Administration of Saccharomyces boulardii mafic-1701 improves growth performance, promotes antioxidant capacity, alleviates intestinal inflammation and modulates gut microbiota in weaned piglets

Background: Probiotics seem to be an alternative to antibiotics for improving animal's health and intestinal development. Saccharomyces boulardii ( S. boulardii ) is a well-known probiotic. However, only few studies have been performed examining the effects of S. boulardii on weaned piglets . Therefore, this study was conducted to investigate the effects of dietary S. boulardii mafic-1701 on growth performance, antioxidant parameters, inflammation and intestinal microbiota in weaned piglets, using aureomycin as positive control. One hundred and eight piglets were randomly divided into three dietary treatment groups: (1) basal diet (CON); (2) basal diet supplemented with 75 mg/kg aureomycin (ANT); (3) basal diet supplemented with 1 × 10 8 CFU/kg S. boulardii mafic-1701 (SB). Results: Compared to CON, the supplementation with S. boulardii mafic-1701 improved feed efficiency over the entire 28 days ( P < 0.01) and decreased the rate of diarrhea during the first week ( P < 0.05). Total superoxide dismutase concentration was markedly increased in piglets with S. boulardii mafic-1701 ( P < 0.01). Moreover, compared with CON, SB increased the concentration of interleukin-4 in ileum ( P < 0.05), while the level of pro-inflammatory cytokines interleukin-6 ( P < 0.01) and tumor necrosis factor ( P < 0.01) were decreased in jejunum. SB increased the abundance of Bacillus and Ruminococcaceae ( P < 0.05), whereas the population of Clostridiaceae were decreased ( P < 0.05). Furthermore, the analysis of microbiota metabolites showed that S. boulardii mafic-1701 administration increased the concentration of formate and isobutyrate in cecum to maintain a stable microbiota and gut health ( P < 0.05). Conclusion: This study indicated that S. boulardii mafic-1701 supplementation could improve growth performance, alleviate the severity


Background
In order to shorten the slaughter cycle of pigs and get sows reproductive better, the early-weaning strategy has been applied in commercial pig production [1], with weaning age has been decreasing [2]. It is one of the most stressful matters in pig's life [3]. To relieve weaning stress, traditional antibiotics have been used markedly in recent decade. While, considering the intensifying substantial environmental pollution, arise antimicrobial resistance and global public health crisis, several replacements have been developed over the past years, including antimicrobial peptides, prebiotics, new antibiotics, anti-virulence molecules, antibodies and probiotics [4].
Probiotics are defined as "friendly" live microorganisms and when administered probiotics in adequate amount they will confer a health benefit on host [5]. For many years since their introduction, the major use has been focused on animal feeds [6], the most commonly used probiotic microorganisms are bacteria, for instance Lactobacillus and Bifidobacterium. Species from other bacterial genera such as Enterococcus, Streptococcus and Bacillus are also widely used as probiotic [7]. The most usual application and the advantageous effects of probiotics should be associated with some widespread mechanisms include: 1) Colonization resistance, 2) Normalization of perturbed microbiota, 3) Acid and short chain fatty acids (SCFAs) production, 4) Increased turnover of enterocytes, 5) Regulation of intestinal transit, 6) Competitive exclusion of pathogens [5]. This is also true for the yeast probiotic, namely, Saccharomyces boulardii (S. boulardii). S. boulardii is one of the most commonly employed probiotics in recent years, which is a safe, efficacious and non-pathogenic yeast isolated from lychee fruit in Indochina and it belongs to the group of species Saccharomyces cerevisiae (S. cerevisiae) [8,9]. Previous study found that S. cerevisiae have probiotic capacity [10].
However, S. boulardii possesses a superior probiotic efficiency than S. cerevisiae because S. boulardii exhibits several distinct physiological and metabolic characteristics considerably different from S. cerevisiae [8]. Notably, S. boulardii could tolerant and growth at high temperature of 37 ℃. It's a unique advantage of S. boulardii that do best at piglets. Moreover, S. boulardii has a great resistance to gastric acidity, bile and proteolysis [11,12]. As it good at tolerating vary pH levels and enzymes, it could better survival in the intestinal surface than that of bacterial probiotics. S. boulardii is naturally resistant to antibiotics, so it can be prescribed during antibiotic treatment. In addition, it is the only yeast strain that is described as a probiotic against gastrointestinal diseases [13]. As we expected, accumulating evidence suggested that oral administration S. boulardii may be protected against antibiotic associated diarrhea and improved clostridium difficile (C. difficile) associated colitis in animal experiments [8,14]. In human studies, administration S. boulardii protected C. difficile infection, mitigated intestinal microbiota disorder and reduced antibiotic associated diarrhea [15][16][17].
The beneficial properties mentioned here would indicate S. boulardii is a promising probiotic as feed additive in animal production. However, intervention studies and investigations of long term S. boulardii effects on weaned piglets remains less clear. Therefore, the objective of this study was to determine the hypothesis, if S. boulardii mafic-1701 supplementation to diets will promote growth performance, antioxidant capacity in serum, gut anti-inflammatory responses, microbiota composition and fermentation metabolites products in weaned piglets.

Materials And Methods
Experimental protocols of this study include piglets handling and treatment 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 (YPD) agar plates to screen single colonies. Colonies of S. boulardi mafic-1701were inoculated in YPD 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; (NH 4 ) 2 SO 4 , 4; KH 2 PO 4 , 2; MgSO 4 , 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 M NaOH. Fermentation was processed at 37 ℃ at 250 rpm with an aeration rate of 5 L/min of air. The pH was maintained at 6.5 by the addition of 3 M NaOH and anti-foaming agents was automatically added when each time foam was generated. To measure the biomass of S. boulardii mafic-1701 fermented samples were collected every 12 h. The actual yeast product used in this present study was obtained by mixing the precipitated of fermentation broth with 21.57 kg wheat brans and the product moisture content finally controlled at 2% by heat drying.

Experimental design and diets
A randomized controlled experiment was undertaken on 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), mean body weight: 8.5 ± 1.1 kg) were randomly assigned to three dietary treatment groups, based on their gender and initial body weight. Each treatment involved 6 replicates and each replicate consisted 6 piglets. All piglets were housed in identical conditions and they had ad libitum to access water and food. Basal diets (Table 1) in this study were formulated to meet or exceed the nutritional requirements of sucking piglets at each stage of growth as recommended by the NRC (2012). Dietary treatment groups consisted of basal diet (CON), basal diet supplemented with 75 mg/kg aureomycin (ANT) and basal diet supplemented with 1 × 10 8 CFU/kg S. boulardii mafic-1701 (SB).

Performance and diarrhea incidence
Piglets were weighted and recorded individually on days 0, 14 and 28 while feed consumption of each pen were determined on days 0, 14 and 28. The average daily gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (F:G) were calculated by pens. To evaluate the rate of diarrhea, fecal consistency was visually assessed three times per day by fixed observers blind to the treatment according to the method described by Hart and Dobb [18]. 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 [19].

Sample collection and processing
One piglet was selected from each pen with the intermediate body weight and piglets were slaughtered on day 28 of the experiment. Prior to slaughter, 7 ml blood samples were collected from jugular vein using vacuum blood tube without anticoagulant. Samples of blood were centrifuged at 3,000× g for 15 min. Serum samples were separated and stored at -20 ℃ for further analysis. Various subsamples from cecum and colon digesta were immediately taken and stored at liquid nitrogen for different analysis. One aliquot of digesta samples were obtained for microbial composition analysis and additional subsamples were taken to determine the bacterial metabolites in the gut. Intestinal samples from jejunum and ileum without gut contents were taken and kept at -80 ℃ for antiinflammatory analysis.

Determination of serum immune parameters and antioxidant indexes
The concentration of serum immune indices including IgA and IgG were detected according to protocols provided by the commercially available piglet serum ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Assessment of antioxidant parameters were performed by determination the concentration of total superoxide dismutase (T-SOD), malondialdehyde (MDA), total antioxidant capacity (T-AOC) and glutathione Peroxidase (GSH-P X ) in the serum using commercially available piglet serum ELISA kits according to instructions described by 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 methods. 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, the raw data were quality-filtered by Trimmomatic and merged by FLASH software with the following criteria: (i) the average quality score less than 20 were truncated at any site. 50 bp sliding window was set and the reads, which shorter than 50 bp or containing ambiguous were discarded; (ii) sequences with longer than 10 bp were assembled in light of their overlapped sequence. The maximum mismatch ratio of overlap area is 0.2. Unassembled reads were discarded; (iii) Samples were noticed and understood according to their barcode and primers besides the sequence was adjusted to a right direction.

Quantification of fermentation products
Approximately 0.5 g of intestinal digesta was weighed into a 10 mL tube and diluted 1:16 with ultrapure water (8 mL). Glass spheres were added and vortexed to homogenize the contents. After ultrasonic wave, samples were 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. Finally, the concentrations of SCFAs in extracted sample solution were determined by high-performance ion chromatography.

Statistical analysis
Data on growth performance, serum immune and antioxidant indices, inflammatory cytokines and SCFAs were analyzed by one-way ANOVA using SPASS19.0 software (SPSS Inc., Chicago, IL, USA).
Least-significant difference (LSD) tests were used to determine the differences between CON, ANT and SB. Treatment effects were considered statistical significance if P less than 0.05.

Diarrhea incidence and Growth performance
In commercial pig production, piglets are abrupt separated from the sows and characterized by stressful changes such as diarrhea. The first step of our study was to investigate whether S. boulardii mafic-1701 administered could relieve the diarrhea of piglets (Fig. 1). During the first week, SB markedly decreased the diarrhea incidence compared to CON (P < 0.05). Similarly, ANT also attenuated the diarrhea incidence (P < 0.05) compared with CON. Whereas, no significant difference in diarrhea incidence was observed between SB and ANT (P > 0.05). During the last week as well as over the entire four weeks, there were no significant differences in the rate of diarrhea among three treatment groups. Growth performance data ( Table 2) were shown that there were no significant changes in ADFI and ADG among three treatment groups (P > 0.05). During d 0 to 14 and d 15 to 28 of the experiment, Compared with CON, SB showed a decreased F:G (P < 0.05). In the whole experiment period, administration of aureomycin and S. boulardii mafic-1701 to the piglets significantly reduced F:G compared with CON (P < 0.01).

Serum immune and antioxidant parameters
The concentration of IgA and IgG among three treatment groups were analyzed, we found that oral aureomycin and S. boulardii mafic-1701 did not significantly impact serum immune indices compared with oral basal diet ( Table 3). The concentration of T-SOD was increased in the serum of piglets with SB compared to piglets with CON (P < 0.01), whereas piglets with or without antibiotic maintained similar results (P > 0.05). For the concentration of MDA, relative to CON, SB was markedly lower in the serum of piglets (P < 0.05). In addition, oral aureomycin and S. boulardii mafic-1701 did not significantly effect on the concentration of T-AOC and GSH-P X in the serum ( Table 3).

Effect of S. boulardii mafic-1701 administration on intestinal inflammatory responses
Indices related to gut inflammation were evaluated in intestinal tissues including jejunum and ileum (Table 4). In this study, the concentration of TNF-α and IL-6 in the jejunum decreased significantly in ANT and SB compared to CON (P < 0.01). In contrast the concentration of TNF-α and IL-6 in the ileum did not differ among three treatment groups. As shown by Table 4 the secretion of IL-8 in the jejunum decreased significantly only in ANT compared with CON (P < 0.05). While the concentration of IL-8 was decreased in both ANT and SB compared with CON in the ileum (P < 0.05). Moreover, the results showed that differences on the concentration of IL-4 in the jejunum were not observed among three treatment groups (P > 0.05). However, supplementation with a single dose of S. boulardii mafic-1701 was significantly increased the production of IL-4 compared with CON in the ileum (P < 0.05).

Intestinal microbiota composition
The OTUs were classified for bacteria community on the basis of usable sequence at a 97% similarity level. The analysis of OTUs were primary shown in Fig. 2 corresponding to cecum and colon digesta samples of three treatment groups including CON, ANT and SB, there were 42, 66, 268 core OTUs specifically each of these three individual groups, respectively and a total of 325 core OTUs were common to all treatment groups. Total 712 OTUs were shared in colon digesta samples among three treatment groups with 419, 318, 799 OTUs confined to CON, ANT and SB, respectively. Expect that, the vast majority of ANT core OTUs were shared with SB OTUs but only 152 core OTUs that were shared between CON and SB and only 138 OTUs were shared between CON and ANT. These results indicated that ANT and SB were more similar in terms of OUT analysis.  (Fig. 4). PCA analysis was conducted, the results indicated that SB was obviously separated in comparison to CON and ANT in cecum microbiota. The PCA analysis of microbiota from colon digesta samples showed that CON was relatively distinct compared to those in ANT and SB. In contrast, we found that the colon microbiota of ANT and SB was more similar. The differences in the relative abundance of microbiota in cecum and colon digesta samples among three treatment groups were shown in cladograms, and the linear discriminant analysis (LDA) scores greater than or equal to 2.0 were confirmed by the linear discriminant analysis effect size (LEfSe). In cecum digesta samples (Fig. 5), the proportion from the Bacillaceae family to Bacillales order was significantly increased in SB (P < 0.05). Moreover, Ruminococcaceae at genus level was enriched in SB (P < 0.05). In colon digesta samples (Fig. 6), the proportion of Bacillus at the genus level was significantly increased by combined with S. boulardii mafic-1701 (P < 0.05), while greater relative abundance of Lactobacillales order and Prevotella genus were observed in CON (P < 0.05). In addition, the abundance of Clostridiaceae family and Clostridiaceae genus was significantly enriched in ANT (P < 0.05).

Concentrations of fermentation metabolites products
SCFAs in the cecum and colon digesta were presented in Table 5. There were no significant differences by the experimental treatments on the cecum digesta including acetate, butyrate, and propionate (P > 0.05). However, a significant increase in the concentration of colonic propionate in ANT compared with CON (P < 0.01). This pattern was also found on acetate and butyrate in the colon digesta with numerical differences (P < 0.05). Furthermore, we found no significant differences for colonic formate among three treatment groups (P > 0.05) but the level of cecal formate was significantly increased in SB compared with CON (P < 0.05). In the cecum digesta, the aureomycin as well as S. boulardii mafic-1701 administration significantly increased the concentration of valerate compared with CON (P < 0.05). Additionally, only the concentration of isobutyrate in SB significantly increased more than CON and ANT in the cecum digesta (P < 0.05).

Discussion
S. boulardii is an important microorganism, which has been the most frequently used as a safe probiotic additive in weaning piglets. Weaning is a critical period in the pig production cycle with a sudden, stressful, short and complex event due to the change in diet and a new living environment conditions [2], leading to reduce feed intake, increase the risk of environment result in gut inflammation, which affect morphological and function of intestinal, humoral immunity in the gut, tissue damage, intestinal microbial ecosystem and increase the incidence of diarrhea [20]. Previous study demonstrated more than two weeks must be required for weaning piglets to adapt to these changes. Therefore, in this study we investigated the effects of dietary S. boulardii mafic-1701 supplemented in the diet on piglet health and gut microbiota composition in a long term intervention.
In the present study, ADFI and ADG were not changed in the whole feeding trials. However, the result showed that weaned piglets supplemented with S. boulardii mafic-1701 significantly improved feed efficiency. In addition, as a positive control, in the whole period of the experiment, supplement aureomycin with the diet had significantly decreased F:G. these results suggested that the inclusion of S. boulardii mafic-1701 in diet could maintain similar feed efficiency as piglets fed the antibiotic diet.
Weaning piglets in CON exhibited diarrhea with semi-liquid or watery faces. During 0-14 days, compared with CON, ANT and SB were significantly decreased the rate of diarrhea. Interestingly, on days 15-28 and the overall experiment period did not markedly differ across three treatment groups.
Obviously, diarrhea of piglets was not caused by the use of the antibiotics, in other word it was not antibiotic associated diarrhea. Thus it can be speculated that the diarrhea was relevant to postweaning stress, although the exact underlying mechanisms remain unclear [21]. However, oral administration aureomycin or S. boulardii mafic-1701 could minimize the adverse effects of weaning stress during the first week, but the incidence and frequency of diarrhea was all decline and did not differ among three treatment groups during the whole 28 days. These results suggested that the piglets may be adapt to the new social and physical environment regardless of the diets. . It is associated with the activation of innate immunity and inflammatory responses. In this study, we found that the concentration of pro-inflammatory cytokines such as TNF-α, IL-8 and IL-6 were decreased in ANT and SB compared with CON. We have also found that the production of IL In our study, we observed that S. boulardii mafic-1701 and aureomycin supplementation had no effect on increasing the levels of IgA and IgG in the serum. It was inconsistent with the previous observation by JEAN-PAUL, who reported that secretory IgA and secretory constituent of immunoglobulins in the rat small intestinal was increased by treated with S. boulardii [28]. Indeed, different probiotic strain could exert different physiological effects, therefore the differential immune response of piglets may be related to the type and does of probiotic strains we used. Besides, it is also associated with the animal models we chose to use in this study. In the future, the verdict need to be validated.
Furthermore, in terms of antioxidant analysis. In our study, we found that T-SOD was increased in SB in the serum of the piglets, which suggested S. boulardii mafic-1701 plays a critical role to improve antioxidant capacity and protect intestinal mucosa [27].
The greatest and most diverse cluster of microorganisms was inhabited in the gut [29]. The gut microbiota has symbiotic relationship with the host, which provides an ideal survival environment for microbes while the microbes provide a broad range of functions for the host such as defense against pathogens, digestion of complicated dietary nutrients, production of beneficial metabolisms and maintenance of the immune system [29]. Diet can drive gut microbiota composition, for instance, Antibiotic treatment can alter the balance of compositional in the intestinal microbiota. In contrast, oral ingestion of probiotics can enhance this delicate balance between host and gut microbes. In fact, the core advantage of the probiotic is considered by supporting a stable immune system. Oral administration probiotics can improve gut health by stimulating the growth of some beneficial microbes and inhibiting pathogens invasion [30]. In present study, PCA indicated that diet is the most crucial factor contribute to changes in microbiota structure, considering that groups were matched for age, had no difference in environment and received no recent medication. Compared with the other regimens, oral administration of S. boulardii mafic-1701 was showed that the largest variety in microbiota in the cecum and colon samples. Namely, the composition and structure of the microbiota was greatly affected by diet. From the results of phylum analysis, we found that the cecum microbial floras were dominated by Firmicutes, this was consistent with previous finding reported by Lei Yu [31]. Whereas colon microbiota communities were dominated by Firmicutes and Proteobacteria, regardless of diet treatment. The colonization of Proteobacteria was increased in the colon that was expected because the members of proteobacteria are facultative anaerobes [32] and the colon is a completely anaerobic environment which could provide a favorable living environment for them [33].
From the results, we found higher bacterial diversity in cecum and colon of the piglets fed with S. Ruminococcaceae species may be have a wonderful ability to utilize simple and complex sugars and polysaccharides then the production of a certain amount of volatile fatty acids as an energy sources which can be utilized by the host [34].The result investigated that S. boulardii mafic-1701 inclusion had beneficial effects on giving a balanced gut microbiota with high stability in a long term intervention.
In the colon, S. boulardii mafic-1701 inclusion showed some alterations in regard to microbiota communities in weaned piglets. Bacillus is identified as a helpful microbe to modulate gut health, they can germinate to form metabolically active and with the ability to survive hostile environments [35].
Bacillus genus. Previous study reported that several Bacillus species, which are considered to reduce pathogen colonization by unclear mechanisms [35]. Notably, the relative abundance of Clostridiaceae family was significantly increased in ANT compared with SB. C. difficile belongs to Clostridiaceae and is generally regarded as the most major etiologic factor for antibiotic associated diarrhea and colitis, C. difficile mediates intestinal disorder by releasing two potent exotoxins, toxin A and toxin B [37].
The diagnosis was further confirmed that antibiotic treatment altered the composition of the gastrointestinal microbiota, cause the gut dysbiosis that manifest the host susceptible to pathogen infection and trigger the immunological dysregulation [38]. In contrast, S. boulardii mafic-1701represents one of the most effective probiotic that have ability to treat antibiotic associated diarrhea and prevent pathogen infection [37]. Lactobacillus belongs to the phylum Firmicutes, class Bacilli, order Lactobacillales and family Lactobacillus [39], which is characterized by antibacterial and anti-inflammation activities. Previous study has reported that Lactobacillus spp. are beneficial to the host due to their underlying effects on gut function and health [40]. Nevertheless, the present study showed that the relative abundance of Lactobacillus genus was decreased in SB compared with CON.
The reason for this result might be associated with the increased diversity of the microbiota induced by inclusion of S. boulardii mafic-1701 in diet [41].Moreover, the community of Lactobacillus in porcine intestinal would decreased in time [42]. Prevotella strains are negatively linked with chronic inflammatory conditions [43]. As we expected, a reduction on the population of Prevotella was observed in piglet supplied S. boulardii mafic-1701. Taken together, these observations further point to the beneficial effect of S. boulardii mafic-1701 on regulating the gut microbial ecology and S. boulardii mafic-1701 may be contribute to a lifetime health of piglets.
The intestinal microbiota is a signaling hub that integrates diet [44], meanwhile, diet drives intestinal microbiota composition and metabolism. A growing body work demonstrated that microbial produced metabolites as crucial executors to regulate the health on the host [29]. SCFAs play a central role in gut metabolism [45]. A previously published report indicated that probiotics can enhance SCFAs production [46]. In this study, we found that the production of valerate was significantly increased in ANT and SB compared with CON in the cecum. The finding demonstrated that S. boulardii mafic-1701 could enhance SCFAs levels and provided an evidence that S. boulardii mafic-1701 can substitution antibiotics to a certain extent. In addition, previous study showed that the major metabolites from the microbial fermentative activity are acetate, propionate, and butyrate [29]. In the present study, it's worth noting that the production of colonic acetate, propionate and butyrate were significantly increased only in ANT, which might be associated with a number of factors, for instance, an increase in dietary intake is the most important element [46] and this hypothesis is accord with the results of growth performance which we investigated. And beyond that, the principal site of fermentation is proximal colon thus the production of SCFAs are depended on the numbers and the types of microbes which colonized in the colon [46].

Conclusion
In conclusion, weaner diet containing S. boulardii mafic-1701 promoted the growth performance,

Availability of data and materials
The data analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
This study was approved by Committee of China Agricultural University Laboratory Animal Care and Use (Beijing, China).

Consent for publication
Not applicable.      The bacterial core OUT community composition of the cecum and colon in weaned piglets.