GC-MS Analysis of Fecal Metabolites in Sows

The metabolites concentrations were evaluated with GC-MS in sows fecal samples. In accordance with He et al. [1 (link)], 100 mg of fresh fecal sample was transferred to 5- mLcentrifuge tubes, mixed with 500 µL distilled water, and was vortexed for 60 s. Then, 1000 μL methanol was added to be an internal quantitative standard and vortexed for 30 s. The ultrasound machine was used to hold samples at 25 °C for 10 min, then the centrifuge process (5000 r/min; 5 °C; 15 min) was performed after 30 min incubation on ice. All the supernatants were placed in 2 mL centrifuge tubes and dried. Then, the dried samples were mixed with 60 μL of methoxyamine solution in pyridine and vortexed (30 s) to be reacted for 120 min at 37 °C. A 60 μL trifluoroacetamide reagent (containing 1% FMCS) was added for 90 min (37 °C) and centrifuged (5000 r/min; 5 °C; 15 min). The produced supernatant was moved to a sample bottle to be analyzed by Agilent 7890A/5975C GC-MS (Agilent Technologies, Santa Clara, CA, USA).