Adiabatic oxygen bomb calorimetry

Feces were oven dried at 65 °C for 72 h and finely ground to pass through 60-mesh screen before chemical analysis. The chromium contents was measured using an absorption spectrophotometer (HitachiZ-5000, Hitachi High-Technologies Corp., Tokyo, Japan) [18 (link)]. Determination of DM in diet and fecal samples by oven drying at 135 °C for 2 h [19 ]. DM of carcass, viscera and blood was calculated to constant weight by freeze-drying, and the value was used to calculate the energy, protein and fat concentration of the whole body. The GE of diets, feces, carcass, viscera and blood were measured using an adiabatic oxygen bomb calorimetry (Parr Instrument Co., Moline, IL, USA). Nitrogen in diets, fecal samples and in freeze-drying samples of body components was determined by a Kjeldahl procedure and protein was calculated as N × 6.25 (method 990.03, AOAC, 1995). Crude fat in diets, fecal samples and freeze-drying samples of body components was determined using the ether extraction method (method 945.16, AOAC, 1995). The ash in diet and fecal samples was determined (method 924.05, AOAC, 1995) [20 ].
Total starch and amylose were determined by assay kits (k-AMYL, Megazyme International Ireland Ltd., Wicklow, Ireland).

The apparent total tract digestibility (ATTD) was determined using acid insoluble ash (AIA) as a digestibility indicator. AIA in feed and feces were measured by the method of Chinese National Standard (GB/T 23742). Chemical analysis of feed and fecal samples was detected as follows. DM (method 930.15), Ash (method 942.05), EE (method 945.16), CP (method 990.03), and CF (method 920.98) were assessed according to the procedures of AOAC (1995) [27 ]. The GE of feed and fecal samples were determined using an adiabatic oxygen bomb calorimetry (Parr Instrument Co., Moline IL). The digestibility was calculated by the following formula: ATTD (%) = (100 − A1/A2 × F2/F1 × 100), in which A1 represents the AIA content of the feed; A2 represents the AIA content of the feces; F1 represents the nutrient content of the feed; F2 represents the nutrient content of the feces.

The enzyme preparation used in this experiment was obtained from Guangdong VTR Bio-Tech Co., Ltd., and the strains were purchased from China General Microbiological Culture Collection Center (CGMCC).
The composition of fermented feed was consistent with the basal feed at each stage. The fermentation process was developed according to patent (no.201910736307.0).
The basel diet and fermented diet were dried at 65 °C for 72 h, after which they were ground to pass through a 40-mesh screen. All feed samples were measured for dry matter, crude protein and crude fat [15 ], crude fiber [16 ], Ca [17 ] and P [18 ]. Gross energy was determined using a specific adiabatic oxygen bomb calorimetry (Parr Instrument Co., Moline, IL, USA).
Approximately 1 g feed samples were used to determine pH. Briefly, the supernatants of feed samples were centrifuged at 6000× g for 5 min after adding 10 mL distilled water (PHS-3C, INESA, Shanghai, China).
Lactic acid in feed were analyzed using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) combined with a UV-VIS Spectrophotometer (UV1100, MAPADA, Shanghai, China) according to the manufacturer’s instructions. Acid-soluble proteins were determined [19 ].