Fecal Amino Acid Digestibility Analysis

At the end of 12 weeks, 3 birds were chosen from each replicate and put in a metabolic cage with collection tray for fecal samples. The fecal samples were collected on replicate basis at 12 h intervals daily for 3 days. Contamination of fecal samples were avoided by ensuring that feed, feathers and other foreign materials were completely excluded from fecal samples during collection. The fecal samples were then weighed, pooled together for each treatment group, and kept in sealed bags at –20°C (Zhou et al., 2021 (link)). Prior to amino acid analysis, the fecal samples were thawed and oven-dried at 65°C for 72 h, then weighed again and pulverized into finely ground powder that can pass through a 0.05 mm mesh. Amino acids determinations from samples of feed and feces was performed by HPLC, following an established method by Varzaru et al. (2013) . The HPLC system was Finnigan Surveyor Plus and HyperSil BDS C18 column, size 250 × 4.6 mm, 5 μm (Thermo-Electron Corporation, Waltham, MA). For apparent fecal amino acid digestibility analysis, the feed intake and the fecal weight (dry matter basis) from each metabolic cage were calculated. The apparent fecal amino acid digestibility was calculated as: Apparent Amino acid Digestibility = 1 ˗ (amino acid concentration in the feces ×feces weight)/(amino acid concentration in the feed × feed intake) × 100%.