Digestive Enzyme Assay

Duodenal digesta and the pancreas was collected from 1 bird per replicate with median BW day 21 posthatching, frozen in liquid nitrogen, and stored at ‒80°C until required for assay. Enzymes activities were determined using a commercially available assay kit (Sigma Chemical Co, St. Louis, MO). The absorbance of the colorimetric final product was measured in a UV/visible spectrophotometer, and the concentration of the respective enzymes was calculated accordingly. For duodenal digesta, the samples were centrifuged at 13,000 rpm at 4°C for 10 min, and aliquots of the supernatant was used for enzyme assay. The activity of the pancreatic enzymes was determined after the whole organ was homogenized in appropriate buffers and centrifuged at 13,000 rpm at 4°C for 10 min to get a clear supernatant. Trypsin activity (EC 3.4.21.4) was determined with benzoyl-DL-arginine-p-nitroanalide as substrate (Sigma Aldrich, CN MAK290). The product of p-nitroaniline was measured at an absorbance of 405 nm. One activity unit of trypsin was expressed as nanomoles of p-nitroaniline released per minute per milligram of protein. Chymotrypsin activity (EC 3.4.4.5) was determined with N-Benzoyl-L-tyrosine ethyl ester as the enzyme substrate and absorbance was measured at 405 nm. Amylase activity (EC 3.2.1.1) was determined using a coupled enzyme assay, and absorbance of ethylidene-pNP-G7 cleaved by the amylase was measured at 405 nm. One unit is the amount of amylase that cleaves ethylidene-pNP-G7 to generate 1.0 μmol of p-nitrophenol per minute at 25°C. Lipase activity (EC 3.1.1.3) was determined at an absorbance of 570 nm using a coupled enzyme reaction. One unit of lipase is the amount of enzyme that will generate 1.0 μmol of glycerol from triglycerides per minute at 37°C.