2.7. Evaluation of fermentation broth against glucocorticoid-induced osteoporosis using Zebrafish larva

All the procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Adult zebrafish AB strains were kept at 28.5°C under a normal light/dark cycle of 14 h/10 h at 28°C for natural maturation. Zebrafish embryos were cultured in 6-well plates. The collected embryos were placed in fish water containing 5.0 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, and 0.33 mM MgSO₄ and treated with 0.1% DMSO (vehicle), 25 μM prednisolone (model), or co-incubated 3.3–30 μM CBP, or 0.308 μM alendronate (positive control) after 3 pdf till 7 dpf. On pdf 7, larvae were collected for skeletal staining and behavioral analysis.

The fish at 7 dpf were vitally stained with 0.2% calcein solution (pH adjusted to 7.0–7.5) for 5 min in the dark. Next, fish were rinsed thrice for 5 min with the system water, anesthetized with 0.016% MS-222, and fixed in 3% methylcellulose. At the end of the treatment, the regenerated bone areas on the larvae were visualized using a fluorescence microscope (Leica DMi8, Wetzlar, Germany), and images were captured using digital cameras (10X objective, and Retiga 2000R). The relative fluorescence intensity (RFI) of skull bone mass per zebrafish was determined using ImageJ software (n = 6).