Thermo ultimate 3000 system

The samples were analyzed using a Thermo Ultimate 3000 system (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a Waters Acquity UPLC^® BEH C18 column (1.7 μm, 100 × 2.1 mm, Waters Co., Milford, MA, USA) at 35 °C with a wavelength detector set to 440 nm. The mobile phases of acetonitrile comprising 0.1% formic acid (A) and water comprising 0.1% formic acid (B) were used for UPLC. At a flow rate of 0.2 mL/min, the following gradient elution program was used: 0–8 min, linearly increasing from 10% A to 50% A; 8–12 min, linearly increasing from 50% A to 90% A; 12–13 min, linearly increasing from 90% A to 100% A; sustained 30 min.
The qualitative analysis of each product was performed using Agilent Technologies 1290 Infinity II and 6545 Q-TOF, together with Dual Agilent Jet Stream Electrospray Ionization sources (Agilent Technologies, Santa Clara, CA, USA). The drying gas was set at 350 °C and 8 L/min; the sheath gas was set at 350 °C, with a gas flow rate of 11 L/min. The nebulizer was set at 35 PSIG; the VCap was set at 3500 V. The data were analyzed using MassHunter (version B.07.00).

The longissimus dorsi and semimembranosus muscles were cut to measure the meat quality traits according to previous reports (3 (link), 16 (link)). Briefly, the pH values were measured at 24 h (pH_24h) after slaughter. Meat color measurements cover indicators L* (lightness), a* (redness), and b* (yellowness) after slaughter 24 h (L_24h, a_24h, and b_24h). Drip loss, cooking loss, crude protein, and ash content were determined as described previously (3 (link), 17 (link)). Amino acid profile and fatty acid composition were determined using gas chromatography–mass spectrometry (GC–MS 7890B-5977A, Agilent, Palo Alto, CA, United States) and liquid chromatography–mass spectrometry (Liquid phase was performed on Thermo Ultimate 3,000 system, Thermo Fisher Scientific Inc., Waltham, MA, United States; Mass Spectrometry was performed on Thermo Q Exactive Focus mass spectrometer, Thermo Fisher Scientific Inc., Waltham, MA, United States), respectively.

After the samples were slowly thawed at 4°C in a salt-ice bath, 100 µl serum sample was precisely transferred by a pipettor to the 1.5 ml centrifuge tubes. Metabolites were extracted using a 400 µl methanol: water (4:1, v/v) solution with 0.02 mg/ml L-2-chlorophenylalanin as an internal standard, vortexed for 30 s, and extracted with low-temperature ultrasound for 30 min (5°C, 40 kHz). After extraction, the samples were left to stand at −20°C for 30 min. The sample was centrifuged (13000 rpm, 4°C, 15 min), and the supernatant was transferred to an UPLC-MS/MS injection vial for detection. Liquid chromatographic separations were performed on a Thermo Ultimate 3000 system (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) equipped with a Waters ACQUITY UPLC® HSS T3 column (150 × 2.1 mm, 1.8 μm). Mass spectrometry operations were performed on a Thermo Q Exactive Focus mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) with spray voltages of 3.8 kV in positive ion mode (ESI+) and −2.5 kV in negative ion mode (ESI−), respectively.
A pooled quality control (QC) sample was prepared by mixing equal volumes of all samples. Metabolomics data have been deposited to the EMBL-EBI MetaboLights database (DOI: 10.1093/nar/gkz1019, PMID:31691833) with the identifier MTBLS4245. The complete dataset can be accessed here https://www.ebi.ac.uk/metabolights/MTBLS4245.