The experimental diets and supplemented AA were analyzed for dry matter [method G-16 (oven)] (CRA, 1999 ), CP (method 990. 03) (AOAC, 2012c ), crude fat (method 942. 16) (AOAC, 2012 ), crude fiber (method 978. 10) (AOAC, 2012b ), and calcium and phosphorus (method 985. 01) (AOAC, 1996 ) and nitrogen (Gavlak et al., 2005 ) by Servi-Tech laboratory (Dodge City, KS; Table 1 ; Appendix Table 1 ) as we previousely described (Shili et al., 2020 (link)). Dietary AA concentration was quantified at Molecular Structure Facility, Proteomics Core of Genome Center (Davis, CA) with Na-based Hitachi 8800 according to established protocols (Cooper et al., 2001 ). Briefly, approximately 10 mg of feed sample was transferred into the glass hydrolysis tube (glass culture tube, VWR #47729-568) and dried under vacuum for 3 to 4 h. Then, liquid phase hydrolysis was performed in vacuo using 6 mol/L HCl and 1% phenol at 110 °C for 24 h. Next, the sample was cooled, unsealed, dried and then was dissolved in the Pickering Diluent containing 40 nmol/mL NorLeu (part #Na220). A volume of 50 μL of sample was injected and subjected to strong cation exchange to separate the AA (AminoSep Beckman Style Na+, 4 × 120 mm, part #AAA-99-6312, Concise, CA). Norleucine (CalBioChem #4890) was included as internal standard to allow correction of the results for any variations in injection volume and other chromatography variables.
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