Result-X101SC21112124-Z01-J001-B1-42

At 60 days of age, 27 Xiangdong black male goats with an average initial BW of 10.88 ± 1.22 kg were randomly allocated to three treatments: high protein [NP; 12.4% protein]; low protein [LP; 8.3% protein; supplement rumen-protected lysine (RPLys) and rumen-protected methionine (RPMet) to the same level of NP]; and low protein with rumen protected Leu [LP+RPLeu; 8.3% protein; supplement RPLys and RPMet to the same level of NP].The animal trial lasted for 110 days, consisting of 20 days adaptation and a 90-day experimental period.Prior to feeding, blood samples were collected from the goats via the jugular vein into a 10 ml vacuum tube containing Na-heparin on the morning of day 90. After centrifugation at 3,000 <i>g</i> for 20 min at 4°C, plasma samples were split and frozen at -80°C until analysis.A volume of 100 mL plasma sample was mixed with 400 mL methanolewater (4:1, vol:vol) solution and then settled at -20℃. The mixture then went through vortex for 30 s and ultrasound at 40 kHz for 30 min at 5℃. Next, the samples were placed at 20℃ for 30 min to precipitate proteins. After being centrifuged at 13,000 g and 4℃ for 15 min, the supernatant was transferred to a specialized vial for UHPLC-MS/MS analysis. A Thermo UHPLC system equipped with an ACQUITY BEH C18 column (length: 100 mm; inner diameter: 2.1 mm; grain diameter: 1.7 mm; Waters, Milford, USA) was used to perform chromatographic separation of the metabolites.The mass spectrometric data were collected using a ThermoUHPLC-Q Exactive Mass Spectrometer equipped with an electrospray ionization (ESI) source operating in either positive or negative ion mode. The optimal conditions were set as followed: heatertemperature, 425 ℃; Sheath gas flow rate 50 arb (arbitrary unit);Aux gas flow rate 13 arb; ion-spray voltage floating (ISVF), -3,500 Vin negative mode and 3,500 V in positive mode, respectively;Normalized collision energy, 20 to 40 to 60 V rolling for MS/MS.Data acquisition was performed with the Data Dependent Acquisition (DDA) mode. The detection range was 70 to 1,050 m/z.