Ika calorimeter c1

On 6 and 14 dpi (18 and 26 d, respectively), 4 birds/replicate were randomly selected, euthanized by cervical dislocation, and digesta were collected from an ileal section starting from 2 cm below the Meckel's diverticulum until 2 cm from the ileo-cecal junction. The samples from the same replicate were pooled, dried in a ventilated oven at 75°C for 48 h, and finely ground using a Kitchen aid coffee grinder. Gross energy was measured using a bomb calorimeter (IKA Calorimeter C1, IKA Works Inc., Wilmington, NC), and the chromium oxide concentration was measured following the methodology described by Dansky and Hill (1952) (link) at the University of Georgia. The crude protein (CP) was analyzed in the Agricultural Experiment Station Chemical Laboratories at the University of Missouri-Columbia (N × 6.25, LECO). The apparent ileal digestibility of CP, energy, and dry matter were calculated according to the following equation: $AID,\%=\left\{\left[{\left(nutrient/C{r}_2{O}_3\right)}_{Diet}-{\left(nutrient/C{r}_2{O}_3\right)}_{digesta}\right]/{\left(nutrient/C{r}_2{O}_3\right)}_{diet}\right\}\times 100$ where (nutrient/Cr₂O₃) is the ratio of dry matter, CP, and energy to Cr₂O₃ in the diet or ileal digesta.

Oven-dried feed and ileal digesta were ground to measure gross energy and minerals. For analysis of gross energy, the ground samples were measured by a calorimeter (IKA Calorimeter C1, IKA Works Inc., Wilmington, NC). Macromineral levels of feed and ileal digesta were determined by the Soil Laboratory, University of Georgia. The chromic oxide was analyzed according to Dansky and Hill (1952) (link). Briefly, 0.3 g of sample was ashen in a nickel crucible at 600°C overnight to burn out organic materials. Additional 5.8 g of fusion mixture (190 g KNO₃ to 100 g Na₂CO₃) and 5.6 g NaOH was added in the nickel crucible and burned at 600°C for additional 2 h. The fusion mix was dissolved in water, and chromite was oxidized to chromate by H₂O₂. The concentration of chromate was determined at 400 nm on a spectrophotometer (Spectramax M5, Molecular Devices, San Jose, CA). The ileal digestible energy (IDE) and apparent ileal digestibility (AID) of minerals were calculated according to the following equations. $IDE=\raisebox{1ex}{$G{E}_{Diet}-\left(G{E}_{Digesta}xC{r}_2{O}_{3Diet}\right)$}\!\left/ \!\raisebox{-1ex}{$C{r}_2{O}_{3Digesta}$}\right.$ $AID,\%=\frac{1-\left(Nutrien{t}_{Digesta}\right)x\left(C{r}_2{O}_{3Diet}\right)x100}{\left(Nutrien{t}_{Diet}\right)x\left(C{r}_2{O}_{3Digesta}\right)}$

Feed and ileal digesta were dried in an oven before analyses. The dried digesta were ground, and the gross energy of the samples was determined by using a calorimeter (IKA Calorimeter C1; IKA Works Inc., Wilmington, NC). The digesta and feed samples were sent to the Soil Laboratory in the University of Georgia for measurement of macrominerals. The chromic oxide was analyzed according to the methods described by Dansky and Hill (1952) (link). Briefly, 0.3 g of sample was ashed in a nickel crucible at 600°C overnight. Then, 5.8 g of fusion mixture (190 g KNO₃–100 g Na₂CO₃) and 5.6 g of NaOH were added and heated for 2 additional hours. The chromite was dissolved in 150 mL of distilled water with 1 mL of H₂O₂. The solution was filtered and diluted by adding distilled water to 250 mL. The samples and standard solution were measured at 400 nm by using a spectrophotometer (SpectraMax M5). The calculations of ileal digestible energy (IDE) as well as AID of minerals and nitrogen were performed by using the following equations: $\mathrm{I}\mathrm{D}\mathrm{E}=G{E}_{\text{Diet}}-\left(G{E}_{\text{Digesta}}\times C{r}_2{O}_{3\text{Diet}}\right)/C{r}_2{O}_{3\text{Digesta}}$ $\mathrm{A}\mathrm{I}\mathrm{D},\%=\frac{1-\left({\text{Nutrient}}_{\text{Digesta}}\right)\times \left(C{r}_2{O}_{3\text{Diet}}\right)\times 100}{\left({\text{Nutrient}}_{\text{Diet}}\right)\times \left(C{r}_2{O}_{3\text{Digesta}}\right)}$