Samples were divided into 3 portions; 1 portion was used for NH3–N analysis with 5ml of 1MH2SO4 added to 45 ml of rumen fluid. The mixture was centrifuged at 16,000×g for 15 min, and the supernatant was stored at−20 °C before NH3–N analysis using the Kjeltech Auto 1030 Analyzer and VFA analysis using HPLC.