Rumen Fluid Analysis for VFA, LPS, and Lactate

In both in vitro and in vivo experiments, the rumen fluid was centrifuged at 10,000 × g for 10 min at 4°C and filtered through a 0.22 μm filter. A total of 0.2 mL of 20% metaphosphoric acid stock solution (Sinopharm Group, China) containing 60 mM crotonic acid (Sinopharm Group, China) was added to 1 mL of the supernatant for volatile fatty acid (VFA) determination. The VFAs were determined through capillary column gas chromatography (GC-14B, Shimadzu, Kyoto, Japan; film thickness of the capillary column, 30 m × 0.53 m × 1 μm; column temperature, 110°C; injector and detector temperature, 200°C) (9 ). The serum and rumen fluid LPS concentrations were measured using a Chromogenic End-point Tachypleus Amebocyte Lysate Assay Kit (Chinese Horseshoe Crab Reagent Manufactory Co., Ltd., Xiamen, China) as described previously (7 (link), 10 (link)). The lactate concentrations of rumen fluid and serum were measured using a lactate Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions protocol. The LDH release of the serum was detected using a lactate dehydrogenase (LDH) kit (Jiancheng Bioengineering Institute, Nanjing, China).