2.2. Bone Calcium Solution and Total Ash Determination

The preparation of antler-bone calcium was evaluated by applying the method of Weaver et al. with a few adjustments [35]. The antler bone was sliced using a bone slicer, cleaned in deionized water to neutral pH, and then dried at room temperature (RT). After drying, the bone was ground into powder. Antler bone powder (1 g) was weighed and combined with 10 mL malic acid solution, which was prepared at a concentration of 5%, then stirred at 55 °C for 70 min. The solution was centrifuged at the process of 10,000× g for 10 min, and RT, yielding a clear supernatant containing the bone calcium solution (AB−Ca). AB−Ca was then lyophilized and preserved at −20 °C for use as CaCl₂ replacement. We weighed the antler bone section (2 g), placed it into a muffle furnace (SGM-M16/10, SIGMA, Shanghai, China) and dried for over 12 h at 550 °C until it reached a constant weight. The ash weight was calculated, the sample was diluted with deionized water, and calcium was tested using AAS (PE-700, Agilent, Santa Clara, CA, USA).