Figure 2

Calcium supplementation from tuna bone, tuna bone with tuna head oil and tuna bone with 25(OH)D₃ enhanced fractional calcium absorption. (A) Experimental design, 4-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 (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). Thee-day calcium balance study was conducted in 3 time points, i.e., 2 weeks (baseline, before calcium supplement), 4 weeks and 11 weeks after calcium supplementation (animal were on age of 6, 10 and 15 weeks, respectively), (B) body weight, (C) relative fractional calcium absorption at rat’s age of 6, 10 and 15 weeks, (D) Representative images of immunostaining against PMCA protein, (E) quantitative data of fluorescent intensity of PMCA1 (relative expression to S1), and (F) 3-day urinary calcium excretion at rat’s age of 6, 10 and 15 weeks. Results are expressed as means ± SE. The different between two sets of data was determined by two-tailed unpaired Student’s t test. The differences between five experimental groups were determined by one-way ANOVA followed by Tukey post hoc test. ***P < 0.001 compared with L.