2.3.1. Fermentation Profile and Enzyme Activity

The rumen pH was immediately determined after sample collection using a pH electrode (Model pH B-4; Shanghai Chemical, Shanghai, China). The NH₃-N concentration of rumen fluid was measured using the phenol-sodium hypochlorite colorimetry method described by Broderick and Kang [19]. The MCP concentration was detected according to Makkar et al. [20]. Then, 0.2 mL of 25% metaphosphoric acid was added to 1.0 mL rumen fluid samples, to wipe off the albumen precipitation, before the quantification of VFAs, which were measured by gas chromatography (6890 N; Agilent technologies, Avondale, PA, USA) according to Cao et al. [21]. The urease [22], protease [23], amylase [24], lipase [25], xylanase [25], and dehydrogenase [26] were measured using a SpectraMax 190 Microplate Reader (MD., New York, NY, USA) with the commercial kits (Suzhou Grace Biotechnology Co., Ltd., Suzhou city China); specifically, the rumen fluid was centrifuged at 2500× g for 10 min at 4 °C temperature, and the supernatant fluid was ultrasonically broken for 3 min and then centrifuged at 12,000× g for 5 min. The measured wavelength of the urease, protease, amylase, lipase, xylanase, and dehydrogenase were 578, 680, 540, 405, 540, and 460 nm, respectively.