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

Abstract

Background: Probiotics are used as a means to improve animal health and intestinal development. Saccharomyces boulardii ( S. boulardii ) is a well-known probiotic, however, few studies have examined the effects of S. boulardii on weaned pig let performance. Therefore, this 28-day study compared the effects of S. boulardii mafic-1701 and aureomycin in weaned piglet diets on growth performance, antioxidant parameters, inflammation and intestinal microbiota. One hundred and eight weaned 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 group, SB group had higher feed efficiency ( P < 0.01) and lower diarrhea rate ( P < 0.05) over the entire 28 days. Total superoxide dismutase concentration in serum was markedly increased in SB group ( P < 0.05). Moreover, compared with CON group, SB group decreased the level of pro-inflammatory cytokines interleukin-6 ( P < 0.01) and tumor necrosis factor ( P < 0.05) in jejunum. Supplementation with S. boulardii mafic-1701 increased abundance of Bacillus and Ruminococcaceae_UCG_009 ( P < 0.05), whereas abundance of unclassified _Clostridiaceae_4 was decreased ( P < 0.05). Furthermore, S. boulardii mafic-1701 administration increased cecal concentration of microbial metabolites , isobutyrate and valerate ( P < 0.05).

Conclusions: The improvement in feed conversion ratio, reduction in diarrhea rate in weaned piglets provided diets supplemented with S. boulardii mafic-1701 may be associated with enhanced antioxidant activity, anti-inflammatory responses and improved intestinal microbial ecology.

Background

In order to market pigs sooner and to improve sows’ reproductive performance, the early weaning strategy has been applied in commercial pig production [1]. Weaning is the most stressful period in pig’s life [1]. Some non-antibiotic solutions, including antimicrobial peptides, prebiotics, anti-virulence molecules, antibodies and probiotics, have been developed to maintain the health status of newly weaned piglets [2-4].

Probiotics are defined as “friendly” live microorganisms. When administered probiotics in adequate amounts, they confer a health benefit to the host before any health issue is present [5]. Saccharomyces boulardii (S. boulardii) is a safe, efficacious and non-pathogenic yeast isolated from lychee fruit in Indochina; S. boulardii belongs to Saccharomyces cerevisiae species [6]. However, S. boulardii possesses a superior probiotic efficiency than Saccharomyces cerevisiae by exhibiting several distinct physiological and metabolic characteristics [6]. In particular, characteristics of S. boulardii that make it suitable for use in weaned piglet diets include heat tolerance and resistance to gastric acidity, bile and proteolysis [7, 8]. The degree of pH tolerance and resistance to enzyme digestion suggest that S. boulardii may be suited for survival in the intestines. In addition, it is a yeast strain that is described as a probiotic against gastrointestinal diseases [9]. Accumulating evidence suggests oral administration S. boulardii may protect against antibiotic-associated diarrhea and improve Clostridium difficile-associated colitis in animal models [10, 11]. In human studies, administration of S. boulardii protected against Clostridium difficile infection, mitigated intestinal microbiota disorder and reduced antibiotic-associated diarrhea [12, 13].

The beneficial properties mentioned here would indicate S. boulardii is a promising probiotic-based feed additive in animal production. However, the effects of S. boulardii on weaned piglets remain unclear. Therefore, the objective of this study was to determine whether S. boulardii mafic-1701 supplementation to weaned piglet diets would improve feed conversion ratio, antioxidant capacity in serum, gut anti-inflammatory responses, microbiota composition and fermentation metabolites products in weaned piglets.

Materials And Methods

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 [14]. 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 randomized complete block 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) [15] 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 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 [16]. 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 [17]. The occurrence of diarrhea was defined as maintaining fecal scores of 3 or 4 for 2 consecutive days [17]. 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 [17].

Sample collection and processing

On the day 28, one piglet from each replicate pen with intermediate body weight was selected for sampling. Blood was collected via jugular venipuncture using vacutainer without anticoagulant (Greiner Bio-One GmbH, Kremsmunster, Austria) [18], 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 intermediate body weights [15]. 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 [14]. 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 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). Assessment of antioxidant parameters were based on serum concentrations of total superoxide dismutase (T-SOD), malondialdehyde (MDA), total antioxidant capacity (T-AOC) and glutathione Peroxidase (GSH-P_X) using commercially available ELISA kits according to manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Cytokine measurement

Tissue 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, tissues of Jejunum and ileum were thawed and homogenized in PBS (1:9 w/v, pH 7.4) and centrifuged at 2000 r/min for 20 min. The supernatant was collected for the determination.

Microbiota analysis

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 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 ℃ [19]. 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 reads including ambiguous bases were removed [19].

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% [14].

Quantification of fermentation products

The concentrations of SCFAs were assayed as literature reported [4, 15]. 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 r/min 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.

Statistical analysis

Replicate (pen) was considered an experimental unit for analysis of differences in growth performance and diarrhea rate. Individual piglets were considered an experimental unit for analysis of serum immune, antioxidant parameters, inflammatory parameters, microbial analysis 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) [20]. The bacterial community at the level of phylum, family and genus were analyzed by Kruskal-Wallis method followed by Welch’s test [14]. Probability values of P < 0.05 were considered statistical significance.

Results

Growth performance and diarrhea incidence

The effects of dietary treatment on ADFI, ADG and F:G are presented in Table 2. There were no significant differences in ADFI and ADG among three treatment groups (P >0.05). However, compared with CON group, SB group had lower F:G during d 15 to 28 (P < 0.05) and d 0 to 28 (P < 0.01). The rate of diarrhea was significantly associated with the dietary treatment (Table 3). Over the entire 28 days, SB group markedly decreased the rate of diarrhea compared to CON group (P < 0.05).

Serum immune and antioxidant parameters

The serum concentration of T-SOD was increased in SB group than that of piglets in CON group (P < 0.05) (Table 4). Moreover, the serum concentrations of T-AOC, MDA and GSH-P_X did not significantly differ among three treatment groups. There were no significant differences in the serum concentrations of IgA and IgG among three treatment groups (Table 4).

Intestinal inflammatory responses

The levels of TNF-α (P < 0.01) and IL-6 (P < 0.05) in the jejunum were decreased significantly in ANT group compared to CON group (Table 5). Similarly, SB group markedly decreased the levels of TNF-α (P < 0.05) and IL-6 (P < 0.01) in the jejunum. In addition, no significant differences were observed on the levels of IL-8 and IL-4 among three treatment groups (P > 0.05).

Intestinal microbiota composition

The OTUs were classified for bacterial community on the basis of usable sequence at 97% similarity. The analysis of OTUs in the cecal and colonic digesta are shown in Fig. 1. There were 42, 66, 268 unique OTUs in the cecal digesta of the CON group, ANT group, and SB group respectively and a total of 325 OTUs were common to all treatment groups. In the colonic digesta, 712 OTUs were common among the three treatment groups with 419, 318, 799 OTUs unique to CON group, ANT group and SB group, respectively. Fig. 2 depicts the microbial composition of cecal and colonic digesta across three treatment groups. In the cecal digesta, Firmicutes was the most predominant phylum among the three treatment groups, and Bacteroidetes was the second abundant phylum in ANT group and SB group. Fig. 2 also shows that Firmicutes and Proteobacteria were the dominant phyla in the colonic digesta.

Principal component analysis (PCA) based on Bray-Curtis distances indicated that SB group was distinctly separated in comparison to CON group and ANT group in the cecal microbiota (Fig. 3). Whereas, the colonic digesta of SB group were clustered with ANT group, which indicated that the colonic microbiota composition of ANT group and SB group was more similar.

Differences in the relative abundance of microbiota in the cecal and colonic digesta among three treatment groups are shown in cladograms, and the linear discriminant analysis (LDA) scores of 2.0 or higher were confirmed by the linear discriminant analysis effect size (LEfSe). In the cecal digesta (Fig. 4), Ruminococcaceae_UCG_009 and Turicibacter genus were enriched in SB group (P < 0.05). In the colonic digesta (Fig. 5), the proportion of Bacillus genus was significantly increased in SB group (P < 0.05). In addition, the abundance of unclassified_Clostridiaceae_4 genus and its family Clostridiaceae_4 were significantly enriched in ANT group (P < 0.05).

Concentrations of fermentation metabolites products

SCFAs in the cecal and colonic digesta are presented in Table 6. The results showed that SB group had higher concentrations of isobutyrate and valerate in the cecal digesta than piglets in CON group (P < 0.05). Additionally, the concentrations of propionate (P < 0.01) and butyrate (P < 0.05) in the colonic digesta of ANT group were higher compared to CON group.

Discussion

S. boulardii is an important microorganism, which has known positive effects on humans [12, 13]. Unfortunately, data on the effect of S. boulardii on weaned piglets are limited. Therefore, in this study we investigated the effects of dietary S. boulardii mafic-1701 supplemented in the diet on weaned piglet health and gut microbiota composition over 4 weeks. The dose of S. boulardii mafic-1701 was selected according to the studies by Kamm et al. [21] and Hancox et al. [22] The two doses of S. boulardii in their studies were 1 × 10⁷ CFU/kg and 1 × 10⁹ CFU/kg, respectively. We selected 1 × 10⁸ CFU/kg, the middle dose of S. boulardii of the two studies as the experimental treatment in this study.

In the present study, supplementation with S. boulardii mafic-1701 improved feed conversion ratio compared with CON group. A previous study identified that administration of yeast improved feed conversion ratio of weaned piglets [23], which corresponds with our results. Reports on the effect of dietary supplementation with S. boulardii on the rate of diarrhea of weaned piglets are limited. The current study demonstrated that the dietary supplementation with S. boulardii mafic-1701 significantly decreased the rate of diarrhea over the entire 4 weeks.

It is generally known that weaning could lead to breakdown of intestinal barrier functions [24, 25]. When the intestinal barrier is damaged, microbial colonization increases the risk of inflammation [26]. In this study, we found that the level of pro-inflammatory cytokines TNF-α and IL-6 were decreased in SB group compared with CON group, but there were no significant differences on the levels of IL-8 and IL-4 among three treatment groups. A previous study also reported that S. boulardii could reduce TNF-α and IL-6 levels in mice ulcerative colitis carcinogenesis model [27]. These results indicated that S. boulardii mafic-1701 has beneficial effects on intestinal health by decreasing inflammation. Previous studies showed that S. boulardii blocked nuclear factor kappa B activation and reduced colonic inflammation [28, 29]. Thus, we speculate that S. boulardii mafic-1701 altered the level of pro-inflammatory cytokines through modulation of the signaling pathway involved in inhibition of nuclear factor kappa B activated pathways. In addition, other previous studies have reported that mucus is composed of many immunomodulatory molecules with mucins forming the basic skeleton, which protect intestinal epithelial barrier integrity and reduce pro-inflammatory responses [4, 30]. Caballero-Franco et al. demonstrated that oral administration of probiotic increased mucin gene expression and secretion [31]. Therefore, it is speculated that S. boulardii mafic-1701 has a modulatory effect on inflammatory responses that correlates with regulation of mucin transcription.

Probiotics can activate the local mucosal protective mechanisms and exert beneficial effects on the host such as modulate anti-oxidation and immune responses [32, 33]. In our study, we observed that S. boulardii mafic-1701 and aureomycin supplementation had no effect on IgA and IgG levels in the serum. In terms of antioxidant analysis, we found that T-SOD was increased in SB group of the piglets, which suggests S. boulardii mafic-1701 plays a role in improving antioxidant capacity and protecting intestinal mucosa [33].

The diversity of the microorganisms in the mammalian gut is very large. It has been estimated that 500-1000 bacterial species inhabit the gut [34]. The gut microbiota has a symbiotic relationship with the host. Oral ingestion of a feed additive can regulate the delicate balance between host and microbes. From the results of phylum analysis, we found that the cecum microbial floras were dominated by Firmicutes, which is consistent with previous findings reported by Yu et al. [35]. Wang et al. report a significant increase in abundance of Firmicutes and decrease in abundance of Bacteroidetes in the probiotics group [15]. In the present study, compared to CON group, we found an increased abundance of Proteobacteria and decreased abundance of Firmicutes and Bacteroidetes in the colon in SB group. Difference between the Wang et al. study and this study may be attributed to the use of different probiotic strains. Indeed, different probiotic strains could exert different physiological effects. Much work remains to be done to understand the function of different gut microbiota populations.

From current study, S. boulardii mafic-1701 inclusion resulted in higher bacterial diversity in cecum and colon of piglets. The population of Ruminococcaceae_UCG_099 and Turicibacter genus were significantly increased in cecum of SB group compared to CON group. These bacteria are believed to be significant producers of SCFAs, which are intestinal epithelial energy components that have anti-inflammatory properties and protect intestinal epithelial cells [36-38]. In addition, Ruminococcaceae can utilize diverse polysaccharides [39]. Indeed, the yeast cell wall consists of mannose, chitin, 1,3-β-glucan and 1,6-β-glucan [6]. Therefore, the increased population of Ruminococcaceae_UCG_099 in the cecum might be due to S. boulardii mafic-1701 being used as a substrate source to stimulate proliferation of Ruminococcaceae_UCG_099. S. boulardii mafic-1701 inclusion showed some alterations with regard to microbiota communities. In the colon, S. boulardii mafic-1701 inclusion increased the abundance of Bacillus genus, which have excellent immunomodulatory and anti-inflammatory efficacy [40, 41]. In addition, a previous study reported that several Bacillus species, reduced pathogen colonization but the mechanisms by which this occurs is unclear [42]. Notably, the relative abundance of Clostridiaceae_4 family, which are negatively linked with antibiotic-associated diarrhea and colitis, was significantly increased in ANT group compared with SB group. It has been demonstrated that antibiotic treatment alters the composition of gut microbiota, manifesting the host susceptible to pathogen infection [24, 43].

Microbially-produced SCFAs as crucial in regulating health of the host and play a central role in gut metabolism [44]. A previously published report indicated that probiotics can increase SCFAs production [15]. In this study, S. boulardii mafic-1701 supplementation increased the concentrations of cecal isobutyrate and valerate. Compared with the other two groups, ANT group increased the concentrations of colonic propionate and butyrate. The increase of SCFAs production may be associated with a lot of factors and the dietary intake is the most significant variable [45]. In additional, the principal site of microbial fermentation is proximal colon [45]. Thus, the production of SCFAs is determined by the numbers and types of microbes colonizing the colon.

Conclusion

In conclusion, diets containing S. boulardii mafic-1701 promoted feed conversion ratio, improved antioxidant activity and anti-inflammatory responses in weaned piglets. The increased diversity of intestinal microbiota and their fermentation products and the higher abundance of Ruminococcaceae_UCG_099 and Bacillus with S. boulardii mafic-1701 supplementation may facilitate maturation of the digestive system of piglets in the subsequent growing phases.

Abbreviations

S. boulardii: Saccharomyces boulardii; ADG: Average daily gain; ADFI: Average daily feed intake; F:G: Feed to gain ratio; SCFAs: Short chain fatty acids; T-SOD: Total superoxide dismutase; MDA: Malondialdehyde; T-AOC: Total antioxidant capacity; GSH-P_X: Glutathione Peroxidase; IL-8: Interleukin-8; IL-4: Interleukin-4; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-α; OTUs: Operational taxonomic units; PCA: Principal component analysis; LDA: The linear discriminant analysis; LEfSe: The linear discriminant analysis effect size.

Declarations

Acknowledgements

The authors would like to express special appreciate to Dr. Crystal Levesque, the professor of Department of Animal Science, South Dakota State University for providing many insight comments and correcting the language.

Author’s contributions

WXZ and YHC designed the experiment. WXZ, CLB and JW performed the experiment. JJZ supervised the whole experiment. WXZ wrote the paper, YHC edited the paper. All authors read and approved the final manuscript.

Funding

This work was supported by National Key R&D Program of China (No.2018YDF0500604) and the Key Research & Development Program of Shandong Province (2019JZZY020308).

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.

Competing interests

The authors declare that they have no competing interests.

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Tables

Table 1 Composition and nutrient levels of basal diets (as-fed basis)

¹Experimental diets were control diet (CON), CON + 75 mg/kg aureomycin (ANT), CON + 1 × 10⁸ CFU/kg S. boulardii mafic-1701 (SB). ²The Vitamin-mineral premix contained (per kilogram of complete diet): vitamin A, 9000 IU; vitamin D₃, 3000 IU; vitamin E, 20.0 IU; vitamin K₃, 3.0 mg; vitamin B₁, 1.5 mg; vitamin B₂, 4.0 mg; vitamin B₆, 3.0 mg; vitamin B₁₂, 0.2 mg; niacin, 30.0 mg; pantothenic acid, 15.0 mg; folic acid, 0.75 mg; biotin, 0.1 mg; Fe (FeSO₄·H₂O), 75.0 mg; Cu (CuSO₄·5H₂O), 150 mg; Zn (ZnSO₄·7H₂O), 90 mg; Mn (MnSO₄), 60.0 mg; I (KI), 0.35 mg; Se (Na₂SeO₃), 0.30 mg. ³Values were calculated according to NRC (2012). ⁴SID: standardized ileal digestible

Table 2 Effect of S. boulardii mafic-1701 on growth performance in weaned piglets¹

¹n = 6 per pen, In the same row, experimental diets were control diet (CON), CON + 75 mg/kg aureomycin (ANT), CON + 1 × 10⁸ CFU/kg S. boulardii mafic-1701 (SB). In the same row, values with different small letter superscripts mean significant difference (P < 0.05)

Table 3 Effect of S. boulardii mafic-1701 on the rate of diarrhea (%) in weaned piglets¹

¹n = 6 per pen, experimental diets were control diet (CON), CON + 75 mg/kg aureomycin (ANT), CON + 1 × 10⁸ CFU/kg S. boulardii mafic-1701 (SB). 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. In the same row, values with different small letter superscripts mean significant difference (P < 0.05)

Table 4 Effect of S. boulardii mafic-1701 on serum immune and antioxidant parameters in weaned piglets¹

¹Experimental diets were control diet (CON), CON + 75 mg/kg aureomycin (ANT), CON + 1 × 10⁸ CFU/kg S. boulardii mafic-1701 (SB). Blood was collected from one piglet selected from each replicate. In the same row, values with different small letter superscripts mean significant difference (P < 0.05)

Table 5 Effect of S. boulardii mafic-1701 on inflammatory parameters in jejunum and ileum in weaned piglets¹

¹Experimental diets were control diet (CON), CON + 75 mg/kg aureomycin (ANT), CON + 1 × 10⁸ CFU/kg S. boulardii mafic-1701 (SB). Intestinal tissues were collected from three piglets per treatment. In the same row, values with different small letter superscripts mean significant difference (P < 0.05)

Table 6 Effect of S. boulardii mafic-1701 on the concentration of SCFAs (mg/kg) in weaned piglets¹

¹Experimental diets were control diet (CON), CON + 75 mg/kg aureomycin (ANT), CON + 1 × 10⁸ CFU/kg S. boulardii mafic-1701 (SB). Cecal and colonic digesta were collected from three piglets per treatment and the concentrations of SCFAs were measured. In the same row, values with different small letter superscripts mean significant difference (P < 0.05)