Generalizability of 3-day refined handling across mouse strains: consistent effects on experimenter interaction and strain- and sex-dependent effects on anxiety-related behavior

Refined handling methods can improve laboratory mouse welfare and reduce handling-related variability. Yet their generalizability across genetic backgrounds and sexes remains incompletely understood. We previously developed a rapid 3-day refined handling method, termed 3D handling, designed to habituate mice to direct human contact. Here, we tested whether the benefits of 3D handling extend beyond C57BL/6 J mice to three additional commonly used mouse strains with distinct behavioral profiles: BALB/cJ, 129×1/SvJ, and J: ARC(S)/CD1-derived mice, and confirmed efficacy in C57BL/6 J. Male and female mice from each strain were assigned to either 3D handling or tail handling for three consecutive days, followed by assessment of experimenter interaction, open field behavior, elevated plus maze performance, novelty suppressed feeding, and handling-induced corticosterone levels. Across strains, 3D handling consistently improved voluntary interaction with the experimenter, with mice approaching the hand more closely, spending more time near it, or maintaining shorter mean distances from it. Effects on anxiety-related behavior were more variable, with benefits most evident in the open field and elevated plus maze in J: ARC(S), C57BL/6 J, and 129×1/SvJ mice, and limited effects in the novelty suppressed feeding test. Composite z-score and forest plot analyses supported an overall beneficial effect across strains, while revealing strain- and sex-dependent variability. 3D handling also reduced corticosterone levels in J: ARC(S) mice and showed a trend toward reduction in C57BL/6 J mice. These findings demonstrate that 3D handling broadly reduces experimenter-directed avoidance across diverse mouse genetic backgrounds, with more context-dependent effects on anxiety-related outcomes. This rapid, practical refinement may complement other non-aversive handling approaches and help improve both animal welfare and the reproducibility of behavioral research.