Rumen Metabolome Analysis by UPLC-MS/MS

Fifty milligrams of rumen contents were thawed on ice, and 500 µL of 70% methanol internal standard extract was added at 4 °C. After shaking for 3 min, the mixture was left to stand at −20 °C for 30 min, followed by centrifugation at 12,000 r/min for 10 min at 4 °C. Next, 250 µL of the supernatant is centrifuged at 12,000 r/min for 5 min at 4 °C. Next, 150 µL of this supernatant is taken in the liner of the corresponding injection bottle for data acquisition and further analysis. The metabolome of rumen contents was analyzed by ultra-performance liquid chromatography (UPLC) and Tandem mass spectrometry (MS/MS) (QTRAP® 6500+, SCIEX, Framingham, MA, USA) [33 (link)–35 (link)]. After obtaining the LC/MS data of different samples, the extracted ion chromatographic peaks of all metabolites were integrated using MultiQuant software (Applied Biosystems, Foster, MA, USA) and the MetWare database (MWDB) database, respectively. The chromatographic peaks of the metabolites in different samples were corrected by integration [36 –38 (link)]. The relative concentrations of rumen metabolites were screened by FC (FC ≥ 2 and FC ≤ 0.5) and VIP (VIP ≥ 1) to identify the different metabolites. The identified metabolites were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) compound database, and the annotated metabolites were then mapped to the KEGG Pathway database [39 (link)].