Animals, diets, and management
The study was approved by the Animal Care and Use Committee of Northeast Agricultural University Institutional Animal Care and Use Committee (NEAU- -9). The animal experiment was conducted in Acheng Experimental Base of Northeast Agricultural University. A total of twenty-eight castrated weaned male piglets [Duroc × (Landrace × York-shire)] with a similar body weight of 10.19 ±1 kg was randomly assigned to 4 treatments with seven replications per treatment and 1 piglet per replicate for 28 days feeding trial. Treatments were arranged as a 2×2 factorial with oil type [FSOs vs. OSOs] and dietary RES (0 vs. 300 mg/kg).
FSOs were purchased from Jiusan Grain and Oil Industry Group Co., Ltd. (Harbin, China). One part of fresh soybean oils was stored at −20°C to prevent automat oxidation. The oxidization degree of soybean oils in different treatments was evaluated mainly by examining the peroxide value (POV). The OSOs were continuously treated with FSOs (POV=11.7 mEqO2/kg) at 65°C and bubbled air at a rate of 4 L/min until the POV reached approximately 384 mEqO2/kg, respectively. The fresh and oxidized soybean oils were stored at −20°C for further experiments. POVs were examined as described by the American Oil Chemists Society22. Nutrient and energy densities of experimental diets, which were shown in Supplementary Table 1, were set to meet, or exceed the nutritional requirements of swine from NRC (2012)2. All piglets were fed separately in a single stainless-steel metabolic cage containing a water dispenser and feeding tank for free drinking and feeding. During experiment, the pigpen temperature was kept between 20 and 23 ℃. Meanwhile, the relative humidity was kept between 65 and 75%. The light regime was a 12 h light/12 h dark cycle. The piglets had no access to probiotics and/or antibiotics throughout the entire experiment.
Performance and diarrhea incidence
After 28 days of feeding, piglets were fasted for 12 h before sacrifice. Body weights (BW) of piglets were individually evaluation on the mornings of day 1, and day 29 of the feeding trial. Deed intake per pen was recorded daily throughout the trial to calculate average daily feed intake (ADFI), average daily gain (ADG) and average daily feed intake/average daily gain (F/G). Clinical signs of diarrhea were visually assessed each morning by observers blinded to the treatments using a five-grade scoring system23,1 = well-formed feces, 2 = slightly soft feces, 3 = soft and partially formed feces, 4 = loose and semiliquid feces, and 5 = watery feces. Then the average daily diarrhea index per replicating was calculated.
Sample collection and processing
On the 29th day of the trial, blood samples (10 mL) were collected using heparin tubes from piglets by venous puncture and then centrifuged at 3 000 g for 15 min at 4°C. The plasma was immediately collected and stored at −20°C pending further analyses. After blood sampling, the piglets were sacrificed using electricity (250 V, 0.5 A, for 5 to 6 s), with subsequent jugular exsanguination. After opening the abdominal cavity, the gastrointestinal tract was removed to obtain portions of the jejunum, and the colon measuring approximately 20 cm was cut longitudinally and cleaned with ice-cold phosphate buffer solution (PBS). Mucosa samples were collected using scraping by sterile glass microscope slides. They were then snap-frozen in liquid nitrogen and stored at −80°C for subsequent analysis. Two continuous segments were carefully cut from the middle of the whole jejunum and colon for histological assay and mucosa collection. Sections of approximately 1.5 cm in length were fixed in fresh, chilled 4% paraformaldehyde for morphometric evaluation and histochemical staining.
Determination of apparent nutrient digestibility
At the end of the experiment (day 24 to 27), feces from each group were collected for nutrient apparent digestibility determination, which was determined by the acid-insoluble ash method24. All the feces of piglets were collected twice a day at 8 h and 20 h, put in plastic bags, weighed, and recorded. To prevent the loss of fecal ammonia, 10 mLH2SO4 were added and the feces was stored in a -20℃ refrigerator. Before the samples were determined, the feces samples were thawed from the refrigerator and mixed evenly for four days from each pig. About 200 g of the mixed feces samples were taken out and dried in an oven at 60± 5℃ for 48-72 h. Then the dried feces samples were crushed with a grinder and passed through a 40-mesh sieve for testing. Acid insoluble ash, dry matter (DM), crude protein (CP), crude fat (EE), gross energy (GE) in feces and feed were determined according to GB/T23742-2009 internal indicator method.
Determination of digestive enzymes in the jejunum
The sample preparation and the activities of jejunum digestive enzymes, including amylase (#C016-1-1), lactase(#A082-1-1), trypsin(#A080-3-1), and lipase(#A054-1-1), (#A080-2-2), maltase(#A082-3-1), sucrase(#A082-2-1) of the jejunum were determined using commercial kits provided by Nanjing Jiancheng Bioengineering Institute (Jiangsu, China).
Intestinal oxidative stress status and plasma cytokines analyses
Based on the manufacturer’s instructions, commercial kits were used to determine total superoxide dismutase (T-SOD # A001-1-1), glutathione peroxidase (GSH-Px #A005–1-2), hydrogen peroxide (H2O2 # A064-1-1), as well as total antioxidant capacity (T-AOC # A015-1-2) and malondialdehyde (MDA #A003–1-2) were determined using commercial kits provided by Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Inflammatory cytokines in the plasma were determined with an ELISA kit (Beijing Sino-UK Institute of Biological Technology.) according to the manufacturer’s instructions.
Determination of diamine oxidase (DAO) activity and D-lactate content in the plasma
Plasma D-lactate and the diamine oxidase (DAO, #A088-1-1) activity concentrations were determined using commercial ELISA kits provided by Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). All procedures were performed with strict adherence to the manufacturer’s guidelines.
Mucosal morphometry and epithelial proliferation in jejunum and colon
For intestinal morphological analysis, after being fixed in paraformaldehyde solution at room temperature for 24 h, jejunal tissue specimens were dehydrated using an upgraded series of ethanol and xylene and then processed into paraffin blocks. A cross-section with a thickness of 5µm was cut from each specimen and stained with hematoxylin and eosin. Three of each group were randomly selected well-oriented and intact villi and adjacent crypts were randomly selected for the measurements of villus height (VH) and crypt depth (CD) per slide by an assessor blinded to the treatments using optical microscopy (Nikon Eclipse 80i Nikon, Tokyo, Japan) and NIS-Elements 3.0 Imaging Software. Villus height needed to be evaluation in at least 10 villi from each pig and averaged. Data were analyzed using a digital microscope M8 and photographed at 40× magnification. The adjacent jejunum and colon were also fixed overnight in a 2.5.% glutaraldehyde solution at 4 ℃, and then, these samples were treated for observation by electron microscopy. Epithelial proliferation was assessed using immunofluorescence in combination with a standardized quantification pipeline in jejunum and colon mucosa. To determine the relative amount of proliferative epithelial cells in the jejunum and colon mucosa samples, tissues were stained with nuclei stain PCNA and Hoechst (n = 3 randomly selected piglets from each treatment group).
The jejunum and colon with 5% blank goat serum wax block sample complete coverage, slice to be placed in the wet box, at 37 ℃ constant temperature and humidity incubator incubation for 30 min. The blocking solution was removed, and the primary antibody working solution (ABclonal ZO-1, Occludin, Bioss, BSM-33070m, Ki67) prepared with antibody diluent was directly added to the samples. The samples were completely covered, and the sections were placed in a wet box and incubated at 4℃ overnight. Rewarming: the samples were placed at room temperature, rewarming for a 15min, antibody working solution was removed, and buffer solution in TBST was washed once for 5 minutes. Buffer solution TBS was washed for 3 times, 5 minutes each time, and the second antibody was incubated: the fluorescent second antibody working solution corresponding to the primary antibody species was dropped on the samples. The samples should be completely covered, protected from light, and incubated at 37℃ for 1 hour. Remove the working solution of the secondary antibody and wash it with buffer TBST once for 5 minutes. Wash with buffer TBS 3 times, 5 minutes each time, dye the core: drop DAPI working solution on the sample, prepare with 0.01M pH7.2 TBS buffer solution, DAPI solution: 0.01M pH7.2 TBS buffer liquid volume ratio 1:500, avoid light, room temperature, incubated for 10 minutes; DAPI working solution was removed and washed with buffer TBST once for 5 minutes. Wash with buffer solution TBS for 3 times, 5 minutes each time, drop anti-fluorescence attenuation sealing tablets, then cover the cover glass sealing tablets, and observe under a fluorescence microscope and collect images.
RNA extraction and RT-PCR
The reaction system and the thermal cycling conditions used for RT-PCR were adjusted according to our previous study25. After retrieving the snap-frozen jejunum mucosal samples, we isolated total RNA using TRIzol Reagent (#9109), as suggested in the manufacturer’s manual (TaKaRa Biotechnology, Dalian, Liaoning, China). Extracted RNA was removed in 50 µL of ultra-pure water. A NanoDrop ND-1000UV spectrophotometer (NanoDrop Technologies) was used to measure the purity and concentration of total RNA at 260 and 280 nm. Electrophoresis on a 1.5% agarose gel that had been stained Ultra GelRed™ (#GR501–01; Vazyme Biotech Co., Ltd.) was used to verify RNA integrity. One microgram of total RNA was then reverse-transcribed into complementary DNA using the Prime-Script™ RT Reagent Kit (#RR036A; TaKaRa Biotechnology). A real-time polymerase chain reaction (PCR) was performed on a QuantStudio 5 Real-Time PCR System (Applied Biosystems, Life Technologies, CA, USA) using the ChamQTM SYBR® qPCR Master Mix Kit (#Q311–02; Vazyme Biotech Co., Ltd.), as recommended in the manufacturer’s guidelines. The PCR process involved a 30-s pre-run at 95°C, 40 cycles of denaturation at 95°C for 5 s, and a 60°C annealing step for 30 s. For the melting curve conditions, one denaturation cycle was performed at 95°C for 10 s; the temperature was then increased from 65 to 95°C at a rate of 0.5°C/s. The primer sequences are shown in Supplementary Table 2. The average of β-actin and GAPDH was used as the internal control. The relative expression abundance of each target gene was calculated by the 2−Δ Δ Ct method, as elucidated previously26.
Gut microbiota analysis
Colonic digesta samples were collected from slaughtered piglets and immediately stored at −80 ℃. Bacterial genomic DNA was extracted from each sample (Qiagen DNA Stool Mini Kit, Duesseldorf, Germany). DNA was quantified with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, NC, USA) and further assessed by running on 1% agarose gels. The V3–V4 hypervariable region of 16S rRNA genes was amplified using specific primer pairs (forward 50-ACTCCTACGGGAGGCAGCA-30 and reverse 50-GGACTACHVGGGTWTCTAAT-30) with barcodes to construct the sequencing libraries (TruSeq ® DNA PCR-Free Sample Prep Kit, Illumina, San Diego, CA, USA). The qualified DNA libraries were loaded in a NovaSeq platform with 2 × 250 bp paired-end sequencing. The paired-end reads were obtained and merged using FLASH software (V1.2.7, http://ccb.jhu.edu/software /FLASH/). Operational taxonomic units (OTUs) with a 97% identity were gathered with Unparsed (ver. 7.1, http://drive5.com/uparse /). Taxonomic annotation was performed using the Mothur algorithm (70% confidence) with the Silva Database (http://www.arb-silva.de/). The taxonomic composition of the bacterial community was then analyzed. RDA analyses were performed to identify the relationship among the identified differential genera of microbiota, growth performance, apparent digestibility, intestinal histomorphology, inflammatory cytokines, intestinal enzymatic activities, SCFAs, and treatments. Furthermore, according to the Pearson correlation analyses, several genera that were separately correlated with apparent digestibility, intestinal enzymatic activities and inflammatory cytokines, etc were also further identified.
Quantification of SCFAs
Short-chain fatty acids (SCFAs), including acetate, propionate, butyrate, valerate, is butyrate, and isovalerate, were quantified by an external standard method using gas chromatography (GC) as described by a previous study. Briefly, 1.5 g colonic chyme samples were added to screw-capped tubes with 6 mL of distilled water. After mixing overnight at 4 ℃ and centrifugation at 1500 r/min for 10 min at 4 ℃, 2 mL of supernatant from each sample were transferred to another centrifuge tube, and 400 µL of meta-phosphoric acid (25% v/v) were added to remove the protein. The samples were then centrifuged at 12,000 r/min for 10 min at 4 ℃. The resulting supernatants (1 mL each) were transferred into gas chromatography sample bottles and analyzed using an Agilent 6890N GC (Palo Alto, CA, USA) coupled to a flame ionization detector with helium used as the carrier gas. An Agilent FFAP column (30 m × 0.53 mm i.d. × 1.00 µm (film thickness)) was installed for analysis, with a constant flow rate of 4.0 mL/min. Spitless injection volume was 0.2 µL of the sample. The injector and detector temperatures were 220 ℃ and 240 ℃, respectively. The GC oven temperature was held at 90 ℃ for 1 min and then increased to 190 ℃ at a rate of 20 ℃/min and held for 3 min. Samples were run in triplicate, with a coefficient of variation less than 15% within triplicate samples used for quality control.
An individual pig was treated as the experimental unit. The statistical model included the fixed main effects of OSOs and RES and their interaction effects. Diarrhea score of piglets was analyzed using univariate ANOVA, data were analyzed using two-factor ANOVA for all other indicators (Version 23.0, SPSS Inc, Chicago, Illinois, USA), and visualized using GraphPad Prism. Semi-quantification of fluorescence was performed by Image J software. The data were expressed as the means ± standard error of measurement (SEM), and a value of (P< 0.05) was considered statistically significant. Statistical differences were separated by Duncan’s multiple range test. The significance level was set at (P< 0.05). Whereas (0.05< P< 0.10) was considered as a tendency.