Sigma a3306

The dry matter content of each ration was detected after drying at 135°C for 3 h, and the ash content was detected following combustion at 550°C for 6 h according to the protocols of the AOAC [26 ]. The Kjeldahl method [27 (link)] was used to determine the levels of crude protein (CP, 6.25×N) using an automated nitrogen analyzer (FOSS, DK-3400 Hillerød, Denmark). The total starch content in the TMR was determined via an enzymatic method (α-amylase and amyloglucosidase) using a commercial kit (Megazyme, Megazyme International Ireland Ltd., Bray, Ireland). To analyze the neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents, heat-stable α-amylase (Sigma A3306; Sigma–Aldrich, Shanghai, China) and sodium sulfite were used according to the methods previously established by Van Soest et al. [27 (link)]. The total calcium (Ca) and phosphorus (P) levels were determined according to the National Measurement Principles of China (GB/T 5009.92–2003 and GB/T 5009.87–2003, respectively). The dietary lysine to methionine ratio (Lys: Met) and the net energy for lactation (NE_L) of each ration were estimated based on the included ingredients using the Cornell-Penn-Miner Dairy (CPM-Dairy, version 3.0.8.1) software.

The in vitro digestibility values of the DDGS samples were analyzed at the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences as slightly modified by (Zhao et al. 2014 (link)). A computer-controlled, simulated digestion system was applied to accurately predict the digestibility of monogastric animals. Briefly, simulated gastric fluid was composed of approximately 1550 U/mL pepsin (Sigma 10070; Sigma-Aldrich Co., St. Louis, MO). The small intestinal fluid was simulated with 4730 U/mL of amylase (Sigma A3306; Sigma-Aldrich Co.), 550 U/mL of trypsin (Amresco 0785; Amresco Inc., Solon, OH), and 154 U/mL of chymotrypsin (Amresco 0164; Amresco Inc.). Before in vitro intestinal digestion, 2 mL of small intestinal fluid was added to a digestion chamber. The small intestinal fluid was diluted by 20 mL residual simulated gastric fluid, which reached a neutral pH after 3 washing procedures during in vitro gastric digestion. The residues were centrifuged at 3000×g for 15 min, and the sediments were dried at 105 °C for 5 h and tested in subsequent DM, CP, gross energy (GE) and AA contents.