Nutrient and Mycotoxin Analysis of Animal Diets

Diet samples were analyzed for dry matter by oven drying at 135°C for 2 h (method 930.15; AOAC Int., 2007 ) and for ash (method 942.05; AOAC Int., 2007 ). The gross energy in diets was measured using an isoperibol bomb calorimeter (model 6400, Parr Instruments, Moline, IL, USA) with benzoic acid used as the standard for calibration. Total starch was analyzed by the glucoamylase procedure (method 979.10; AOAC Int., 2007 ), which yields the enzymatically hydrolyzed starch in the sample. Diets were analyzed for N (method 990.03; AOAC Int., 2007 ) using a Leco Nitrogen Determinator (model FP628, Leco Corp., St. Joseph, MI, USA) and crude protein was calculated as N × 6.25. Diets were analyzed for insoluble dietary fiber and soluble dietary fiber on an Ankom Total Dietary Fiber Analyzer (Ankom Technology, Macedon, NY, USA) using method 991.43 (AOAC Int., 2007 ). Total dietary fiber was calculated as the sum of analyzed insoluble and analyzed soluble dietary fiber. Acid-hydrolyzed ether extract in diets was measured by crude fat extraction using petroleum ether (Ankom^XT15, Ankom Technology, Macedon, NY, USA) following hydrolysis using 3N HCl (Ankom^HCl, Ankom Technology, Macedon, NY, USA). Amino acids were analyzed at the University of Missouri Agricultural Experiment Station (Columbus, MO). Diets were analyzed for amino acids on a Hitachi Amino Acid Analyzer, Model No. L8800 (Hitachi High Technologies America, Inc; Pleasanton, CA, USA) using ninhydrin for post-column derivatization and norleucine as the internal standard. Prior to analysis, samples were hydrolyzed with 6N HCl for 24 h at 110°C [method 982.30 E(a); AOAC Int., 2007 ]. Methionine and Cys were determined as Met sulfone and cysteic acid after cold performic acid oxidation overnight before hydrolysis [method 982.30 E(b); AOAC Int., 2007 ]. Tryptophan was determined after NaOH hydrolysis for 22 h at 110°C [method 982.30 E(c); AOAC Int., 2007 ]. The corn and hybrid rye used in the experiment were analyzed for mycotoxins at Trilogy Analytical Laboratories (Washington, MO, USA) using liquid chromatography-tandem mass spectroscopy. The detection limit was 0.1 mg/kg for 15-acetyl deoxynivalenol, 3-acetyl deoxynivalenol, deoxynivalenol, fumonisin (B1, B2, and B3), fusarenon X, and nivalenol. Citrinin and diacetoxyscirpenol had a detection limit of 0.05 mg/kg, neosolaniol had a detection limit of 0.02 mg/kg. The detection limit for zearalenone was 0.0125 mg/kg, the detection limits for HT-2 and T-2 toxins were 0.005 mg/kg, and detection limits for aflatoxin B1, B2, G1, and G2 and for ochratoxin A was 0.001 mg/kg. Analysis of ergot alkaloids in hybrid rye was conducted by refractive index high-performance liquid chromatography using Phenomenex Strata-X-CW weak cation exchange and a reversed-phase column with a detection limit of 10 µg/kg (Phenomenex, Inc., Torrance, CA, USA).