Major Physiological Differences Exist Between C57BL/6 Substrains

The C57BL/6 inbred mouse strain is the most widely used strain in the world. However, there is currently no source to obtain C57BL/6 mice, as this strain diverged in the mid-20th century into two main substrains, C57BL/6J and C57BL/6N. Still, many researchers refer to mice from C57BL/6 substrains as C57BL/6 mice. This is incorrect and may influence reproducibility. Here, we examined physiological differences in male and female C57BL/6J and C57BL/6NCrl mice, as well as UM-HET3 mice, a genetically diverse mice strain created by crossing four inbred strains: BALB/cByJ, C57BL/6J, C3H/HeJ, and DBA/2J. Body mass was higher in UM-HET3 compared to C57BL/6 substrains (UM-HET3: 32.1±1.0 vs. C57BL/6J: 27.5±0.8 or C57BL/6NCrl: 27.8±0.7 g; p<0.05). Fasting blood glucose was lower in UM-HET3 compared to C57BL/6 substrains (UM-HET3: 166±4 vs. C57BL/6J: 182±3 or C57BL/6NCrl: 171±3 mg/dL; p<0.05) and differed between C57BL/6 substrains (p<0.05). Blood glucose incremental area under the curve (iAUC) during glucose tolerance test was similar in UM-HET3 and C57BL/6NCrl (P>0.05), which were lower than C57BL/6J (UM-HET3: 8.5±0.8 or C57BL/6NCrl: 8.9±0.7 vs. C57BL/6J: 12.2±0.9 AU; p<0.05). Blood glucose iAUC during insulin tolerance test was similar between UM-HET3 and C57BL/6J (P>0.05), which were lower than C57BL/6NCrl (UM-HET3: -5.0±0.5 or C57BL/6J: -6.4±0.6 vs. C57BL/6NCrl: -3.6±0.5 AU; p<0.05 for C57BL/6J vs. C57BL/6NCrl). Systolic blood pressure was lower in C57BL6NCrl compared to C57BL/6J and UM-HET3 (C57BL/6NCrl: 101±1 vs. C57BL/6J: 112±1 or UM-HET3: 112±1 mmHg; p<0.05). Aortic stiffness, measured by pulse wave velocity, was lower in UM-HET3 compared to C57BL/6 substrains, which also differed from each other (UM-HET3: 263±4 vs. C57BL/6J: 322±4 or C57BL/6NCrl: 295±4 cm/sec; p<0.05). Aortic histology revealed ~60% lower collagen and ~20% higher elastin content in UM-HET3 compared to C57BL/6 substrains (p<0.05). Ex vivo arterial function in isolated carotid arteries showed higher peak acetylcholine-mediated vasodilation in UM-HET3 than C57BL/6 substrains (UM-HET3: 89±2 vs. C57BL/6J: 84±1 or C57BL/6NCrl: 83±1%; p<0.05). Peak flow-mediated vasodilation was similar between UM-HET3 and C57BL/6NCrl (P>0.05), which were higher than C57BL/6J (UM-HET3: 33±4 or C57BL/6NCrl: 33±5 vs. C57BL/6J: 18±5%; p<0.05). Perfused boundary region (PBR), a marker of glycocalyx function, was lower in UM-HET3 compared to C57BL/6 substrains (UM-HET3: 2.26±0.04 vs. C57BL/6J: 2.41±0.04 or C57BL/6NCrl: 2.43±0.03 μm; p<0.05), indicating glycocalyx dysfunction in C57BL/6 substrains. This study exposes major physiological differences in C57BL/6 substrains with C57BL/6NCrl exhibiting a healthier cardiometabolic profile than C57BL/6J. These findings challenge the notion that ‘C57BL/6’ is a useful descriptor for mice and that failing to identify by substrains would lower reproducibility and cause misinterpretation of results. For example, in aging studies, young C57BL/6 mice are often described as being purchased from Charles River, which also houses old C57BL/6 mice in the NIA aging colony. However, mice purchased from Charles River and mice in the aging colony are from C57BL/6N and C57BL/6J substrains, respectively. Thus, in this example, age-related differences could simply be substrains-related differences. Notably, UM-HET3 mice demonstrate a healthier cardiometabolic profile than each C57BL/6 substrains. This may make UM-HET3 mice more attractive for studies that induce dysfunction, as they may accrue greater dysfunction than inbred mice that are dysfunctional at baseline. In summary, these results underscore the need for careful strain selection and notation in biomedical research. This study was funded in part by a grant from the National Institutes of Health (R00 AT010017). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.