Experimental protocols of animal handling and dietary treatments were approved by the “Institutional Animal Care and Use Committee of China Agricultural University” (ICS 65.020.30). All animal procedures were carried out in accordance with the specifications of the National Research Council’s Guide for the Welfare and Ethics of Laboratory Animals.
Probiotic strain and culture conditions
The yeast S. boulardii mafic-1701 was isolated by our laboratory and kept on yeast extract peptone dextrose agar plates to screen single colonies. Colonies of S. boulardi mafic-1701 were inoculated in yeast extract peptone dextrose medium for 16 h at 37 ℃ to prepare seed cultures. High density fermentation cultivation was performed using a fermentor (30 L) with an initial volume of 15 L of medium with the following composition (g/L): dextrose, 50; corn steep liquor powder, 25; (NH4)2SO4, 4; KH2PO4, 2; MgSO4, 0.5. 750 mL of seed cultures were added into medium. The initial dissolved oxygen concentration was adjusted to 30%. The pH was set at 6.5 using 3 mol/L NaOH. Fermentation was processed at 37 ℃ at 250 r/min with an aeration rate of 5 L/min of air. The pH was maintained at 6.5 by the addition of 3 mol/L NaOH and anti-foaming agents were automatically added when each time foam was generated. Samples were collected every 12 h to measure the biomass of S. boulardii mafic-1701 fermented. The yeast product used in this present study was obtained by mixing the precipitate of the fermentation broth with 21.57 kg wheat bran. The final product moisture content was controlled at 2% by heat drying.
Experimental design and diets
A randomized controlled experiment was undertaken at Feng Ning Swine Research Unit of China Agriculture University (Academician Workstation in Chengdejiuyun Agricultural & Livestock Co., Ltd). A total of 108 piglets (Duroc × Landrace × Yorkshire) were weaned at 28 d of age (8.5 ± 1.1 kg), and randomly assigned to one of three dietary treatment groups, based on their gender and initial body weight. Dietary treatment groups consisted of basal diet (CON), basal diet supplemented with 75 mg/kg aureomycin (Chia Tai Group, Henan, China) (ANT) [14] and basal diet supplemented with 1 × 108 CFU/kg S. boulardii mafic-1701 (SB). Basal diets (Table 1) in this study were formulated to meet or exceed NRC (2012) nutritional requirements of piglets in 2 phases (d 0-14 and d 15-28) after weaning. Each treatment group consisted of 6 replicate pens and each pen consisted 3 male and 3 female piglets. All piglets were housed in identical conditions (Room temperature setpoint was 26 ℃ on the day of weaning and gradually decreased to 22 ℃ within day 7 after weaning. The humidity was held constant at 65-75%) and piglets had ad libitum to access water and feed.
Performance and diarrhea incidence
Piglets were weighed individually on days 0, 14 and 28; pen feed disappearance was also determined on days 0, 14 and 28. Average daily gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (F:G) were calculated on a pen basis. To evaluate the rate of diarrhea, fecal consistency was visually assessed three times per day throughout the experiment by fixed observers blind to the treatment according to the method described by Hart and Dobb [15]. The scoring system was applied to determine the rate of diarrhea as following: 1 = normal feces; 2 = possible slight diarrhea; 3 = fluid feces; 4 = very watery diarrhea. The occurrence of diarrhea was defined as maintaining fecal scores of 3 or 4 for 2 consecutive days. The rate of diarrhea was calculated according to the following formula: the rate of diarrhea (%) = (number of piglets with diarrhea × diarrhea days)/(number of piglets × total observational days) × 100 [16].
Sample collection and processing
One piglet was selected from each pen with intermediate body weight and piglets were slaughtered on day 28 of the experiment. Prior to euthanasia, blood sample (7 ml) was collected via jugular venipuncture using vacutainer without anticoagulant. Blood was centrifuged at 3,000 ×g for 15 min [14]. Serum was separated and stored at -20 ℃ until analysis for serum immune parameters and antioxidant indexes. Approximately 10 g digesta from mid cecum and colon of each piglets were collected in sterile tubes, flash frozen in liquid nitrogen and stored at -80 ℃ until further analysis [17]. One aliquot of digesta samples were obtained for microbial composition analysis and additional subsamples were taken to determine the short chain fatty acids (SCFAs) in the gut. Intestinal tissue samples (3.0 cm) taken from jejunum and ileum, washed with normal saline to remove gut contents, immediately preserved in liquid nitrogen and kept at -80 ℃ for anti-inflammatory analysis.
Determination of serum immune parameters and antioxidant indexes
The concentration of serum immune indices, IgA and IgG, were detected using commercially available ELISA Kits according to manufacturer protocols (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Assessment of antioxidant parameters were based on serum concentration of total superoxide dismutase (T-SOD), malondialdehyde (MDA), total antioxidant capacity (T-AOC) and glutathione Peroxidase (GSH-PX) using commercially available piglet serum ELISA kits according to instructions described by the manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Cytokine measurement
The levels of interleukin-8 (IL-8), interleukin-4 (IL-4), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined in intestinal tissues. Jejunum and ileum samples were thawed and homogenized in PBS (1:9, w/v) with pH 7.4, then centrifuged at 2000 r/min for 20 min, and supernatant collected. The levels of cytokines were determined using commercially available piglet ELISA kits according to instructions of the manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Microbiota analysis
Microbial community genomic DNA was isolated from cecum and colon digesta samples, using the E.Z.N.A.® stool DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to the manufacturer’s specifications. The V3-V4 regions of the bacterial 16S rRNA gene were amplified by PCR using universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') with following procedures: initial denaturation at 95 ℃ for 3 min, followed by 27 cycles of denaturing at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 45 s, single extension at 72 ℃ for 10 min and end at 4 ℃. Illumina sequencing was performed, raw data were quality-filtered using Trimmomatic and merged by FLASH software with the following criteria: (i) average quality score less than 20 were truncated. 50 bp sliding window was set and reads shorter than 50 bp or containing ambiguous reads were discarded; (ii) sequences longer than 10 bp were assembled based on their overlapped sequence. The maximum mismatch ratio of overlap area was 0.2. Unassembled reads were discarded; (iii) Samples were distinguished according to their barcode and primers, and reads including ambiguous bases were removed.
Using UPARSE (version 7.1, http://drive5.com/uparse/) operational taxonomic units (OTUs) with 97% similarity cutoff were clustered and chimeric sequences were filtered out. Each 16S rRNA representative gene sequence was categorized and analyzed by RDP Classifier (http://rdp.cme.msu.edu/) against the Silva (SSU128) 16S rRNA database using confidence threshold of 70%.
Quantification of fermentation products
Approximately 0.5 g of intestinal digesta was weighed into a 10 mL polypropylene tube and diluted 1:16 with ultrapure water (8 mL). Glass spheres were added and vortexed to homogenize the contents. Polypropylene tubes were paced in ultrasonic bath (KQ5200DE; Kunshan Ultrasonic Instrument, Jiangsu, China) at room temperature for 30 min. Then, the mixture was centrifuged at 4000 r/min for 15 min. One hundred and sixty microliters supernatant was transferred into a 10 mL tube with 7.84 mL ultrapure water then filtered through a 0.22 μm filter. 25 μl extracted sample solution was determined by high performance ion chromatography (ICS-3000; Dionex, USA) with a conductivity detector. Finally, the concentration of SCFAs were calculated and normalized to intestinal content weight as milligram per kilogram.
Statistical analysis
Replicate (pen) was considered the experimental unit for analysis of the difference in growth performance, the rate of diarrhea. For serum immune, antioxidant parameters, inflammatory parameters, microbial analysis and SCFAs, individual piglets were considered the experimental unit for statistical analysis. Data were analyzed by one-way ANOVA using SPASS 19.0 software (SPSS Inc., Chicago, IL, USA). Bonferroni tests were used to determine differences between CON, ANT and SB. Treatment effects were considered statistical significance if P < 0.05.