Experiment birds and treatment
The experiment birds and treatment was conducted in accordance with our previously described methods . One thousand five hundred and thirty 1-day-old Lingnan Yellow broiler chickens were obtained from a local commercial hatchery. During the whole experiment, the birds were housed in 15 concrete floor rooms (8 m2) bedded with 6 cm rice hulls. Pre-experiment lasted for 21 days and during this period all birds were reared at the same stocking density (12.75 birds/m2). Sick and weak chicks observed during this period were removed. On day 22, one thousand four hundred and forty broilers with a similar body weight were randomly allotted into 5 treatments, each with three replicates. Meanwhile, all rooms were filled with new rice hulls. The stocking densities of these 5 treatments were 8, 10, 12, 14, and 16 birds per m2 (64, 80, 96, 112, and 128 birds per room, respectively). The birds had free access to feed and water throughout the experimental period with light-dark cycles per day. During the experiment, all rooms were environmentally controlled at 25°C. The relative humidity was maintained at 55 ± 5%. A corn-soybean meal basal diet was formulated to meet the nutrient requirement reference to the NRC (1994) (Table 1).
Sampling and processing
At the age of 50 days old, 12 male broilers (4 birds per replication) were randomly selected from each treatment. Feed was removed 12 hours before slaughter, while water was available. Blood samples by the vein under the wing (5 mL/bird) were immediately collected into a coagulant tube and centrifuged at 3000 rpm/min for 10 min at 4°C to obtain serum. Then the birds were slaughtered to obtain trachea. All samples were frozen directly in liquid nitrogen and stored at − 80°C for further analysis.
Trachea samples were embedded in paraffin wax after dehydration and sectioned at 5 μm on a rotary microtome. The sections were further stained with haematoxylin and eosin [H&E] for morphological analysis and digital images were obtained under a light microscope [Olympus].
Relative protein expression by western blot
The protein expression of claudin1, muc2 and caspase3 in the trachea was determined by western blot. Total protein extraction was performed using T-PER Tissue Protein Extraction Reagent (Thermo Pierce, 78510), protein quantification was then performed using the BCA Quantitation Kit. After SDS-PAGE and membrane transfer, T-TBS (containing 5% non- fat dry milk or BSA) was added to the membrane for blocking at room temperature for 1 h. The antibody (1:100) was then added and incubated overnight at 4°C, followed by washing the membrane. Secondary antibody [Goat anti-Mouse IgG (H + L)] was added and incubated at room temperature for 1 h, and then washed. SuperSignal® West Dura Extended Duration Substrate was used for western blot detection. The optical density of the bands was analyzed using Image J software. β-actin was used as an internal control and was found to exhibit no difference between groups. The relative abundance of each target protein was expressed as the ratio of target protein/β-actin.
Determination of immune status
The commercial kits for all the cytokine indexes were purchased from Nanjing Jiancheng Bioengineering Institute. The trachea mucosal supernatant was analyzed for the content of interleukin-1β, 4, 10 (IL-1β, 4, 10). The procedures were carried out according to the description of the assay kits.
Sample Preparation for GC-TOF/MS Analysis
The samples were prepared for metabolomics analysis as described previously . Briefly, 100 μL plasma was added with 350 μL of extraction liquid (methanol:acetonitrile:water = 2:2:1, volume ratio) and 20 μL L-2-chlorophenylalanine. The sample mixture was maintained at 4oC for 30 min and subsequently centrifuged at 13,000 rpm for 15 min. The supernatant (400 μL) was collected into a 1.5 mL tube, and then transferred to a glass vial and dried in a vacuum. Methoxyamine salt reagent (methoxyamine hydro-chloride, dissolved in pyridine 20 mg/mL) was added and the mixture incubated in an oven at 80oC for 30 min, then derivatized using N, O Bis (trimethylsilyl) trifluoroacetamide (BSTFA), incubated for 1 hour at 70oC. The final mixture was strongly vortexed for 1 min and used for analysis .
As our previously described methods , the derivatized samples were analyzed using an Agilent 7890 GC system equipped with a Pegasus 4D TOFMS (LECO, St. Joseph, MI) with a DB-5MS capillary column (30 m × 250 μm inner diameter, 0.25 μm film thickness coated with 95% dimethyllpolysiloxane cross-linked with 5% diphenyl under the following conditions: initial temperature was kept at 80oC for 12 seconds, increased to 180oC at a rate of 10oC /min, to 240oC at a rate of 5oC /min, and further to 290oC C at a rate of 20oC /min and maintained for 11 min. One μL of sample solution was injected with helium as the carrier gas at a flow rate of 1 mL/minute. The temperatures of transfer line and ion source were 245oC and 220oC respectively. The MS data were acquired with a mass-to-charge ratio (m/z) range of 20-600 in a full-scan mode.
GC-TOF/MS Data Acquisition and Processing
The raw peaks extraction, data baselines filtering and calibration, peak alignment, deconvolution analysis, peak identification, and integration of the peak area were operated using the Chroma TOF4.3X software (LECO) and LECO-Fiehn Rtx5 database. The peak identification was tested using the RI (retention time index) method. The missing values of the original data were filled by half of the minimum value via a numerical simulation method. Noise removal was performed based on an interquartile range to filter data and then normalized by area normalization methods. The SIMCA14.1 software package (Umetrics, Umea, Sweden) was used for multivariate variable pattern recognition analysis: principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least-squares discriminant analysis (OPLS-DA). PCA was used to show the internal structure of the data and display the similarity and difference. OPLS-DA was applied to obtain a higher level of group separation and better explain the variables. To evaluate the predictive ability and fitting level of the model, the parameters R2Y and Q2 were applied. The metabolites responsible for differentiating two groups were filtered with the following requirements: variable importance in the projection (VIP) >1 and P-values of 0.05 (threshold) with 95% Hotelling’s T-squared ellipse .
The data was examined by one-way analysis of variance (ANOVA) using IBM SPSS 19.0 and expressed as means with SEM derived from the ANOVA error mean square. Differences between means were examined through Duncan’s multiple range tests using P < 0.05. Moreover, Student’s t-test was used to profile metabolite differences between two groups.