Rumen Metabolite Analysis by Spectrophotometry

Determination of ammonia nitrogen by spectrophotometer colorimetric method [16 (link)]. Instantly measured rumen pH with a portable pH meter (Model PHB-4, Shanghai Leica Scientific Instrument Co., Ltd., Shanghai, China). Volatile fatty acid (VFA) concentrations were determined as described previously [17 (link)]. In brief, 2 mL of thawed rumen content was centrifuged at 5400 rpm at 4 °C for 10 min, and then 0.2 mL of 25% metaphosphoric acid was added to 1 mL of the supernatant for homogenization. The mixed solution was centrifuged at 10,000 rpm for 10 min at 4 °C, and VFAs (acetate, propionate and butyrate) were determined by a gas chromatograph (HP6890N, Agilent Technologies, Wilmington, DE, USA) equipped with an AT-FFAP capillary column (50 m × 0.32 mm × 0.25 µm). The column temperature was held at 60 °C for 1 min, then ramped unrestricted at 5 °C/min to 115 °C, then to 180 °C at 15 °C/min.