Oxygen bomb calorimetry

Approximately 25 g of feces from a single stool was collected and stored at −80 °C (14 (link)). Specimens were thawed at 4 °C for 24 hours, homogenized, frozen at −80 °C, and lyophilized (Labconco Freeze Dryer). A 0.1 to 0.3 g aliquot of dried stool was pelletpressed, spiked with a benzoic acid tablet of known energy content, and bomb calorimetry was performed in duplicate according to the manufacturer’s instructions (Oxygen Bomb Calorimetry-Parr Instrument Company) (15 (link)).
See Supplemental Digital Content 1 (http://links.lww.com/MPG/B879) for methods for anthropometric measurements, urine metabolomics, stool microbiology, diarrheal morbidity, and adverse events.

Feces from the last 4 days of one medium-weight piglet from each pen were mixed thoroughly and dried at 65 °C for 72 h, after which they were ground to pass through a 40-mesh screen. The Apparent total tract digestibility (ATTD) was evaluated by using acid insoluble ash (AIA) as the digestibility indicator. The AIA in diet and fecal samples were determined by a method described by Mccarthy et al. [16 (link)] with modifications. The AIA content of the basal diet averaged 0.20 ± 0.002% DM. After AIA analysis, all feed and fecal samples were analyzed for DM (method 930.15, Association of Official Analytical Chemists (AOAC), 1995), ash (method 924.05, AOAC, 1995), EE (method 945.16, AOAC, 1995), CP (method 990.03, AOAC, 1995), and GE [17 ]. GE was determined using a specific adiabatic oxygen bomb calorimetry (Parr Instrument Co., Moline, IL, USA). The digestibility of chemical constituents was calculated using the following formula: $\mathrm{ATTD}(\%)=100-\mathrm{A}1\mathrm{F}2/\mathrm{A}2\mathrm{F}1\times 100$
in which A1 = AIA content in diet, A2 = AIA content in feces, F1 = nutrient content in diet, and F2 = nutrient content of feces [18 (link)].
Total starch and AR were determined by assay kits (k-AMYL, Megazyme International Ireland Ltd., Wicklow, Ireland).