VD Knickerbocker1, T Laster1, J Osgood1, K Perron1, N Stroud1, J Suckovic1, C Wise2, JM Wotton2, and Z Bichler1

C57BL/6J mice represent the predominant inbred background in preclinical research, with extensive phenotypic characterization available through resources such as the Mouse Phenome Database (https://phenome.jax.org/). Despite this breadth, inter‑laboratory variability in experimental design, protocol implementation, and under‑powered designs frequently limit the reliability and reproducibility of reported phenotypes. At the same time, many additional mouse strains are routinely used, further highlighting the need for harmonized reference data.

To address this, we leveraged the large, continuously expanding dataset generated at The Jackson Laboratory’s Neurobehavioral Phenotyping Core since 2019, comprising mice tested under harmonized protocols, controlled environmental parameters, and validated operator procedures. Using aggregated multi‑assay datasets, we derived strain‑specific and age‑stratified reference ranges for key motor and activity‑related phenotypes. Analytical efforts included composite score generation, inter‑assay correlation matrices, and cross‑modal concordance analyses to evaluate redundancy and discriminative sensitivity across widely used motor assays.

Our initial objective was to develop recommendations for aging studies using C57BL/6J as a benchmark strain. We identified assays most sensitive to age- and sex-related differences, estimated empirically supported group‑size thresholds, and examined correlations across assays to highlight complementary or redundant motor measures. These analyses provide a framework for selecting reliable phenotypes and designing efficient behavioral pipelines.

Ultimately, we hope this effort will contribute to improved reproducibility, more strategic and ethical study design, and broader alignment with the 3Rs (Replacement, Reduction, Refinement) principles in in vivo behavioral research.

1Neurobehavioral Phenotyping Core at the Center for Biometric Analysis, 2Center for Biometric Analysis, The Jackson Laboratory, Bar Harbor, Maine, USA.

Acknowledgement: The authors would like to extend the co-authorship to all former staff

members of the Neurobehavioral Phenotyping core at the Center for Biometric Analysis at The Jackson Laboratory as they have generated or help generate essential data needed for this work.