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Phenotyping spontaneous locomotor activity in inbred and outbred mouse strains by using Digital Ventilated Cages


Mouse strains differ markedly in all behaviors, independently of their genetic background. We undertook this study to disentangle the diurnal activity and feature key aspects of three non-genetically altered mouse strains widely used in research, C57BL/6NCrl (inbred), BALB/cAnNCrl (inbred) and CRL:CD1(ICR) (outbred). With this aim, we conducted a longitudinal analysis of the spontaneous locomotor activity of the mice during a 24-h period for 2 months, in two different periods of the year to reduce the seasonality effect. Mice (males and females) were group-housed in Digital Ventilated Cages (Tecniplast), mimicking standard housing conditions in research settings and avoiding the potential bias provided in terms of locomotor activity by single housing. The recorded locomotor activity was analyzed by relying on different and commonly used circadian metrics (i.e., day and night activity, diurnal activity, responses to lights-on and lights-off phases, acrophase and activity onset and regularity disruption index) to capture key behavioral responses for each strain. Our results clearly demonstrate significant differences in the circadian activity of the three selected strains, when comparing inbred versus outbred as well as inbred strains (C57BL/6NCrl versus BALB/cAnNCrl). Conversely, males and females of the same strain displayed similar motor phenotypes; significant differences were recorded only for C57BL/6NCrl and CRL:CD1(ICR) females, which displayed higher average locomotor activity from prepuberty to adulthood. All strain-specific differences were further confirmed by an unsupervised machine learning approach. Altogether, our data corroborate the concept that each strain behaves under characteristic patterns, which needs to be taken into consideration in the study design to ensure experimental reproducibility and comply with essential animal welfare principles.

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Fig. 1: Heatmaps of spontaneous locomotor activity.
Fig. 2: Activity pattern over 24 h of the three analyzed strains.
Fig. 3: Day and night activity of male and female cages of each strain.
Fig. 4: Average of diurnal activity (diurnality).
Fig. 5: Behavioral responses during the lights-on phase.
Fig. 6: Behavioral responses during the lights-off phase.
Fig. 7: Acrophase and activity onset.
Fig. 8: Day and night RDI.
Fig. 9: Response to cage change.
Fig. 10: Cluster analysis.

Data availability

Datasets and codes used in the analyses are stored at the authors’ home institution and will be provided upon request.


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The authors are grateful to Alessandro Grop (CNR-IBBC/EMMA/Infrafrontier/IMPC) and Giampaolo D’Erasmo (CNR-IBBC/EMMA/Infrafrontier/IMPC) for technical assistance with mouse colonies. Further acknowledgements go to (i) the whole management of Charles River Laboratories Italy for the support of Sara Fuochi’s PhD program, providing mice to support the studies described in this paper; (ii) University of Naples Federico II for coordinating the PhD project; and (iii) Tecniplast SpA for providing DVCs.

Author information




S.F., L.D’A., P.d.G. and F.I. designed and supervised the research. M.Ra. and F.S. were responsible for mouse maintenance and prepared a first draft of Methods. M.Ri. and F.I. analyzed and generated the datasets. L.D’A., P.d.G. and S.F. provided a first draft of the manuscript. All the authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Livia D’Angelo.

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Competing interests

F.I. and M.Ri. were employed by Tecniplast SpA, which provided support in the form of salaries for authors F.I. and M.Ri. Tecniplast SpA did not have any additional role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. S.F. was employed by Charles River Laboratories Italy, which provided support in terms of animal models, salary for the author and final review and approval of the manuscript. Charles River Laboratories did not have any additional role in the study design, data collection, analysis or interpretation.

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Peer review information Lab Animal thanks Rasneer Sonia Bains and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary methods, Supplementary Figures 1–5, activity heatmaps

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Supplementary Data 1

Statistical models and test results

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Fuochi, S., Rigamonti, M., Iannello, F. et al. Phenotyping spontaneous locomotor activity in inbred and outbred mouse strains by using Digital Ventilated Cages. Lab Anim 50, 215–223 (2021).

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