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Effect of colonization on bone phenotype of germ-free mice is independent of age of donor microbiome. Effect of colonization for 1 month on trabecular bone volume fraction (Tb. BV/TV), cortical thickness (Ct. Th), cortical area (Ct. Ar), serum P1NP and CTX-1 in young adult (2-month-old, a–e) and skeletal immature (1-month-old, f–j) females is shown. Consequences of 8 months colonization on Tb. BV/TV, Ct. Th, and Ct. Ar are shown for (k–m) female and (p–r) male mice. n, o Serum P1NP and CTX-1 is shown in female after 8 months colonization. s Representative 3D images of femur trabecular bone are shown. Data are represented as mean ± SEM. One-way ANOVA or Kruskal-Wallis test with Tukey post hoc was performed. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not statistically significant. For (a–e): GF, n = 11; Col with 3-month, n = 14; Col with 24-month, n = 14. For (f–j): GF = 8; Col with 3-month, n = 9; Col with 24-month, n = 8. For (k–o): GF, n = 12; Col with 3-month, n = 7; Col with 24-month, n = 8. For (p–r): n = 8 per group. 4 data in (e) and 5 data in (n) and (o) were missing due to insufficient sample collection.