Similarly, mean bodyweight was significantly greater in 0

Similarly, mean bodyweight was significantly greater in 0.5Gln to 2.0Gln than in DPV, DPV + 1.0Gln, and DPV + 2.0Gln at day 2 ( .05). considered a mechanism Paullinic acid by which immunonutrition can assist in the recovery from DPV contamination. at 4C for 15 minutes to separate the plasma. All plasma samples were stored at ?70C until assayed. After thawing, Gln levels in the plasma and duodenal mucosa were determined using a commercial Gln assay (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the manufacturers protocol. Mean values are reported as micromoles of Gln per gram of tissue and as nanomoles of Gln per milligram of protein, respectively. Intestinal protein concentrations were decided as described elsewhere.14 Measurement of Immunoglobulins Frozen duodenal tissue samples were weighed and homogenized in PBS containing Robo2 1% IGEPAL nondenaturing detergent (Sigma-Aldrich Corporation, St. Louis, Missouri), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and complete (ethylenediaminetetraacetic acid-free) protease inhibitor cocktail tablets. The homogenates were kept on ice for 30 minutes and then centrifuged at 10 000at 4C for 10 Paullinic acid minutes. Concentrations of immunoglobulin (IgG) and IgA in plasma and secretory IgA (s-IgA) in supernatants of mucosal tissue were measured using an automated biochemical analyzer (SYNCHRON CX PRO model; Beckman Coulter, Inc, Brea, California) with a commercially available kit (Biosino Bio-Technology & Science Inc, Beijing, China). Enzyme-Linked Immunosorbent Assay Measurement Paullinic acid of Plasma Cytokine Levels and TLR4 Pathway Plasma interleukin 6 (IL-6) and IL-10 concentrations and duodenal tissue TLR4, MYD88, TRAF6, and NF-B concentrations were decided using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Jianglai Biotech, Shanghai, China) in accordance with the manufacturers instructions. Optical densities were measured using a GENios microplate reader (Tecan Austria GmbH, Gr?dig, Austria) at a wavelength of 450 nm. Data were analyzed against the linear portion of a generated standard curve. Total RNA Extraction and Real-Time Polymerase Chain Reaction Analysis Total RNA was isolated from the organs of all ducklings using the E.Z.N.A. Total RNA kit (Omega Bio-tek, Inc, Doraville, Georgia) in accordance with the manufacturers protocol. RNA concentrations were measured using a spectrophotometer at an absorbance of 260 nm. RNA integrity and quality were determined by agarose gel electrophoresis and confirmation that this A260 nm/A280 nm absorbance ratio was between 1.8 and 2.0. Expression levels of TLR4, MyD88, TRAF6, NF-B, and glyceraldehyde-3-phosphate dehydrogenase, as an internal control, were determined by real-time polymerase chain reaction (PCR) analysis using the iQ5 real-time PCR detection system (Bio-Rad Laboratories, Hercules, California). The first strand was synthesized from DNase-treated total RNA using random hexamers and murine Moloney leukemia virus reverse transcriptase with a high-capacity complementary DNA (cDNA) reverse transcription kit (Applied Biosystems China, Inc, Beijing, China) in accordance with the manufacturers instructions. Primers were designed using Primer Express software (Applied Biosystems, Waltham, Massachusetts) showed in Table 1. Briefly, the PCR reactions consisted of 50 ng of first-strand cDNA and 500 nmol/L of primers in a final volume of 20 L using SYBR Green IPCR core reagents included in the SYBR real-time PCR kit (TaKaRa Biotechnology Co Ltd, Dalian, China). The expression values were calculated as 2??Ct.15 Table 1. Primers Used for Quantitative Real-Time PCR. .05) in 1 to 2 2 days, 3 to 4 4 days, and 5 to 6 days. In control, 0.5Gln, 1.0Gln, and 2.0Gln, mean feed consumption sharply raised with Gln supplementations. Otherwise, mean feed consumption of DPV, DPV + 0.5Gln, and DPV + 1.0Gln decreased in 3 Paullinic acid to 4 4 days compared with 1 to 2 2 days and then increased in 5 to 6 days. Open in a separate window Physique 1. Mean feed consumption per duckling during 6 days of ducklings with and without Gln or DPV injection. DPV indicates Duck plague virus; Gln, glutamine. The mean body weight of each duckling was presented in Physique 2. The average weights of the animals in the 8 groups were similar at the beginning and 12 hours ( .05). Mean body weight was significantly greater in 1.0Gln and 2.0Gln than in DPV to DPV + 1.0Gln at day 1 ( .05)..