher recognition index in the novel object recognition test and longer center-zone time in the elevated plus maze than mineral-acid casein. OSD increased cecum weight compared with AIN-93G. CONCLUSIONS: These findings suggest that dietary casein source is associated with differences in water intake and selected behavioral outcomes in mice, with mineral-acid casein increasing water intake and lactic-acid casein improving memory performance. The choice of protein source in semi-purified diets should be considered when designing nutritional and behavioral experiments.