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 maintained 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 drying at the temperature of 37 ℃.
Experimental design and diets
The experiment was conducted at Feng Ning Swine Research Unit of China Agriculture University (Academician Workstation in Chengdejiuyun Agricultural & Livestock Co., Ltd). The experiment was conducted as a completely randomized design. 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. Treatment diets included basal diet (CON), basal diet supplemented with 75 mg/kg aureomycin (Chia Tai Group, Henan, China) (ANT)  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 1.2 × 2.1 m pens equipped with plastic leakage dung floors and were allowed ad libitum access to water and feed. Room temperature setpoint was 26 ℃ on the day of weaning and gradually decreased to 22 ℃ within the first week after weaning. The humidity was held constant at 65-75%.
Performance and diarrhea incidence
Piglets and feeders were weighted 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 . 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 .
Sample collection and processing
On the day 28, one piglet from each replicate pen close to the median body weight was selected for sampling. Blood (7 mL) was collected via jugular venipuncture using vacutainer without anticoagulant (Greiner Bio-One GmbH, Kremsmunster, Austria) , which was subsequently centrifuged at 3,000 ×g for 15 min for serum preparation and stored at -80 ℃ until further analysis.
Three piglets per treatment group were randomly selected for slaughter. The selected piglets were from the different pens and their body weights were close to the median body weight . Approximately 10 g digesta from the mid cecum and colon of each piglet were collected in sterile tubes, flash frozen in liquid nitrogen and stored at -80 ℃ until further analysis . 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 tissues (3.0 cm) were respectively 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.
Serum immune and antioxidant parameters
Serum immunoglobulins (IgA and IgG) were analyzed using commercially available ELISA kits following manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The antioxidant capacity based on serum concentrations of total superoxide dismutase (T-SOD), malondialdehyde (MDA), total antioxidant capacity (T-AOC) and glutathione Peroxidase (GSH-PX) were assessed using commercially available ELISA kits according to manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
The tissue concentrations of interleukin-8 (IL-8), interleukin-4 (IL-4), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined with commercially available ELISA kits following the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, samples of the jejunum and ileum tissues were thawed and homogenized in PBS (1:9 w/v, pH 7.4) and centrifuged at 2000 ×g for 20 min. The supernatant was collected for the determination.
Microbial community genomic DNA was isolated from cecal and colonic digesta, 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 the 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. A 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 the reads with 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
The concentrations of SCFAs were assayed as literature reported [4, 14]. Briefly, 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 an ultrasonic bath (KQ5200DE; Kunshan Ultrasonic Instrument, Jiangsu, China) at room temperature for 30 min. Then, the mixture was centrifuged at 4000 ×g for 15 min. Next, 0.16 mL of supernatant transferred into a 10 mL tube with 7.84 mL ultrapure water and filtered through a 0.22 μm filter. The SCFAs in a 25 μL extracted sample solution were determined by high performance ion chromatography (ICS-3000; Dionex, USA) with a conductivity detector. Finally, the concentrations of SCFAs were calculated and normalized to intestinal digesta weight as milligrams per kilogram.
Replicate (pen) was considered the experimental unit for analysis of differences in growth performance and diarrhea rate. Individual piglets were considered the experimental unit for analyses of serum immunoglobulins, antioxidant parameters, gut inflammatory parameters, microbiota, and SCFAs. Growth performance, serum immune, antioxidant parameters, inflammatory parameters and SCFAs were analyzed by one-way ANOVA using Bonferroni test (SPSS Inc., Chicago, IL, USA). Diarrhea rate were analyzed by Chi-square test (SPSS Inc., Chicago, IL, USA) [19, 20]. The bacterial community at the level of phylum, family and genus were analyzed by Kruskal-Wallis method followed by Welch’s test [13, 20]. Probability values of P < 0.05 were considered statistical significance.