2.4. In Vitro Neutral Detergent-Cellulase Plus Amylase Method

The in vitro organic matter digestibility was determined by NDC plus amylase method using small nylon bags (37 µm, 25 × 60 mm). Samples (0.50 ± 0.001 g) were transferred to small nylon bags, sealed (FR-300B, Blueberry, Shanghai, China), boiled in neutral detergent solution with heat-stable α-amylase for 75 min (ANKOM A220, ANKOM Technology Corp., Macedon, NY, USA), then oven-dried at 65 °C for 48 h. For the cellulase buffer solution preparation, we incubated 20 g cellulase [17] in a 1 L acetic acid buffer (pH 4.8) for 1 h at 40 °C. Two small nylon bags were randomly placed in 100-mL culture tubes and pre-warmed overnight at 39 °C. The next morning, cellulase buffer solution (80 mL) was added into each preheated 100-mL culture tube using an automatic pump and then incubated for 24 h in a 40 ± 2 °C water at 40–60 rpm (DSHZ-300A, Jiangsu, China). Following incubation, the bags were immersed in cold water to stop fermentation, washed as in situ nylon bag technique, and oven-dried at 65 °C for 48 h.