All experimental procedures used in this study were approved by the Animal Care and Use Committee of Jiangxi Agricultural University (permit No. JXAULL-20190098).
Animals, diets and experimental design
Two hundred and forty pigs (Duroc × Landrace × Yorkshire), weaned at 24 days with an initial average body weight (BW) of 6.79 ± 0.04 kg, were randomly assigned into one of four dietary treatments with six replicate pens per treatment (five barrows and five gilts per pen) on the basis of initial BW, sex and litter. The dietary treatments consisted of the basal diet (control group, CON) and basal diet supplemented with 200, 400 or 800 mg/kg SDP (provided by Qingdao Haida Biotechnology Co., Ltd, Qingdao, China). The SDP were produced from the seaweed Enteromorpha prolifera, which contains 4.82% protein, 2.9% moisture, 17487.6 mg/kg calcium, 15362.7 mg/kg magnesium, 24262.4 mg/kg potassium, 3.93 mg/kg arsenium, 1.22 mg/kg plumbum, and 0.86 mg/kg cadmium. The content of polysaccharides is more than 48%. According to the analysis of polysaccharide composition by High Performance Liquid Chromatography (HPLC), the SDP consisted of five monosaccharides including rhamnose (Rha), glucose (Glc), glucuronic acid (GlcA), xylose (Xyl) and galactose (Gal). The molar percentage of monosaccharides is Rha: 40.6%, Glc: 38.2%, GlcA: 9.3%, Xyl: 6.3%, Gal: 5.6%. The basal diet was formulated to meet the National Research Council (2012)-recommended nutrient requirements for pigs at corresponding growth period, and the ingredient and nutrient composition were shown in Table 1. Pigs were fed isonitrogenous diets containing various levels of SDP. Although the additional SDP was added to the basal diet by substituting for the same amount of corn, the protein level is still low, which is basically consistent with protein in basal diet. All pigs were housed in a temperature-controlled room and had free access to water and feed throughout the trial. The experimental period lasted 21 days.
The pigs were weighed individually after 12-h fasting in the morning of day 1 and day 22, and feed consumption per pen was recorded daily to calculated average daily feed intake (ADFI), average daily gain (ADG) and feed: gain (F/G).
Animals, diets and experimental design
A total of twenty healthy male pigs (Duroc × Landrace × Yorkshire), weaned at 24 days with an initial average body weight of 6.77 ± 0.10 kg, were randomly assigned into two dietary treatments (n = 10) based on their BW and litter, consisting of the basal diet (CON) or the basal diet supplemented with 400 mg/kg SDP. This selection of SDP level in Exp. 2 was based on the results of Exp. 1, which suggested that 400 mg/kg SDP resulted in lower F/G ratio and higher ADG (P < 0.05) than CON group. The basal and experimental diet were the same as described for Exp. 1, belonging to the same production batch. All pigs were housed in individual metabolism cages (0.7 × 1.5 m) in a temperature-controlled conditions (26 ± 2℃) with free access to food and water. The experimental period lasted for 21 days, and experimental procedures used were similar to those previously described for Exp. 1. Individual pig body weight was recorded after 12-h fasting in the morning of day 1 and 22, and feed consumption was recorded daily. ADFI, ADG and F/G were calculated. The pigs’ diarrhea was observed at the same time of each morning throughout the experimental period as previously described . The fecal consistency of each pig was assessed with a score from 0 to 3 (0 = normal feces, 1 = pasty feces, 2 = semiliquid feces, and 3 = liquid feces). The occurrence of diarrhea was defined as daily fecal consistency score of ≥ 2. Diarrhea rate per day in each treatment group was expressed as follows: diarrhea rate (%) = (number of pigs with diarrhea/total number of pigs) × 100, and the cumulative rate of diarrhea was calculated.
Sample collection and preparation
At the end of the experiment, after 12-h fasting, eight pigs with weight close to the average level of each group were selected for bleeding via the anterior vena cava. Blood samples were centrifuged at 3,000 × g for 15 min at 4℃ to collect serum. After blood collection, the same pigs were then euthanized by intravenous injection of pentobarbital sodium. The gastrointestinal tract was immediately removed and divided into duodenum, jejunum, ileum and cecum. Approximately 2 cm segments of the middle of duodenum, jejunum and ileum were isolated, washed and fixed in 4% paraformaldehyde for morphological analysis. The mucosa samples of small intestine were collected by scraping the intestinal wall with glass microscope slides, frozen in liquid nitrogen and stored at -80℃ until analysis. Finally, caecal digesta were snap frozen and stored at -80℃ for microbial population measurements.
Serum parameters analysis
Serum antioxidant parameters including glutathione peroxidase (GSH-Px) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, total antioxidant capacity (T-AOC), and malondialdehyde (MDA) concentration were determined by the commercial kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China) according to the manufacturer’s instructions. The level of interleukin (IL)-6, IL-10, IL-1β, tumour necrosis factor (TNF)-α, D-lactate and immunoglobulin (IgA, IgG and IgM) and diamine oxidase (DAO) activity were measured using commercially porcine-specific ELISA kits (Beijing Winter Song Boye Biotechnology Co. Ltd, Beijing, China).
The fixed intestinal segments were dehydrated, and then embedded in paraffin, and cut into 5-μm thick sections. The sections were deparaffinized, rehydrated and stained with hematoxylin and eosin. At least four images per section and five sections from each pig were obtained. The villus height and crypt depth were determined on the images using an Olympus CK 40 microscope (Olympus Optical Company, Tokyo, Japan), and villi-crypt ratio (VCR) was calculated.
Small intestine biochemical analysis
The frozen small intestinal mucosa samples (approximately 0.5 g) were weighed and homogenized in ice-cold physiological saline solution (1:9, weight/volume), and then were centrifuged at 3,500 ×g for 15 min at 4℃ to collect the supernatant. The secreted immunoglobulin A (sIgA) content in each section of small intestine was determined by the porcine ELISA assay kit (mlbio Biotech, Shanghai, China), and the results were expressed as mg sIgA/g intestinal protein. The activities of disaccharidase (sucrase, lactase and maltase) in jejunal mucosa were analysed using commercial assay kits (Nanjing Jiancheng Bioengineering Institute), and expressed as on a per milligram protein basis. The protein concentration was determined by Pierce BCA Protein Assay kit (Thermo Scientific, Waltham, MA, USA).
Quantitative real-time PCR (qRT-PCR) analysis
Total RNA was extracted from the small intestinal mucosa using TRIzol reagent (Sigma-Aldrich, Saint Louis, MO, USA) in accordance with the manufacturer’s instructions. The concentration and integrity of RNA was determined by nucleic-acid/protein analyzer (Beckman Coulter DU800, Beckman Coulter Inc, Fullerton, CA, USA) and 1% agarose gel electrophoresis, respectively. Reverse transcription reactions were conducted using iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). qRT-PCR was performed on the CFX96 RT-PCR Detection System (Bio-Rad) as described previously . The relative mRNA expression of target genes was calculated and normalized with β-actin reference using the method of 2−ΔΔCT . The used primer sequences are shown in Table 2.
Immunoblotting analysis was performed as previously described . Briefly, protein extracts from jejunal mucosa were separated by 10% SDS-PAGE and then transferred to PVDF membrane. Membranes were blocked with 5% non-fat dry milk in TBST for 1 h and overnight incubated at 4℃ with the primary antibodies against zonula occludens-1 (ZO-1), Claudin-1, Occludin, and β-actin (Proteintech, Chicago, IL, USA), followed by incubation with HRP-linked secondary antibody anti-rabbit IgG or anti-mouse IgG (Cell Signaling Technology, Danvers, USA) in TBST for 1 h at room temperature. Finally, membranes were visualized using Bio-Rad ChemiDocTM imaging system (Bio-Rad). Band density of target protein was quantified after normalization to β-actin.
Quantification of microbial population
Bacterial DNA was extracted from caecal digesta using the Stool DNA kit (Omega Bio-Tek, Norcross, GA, USA) in accordance with the manufacturer’s instruction. The microbial qRT-PCR was analysis on the CFX-96 RT-PCR Detection System (Bio-Rad) as described previously . Briefly, the number of total bacteria was determined using SYBR Premix Ex Taq reagents (TaKaRa Biotechnology, Dalian, China), and the PCR system was composed of 12.5 μl SYBR Premix Ex Taq (2 ×), 1 μl of forward and 1 μl of reverse primers (100 nM), 9.5 μl ddH2O and 1 μl cDNA. Cycling conditions were as follows: 95℃ for 30 s, followed forty cycles of denaturation at 95℃ for 5 s, annealing at 60℃ for 30 s and extension at 72℃ for 60s. The number of Lactobacillus, Bacillus, E. coli and Bifidobacterium were measured using PrimerScriptTM PCR kit (TaKaRa), and the PCR system was composed of 8 μl RealMasterMix (2.5 ×), 1 μl of forward and 1 μl of reverse primers (100 nM), 1 μl 20 × probe enhancer solution, 0.3 μl probe (100 nM), 1 μl DNA and 7.7 μl ddH2O. Cycling conditions were as follows: 95℃ for 2 min, followed fifty cycles of denaturation at 95℃ for 15 s, annealing at 60℃ for 30 s and extension at 72℃ for 50 s. For the quantification of bacteria in test samples, specific standard curves were generated by constructing standard plasmids as presented by Chen et al . The bacterial copies were transformed (log10) before statistical analysis. The used primers and probes were listed in Table 3.
Determination of caecal short-chain fatty acid (SCFAs) concentrations
The concentration of main SCFAs in caecal digesta was determined by gas chromatography as previously described . Digesta samples were weighed and then centrifuged to obtain the supernatants after adding distilled water. The supernatant (1 mL) was mixed with 0.2 ml metaphosphoric acid, followed incubation at 4℃ for 30 min. The mixture was centrifuged at 12,000 g for 10 min at 4℃ and 1 μl of the supernatant was measured by injection into the gas chromatograph system (GC-2014, Shimadzu Corporation, Kyoto, Japan).
The experimental design of Exp.1 was a completely randomized block design based on initial BW, sex and litter. For the growth performance in Exp. 1, data were analyzed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA), with pen as the experimental unit. Significant differences among groups were determined by Duncan’s multiple range test. Mortality rate of pigs were analyzed using chi-square test. For all other variables in Exp. 2, data were analyzed by using an unpaired two-tailed Student’s t-test, with the selected pig as the experimental unit. Results were presented as means and standard error (SE) except for the mortality rate of pigs as percentage. A significant difference was considered as P＜0.05.