Figure 3

Calcium supplementation from tuna bone, tuna bone with tuna head oil added 25(OH)D₃ and tuna bone with 25(OH)D₃ mitigated calcium insufficiency-induced osteoporosis. Four-week female rats were received calcium-replete diet (0.55% w/w as a CaCO₃) and daily oral given 25(OH)D₃ in the dose of 20 IU/kg until age of 15 weeks as normal baseline control (denoted as S1, n = 8). Another set of 4-week female rats (n = 32), they were all challenged with low calcium diet (0.15% w/w) for 2 weeks, thereafter, rats were randomly divided into 4 groups, i.e., (i) stayed on low calcium diet (L, n = 8) or (ii) switched to calcium repletion diet (0.55% w/w) in which extra calcium was from tuna bone (S2, n = 8), (iii) tuna bone calcium repletion diet with tuna head oil added 25(OH)D₃ (S3, n = 8) and (iv) tuna bone calcium repletion diet with commercial 25(OH)D₃ (S4, n = 8). (A) Representative images of three-dimensional reconstruction of metaphyseal distal femur (cross-sectional view), Scale bars, 1 mm, (B) bone mineral density and content (BMD and BMC, respectively) of total bone (TOT), trabecular (Tb) and cortical compartment (Ct). Cortical area (Ct.A) thickness (Ct.Th), periosteal perimeter (Ct.Ps.Pm) and endosteal perimeter (Ct.Es.Pm), all parameters were analyzed by pQCT at distal metaphyseal femur, (C) representative images of three-dimensional reconstruction of mid-shaft diaphyseal femur (cross-sectional view), Scale bars, 1 mm, and (D) cortical bone parameters analyzed by pQCT at mid-shaft femur. Results are expressed as means ± SE. The differences between five experimental groups were determined by one-way ANOVA followed by Tukey post hoc test. *P < 0.05, ***P < 0.001 compared with L.