2.4. Pretreatment, Intestinal Incubation, and Total-Tract Digestibility Determination

The mobile nylon bag incubation procedure followed the protocol proposed by Kaitho et al. [55]. In the present study, the rumen passage rate (kp) for CP was calculated to be 4.18 according to the prediction models recommended by NRC (2001) [51]. The ruminal incubated residues (12 h for CP, 24 h for NDF and ADF) were ground through a 1 mm sieve and weighed at a rate of 0.5 g per mobile nylon bag (3 × 6 cm bag size; 25 μm pore size) which was subsequently heat-sealed. Twelve duplicated bags per forage were placed in a shaking bath filled with pepsin/HCl solution for 1 h at 39 °C to simulate abomasal digestion before intestinal incubation. Pepsin/HCl solution was prepared by dissolving 1 g of pepsin powder (activity 1:10,000, Beijing Aoboxing Biotech Co., Ltd., Beijing, China) in 1 L of 0.01 mol/L HCl. The three Holstein cows fitted with three-site (rumen, anterior duodenum, and terminal ileum)-cannula were used for the intestinal incubation. Each cow was regarded as a replicate, each forage had three replicates, and each forage had 4 parallel replicates per cow (n = 12). Four bags of each forage were individually inserted in random order into the anterior duodenum cannula of each cow at a rate of two bags every 30 min starting from the morning feeding. A maximum of 12 bags can be inserted into the duodenum cannula per cow per day. Bags were collected in the feces from 8 h after incubation. The recovered bags were rinsed and manipulated in cold water until the water ran clear, then dried to a constant weight at 65 °C for 48 h. Only bags recovered within 24 h after insertion were used for further analysis.