Fiber analyzer

Diet TMR samples were collected weekly throughout the experiment and composited over the feeding period. The composited sample was dried in a 55 °C oven and ground to pass through a 2 mm screen. Samples were analyzed for dry matter, ash, N (Kjehldahl method), Ca, P, and ether extract by standard procedures (AOAC, 1990). Crude protein was determined by multiplying N by 6.25. Neutral detergent fiber and acid detergent fiber concentrations were determined by the modified method of Van Soest et al. [25 (link)] using a fiber analyzer (Ankom Technology Corp., Fairport, NY, USA). The TMR was also analyzed for concentrations of Cu, Zn, Mo, Fe, and S using inductively coupled plasma optical emission spectroscopy and concentrations of Co and Se via inductively coupled plasma mass spectrometry by the Veterinary Diagnostic Laboratory at Michigan State University.

Crude fiber was determined by first weighing the filter bag (W₁), then 0.5 g of dried sample (W₂), that was ground to pass a 1 mm screen, and directly added into a filter bag. A blank bag was weighed and included in digestion, to determine blank bag correction (C₁). The bags were sealed within 0.5 cm from the pen edge, using the heat sealer. The bags were placed in a bag suspender. About 2000 mL of temperature acid detergent solution was added into the ANKOM Fiber Analyzer vessel and the suspender was covered. The Fiber Analyzer vessel was set for 60 min. Approximately 2000 mL of hot water was added, and the samples were agitated again. The samples were rinsed for 3–5 min with water until a neutral pH was reached. Excess water was pressed out from the bags. The bags were then placed in a beaker and covered with acetone for 3 min. The bags were removed and pressed again to remove excess acetone. The bags were dried in the oven at 105 °C, for at least 2 h. The bags were cooled to ambient temperature and weighed (W₃).
$\%\mathrm{ADF}\left(\mathrm{as}-\mathrm{is}\mathrm{basis}\right):=\frac{\left[{\mathrm{W}}_3-\left({\mathrm{W}}_1\times {\mathrm{C}}_1\right)\right]\times 100}{{\mathrm{W}}_2}$

All chemical analysis were conducted in duplicate and repeated if the results differed by more than 5%. The ingredients used in this experiment were analyzed for dry matter (DM) [16 ], ether extract (EE) [17 (link)], ash [16 ], calcium [16 ], and phosphorus [16 ]. Kjeldahl N was determined according to the method used by Thiex et al. [18 (link)]. The content of neutral detergent fibre (NDF) and acid detergent fibre (ADF) were determined using filter bags and Fiber Analyzer equipment (Fiber Analyzer, Ankom Technology, Macedon, NY) following a modification of the procedure of Van Soest et al. [19 (link)]. Starch content was determined after converting starch to glucose using an enzyme assay kit (Megazym International Ireland, Wicklow, Ireland). The GE of feces, diets and corn samples were measured using an automatic adiabatic oxygen bomb calorimeter (Parr 6300 Calorimeter, Moline, IL). The GE of urine was measured by injecting 4 ml of the sample into 2 filter papers in a special crucible, and dried for 8 h in a 65°C drying oven to determine the energy. The 1,000-kernel weight (g/1,000 seeds) was measured in each sample of test corn by first cleaning it of all foreign materials and then counting 1,000 seeds.

Chemical analyses of all samples were conducted in duplicate. The diet was analyzed including dry matter (DM), crude protein (CP), calcium, total phosphorus (16 ) and ether extract (EE) (17 (link)). Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were measured with filter bags using a Fiber Analyzer (Ankom Technology, Macedon, NY, USA). Gross energy (GE) was determined by an automatic adiabatic oxygen bomb calorimeter (Parr 1281, Automatic Energy Analyzer; Moline, IL, USA) according to previous study (18 (link)).
Blood samples were centrifuged (Biofuge22R; Heraeus, Hanau, Germany) at 3,000 × g for 10 min at 4°C, then the supernatant was transferred to storage tubes and stored at−80°C. After the frozen samples were thawed at 4°C, plasma levels of triglyceride, total cholesterol, high-density lipoprotein, low-density lipoprotein, total protein, albumin, and urea nitrogen were quantified using a biological analyzer (7600 Automatic Biological Analyzer; Hitachi, Tokyo, Japan) at the Kangjia Hongyuan Biotech Company (Beijing, China). Sample preparation for metabolomics, UPLC-MS analysis and data mining and processing according to previous study (1 (link)).