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Experiments done in Black-6 mice: what does it mean?


Low replicability of animal experiments is perceived as a major hurdle in the field of biomedicine. Attempts to enhance the replicability and to reduce the variability in basic research has led to the recommendation to use isogenic mice. The C57BL/6 strain has evolved as a gold standard strain for this purpose. However, C57BL/6 mice are maintained as substrains by multiple vendors. Evidence exists that the subtle differences between these mouse lines have not been systematically investigated and are often ignored. In the present study, we characterized the female mice of two closely related substrains (C57BL/6J and C57BL/6N) from three vendors in Europe (Charles River Laboratories, Envigo, Janvier Labs) in a battery of behavioral tests. Our data show and confirm substantial behavioral differences between the C57BL/6J and C57BL/6N mice. Importantly, the substrain differences were largely affected by the origin of the animals, as a significant effect of vendor or interaction between the substrain and vendor occurred in all tests. This work highlights the importance of adhering to precise international nomenclature in all publications reporting animal experiments. Moreover, the generalization of research findings from a single mouse substrain can be seriously limited due to genetic drift and environmental variables occurring at different vendors. However, heterogenization of samples, by including animals of different substrains, can enhance generalizability. These issues need to be seriously addressed to improve reproducibility, replicability, and the translational potential of the mouse models.

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Fig. 1: Experimental design, transport boxes and mouse body weight.
Fig. 2: Elevated plus-maze, light-dark box and open field.
Fig. 3: Social approach, acoustic startle and pre-pulse inhibition, fear conditioning.
Fig. 4: Recording of circadian activity and stress-induced hyperthermia in individually caged mice.


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This study was supported by Jane and Aatos Erkko foundation (VV), Biocenter Finland and Helsinki Institute of Life Science. Professors Iiris Hovatta and Jaan-Olle Andressoo are greatly acknowledged for their valuable comments and discussions during the preparation of this manuscript.

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Correspondence to Vootele Voikar.

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Åhlgren, J., Voikar, V. Experiments done in Black-6 mice: what does it mean?. Lab Anim 48, 171–180 (2019).

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