A3306

We collected diet samples twice per week and stored them at −20 °C. At the end of the experiment, they were used to analyze the composition and nutrient levels of the basal diet. We determined the DM content using oven drying at 105 °C to constant mass (method 930.15, AOAC) [22 ] and calculated the dry matter intake (DMI) using DM. We used Kjeldahl nitrogen analysis to determine the CP (method 945.16, AOAC) [22 ] and used a Soxhlet extractor to determine the EE content (method 945.16, AOAC) [22 ]. NDF and ADF were analyzed using heat-stable amylase (A3306, Sigma Chemical Co., St. Louis, MO, USA) and sodium sulfite according to the procedure of Van Soest [23 (link)]. In addition, we used a muffle furnace to measure the ash content by combustion (method 942.05, AOAC) [22 ]. We used the colorimetric method and atomic absorption spectrometry to analyze phosphorus (Spectrophotometer UV755N, Yoke Instrument Co., Ltd., Shanghai, China) and calcium (PerkinElmer AAS800, Waltham, MA, USA), respectively [24 (link)].

Samples of each diet were collected twice per week, stored at −20°C, and composited weekly for the analysis of DM, CP, ether extract (EE), neutral detergent fiber (NDF), acid detergent fiber (ADF), ash, calcium (Ca), and phosphorus (P). We calculated dry matter intake (DMI) according to the feed intake recorded by Automatic feeding equipment (Insentec Co., The Netherlands) and DM obtained through dietary analysis. The DM content was determined by oven drying at 105°C to constant mass (method 930.15, AOAC) [13 ], the CP content was determined using Kjeldahl nitrogen analysis (method 945.16, AOAC) [13 ], and the EE content was determined using a Soxhlet extractor (method 945.16, AOAC) [13 ]. NDF and ADF were analyzed using heat-stable amylase (A3306, Sigma Chemical Co., St. Louis, MO, USA) and sodium sulfite according to the procedure of Van Soest et al [14 (link)]. The ash content was measured by combustion using a muffle furnace (method 942.05, AOAC) [13 ]. A colorimetric method was used for the analysis of phosphorus (Spectrophotometer UV752N, Yoke Instrument Co. Ltd., Shanghai, China) and calcium was measured using atomic absorption spectrometry (PerkinElmer AAS800, Waltham, MA, USA).

This analysis was performed according to the method described by Shin [20 (link)] with minor modifications. Duplicates of 1 g sample were weighed and suspended in phosphate buffer (0.08 M) and digested sequentially with heat-stable α-amylase (A3306 Sigma) (pH 6; 100°C; 30 min), protease (P3910 Sigma) (pH 7.5; 60°C; 30 min), and amyloglucosidase (A9913 Sigma) (pH 4–4.6; 60°C; 30 min) to remove protein and starch. Enzyme digestates were filtered through glass fritted crucibles. Crucibles containing insoluble dietary fiber were rinsed with dilute ethanol followed by acetone and dried overnight in a 105°C oven and weighed to nearest 0.1 mg. Filtrates plus washings were mixed with 4 volumes of 95% ethanol to precipitate materials that were soluble in the digestates. After 1 h, precipitates were filtered through fritted crucibles. One of each set of duplicate insoluble fiber residues and soluble fiber residues was ashed in a muffle furnace at 525°C for 5 h. Another set of residues were used to determine protein as Kjeldahl nitrogen. Soluble or insoluble dietary fiber residues were obtained through calculation. Total dietary fiber was calculated as the sum of soluble and insoluble dietary fiber.