Cecal mass interferes with normalization of energy expenditure.

(A-B) Comparison of circadian changes in energy expenditure (without normalization) among GF, OligoMM12, and SPF C57B6/J mice. (A) Circadian variation in average energy expenditure per time point and (B) overlayed curves obtained by smoothing function of data obtained every 24 min per mouse over 10 d. (C-E) Energy expenditure values obtained by “classical” ratio-based normalization methods (dividing energy expenditure values per phase by mass). (C) Area-under-curve after normalization by total mass after cecal dissection. (D) Area-under-curve after normalization by lean body mass (EchoMRI). (E) Area-under-curve after normalization by total body mass before cecal dissection. Number of mice per group in all figures unless otherwise specified: GF = 9, OligoMM12 = 8, SPF = 10. p-values obtained by Tukey’s honest significance test. Data underlying this figure are supplied in S1 Data. GF, germ-free; SPF, specific-opportunistic-pathogen-free. (TIF)