Chemical Analysis and Parameter Calculations for Experiment 2

Proximate and amino acid content analyses of corn and soybean meal were conducted before feed formulation of experimental diets by near-infrared spectroscopy (NIRS, DS2500, FOSS, Denmark) using calibration models developed by Evonik Nutrition & Care GmbH (Hanau, Hessen, Germany). All experimental diets were also analyzed with the same NIRS methodology and by wet chemistry as follows.

Dietary treatments (12 starter feeds), excreta, and ileal digesta samples were analyzed for DM, CP, and titanium dioxide content. Excreta samples were further analyzed for gross energy (GE) and ileal digesta samples for starch. DM was determined according to AOAC International (2012) standard methods, CP (N × 6.25) was determined by combustion method (LECO, AOAC International, 2006; method 990.03). The GE analysis was determined using an oxygen bomb calorimeter (IKA C5003; IKA Labortechnik, Staufen, Germany), and AME was corrected to zero N retention (AME_n) using a factor of 8.22 kcal/g (Hill and Anderson, 1958). Total starch, amylose proportion within the starch, resistant starch, and in vitro hydrolyzed starch or damaged starch analyses were done using Megazyme kits: Total Starch (amyloglucosidase/α-amylase method), Amylose/Amylopectin, Resistant Starch, and Starch Damage assay procedures following manufacturer's recommendations (Megazyme Inc., Chicago, IL). Gelatinized starch was conducted based on AACC 76-11.01/AOAC 979.10 method. Amylase activity in pelleted feed was determined using Megazyme kit assay of alpha-amylase using red-starch (Megazyme, Bray, Ireland). Titanium dioxide concentrations were measured in triplicate for dietary feeds and duplicates for excreta and ileal digesta samples on a UV spectrophotometer following the method described by (Myers et al., 2004). Apparent ileal digestibility, total tract utilization, and AME_n were calculated using the following equations (Kong and Adeola, 2014):

where M_i represents the concentration of titanium dioxide in the diet in g/kg DM; M_o represents the concentration of titanium dioxide in the excreta and ileal digesta in g/kg DM output; E_i represents the concentration of DM, CP, energy, or starch in the diet in mg/kg of DM; and E_o represents the concentration of DM, CP, starch, and energy in the excreta and ileal digesta, in mg/kg DM. The GE_i is gross energy (kcal/kg) in the diet; GE_o is the gross energy (kcal/kg) in the excreta; N_i represents nitrogen concentration in the diet; and N_o represents nitrogen concentration in the excreta in g/kg DM.