Raw potato starch (RPS; AVEBE Ltd), a typical Type Ⅱ RS, used in this study and contains 54.72% RS content (dry matter basis) according to our previous analysis . The inhibitor of bacterial SCFAs production β acids extracted from the hops plant (S.S. Steiner, USA). MI-2 (Eternity Bioscience Inc. NJ, USA) was dissolved at a concentration of 30 mM as a stock solution, stored at -20°C.
Animals and management
Day-old male Cherry valley ducklings with an initial average body weight of 48 ± 1.34 g were used in this study. All birds were housed in individual cages (0.16 m2/bird) in a temperature- and humidity-controlled room. Temperature was maintained at 32 ± 1°C for the first week and then the temperature was decreased by 2.75°C at d 7 and 14. The light schedule was 18 L:6 D throughout the experimental period. The diet was formulated in line with the National Research Council requirements (Table S1). RS in RPS and diets was analysed using an RS assay kit (K-RSTAR; Megazyme Ltd), and confirmed proper preparation of experimental diets. At d 7 and 14, after 6 h of fasting, all ducks were weighed, and feed intake was measured on a per cage basis.
Ducklings were randomly allocated to 3 treatments: non-challenged (Ctrl, fed with basal diet), E. coli-challenged (E. coli, fed with basal diet), and E. coli-challenged with RS (E. coli-RS; fed a 12% RPS-supplemented diet and challenged with E. coli). On d 7, except for the Ctrl group, which received 0.6 mL of sterilised Luria-Bertani culture, all birds were orally administrated with 0.6 mL of Luria-Bertani culture containing 6 × 108 CFU/mL of E. coli O88 twice, 8 h apart, as previous description . In addition, to evaluate the role of SCFAs in bone metabolism, ducklings were randomly assigned to E. coli, E. coli-RS, and E. coli-RS with β acids (E. coli-RS + β acids). β acids were added into drinking water at a final concentration of 20 mg/L from 1 d. E. coli and E. coli-RS birds received pH and sodium matched water. To further confirm the potential mechanism of RS interacted inflammatory response to restore a reduction in E. coli-induced bone loss, ducklings were randomly allocated to E. coli, E. coli-RS, and E. coli-MI-2. From d 1 to 14, Both birds of E. coli and E. coli-RS were injected intraperitoneally phosphate buffer saline, where ducks from E. coli-MI-2 were injected intraperitoneally 30 mg/kg MI-2, respectively. Each treatment with 6 replications of 10 birds per replicate.
Sample and data collection
At d 14, 1 duck of average body weight from each cage was blended via jugular vein after fasting 6 h, and centrifuged at 4,000 g/15min at 4°C for serum. Subsequently, birds were sacrificed, the ileal content of each bird was gently removed and the pH value of the content was directly measured using a pH-STAR (Matthuas, Inc.). Then, the weight and length of ileum were quickly determined. Mid-ileal mucosa, cecal contents, left tibia (the proximal end), and bone marrow were collected and stored (-80°C) until analysis. Right tibia (the proximal end) was dissected and rapidly immersed in phosphate-buffered formaldehyde for histology analysis. Another 6 ducks (1 bird per cage) were randomly selected and euthanized. The left tibia was removed for micro computed tomography (Micro-CT) analysis. The right tibia was harvested, length, and width (at 50% of length) of tibia were measured after removal of soft tissues.
In vivo intestinal permeability
For whole intestinal permeability, 15 d-old meat ducks were received orally fluorescein isothiocyanate dextran (FITC-d, 4.16 mg/kg body weight) 2 h prior to the time of blood collection. Serum fluorescence was analysed using a Gemini XPS Microplate Reader (Molecular Devices, LLC. Sunnyvale, CA) at an excitation/emission wavelength of 485/530 nm. The content of FITC-d transfer into the serum was calculated from standard curves generated by the serial dilution of FITC-d.
Sequencing of cecal microbiota
According to our recently description , the total DNA in cecal content was extracted using a DNA stool mini kit (Qiagen, Valencia, CA, United States). After assessing the integrity and size of DNA, the hypervariable V3-V4 regions of the 16S rDNA gene was amplified. Then, the resulting PCR products were sequenced on an Illumina PE250 platform (BGI, Shenzhen, China). The obtained sequences were processed using FLASH (v1.2.11) and USEARCH (v7.0.1090) for alignment and clustering. All effective reads were clustered into operational taxonomic units (OTUs) with a similarity threshold of 97%. The representative sequence of each OTU was aligned against the Greengene database for taxonomy analysis. As for data analysis, principal coordinate analysis (PCoA) was performed based on the Bray-Curtis dissimilarity calculated by QIIME software and displayed using R software.
Cecal SCFAs analysis
Approximately 0.5 g of cecal content was diluted with 2 mL of ultrapure water mixed with a uniform, followed by depositing for 30 min and centrifuging at 3,000 × g for 15 min. 1 mL supernatants were mixed with 0.2 mL ice-cold 25% (w/v) metaphosphoric acid solution and incubated at 4°C for 30 min. After centrifuging at 11,000 × g for 10 min, the SCFAs contents including acetate, propionate, and butyrate were separated and determined by gas chromatograph (Varian CP-3800, USA), as previously described .
Detection of skeletal strength, fat-free weight, and ash
Mechanical strength was performed by the 3-point bending method using the texture analyser (TA. XT Plus; Stable Microsystems) with a constant 50 kg load cell. Loading proceeded at a constant rate (5 mm/min) until a fracture occurred. The load-displacement curve was recorded, and the maximum load of the tibia was directly read from the peak value. Hereafter, fat-free weigh was determined through air-drying for 24 h at room temperature, extracting by ethyl ether for 48 h, and oven dried at 108°C for 24 h. Subsequently, dry-defatted tibia was ashed in a muffle furnace at 550°C for 24 h and the ash was measured based on the percentage of dry-defatted weight.
Micro-CT imaging was performed using a GE Explore Locus Micro-CT (GE Healthcare, Piscataway, NJ, USA) with instrument settings optimized for calcified tissue visualization at 90 kV. The analysis of the trabecular bone in the proximal end of the tibia (metaphysis) was performed starting from 9mm below the surface of the condyles and extending 4 mm distally. To exclude denser cortical regions at the bone surface, the outer 0.5 mm of the bone surface was removed from the region of interest. Bone volume/total volume (BV/TV) and thickness (Tb.Th) of trabecular bone were calculated. The average thickness of the structures was measured using the thickness plugin from Bone J as our previous method following our recent methods .
The proximal end of tibia was fixed in 10% formaldehyde solution for 24 h and decalcified in ethylene diamine tetraacetic acid (Sigma, USA). Tissues were embedded in paraffin and 5 µm sections were stained with tartrate resistant acid phosphatase (TRAP) bone staining using assay kit (Sigma-Aldrich, USA). Histopathological images were collected using a microscope with image analysis software (Nikon Corporation, Tokyo, Japan). The number of osteoclast (N.Oc/BS) was quantified based on the TRAP staining.
Splenocytes were harvested from the spleen. Erythrocytes were lysed with ammonium-chloride-potassium lysing buffer and spleen cells were stimulated for 5 h in RPMI 1640 medium containing 20 ng/mL phorbol myristate acetate, 1 µg/mL ionomycin, and 10 µg/mL brefeldin A (Sigma-Aldrich, St Louis, MO, USA). For T regulatory (Treg) cells weresuccessively stained with anti-CD3, anti-CD4, anti-CD25, and anti-Foxp3 antibodies. The stained cells were rinsed, resuspended, and analysed by BD AccuriC6 flow cytometer with analysis software (BD Biosciences, San Jose, CA, USA).
Cytokines of IL-1β, IL-10, IL-17, TNF-α concentrations were measured using enzyme-linked immunosorbent assay (ELISA, Meimian Biotechnology Co., Ltd, Jiangsu, China). Calcium (Ca) and phosphorus (P) level were measured with Biochemistry Analyzer (Yellow Springs Instrument Co. Inc., Yellow Springs, OH). Serum bone turnover markers including procollagen type I N-terminal propeptide (P1NP) and C-terminal cross-linked telopeptide of type I collagen (CTx), alkaline phosphatase (ALP) and TRAP activity were assayed by ELISA assay (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturers. All samples were tested in triplicate within each assay.
Gene expression assays
Ileal, tibia, and bone marrow were pulverized and RNA was extracted using Trizol (Invitrogen) following the manufacturer’s instructions. Reverse transcription into cDNA and quantitative real-time PCR were performed was performed on ABI 7900HT detection system (Applied Biosystems, CA, USA). The primer sequences for the target genes designed using Primer 3 (Table S2). Relative gene expression was quantified by normalizing to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin.
Bone marrow (approximately 0.3 g) was ground in liquid nitrogen and lysed using 3 mL of lysis buffer. After centrifugation, the protein content of the supernatant was determined by using bicinchoninic acid protein assay kits (Thermo Fisher Scientific Inc.). The protein lysates were separated by 10% SDS-PAGE and subsequently transferred onto a polyvinylidene diflouride membrane (Trans Blot Turbo transfer system; Bio-Rad). Membranes were blocked using 5% non-fat milk in a solution of Tris-buffered salt with Tween-20 for 1 h at room temperature. The blocked membrane was incubated with rabbit anti-pIκBα (catalogue no. mAb2859; dilution 1:1500), mouse anti-p65 (catalogue no. mAb6956; dilution 1:1000), mouse anti-β-actin (catalogue no. mAb3700; dilution 1:2000), and corresponding secondary antibody-conjugated horseradish peroxidase. All antibodies were from Cell Signalling Technology Biotechnology Inc., (Massachusetts, USA). The blots were visualized using Western blotting detection system and the protein concentrations in each specimen were normalised to β-actin abundance
Statistical analyses were performed using GraphPad Prism (GraphPad Software Inc., CA, USA). After checking for normal distribution and equal variance using the Shapiro-Wilk and Levene’s tests, respectively. Two-tailed unpaired t-test or one-way analysis of variance followed by Tukey’s post hoc test was conducted to examine statistical significance. Data were expressed as mean and standard deviation (SD). P-value less than 0.05 was considered significant.