2.5. In vivo test of calcium absorption in rats

The animal experiment program was approved by the institutional animal Ethics Committee of Yunnan Agricultural University. Sixty male Sprague Dawley (SD) rats weighing 60–80 g (Peng et al., ²⁰¹⁷) were obtained from Liaoning Changsheng Biotechnology Co., Ltd. [Certificate number: SCXK (Liao) 2015‐0001]. The rats were housed at a temperature of 22 ± 2°C, a relative humidity of 60 ± 5%, 12 hr of light, and 12 hr of darkness. All diets were purchased from Trophic Animal Feed High Tech Co., Ltd. and prepared according to the AIN‐93 diet. The calcium content in normal and low‐calcium feeds was 4,500 and 140 mg/kg, respectively.

After 7 days of adaptation, the rats were randomly divided into normal, model, and CaCO_3, and three experimental groups (the Ca²⁺ content of CaCO₃ group and a moderate dose of MOLF with calcium content remaining equal) with rats in the low‐Ca, medium‐Ca, and high‐Ca groups were intragastric administration of 0.065, 0.13, and 0.195 g/kg of MOLF according to their body weight (calculated by calcium content) (n = 10/group). During the 4‐week feeding period, the control rats were given ad libitum access to the control diet, and the remaining experimental groups were given ad libitum access to low‐calcium feed. Both MOLF and calcium carbonate were dissolved in deionized water as a solvent (Peng et al., ²⁰¹⁷).

During the experiment, the body weight of the rats was weighed weekly and the feed intake was recorded daily. The experiment was carried out for last 3 days of week 4 for conducting calcium metabolism experiments. In the last 3 days of the experiment, rat feces were collected and weighed daily, and calcium excreted in the feces was measured. The absorption rate calculation was done as follows: