Effects of Subthermoneutral Housing on Bone in Female C57BL/6J Mice
Authors: Sattgast Lara H. , Wong Carmen P. , Olson Dawn A. , Branscum Adam J. , Iwaniec Urszula T. , Turner Russell T. 
Published: May 2022

ObjectiveMice are facultative daily heterotherms with a thermoneutral point of ~32°C and when housed at standard room temperature (~22°C) experience cold stress and premature cancellous bone loss. It is unclear whether housing mice at the maximum recommended temperature of 26°C or group housing affects bone. The objectives of this study were to determine (1) how a 4°C difference in housing temperature (22°C vs. 26°C) and (2) how single versus group housing influence bone metabolism in female mice.HypothesisHousing mice at room temperature (22°C) will result in premature cancellous bone loss in long bones of growing female mice, and housing mice at 26°C or group housing mice at 22°C will attenuate the bone loss.MethodsFive‐week‐old female C57BL/6J mice were randomized by weight in one of 4 treatment groups (N=10/group): 1) baseline, 2) single housed at 22°C, 3) single housed at 26°C, or 4) group housed (2 cages; n=5/cage) at 22°C. Baseline mice were sacrificed and the remaining mice were maintained at their respective temperatures and housing conditions for 13 weeks until 18 weeks of age. Total femur mass and cortical (femur midshaft) and cancellous (distal femur metaphysis) bone microarchitecture were assessed using dual energy absorptiometry and microcomputed tomography, respectively. Osteocalcin, a marker of global bone turnover, was measured in serum using an enzyme‐linked immunoassay.ResultsFemur length, area, bone mineral content, and bone mineral density increased in all groups compared to baseline. Cortical volume, marrow volume, and cortical thickness in midshaft femur increased in all groups compared to baseline. No or minimal differences were observed in total femur mass or midshaft femur microarchitecture among mice single housed at 22°C, single housed at 26°C, or group housed at 22°C. In contrast, there were large reductions in cancellous bone volume fraction, connectivity density, trabecular number and an increase in trabecular separation in the distal femur metaphysis in all groups compared to baseline. Single housing mice at 26°C and group housing mice at 22°C attenuated, but did not prevent, cold stress‐induced bone loss. Cancellous bone volume fraction and trabecular number were higher and trabecular separation was lower in mice single housed at 26°C compared to mice single housed at 22°C, while cancellous bone volume fraction and trabecular thickness were higher in mice group housed at 22°C compared to mice single housed at 22°C. Differences between mice single housed at 26°C and group housed at 22°C were not detected for any of the cancellous endpoints evaluated. Levels of serum osteocalcin were decreased in all groups compared to baseline, but tended to be higher in mice single housed at 26°C (p=0.092) and in mice group housed at 22°C (p=0.076) compared to mice single housed at 22°C.ConclusionsHousing mice at 26°C and group housing mice at 22°C slows, but does not prevent, cold stress‐induced premature cancellous bone loss in distal femur metaphysis, a commonly evaluated anatomical site. These findings demonstrate that small differences in housing conditions can significantly influence cancellous bone microarchitecture.