A019 2

The lambs were weighted on two consecutive days after they were reared in individual units for 50 days. Then, ruminal fluid was collected from each lamb by use of an orogastric tube 2–4 h after morning feeding. The ruminal fluid was mixed thoroughly and filtered through 4 layers of cheesecloth.
The ruminal fluid pH was immediately determined using a pH meter. An 8 mL aliquot of ruminal fluid was preserved with adding 1 mL of metaphosphoric acid (25% wt/vol) to determine volatile fatty acid (VFA) content. The rest of the samples were stored at -20°C for bacteria DNA extraction. For VFA determination, thawed samples of the rumen fluid were centrifuged for 15 min at 10,000 × g at 4°C. Two milliliters of the supernatant were then mixed with 200 μL crotonic acid (1% wt/vol), and the solution was filtered through a 0.45 μm filter. The ruminal VFAs were separated and quantified by using a gas chromatograph (Trace 1300, Thermo Fisher Scientific, United States) as described by Li et al. (2014) (link), using a 30 m × 0.32 mm × 0.33 μm fused silica column (DB-FFAP, Agilent Technologies, United States). Lactate concentrations in the ruminal fluid were determined using commercially available lactate assay kits (A019-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Cells were seeded into 96‐well plates at a density of 3000/well. After being cultured for 24 h, cells were cultured with a serum‐free medium for 12 h and incubated with a complete medium for another 24 h. The supernatant was collected and the concentration of lactate was measured according to the instruction of the lactic acid detection kit (A019‐2, Nanjing Jiancheng) and normalized according to the MTT assay. The absorbance was measured using a microplate reader (MD, SpectraMax 190) at 530 nm.
Ldh activity was determined using commercial kits for Ldh activity assay (A020‐2, Nanjing Jiancheng). Protein concentration was used to adjust the cell quantity. The absorbance was measured using a microplate reader (MD, SpectraMax 190) at 450 nm.

A commercially available kit was used (Cat# A019-2, JianCheng BioTech, Nanjing, PR China) to measured the lactate acid level in cell culture medium that was obtained at the end of each cell experiment. All the data were expressed as fold changes in comparison to controls.