Automated spectrophotometer

A 10 mL blood sample was collected from two randomly selected pigs from each pen on d 14 and 28. The sampling was conducted by jugular vein puncture using a disposable vacutainer tube without anticoagulants (Becton Dickinson, Franklin, NJ, USA). The blood samples were then centrifuged by 3,000×g for 15 min 4°C, and separated serum was stored at −20°C until analysis. Immunoglobulin A (IgA), IgG, and IgM of serum were analyzed with immunoglobulin kits (Sanwei Biological Engineering Co., Ltd., Shandong, China), using an automated spectrophotometer (Shimadzu, Japan).

At the end of each phase, pigs were weighed individually, and feed consumption was measured to calculate average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F). Fecal grab samples were collected during the last 4 d of each phase to determine the apparent total tract digestibility of dry matter (DM), gross energy (GE), crude protein (CP), and Zn. Pens were cleaned before the start of sample collection and the fecal samples were then pooled within the pen, dried in a forced air oven at 60°C for 72 h, and ground in a Wiley mill (Thomas Model 4 Wiley Mill, Thomas Scientific, Swedesboro, NJ, USA) using a 1-mm screen for chemical analysis. Analysis for each sample was done in triplicate for DM (Method 930.15), CP (Method 990.03), ash (Method 942.05), Ca, and P (method 985.01; 16) according to the methods of AOAC [11 ]. The GE of diets and feces were measured using a bomb calorimeter (Model 1261, Parr Instrument Co., Molin, IL, USA), while Cr concentrations were determined [12 (link)] with an automated spectrophotometer (Shimadzu, Japan).

Three birds per pen were transferred to metabolic cage to conduct the trial in nutrient digestibility. The chickens were fed diets with chromic oxide (0.25%) added for the last 10 days (adaptive and collective periods) of each phase to determine the apparent total tract digestibility of dry matter (DM), gross energy (GE), crude protein (CP) and Cu. The excreta samples per replicate were collected into stainless steel trays and then dried in a forced-air oven at 60°C for 72 hours, grounded in a Wiley Mill (Thomas Model 4 Wiley Mill; Thomas Scientific, Swedesboro, NJ, USA) using a 1-mm screen, and used for chemical analysis. Each sample was analyzed in triplicate for DM (Method 930.15) and CP (Method 990.03), according to the methods of AOAC [31 ]. The Cu in the feed, and excreta was determined by inductively coupled plasma emission spectroscopy (ICP) according to the methods of AOAC [31 ]. The GE of the diets and excreta were measured using a bomb calorimeter (Model 1261, Parr Instrument, Molin, IL, USA), and the chromium concentrations were determined with an automated spectrophotometer (Shimadzu, Japan) according to the procedure described by Fenton and Fenton [32 (link)].