Figure 5

(A) Calcein vital staining of scales from treated fish and untreated controls. Control scale show normal profile, while scales from treated fish show bone resorption lacunae after 3 weeks of treatment (T3, white arrowhead). At 4 weeks of SBM treatment, the lacunae started to fill by new matrix deposition (T4, white arrows) and appeared completely repaired in the fifth week (T5) (scale bar: 0.1 mm). (B) Percentage of resorbed scales versus repaired scales. The percentage of resorbed scales decreased as the percentage or repaired scales increased (resorbed scales: CTR vs. T3, p < 0.001; CTR vs. T4, p < 0.05; repaired scales: CTR vs. T4, p < 0.001; CTR vs. T5, p < 0.001). (C) Alizarin Red-calcein double staining shows that the resorbed area was completely filled by new matrix at the end of the fifth week (green signal in T5) of SBM treatment in all scales. Untreated control (CTR) is shown as reference (scale bar: 0.07 mm). (D) Biochemical ALP activity assay indicates a peak in anabolic activity in the scale after 3 weeks of treatment (T3) (CTR vs. T3, p < 0.001). The value of the control at each experimental point was omitted as it was identical to the initial CTR. Significance values were set at less than p < 0.05 (*), and p < 0.001 (***).