PLOS ONE RESEARCH ARTICLE Stimbiotic supplementation improved performance and reduced inflammatory response via stimulating fiber fermenting microbiome in weaner pigs housed in a poor sanitary environment and fed an antibiotic- free low zinc oxide diet a1111111111 a1111111111 Hyun Min Cho1, Gemma González-Ortiz ID2*, Diego Melo-Durán3, Jung Min Heo1, a1111111111 Gustavo Cordero2, Michael R. Bedford ID2, Jae Cheol Kim ID2 a1111111111 1 Department of Animal Science and Biotechnology, Chungnam National University, Daejeon, South Korea, a1111111111 2 AB Vista, Marlborough, Wiltshire, United Kingdom, 3 Departament de Ciència Animal i dels Aliments, Servei de Nutrició i Benestar Animal (SNiBA), Universitat Autonoma de Barcelona, Bellaterra, Spain * Gemma.Gonzalez@abvista.com OPEN ACCESS Citation: Cho HM, González-Ortiz G, Melo-Durán D, Abstract Heo JM, Cordero G, Bedford MR, et al. (2020) Stimbiotic supplementation improved performance This study investigated whether the inclusion of a stimbiotic (STB) can improve perfor- and reduced inflammatory response via stimulating mance, influence intestinal microbiota and fermentation activity, and reduce pro-inflamma- fiber fermenting microbiome in weaner pigs housed in a poor sanitary environment and fed an tory cytokines in piglets fed a low zinc oxide diet without antimicrobial growth promotors antibiotic-free low zinc oxide diet. PLoS ONE 15 compared to fructo-oligosaccharide (FOS) and mannan-oligosaccharide (MOS) when (11): e0240264. https://doi.org/10.1371/journal. housed either in good sanitary (GS) or poor sanitary (PS) environments. One hundred forty- pone.0240264 four male pigs (28-day-old) were sorted by initial body weight (BW) and allocated to one of Editor: François Blachier, National Institute for six experimental treatments: 1) GS environment without any additive (GS-CTR); 2) GS envi- Research on Agriculture, Alimentation and ronment with 0.01% stimbiotic (GS-STB); 3) PS environment (without cleaning and disinfec- Environment (INRAE), FRANCE tion of a previously populated room) without any additive (PS-CTR); 4) PS environment with Received: June 22, 2020 0.01% STB (PS-STB); 5) PS environment with 0.1% MOS (PS-MOS); and 6) PS environ- Accepted: September 22, 2020 ment with 0.2% FOS (PS-FOS). Each treatment had six replicates, with four animals each. Published: November 10, 2020 Three feeding phases, based on corn, wheat, and soybean meal were available ad libitum Copyright: © 2020 Cho et al. This is an open for the 42-days of the study. Housing piglets under PS conditions negatively influenced per- access article distributed under the terms of the formance, increased plasma tumor necrosis factor alpha (TNF-α), affected the fecal micro- Creative Commons Attribution License, which bial populations and increased concentrations of branched-chain fatty acids (BCFA) permits unrestricted use, distribution, and compared to GS. Stimbiotic improved 42-d-BW under PS conditions (P < 0.05) whereas reproduction in any medium, provided the original author and source are credited. MOS or FOS had no effect. On d35, plasma TNF-α was reduced with STB in PS (P < 0.05). The ratio between VFA:BCFA increased (P < 0.05) with STB, MOS or FOS in PS, and Data Availability Statement: All relevant data are within the manuscript and its Supporting under GS condition, STB also increased the ratio. Stimbiotic increased the proportion of information files. Clostridiales Family XIII Incertae Sedis and Clostridiaceae, while MOS and FOS increased Funding: This study was supported by AB Vista, Selenomonadaceae, Catabacteriaceae and Fibrobacteraceae. These results indicate that who supplied the stimbiotic used in this trial. STB shifted the intestinal microbiome to favor fiber fermentation which likely contributed to Additionally, AB Vista provided support in the form of salaries for GGO, GC, MB, and JK. The specific PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 1 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs roles of these authors are articulated in the ‘author reduced inflammatory response and improved performance, particularly in piglets reared in contributions’ section. AB Vista was actively PS conditions. involved in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have read the journal’s policy and have the following competing interests: GGO, GC, MB, and JK are paid employees of AB Vista. This study evaluates Introduction products marketed by AB Vista, Alltech, Inc., and Antimicrobial growth promoters (i.e., antibiotics and zinc and copper; AGP) have long been Dreamfeed, Inc. This does not alter our adherence used as a cost-effective strategy to improve growth performance of piglets through mitigating to PLOS ONE policies on sharing data and enteric disease-associated mortality and morbidity [1]. However, due to concerns of antimi- materials. crobial resistance and environmental pollution, the use of AGP in swine production has been reduced or banned in many countries [2]. Inclusion of AGP controls the overall density of microbial activity in the gastrointestinal tract (GIT) and may suppress pathogenic bacterial challenges. However, they likely also suppress the activity of commensal microbiota that are beneficial for maintaining intestinal integrity and GIT-associated immune system [3]. In this regard, there is considerable interest in monogastric nutrition to increase the fiber fermenting microbiota in the large intestine to minimize dysbacteriosis and improve energy extraction from the fiber fraction of feed, which has long been ignored as a source of energy for growth. The term stimbiotic (STB) has been introduced recently and is defined as non-digestible but fermentable additives that stimulate fiber fermentability but at a dose that is too low that the stimbiotic itself could contribute in a meaningful manner to volatile fatty acid (VFA) pro- duction [4, 5]. Therefore, unlike prebiotics that are quantitatively fermented by the micro- biome, STB simply improves the fermentation of fiber that is already present in the diet [5]. For example, supplementation of broiler diets with 0.1g/kg and piglet diets with 0.2g/kg xylo- oligosaccharides (XOS) improved performance. From an energy contribution viewpoint, 0.1 g XOS only contributes 0.3 kcal/kg of energy to the diet thus highlighting the mechanism cannot involve quantitative fermentation alone [6, 7]. There are several commercialized prebiotics such as fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) and mannan-oligosac- charides (MOS) for the animal and human industry, and these are all thought to be quantita- tively fermented into VFA. However, dietary supplementation with or in vivo creation of XOS in the GIT via the action of supplemental enzymes likely result in trivial increments in VFA directly but significant increments indirectly by preferentially stimulating the growth and activity of beneficial bacteria such as Bifidobacterium and other lactic acid producing bacteria in the hind gut of monogastric animals [8–12]. One of the factors influencing identity of the intestinal microbiota is the environmental microbial challenge present in commercial swine production systems. A poor sanitary (PS) condition in commercial production systems can have an adverse effect on growth rate and efficiency due to increased risk of bacterial infection [13], which could be ameliorated by the use of prebiotics [14, 15]. The introduction of the STB concept is new to the swine industry, and little research has been conducted particularly in piglets at weaning. Therefore, the aim of this study was to compare performance, immune response, and fecal microbiota in pigs housed either in good sanitary (GS) or PS condition and the effects of either conventional prebiotics or STB when pigs were housed under PS conditions. The latter tends to be more relevant for commercial production systems. The hypotheses tested were that (1) pigs housed in PS conditions will have higher plasma concentration of endotoxins and pro- inflammatory cytokines, (2) in PS, a STB will increase fecal VFA to branched-chain fatty acid (BCFA) ratio via stimulation of a fiber fermenting microbiota at the expense of a protein fer- menting microbiota, and hence will reduce pro-inflammatory cytokine production and PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 2 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs improve performance in antibiotic-free, low zinc oxide (ZnO) diets when compared to con- ventional prebiotics such as a MOS or FOS. Materials and methods All experimental procedures received prior approval from the Animal Ethics Committee of Chungnam National University (201909A-CNU-164). Animals and managements A total of 144 male [Duroc × (Yorkshire × Landrace)] pigs were weaned at 28 ± 1 days of age with the initial body weight (BW) of 7.50 ± 0.70 kg (mean ± SEM). Pigs were obtained from a local government experimental farm (Happypork, Sinchang-myeon, Asan-si, Chungchung- namdo, South Korea), and transported to the animal husbandry unit at Chungnam National University. No animal showed any signs of disease at allocation. A total of thirty-six pens (3.23 x 1.75m) were used with four pigs per pen (4 pigs × 6 replicate pens × 6 dietary treatments) and were assigned to their experimental treatments based on initial BW and block within the room in the animal facility. The facility contained two rooms that allowed pigs to be housed separately to avoid any cross-contamination between GS and PS conditions. Each pen was equipped with a nipple bowl drinker and a metal feeder. The ambient temperature was main- tained at 29 ± 1˚C for the initial week and then gradually decreased by 1˚C every week. Pigs were offered the experimental diets in mash form and freshwater on an ad libitum basis. Experimental design and diets Experimental diets formulated based on corn, soybean meal, and wheat to meet or exceed the nutrient recommendations for weaned piglets were fed in three phases: phase 1, from 28 to 42 days of age; phase 2, from 42 to 56 days of age; and phase 3, from 56 to 70 days of age [16]. The composition and nutrient contents of the experimental diets are presented in Table 1. Since AGP and pharmaceutical levels of ZnO were not used, the base diets were formulated to limit crude protein (CP) contents to 19.57, 18.51, 17.25% in Phase 1, 2, and 3 diets, respectively. For each phase, one basal diet with 1% less corn was made, then split into four batches each of which were supplemented with the STB, MOS, FOS or none by direct replacement of corn. Therefore, six experimental treatments were evaluated consisting of (1) in a room with a GS condition (a previously unpopulated room cleaned and disinfected) fed either a control diet (GS-CTR), or (2) a control diet with supplementation of 100 g/tonne of the STB (GS-STB; Signis, β-1,4-endo-xylanase and xylo-oligosaccharides, AB Vista, Marlborough, UK); (3) under PS conditions (without cleaning/disinfection of a previously populated room) fed either a control diet (PS-CTR), or a control diet with supplementation of either (4) 100 g/tonne of the STB (PS-STB), (5) 1 kg/tonne of MOS (PS-MOS; Bio-Mos1, Alltech Inc, Nicholasville, KY, USA), or (6) 2 kg/tonne of FOS (PS-FOS; FOS-MAX1, Dreamfeed Inc., Seoul, Republic of Korea;). Diets did not contain any AGP, and the Zn content was limited to 150 ppm. Experimental procedures Piglets were weighed individually at the end of the study on d42 (70 ± 1 days of age), to mea- sure mean BW and calculate average daily gain (ADG). Feed consumption was determined by pen and used to calculate average daily feed intake (ADFI) and feed conversion ratio (FCR). One pig per pen (n = 36) closest to the average BW was selected at weaning (d0) as the sam- pling observational unit (SOU) throughout the trial. Blood samples were collected from the SOU at weaning (d0) and days 7, 14, 21 and 35 of the study via jugular vein puncture into PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 3 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs Table 1. Ingredient composition and calculated and analyzed composition of the experimental diets. Ingredient (%) Phase 1 Phase 2 Phase 3 (d 0–14) (d 14–28) (d 28–42) Wheat 27.38 20.80 25.00 Wheat bran 5.00 3.87 4.00 Maize (corn) 22.45 35.00 44.39 Soybean meal (crude protein 47%) 10.38 15.11 17.41 Blood plasma 5.00 3.00 - Fishmeal 5.00 3.00 2.00 Lactose powder 6.00 5.00 - Whey (sweet) 13.20 7.43 - Soya oil 1.50 3.00 3.21 Limestone 0.98 0.91 0.69 Dicalcium phosphate (18% phosphorous) 0.77 1.01 1.25 Salt 0.50 0.20 0.20 Zinc Oxide 0.01 0.01 0.01 Choline chloride (50%) 0.05 0.07 0.07 Lysine HCl 0.58 0.56 0.66 DL-Methionine 0.21 0.19 0.19 L-Threonine 0.17 0.16 0.21 L-Tryptophan 0.02 0.01 0.03 L-Valine 0.11 0.09 0.12 L-Isoleucine 0.12 0.07 0.04 L-Leucine 0.08 0.00 0.01 Vitamin-Mineral Premix1 0.50 0.50 0.50 Calculated chemical composition Metabolizable energy, kcal/kg 3,314 3,290 3,366 Lysine, % 1.65 1.48 1.34 Zinc, (mg/kg) 150 150 150 Analyzed chemical composition Gross energy, kcal/kg 3,900 4,040 4,060 Crude protein, % 19.57 18.51 17.25 1 Provided per kilogram of diet: vitamin A, 12,000 IU; vitamin D3, 2,500 IU; vitamin E, 30 IU; vitamin K3, 3 mg; D- pantothenic acid, 15 mg; nicotinic acid, 40 mg; choline, 400 mg; and vitamin B12, 12 μg; Fe, 90 mg from iron sulfate; Cu, 8.8 mg from copper sulfate; Zn, 31.25 mg from zinc oxide; Mn, 54 mg from manganese oxide; I, 0.35 mg from potassium iodide; Se, 0.30 mg from sodium selenite. https://doi.org/10.1371/journal.pone.0240264.t001 5–10 mL vacutainer tubes coated with lithium heparin for analyses of pro-inflammatory cyto- kines and endotoxins (Becton Dickinson, Franklin Lakes, NJ, USA). Fecal samples were col- lected from the SOU at weaning (d0) and days 7, 14, 21 and 35 of the study by rectal palpation, and immediately stored at -80˚C for subsequent VFA analyses and metagenomics. Assessment of fecal consistency and the incidence of diarrhea was carried out daily at 10:00 am for the first 14 days post-weaning. The visual assessment of the incidence of diarrhea was conducted using the procedure described by Heo et al. [17]. The number of antibiotic inter- ventions (intramuscular injection, Amoxyl 150 LA, Green Cross Veterinary Product, Co. Ltd, South Korea; Active ingredient: Amoxicillin trihydrate 150 mg/mL) was recorded during the first two weeks of the study. After 42 days all pigs are moved to grower barns and grown out as per commercial practice. PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 4 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs Sample analyses Diets chemical composition. Diets were analyzed for xylanase activity by ELISA (Enviro- logix, USA). Feed samples were also analyzed for gross energy and CP according to AOAC (1990) [18] Official Methods. Gross energy was determined by using an adiabatic bomb calo- rimeter (model 6300, Parr Instrument, Moline, IL, USA) that was calibrated using benzoic acid as a standard. The nitrogen (N) content in the experimental diets was determined by the combustion technique (method 990.03) [18] using the LECO N analyzer (model CNS-2000; LECO Corp., St. Joseph, MI, USA), and CP was calculated as N× 6.25. Pro-inflammatory and endotoxin markers. The concentrations of interleukin (IL) 1β (IL-1β; R & D Systems, Minneapolis, MN), 6 (IL-6; R & D Systems, Minneapolis, MN) and tumor necrosis factor alpha (TNF-α) (R & D Systems, Minneapolis, MN) in plasma were quantified using commercially available ELISA kits according to the manufacturers’ instruc- tions and as previously described [19]. Plasma concentration of endotoxins was determined using a commercial kit of Chromogenic End-point Tachypleus Amebocyte Lysate (Xiamen Houshiji, Ltd., Xiamen, China), according to the manufacturer’s instructions. Fermentation activity. For VFA analysis, thawed fecal samples were diluted 1:1 (w/v) with distilled water, mixed, centrifuged, and the supernatant fraction was analyzed chromato- graphically. The supernatant fraction (0.1 mL) was added to 1.0 mL internal standard solution containing valeric acid before analysis by capillary gas-chromatography (Hewlett Packard 5890A, Wilmington, DE, USA). The injector and detector FID were set at 260 and 265˚C, respectively, and initial and final oven temperatures were 120 and 240˚C, respectively, with 25 min run time. The carrier gas flow rate was 5.0 mL/min, and the split-flow rate was 70 mL/ min. A working standard and control (distilled water) were included in each run of the analy- sis, with the working standard containing acetic acid (60 mM), propionic acid (20 mM), iso- butyric acid (6.67 mM), butyric acid (20 mM), iso-valeric acid (10 mM), and valeric acid (10 mM). The Hewlett Packard Chemstation integration system was used to calculate the VFA concentrations from the area of the peaks. The acids measured were acetic, propionic, butyric, valeric and BCFA. Metagenomic analyses. Deoxyribonucleic acid (DNA) was extracted from 300 mg of fecal contents using QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) following manufac- turer’s instructions. Deoxyribonucleic acid concentration and quality were assessed in the Nano Drop ND-1000 spectrophotometer (NanoDrop Technologies, DE, USA). Deoxyribonu- cleic acid was stored at −20˚C until further analysis. For the 16S rRNA gene sequencing, the primers 799F-mod6 (5 ‘-CMGGATTAGATACCCKGGT-3’) and 1114R (5 ‘-GGGTTGCGCT CGTTGC-3’) were used to amplify the V5 through V6 hypervariable regions of the 16S rRNA gene [20]. The amplification mix contained 5× PrimeSTAR Buffer (Mg2+) (Takara Bio, Inc., Shiga, Japan), 2.5 mmol/L concentrations of each of deoxynucleotide triphosphates (dNTPs), 2.5 IU/μL of Prime STAR HS DNA Polymerase, a 10 pmol of each primer, and 25 ng of DNA in a reaction volume of 50 μL. The thermal cycling parameters were as follows: initial denatur- ation at 98˚C for 3 min, followed by 35 cycles of 98˚C for 10s, 55˚C for 15s, and 72˚C for 30s, and a final 3-min extension at 72˚C. Polymerase chain reaction (PCR) products were purified using the PCR purification kit, Wizard1 SV Gel, and PCR Clean-Up System (Promega, Wis- consin, USA). The barcoded 16S rRNA gene amplicons were sequenced using the Illumina MiSeq platform at Macrogen Inc. (Seoul, Republic of Korea). For the whole metagenome shot- gun sequencing, DNA representing the fecal microbial communities extracted from the feces was sequenced using paired-end shotgun sequencing using the Illumina Hi-Seq 2000 platform at Macrogen Inc. (Seoul, Republic of Korea). The 16S rRNA gene sequences were processed using the Mothur software to remove low-quality sequences [21]. Briefly, sequences that did PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 5 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs not match the PCR primers were eliminated from demultiplexed sequence reads. Sequences containing ambiguous base calls and sequences with a length of less than 100 bp were trimmed to minimize the effects of random sequencing errors. Chimeric sequences were further deleted using the UCHIME algorithm implemented in Mothur. QIIME (Quantitative Insights into Microbial Ecology) software package (version 1.9.1) was used for de novo operational taxo- nomic unit (OTU) clustering with an OTU definition at an identity cutoff of 97% [22]. Taxo- nomic assignment was performed using the naive Bayesian RDP classifier and the Greengenes reference database. Statistical analysis Animal performance and the VFA results were subjected to one-way analysis of variance using JMP Pro 15 (SAS). The initial BW was included as a covariate for the growth performance analyses. The pen was the experimental unit for all performance measurements, while the pig- let was considered the experimental unit when the pro-inflammatory cytokines, endotoxins, VFA and microbial populations from the feces were analyzed. For the coefficient of variation (CV) of BW at 42 days of age, the individual weights were considered. Means were separated only when the treatment P-value was significant and then by using a protected least significant difference (LSD) test. A contingency test was used to evaluate the diarrhea incidence and the number of antibiotic interventions. The Wilcoxon test was used to evaluate the effect of treat- ments on the blood parameters measured. Biostatistical analysis for microbiota was performed in open source software R-Studio v.3.6.1. Diversity was analyzed at OTU level using the vegan package [23]. The richness and alpha diversity were calculated with raw counts based on Shan- non estimators. Beta diversity was evaluated by multivariate ANOVA based on dissimilarities through anosim and adonis functions. Finally, differential abundance analysis was performed with taxa relative abundances under a zero-inflated log normal (Ln) mixture model, P-values were corrected by the false-discovery rate (FDR) with metagenomeseq package [24]. State- ments of significance were based on P-value of equal to or less than 0.05, and a P-value between 0.05 and 0.10 was considered as a trend. Results Analyzed enzyme activities in feed samples were all close to expected (Table 2). General health All pigs remained healthy to be used in the experiment and performed well throughout the study, and no mortality was observed. From d0 (weaning) to d14, no differences were observed with regards to the incidence of diarrhea or the average number of intramuscular antibiotic interventions required for treatment of a disease condition (P > 0.05). However, housing Table 2. Analyzed xylanase activities (BXU/kg) in feed samples1. Diet phase CTR STB MOS FOS Phase 1 <2,000 14,900 <2,000 <2,000 Phase 2 <2,000 16,900 <2,000 <2,000 Phase 3 <2,000 15,300 <2,000 <2,000 1 One BXU or birch xylan unit is defined as the amount of enzyme that produces one nmol of reducing sugars from birchwood xylan in one second at 50˚C and pH 5.3. https://doi.org/10.1371/journal.pone.0240264.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 6 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs Table 3. Diarrhea incidence and average number of intramuscular antibiotic intervention from d0 to d14 of the study. Treatments Diarrhoea incidence (%) Average number of antibiotic interventions (n)1 NO YES GS-CTR 79.2 20.8 0.92 GS-STB 83.3 16.7 0.75 PS-CTR 62.5 37.5 2.04 PS-STB 70.8 29.2 1.13 PS-MOS 75.0 25.0 1.04 PS-FOS 79.2 20.8 1.08 Pooled SD - 2.13 P-Value 0.627 0.446 1 Average number of intramuscular antibiotic injection per pig over first 14 days. https://doi.org/10.1371/journal.pone.0240264.t003 piglets in PS environment numerically increased the percentage of diarrhea and number of antibiotic interventions that were somewhat ameliorated with the feed additives (Table 3). Growth performance No differences were observed in ADFI (P > 0.05) and FCR (P > 0.05) among treatments (Table 4). Housing piglets under PS-CTR conditions reduced BW after 42-days by 1.74 kg compared to GS-CTR condition (20.14 vs. 21.88 kg; P < 0.05). Supplementation of STB in GS condition did not improve final BW (P > 0.05), but under PS condition, PS-STB piglets were 1.97 kg heavier (P < 0.05) than PS-CTR, while the final BW of PS-FOS and PS-MOS piglets were not different to PS-CTR piglets (P > 0.05). Likewise, supplementation of STB in GS con- dition did not improve ADG. However, in PS condition, ADG of the PS-STB piglets was 47g higher (P < 0.05) than the PS-CTR piglets, while PS-FOS and PS-MOS piglets did not differ from PS-CTR piglets (P > 0.05). Table 4. Effect of experimental treatments1 on growth performance of pigs for 42 days after weaning2. Treatments Body weight, kg ADG, g/pig/d ADFI, g/pig/d FCR, g/g CV3 BW 42 d GS-CTR 21.9ab 343ab 541 1.57 10.30bc a a GS-STB 23.0 370 551 1.48 9.68d c c PS-CTR 20.1 301 529 1.74 11.10a ab ab PS-STB 22.1 348 526 1.51 10.07cd bc bc PS-FOS 21.0 322 512 1.58 10.62ab bc bc PS-MOS 20.9 319 513 1.60 10.64ab 4 Pooled SD 1.25 30 128 0.313 0.861 P-value5,6 0.006 0.006 0.994 0.766 <0.0001 1 Experimental treatments: GS: good sanitary condition, PS: poor sanitary condition, CTR: control diet, STB: Control diet with 0.01% stimbiotics, MOS: Control diet with 0.1% mannan-oligosaccharides, FOS: Control diet with 0.2% fructo-oligosaccharides. 2 Mean values for six replicate pens with four piglets per replicate pen. 3 CV: coefficient of variation. 4 SD, standard deviation. 5abc Values within a column with different superscripts are significantly different. 6 Treatment effect analysed using the initial BW as a covariate. https://doi.org/10.1371/journal.pone.0240264.t004 PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 7 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs Table 5. Effect of experimental treatments1 on levels of plasma pro-inflammatory cytokines (pg/mL) and endotoxins (EU/ng) in weaned pigs2. Item GS-CTR GS-STB PS-CTR PS-STB PS-MOS PS-FOS Pooled SD3 P-Value4 Day 0 IL-1ß 0.18 0.10 0.24 0.19 0.22 0.21 0.42 0.986 IL-6 1.78 1.73 1.81 1.72 1.72 1.79 3.39 1.000 TNF-α 0.56 0.58 0.59 0.57 0.60 0.60 0.86 1.000 Endotoxins 0.30 0.32 0.25 0.26 0.23 0.34 0.69 1.000 Day 7 IL-1ß 4.50 2.20 7.26 5.37 6.25 5.76 10.0 0.902 IL-6 23.5 15.3 36.0 24.7 31.9 24.9 34.6 0.845 TNF-α 1.17 1.14 1.64 1.42 1.52 1.54 1.32 0.896 Endotoxins 0.35 0.45 0.71 0.61 0.68 0.67 0.89 0.927 Day 14 IL-1ß 39 38 56 44 47 49 57 0.996 IL-6 71 61 129 90 109 113 95 0.728 TNF-α 9.6b 9.3b 18.8a 14.1ab 15.7a 15.0ab 7.1 0.012 Endotoxins 0.46 0.42 1.21 0.76 1.02 0.98 1.09 0.349 Day 21 IL-1ß 99 82 174 119 137 159 118 0.481 IL-6 182 161 237 203 212 192 155 0.878 TNF-α 20.9b 20.7b 39.0a 29.9ab 35.5a 36.7a 13.8 0.001 Endotoxins 0.40 0.43 1.61 1.17 1.29 1.26 1.22 0.148 Day 35 IL-1ß 136 131 242 157 207 203 151 0.321 IL-6 347 332 387 355 362 368 199 0.959 TNF-α 42.5c 39.3c 73.9a 55.9bc 68.7ab 69.3ab 22.0 <0.001 Endotoxins 0.73 0.74 2.42 1.72 1.98 2.02 1.77 0.142 1 Experimental treatments: GS: good sanitary condition, PS: poor sanitary condition, CTR: control diet, STB: Control diet with 0.01% stimbiotics, MOS: Control diet with 0.1% mannan-oligosaccharides, FOS: Control diet with 0.2% fructo-oligosaccharides. 2 Mean values for six replicate pens with one piglet per replicate pen. 3 SD, standard deviation. 4abc Values within a row with different superscripts are significantly different. https://doi.org/10.1371/journal.pone.0240264.t005 Plasma levels of pro-inflammatory cytokines and endotoxins The effect of experimental treatments on the levels of plasma cytokines and endotoxins on days 0, 7, 14, 21, and 35 after weaning is presented in Table 5. The experimental treatments did not alter the levels of IL-1β, IL-6 and endotoxins on any of the sampling days (P > 0.05). How- ever, piglets housed in PS-CTR condition showed higher plasma TNF-α levels from d14 onwards compared to piglets housed in GS-CTR condition (P < 0.05). None of the feed addi- tives evaluated under PS condition at d14 and d21 reduced TNF-α levels (P > 0.05), but at d35 PS-STB piglets had lower plasma TNF-α content (P < 0.05) compared with PS-CTR piglets whereas the PS-FOS or PS-MOS treatments had no effect vs the PS-CTR (P > 0.05). Fecal fermentation The effects of experimental treatments on the concentration of VFA in the feces of piglets on days 0, 7, 14, 21, and 35 are presented in Table 6. On d0, no differences were observed in any of the VFA measured (P > 0.05) among treatments with the exception of butyric acid PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 8 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs Table 6. Effect of experimental treatments1 on volatile fatty acids (VFA) and branched-chain fatty acid (BCFA) contents (mmol/kg) in faeces of weaned pigs2. Treatment GS-CTR GS-STB PS-CTR PS-STB PS-MOS PS-FOS Pooled SD3 P-Value4 Day 0 Acetic 51 52 51 54 52 52 6.22 0.965 Propionic 20 18 19 22 18 20 3.93 0.507 ab bc a c a a Butyric 18 15 19 14 18 19 2.81 0.010 Valeric 5.9 5.7 5.1 6.1 5.4 5.9 0.86 0.352 BCFA 13.4 15.3 13.7 13.5 13.4 13.4 1.34 0.128 VFA:BCFA ratio 7.7 6.6 7.6 7.7 7.6 7.8 1.08 0.386 Day 7 Acetic 85a 87a 76b 85a 86a 84a 5.40 0.013 bc ab c a ab Propionic 19 23 18 25 23 24ab 3.54 0.021 Butyric 23a 23a 15b 22a 22a 22a 3.15 0.001 Valeric 6.1 5.0 5.5 4.7 4.7 5.0 1.00 0.145 BCFA 11.1b 9.8bc 12.9a 9.5c 9.3c 9.3c 1.30 0.001 VFA:BCFA ratio 13.1b 15.2ab 9.8c 15.7a 15.6a 15.4ab 2.00 0.001 Day 14 Acetic 91bc 97b 78c 123a 104b 105b 14.67 0.001 Propionic 22 26 20 25 26 22 5.18 0.308 Butyric 23 22 19 22 19 22 2.86 0.092 Valeric 7.4ab 5.3d 8.4a 5.3d 6.8bc 5.9cd 1.10 0.001 b bc a d c bc BCFA 10.7 9.4 13.3 6.8 9.0 9.1 1.42 0.001 VFA:BCFA ratio 14.9b 18.1b 10.3c 27.4a 18.3b 18.3b 3.87 0.001 Day 21 Acetic 85 103 93 107 107 100 13.58 0.058 Propionic 41a 26bc 24c 35ab 28bc 25c 7.96 0.008 Butyric 25 24 22 25 22 21 4.25 0.323 Valeric 8.3a 4.7bc 7.2ab 4.9bc 4.8bc 4.3c 2.16 0.015 BCFA 10.6b 7.8c 13.0a 8.6bc 8.5bc 9.5bc 1.90 0.001 bc a c ab ab bc VFA:BCFA ratio 16.0 23.5 13.1 21.5 20.4 17.3 4.75 0.007 Day 35 Acetic 82c 105ab 90bc 110a 102ab 98abc 14.62 0.028 Propionic 29 28 24 30 27 25 6.47 0.579 Butyric 3.5 3.6 3.5 3.9 4.1 3.4 0.56 0.253 Valeric 5.9a 3.8b 6.4a 4.8ab 5.8a 5.3ab 1.38 0.042 BCFA 11.7b 8.4d 13.3a 8.5d 9.6cd 10.5bc 1.06 0.001 VFA:BCFA ratio 11.1c 17.9a 9.5c 18.5a 15.5b 13.5b 1.70 0.001 1 Experimental treatments: GS: good sanitary condition, PS: poor sanitary condition, CTR: control diet, STB: Control diet with 0.01% stimbiotics, MOS: Control diet with 0.1% mannan-oligosaccharides, FOS: Control diet with 0.2% fructo-oligosaccharides. 2 Mean values for six replicate pens with one piglet per replicate pen. 3 SD, standard deviation. 4abc Values within a row with different superscripts are significantly different. https://doi.org/10.1371/journal.pone.0240264.t006 (P < 0.05). Both treatments containing STB, GS-STB and PS-STB, had lower butyric acid con- centrations compared to GS-CTR and PS-CTR (P < 0.05), respectively. Acetic acid, as the main component of fecal VFAs, was present at lower levels (P < 0.05) in PS-CTR piglets for all days measured from d7 onwards compared to the other treatments. Supplementation of STB under PS condition increased acetic acid concentrations similar to the levels observed in PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 9 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs GS-CTR piglets or even higher for all measurement ages (P < 0.05). Mannan-oligosaccharides and FOS supplementation under PS condition also increased acetic acid concentration on d7, d14 and d35 compared to PS-CTR (P < 0.05). Piglets under PS condition supplemented with STB had higher propionic acid levels on d7 and d21 (P < 0.05) compared with the control whereas this was the case for MOS and FOS fed piglets on d7 only (P < 0.05). Stimbiotic, MOS and FOS increased butyric acid concentrations on d7 under PS conditions compared to CTR piglets (P < 0.05); however, this effect was not observed in the subsequent sampling days. Supplementation of the STB reduced valeric acid concentrations on d14, d21 and d35 com- pared to CTR treatments either in GS or PS conditions (P < 0.10). Mannan-oligosaccharides supplementation reduced valeric acid concentrations on d14 (P < 0.05) whereas FOS reduced it on days 14 and 21 (P < 0.05). Higher BCFA concentrations were observed under PS com- pared to GS conditions from as early as d7 after weaning onwards (P � 0.001). Stimbiotic, MOS, and FOS supplementation all reduced the concentration of BCFA even below the level found in GS-CTR piglets on all days measured (P < 0.05). The VFA:BCFA ratio, which repre- sents the ratio between carbohydrate fermentation versus protein fermentation end products, was calculated to access the overall fermentation response of the experimental treatments. Housing piglets under PS condition and fed the CTR diet reduced VFA:BCFA ratio compared to piglets fed the CTR diet in GS condition from d7 onwards (P < 0.01). In GS condition, sup- plementation with STB did not influence the VFA:BCFA ratio until day 21, after which this ratio was increased compared to the GS-CTR piglets (P < 0.01). Under PS condition, STB, MOS and FOS all resulted in a higher VFA:BCFA than PS-CTR piglets from d7 until the end of the experiment (P < 0.10). Fecal microbiome Beta-diversity changed with age (P = 0.001) as the clustering of observations for each of the sampling days shows in the non-metric multi-dimensional scaling (NMDS) plot (Fig 1A). On d0, Firmicutes (71%) and Bacteroidetes (23%) represented the majority of the bacteria (14 phyla) found in the feces after weaning (Fig 1B). On d7, after one week of feeding the experi- mental treatments, Bacteroidetes (43%) was found to increase in abundance whereas Firmi- cutes (36%) reduced, with other minor phylums such as Spirochaetes (3%) and Proteobacteria (4%) increasing in proportion. On d21, Firmicutes (71%) and Bacteroidetes (20%) represented the majority of the bacteria found (10 phyla). On d35, the proportional representation of Bac- teroidetes (21%) was not changed from d21, but Firmicutes fell somewhat (63%), giving way to the establishment of other phylums such as Spirochaetes (6%), unknown phyla (7%) and Pro- teobacteria (2%). Shannon Index (Fig 2) was not influenced by the experimental treatments on d0 or d7 (P > 0.05). However, on d21 although PS-FOS had the highest biodiversity (3.79), it was not statistically different from PS-CTR (3.69), but it was significantly higher compared with GS-STB (3.31). On d35, STB supplementation under GS conditions increased bacterial diver- sity of the fecal samples compared to GS-CTR (P < 0.05). Under PS conditions, microbial diversity was not influenced by any of the additives evaluated compared to CTR (P > 0.05). The NMDS plots (Fig 3) show the treatment effects on beta diversity as determined among groups using a Bray-Curtis dissimilarity index. Statistical differences were noted between treat- ments on days 0, 7, 21 and 35 (Adonis P-value < 0.01) as a result of the presence and absence of several species depending on the sample age which was reflected in the taxonomical analysis. On d0, no significant differences were observed at the phylum level among different treat- ments (Fig 4). In the immediate period after weaning (d0—d7), the proportion of Firmicutes PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 10 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs Fig 1. Effect of age on beta diversity, represented in the non-metric multi-dimensional scaling (NMDS) plot (A) and on the relative abundance of phyla (B) of fecal samples from piglets at 0, 7, 21 and 35 of the study. Mean values were obtained from 1 animal per replicate from each treatment on each sampling day. https://doi.org/10.1371/journal.pone.0240264.g001 decreased while that of Bacteroidetes increased regardless of experimental treatment. Housing piglets under PS condition, led to an increase in Proteobacteria which was decreased with STB, MOS and FOS supplementation. Such an increase in Proteobacteria abundance under PS con- dition was not observed on d21. On d35, piglets housed under GS condition had a relatively higher proportion of Spirochaetes compared to piglets housed under PS condition. Changes observed for phylum taxa on d35 seems to respond more to the natural evolution of the fecal microbiota influenced by the sanitary conditions more than dietary treatment. At family level (Table 7), Selenomonadaceae, Fibrobacteraceae, Streptococcaceae and Por- phyromonadaceae were 1.24, 0.95, 0.06 and 0.06-fold higher, respectively, in PS condition compared to GS condition (P < 0.05). In contrast, Lactobacillaceae, Clostridiales Family XIII Incertae Sedis and Spirochaetaceae were 0.01, 1.07 and 1.30-fold higher in GS (P < 0.05) com- pared with PS conditions. Under GS conditions, STB supplementation increased Clostridiales Family XIII Incertae Sedis and Clostridiaceae by 2.94 and 0.82-fold, respectively. Stimbiotic supplementation did not increase any family under PS condition, but it kept under control the PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 11 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs Fig 2. Alpha diversity (Shannon Index) of fecal samples from piglets at 0, 7, 21 and 35 of the study from good sanitary condition (GS) or poor sanitary condition (PS) and control-CTR-, stimbiotic -STB-, fructo-oligosaccharide -FOS- and mannan-oligosaccharide -MOS-. Mean values were obtained from 1 animal per replicate from each treatment on each sampling day. https://doi.org/10.1371/journal.pone.0240264.g002 Fig 3. Beta diversity of fecal samples from piglets at 0, 7, 21 and 35 of the study from good sanitary condition (GS) or poor sanitary condition (PS) and control-CTR-, stimbiotic -STB-, fructo-oligosaccharide -FOS- and mannan- oligosaccharide -MOS-. Mean values were obtained from 1 animal per replicate from each treatment on each sampling day. https://doi.org/10.1371/journal.pone.0240264.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 12 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs Fig 4. Mean relative abundance (%) of OTU found at phylum level of fecal samples from piglets at 0, 7, 21 and 35 of the study from good sanitary condition (GS) or poor sanitary condition (PS) and control-CTR-, stimbiotic -STB-, fructo-oligosaccharide -FOS- and mannan-oligosaccharide -MOS-. Mean values were obtained from 1 animal per replicate from each treatment on each sampling day. https://doi.org/10.1371/journal.pone.0240264.g004 population of families such as Mycoplasmataceae, Catabacteriaceae, Fibrobacteraceae, Rumino- coccaceae and Selenomonadaceae, whose presence was higher in PS-CTR compared to PS-STB (P < 0.05). Very few changes were noted with FOS and MOS, but populations of Catabacteria- ceae and Fibrobacteraceae were reduced compared with the CTR pigs by feeding either of these supplements. Mannan-oligosaccharide also reduced Coriobacteriaceae 1.24-fold com- pared with the PS-CTR but Leuconostocaceae, Spiroplasmataceae and Acidaminococcaceae were 3.23, 2.13 and 1.39-fold higher with the supplementation of MOS (P < 0.05). Fructo-oli- gosaccharide supplementation increased the abundance of Acidaminococcaceae, Oxalobactera- ceae, Lachnospiraceae and Oscillospiraceae (P < 0.05). Between 18 to 32 species (see S1 Table) were influenced on d35 depending on the feed addi- tive supplemented (P < 0.05). Among these species, the most relevant fibrolytic species (n = 16) were identified and summarized in Table 8. Three fibrolytic bacteria (Clostridium cel- lobioparum, Butyrivibrio crossotus and Intestinimonas butyriciproducens) were promoted under GS condition with the supplementation of the STB, and no fibrolytic species was found to be depressed. Under PS condition, the supplementation of the STB promoted four fibrolytic species (Cellulosilyticum ruminicola, Fibrobacter intestinalis, Pseudobutyrivibrio ruminis and Faecalibacterium prausnitzii), but one fibrolytic species was found to be depressed (Fibrobacter succinogenes). Mannan-oligosaccharide supplementation under PS condition, also promoted four fibrolytic species (F. intestinalis, P. ruminis, Clostridium cellulolyticum and Coprococcus eutactus) while a reduction in Eubacterium eligens and Roseburia hominis was observed. Finally, FOS supplementation under PS conditions also promoted F. succinogenes, Eubacte- rium rectale and Faecalicoccus acidiformans, while F. prausnitzii, Butyricicoccus pullicaecorum and Roseburia inulinivorans were reduced. PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 13 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs Table 7. Ln changes promoted by treatment1 on family taxa found in the fecal samples of piglets on d 35 of the study2,3. Family GS-CTR VS PS-CTR GS-CTR vs GS-STB PS-CTR vs PS-STB PS-CTR vs PS-MOS PS-CTR vs PS-FOS Acidaminococcaceae -1.39 -1.29 Catabacteriaceae 1.74 2.00 0.70 Clostridiaceae -0.82 Clostridiales Family XIII. 1.07 -2.94 Incertae Sedis Coriobacteriaceae 1.24 Fibrobacteraceae -0.95 1.03 0.36 0.95 Lachnospiraceae -0.42 Lactobacillaceae 0.01 Leuconostocaceae -3.23 Mycoplasmataceae 1.80 Oscillospiraceae -0.33 Oxalobacteraceae -0.77 Others 0.22 -0.87 Porphyromonadaceae -0.06 Ruminococcaceae 0.18 Selenomonadaceae -1.24 1.44 0.67 Spirochaetaceae 1.30 Spiroplasmataceae -2.13 Streptococcaceae -0.06 1 Experimental treatments: GS: good sanitary condition, PS: poor sanitary condition, CTR: control diet, STB: Control diet with 0.01% stimbiotics, MOS: Control diet with 0.1% mannan-oligosaccharides, FOS: Control diet with 0.2% fructo-oligosaccharides. 2 Positive values and negative values indicate greater or lower abundance. Differences presented are based on all taxa detected in samples per treatment. Only significant results of changes in families (adjusted P < 0.05) are presented in the table. 3 Mean values from six replicate pens with one piglet per replicate pen. https://doi.org/10.1371/journal.pone.0240264.t007 Discussion The model used in this experiment to mimic higher microbial loads in commercial swine pro- duction systems resulted in clear differences between the good and poor sanitary conditions. Table 8. Most relevant fibrolytic species promoted or depressed (adjusted P < 0.05) based on the Ln changes compared to control treatments1 in the fecal samples of piglets on d35 of the study2,3. Additives2 STB MOS FOS Sanitary condition2 GS PS PS PS Species promoted Clostridium cellobioparum Cellulosilyticum ruminicola Fibrobacter intestinalis Fibrobacter succinogenes Butyrivibrio crossotus Fibrobacter intestinalis Pseudobutyrivibrio ruminis Eubacterium rectale Intestinimonas butyriciproducens Pseudobutyrivibrio ruminis Clostridium cellulolyticum Faecalicoccus acidiformans Faecalibacterium prausnitzii Coprococcus eutactus Species depressed Fibrobacter succinogenes Eubacterium eligens Faecalibacterium prausnitzii Roseburia hominis Butyricicoccus pullicaecorum Roseburia inulinivorans 1 Species promoted indicate greater abundance than the control treatment. Species depressed indicate lower abundance than the control treatment. 2 Experimental treatments: GS: good sanitary condition, PS: poor sanitary condition, STB: Control diet with 0.01% stimbiotics, MOS: Control diet with 0.1% mannan- oligosaccharides, FOS: Control diet with 0.2% fructo-oligosaccharides. 3 Mean values from six replicate pens with one piglet per replicate pen. https://doi.org/10.1371/journal.pone.0240264.t008 PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 14 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs Rearing pigs under PS conditions, without antibiotics, ZnO and copper sulphate, increased plasma TNF-α, reduced VFA:BCFA ratio in feces, and reduced ADG by 42g compared to pigs housed under GS conditions. In a piglet study, it was reported that rearing piglets under PS conditions reduced ADG of pigs by 11% during 42-day post-weaning feeding trial [13], which is similar in magnitude as found in this study (12%). Given that feed intake did not differ between the sanitary conditions, a numerical reduction in feed efficiency as a result of an increase in nutrient partitioning to the innate immune system (based on the increased TNF-α) can explain the reduced ADG in piglets housed under PS conditions [25, 26]. The results obtained in this study indicate that the PS challenge (i.e., extra environmental microbial load) altered fermentation pattern towards less fiber fermentation and greater protein fermentation likely as a result of contamination of the GIT with pathogenic bacteria. Lactobacillaceae and Clostridiales Family XIII Incertae Sedis were lower in the feces collected from piglets housed under PS compared with GS conditions. Lactobacillaceae family includes several lactic acid producing species, while some members harbored in the Clostridiales Family XIII are related to butyrate-producing bacteria and are considered as gut health biomarkers [27]. Therefore, the environmental microbial load was sufficient enough to change the structure of the micro- biome resulting in a subsequent increased TNF-α response but coupled with no effect on IL- 1β or IL-6. Although the roles of different pro-inflammatory cytokines are not elucidated yet, previous reports indicate that bacterial inflammation such as swine dysentery [28] and myco- plasma hyopneumoniae [29] increases TNF-α consistently while IL-1β and IL-6 responses are variable. Moreover, up-regulation of IL-1β and IL-6 has been noted in pigs experimentally infected with porcine reproductive and respiratory syndrome (PRRS) virus [29] and vesicular stomatitis virus [30], indicating variable pro-inflammatory cytokine responses depending upon the source and degree of inflammation. Given that the occurrence of clinical diarrhea and the levels of plasma endotoxins were not significantly increased in pigs housed under PS conditions, it is likely that the sanitary challenge model used in this experiment was mild and may have only marginally stimulated the innate immune response. Therefore, the first hypoth- esis tested that pigs housed under PS conditions will have a higher plasma concentration of endotoxins and pro-inflammatory cytokines was only partially supported. The stimbiotic used in this experiment was a product designed to deliver XOS to the com- mensal microbiota in the large intestine by co-supplementing a small amount of ex-vivo pro- duced short-chain XOS with a β-1,4-endo xylanase, the latter hydrolyzes the arabinoxylan fraction in the feed and produces XOS in situ in the GIT. Targeting in vivo synthesis of XOS from fiber fractions in feed along with co-supplementation of ex-vivo produced XOS is an ideal approach to stimulate fiber fermenting microbiota. The most abundant non-cellulosic polysaccharides in a corn-soybean meal-DDGS diet, particularly in cereals, are arabinoxylans [31, 32]. However, arabinoxylans and XOS are not fermented in the small intestine but increase fiber fermenting microbiota in the large intestine [5, 9, 33, 34]. For example, a piglet study demonstrated that 20 g/kg XOS stimulates microbial-origin non-starch polysaccharide- hydrolyzing enzymes such as xylanase and cellulase in the large intestine of piglets [35]. Pan et al. [36] reported that a 100 g/ton XOS supplementation resulted in a diversified fibre fer- menting microbial community in grower-finisher pigs (i.e., promoting Bifidobacterium) which could prevent intestinal disorders and increase growth performance. Similarly, in broil- ers and turkeys, xylanase supplementation increased Bifidobacterium populations and other butyrate-producing bacteria [37] likely through generation of XOS or arabinoxylo-oligosac- charides (AXOS) in the GIT. Indeed XOS has been shown to selectively increase the abun- dance of Bifidobacterium and the production of butyrate and acetate [38]. The second hypothesis tested was that supplementation of STB would increase fecal VFA:BCFA ratio via stimulation of fiber fermenting microbiota and hence will reduce PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 15 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs pro-inflammatory cytokine production and improve performance of pigs fed an antibiotic- free, low ZnO diet when compared with conventional prebiotics such as a MOS or FOS. There were no differences in fecal compositions of VFA and BCFA between experimental treatments at weaning, except significantly lower butyric acid content in PS-STB piglets. It is most likely due to variation in microbial community transferred from the sows. Although PS-STB piglets had lower butyric acid content on d0, other indicators such as total VFA, BCFA and VFA: BCFA ratio were not different between treatments, indicating the minimal impact of such vari- ation in microbial composition at the start of the experiment. Fecal Clostridiales Family XIII Incertae Sedis and Clostridiaceae were increased with the STB supplementation. These two families contain fibrolytic and butyrate-producing bacteria, reinforcing the establishment of a more beneficial microbiome to the host. These effects of the STB were consistent regardless of the sanitary conditions. Fibrolytic species such as C. cellobioparum, B. crossotus, I. butyricipro- ducens, C. ruminocola, F. intestinalis, P. ruminis and F. prausnitzii were all promoted with sup- plementation of STB. Most of these species possess genes encoding xylanases which enables then to digest this fibre and ultimately produce VFA [39–41]. However, under PS-CTR condi- tion where a lower VFA:BCFA ratio was observed compared with GS-CTR conditions, the STB did not increase any of these butyrogenic families, but it did keep under control the fami- lies that are not considered to bring any benefit to the host like Mycoplasmataceae, Catabacter- iaceae and Selenomonadaceae. Thus the reduction in the VFA:BCFA ratio with the PS condition challenge was reversed by addition of the STB which likely explains much of the response in performance noted here. Increased protein fermentation and production of harm- ful by-products such as ammonia and hydrogen sulphide can irritate mucosal surfaces [1–3] which facilitates invasion of endotoxins and pathogenic microbiota into the circulatory system and increases inflammatory responses in pigs with a lower faecal VFA:BCFA ratio [17, 25, 42, 43]. The STB, as noted above, increased the VFA:BCFA ratio by increasing fibrolytic species of microbiota, and decreased protein fermentation hence reducing the inflammatory response and enabling more rapid and efficient growth. The search for alternatives to antibiotics and heavy metal based additives (ZnO and cop- per sulphate) is an ongoing problem. One of the keys to success is the balance between fibre and protein fermentation as noted above. The negative effect of bacterial nitrogen fermenta- tion and the roles of a low protein diet in reducing the incidence of post-weaning diarrhea in pigs are well established [17, 42]. The increase of fiber fermentation in the hindgut by increasing fermentable fiber content (8% wheat bran and 5% sugar beet pulp) in diets either with a high or low fermentable protein content has been demonstrated. The fermentable fiber supplemented did not change VFA and BCFA production in a low fermentable protein diet. In contrast, fermentable fiber increased VFA and decreased BCFA in a high fermentable protein diet, along with similar responses in the populations of the selected microbiota [43]. Therefore, it seems that altering large intestinal microbiota and fermentation characteristics in piglets is partly dependent on the substrates available, and also supplementing a large vol- ume of fermentable fiber does not always increase VFA:BCFA ratio. Volatile fatty acid pro- duction is affected by many factors, including the carbohydrate composition and retention time in the large intestine [44]. However, BCFA, which are derived from fermentation of spe- cific amino acids, have been suggested as a critical factor in the induction of diarrhea in pigs [11, 45]. The experimental diet used in this study contained moderate amounts of CP (19.57, 18.51 and 17.25% for phase 1, phase 2 and phase 3, respectively). Although the first phase diet contained highly digestible sources of protein meals such as fish meal, blood meal and milk proteins, more undigested and hence fermentable protein are present in Phase 2 and 3 diets. With the increasing amount of fermentable protein in Phase 2 and 3 diets, inclusion of a large quantity of fermentable fiber or conventional prebiotics would have been required to PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 16 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs maintain a favourable VFA:BCFA ratio, as demonstrated in a previous study [43]. Supple- mentation with the STB, which was included in a rate of 0.01%, resulted in much higher increases in the VFA:BCFA ratio compared to supplementation of the conventional prebio- tics, MOS and FOS, which were included at 10 and 20 times higher doses respectively. This supports the hypothesis that stimbiotics are not prebiotics but have the ability to stimulate fiber fermenting microbiota in the large intestine. However, inferences of beneficial effects cannot be made by analysis of the microbiome in isolation. For example, the stimulation of Lachnospiraceae in FOS-supplemented piglets, a family which clusters several butyrate-pro- ducing bacteria, did not correlate with butyrate levels or to improved performance. This observation highlights the importance of having animal performance data as proof of the “beneficial” effects of treatments above any other parameter. Under the diversity and specific conditions of each experimental, animal performance is the best judge of the success of any feed additive in animal nutrition. Even though FOS and MOS also stimulated some fibrolytic species, according to the results obtained in this study, they were not as effective as STB in improving performance, reducing TNF-α or increasing VFA:BCFA at the dose of 10 to 20 times more than STB. The concentration of VFA and microbiota profile on sampling days from weaning to 35 days post-weaning indicate differences in nutrient flow, microbial colonization and gut devel- opment [46]. From 28 days of age (d0) up to 49 days of age (d21), it was noted that the VFA: BCFA ratio increased regardless of dietary treatments, highlighting the adaptation of the microbiota to ferment complex carbohydrates from the solid feed and the ability of the host to more effectively digest protein and hence reduce the proportion available for fermentation. As the animal ages, the flow of rapidly fermentable carbohydrates is reduced and the diversity of the microbiome in the hindgut increases, reducing the production of lactic acid in favour of other VFA such as acetic, propionic or butyric acids [5]. It is well accepted that a greater microbial diversity in humans has benefits for the mucosal surfaces since it is linked with reduced opportunities for pathogens to colonize the gut [47]. In this study, a greater biodiver- sity was observed at seven days post-weaning, when an imbalance of Bacteroidetes and Firmi- cutes was observed regardless of the experimental treatment, but in contrast, it declined afterwards on d21 and d35 post-weaning. The results of this study demonstrate that biodiver- sity is not a fully reliable parameter to be linked with animal performance and other factors like the taxonomic characterization and specific roles of bacteria should be considered for comparisons. Conclusions Exposure to poor sanitary conditions depressed animal performance, increased inflammatory responses and altered the structure of the microbiome and its fermentation activity. Supple- mentation of the STB, which stimulates arabinoxylan degrading microbiota, mitigated these effects and can thus be considered as a strategy to reduce the negative impact of mild microbial challenges as seen in most commercial production systems. Stimulating such a microbiota increases its resilience to the challenges of poor sanitary conditions, extracts more energy from the feed by fermentation of fiber otherwise voided, and in doing so stabilizes the intestines such that the innate immune system imposes a lower cost on growth rate and efficiency. Among the feed additives evaluated in this study, supplementation with the STB resulted in less protein fermentation, a more fibrolytic microbiome, lowered inflammatory responses and enhanced animal performance compared to FOS or MOS when piglets were housed in a poor sanitary condition. PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 17 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs Supporting information S1 Table. Contrasts of the natural logarithm (Ln) values, only from those significant (P < 0.05), of the fecal microbiota analyzed on day 0, 7, 21 and 35. (DOCX) S1 Raw Data. (XLSX) S1 Data. Microbiota total. (XLSX) Acknowledgments The authors would like to thank following post-doctorate and post-graduate students at Chungnam National University for assisting us with the sample collection and the care of the animals; Dr Samiru Sudharaka, Mr Shemil Priyan Macelline, Mr Yu Bin Kim and Mr Jun Seon Hong. Author Contributions Conceptualization: Jung Min Heo, Gustavo Cordero, Michael R. Bedford, Jae Cheol Kim. Data curation: Hyun Min Cho, Gemma González-Ortiz, Diego Melo-Durán, Jung Min Heo. Formal analysis: Hyun Min Cho, Gemma González-Ortiz, Diego Melo-Durán, Jung Min Heo. Funding acquisition: Jung Min Heo, Jae Cheol Kim. Investigation: Hyun Min Cho, Jung Min Heo, Jae Cheol Kim. Methodology: Hyun Min Cho, Jung Min Heo, Gustavo Cordero, Michael R. Bedford, Jae Cheol Kim. Project administration: Jung Min Heo. Resources: Hyun Min Cho, Jung Min Heo. Supervision: Jung Min Heo, Jae Cheol Kim. Validation: Gustavo Cordero, Michael R. Bedford, Jae Cheol Kim. Visualization: Gemma González-Ortiz, Diego Melo-Durán. Writing – original draft: Hyun Min Cho. Writing – review & editing: Hyun Min Cho, Gemma González-Ortiz, Diego Melo-Durán, Jung Min Heo, Gustavo Cordero, Michael R. Bedford, Jae Cheol Kim. References 1. Pluske JR, Turpin DL, Kim JC. Gastrointestinal tract (gut) health in the young pig. Anim Nutr. 2018. 4: 187–196. https://doi.org/10.1016/j.aninu.2017.12.004 PMID: 30140758 2. Kim JC, Hansen CF, Mullan BP, Pluske JR. Nutrition and pathology of weaner pigs: Nutritional strate- gies to support barrier function in the gastrointestinal tract. Anim Feed Sci Technol. 2012. 173: 3–16. 3. Heo JM, Opapeju FO, Pluske JR, Kim JC, Hampson DJ, Nyachoti CM. Gastrointestinal health and func- tion in weaned pigs: a review of feeding strategies to control post-weaning diarrhoea without using in- feed antimicrobial compounds. J Anim Physiol Anim Nutr. 2013. 97: 207–237. https://doi.org/10.1111/j. 1439-0396.2012.01284.x PMID: 22416941 PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 18 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs 4. Bedford MR. Future prospects for non-starch polysaccharide degrading enzymes development in monogastric nutrition. In: Gonzalez-Ortiz G, Bedford MR, Bach Knudsen KE, Courtin CM, Classen HL editors. The value of fibre—Engaging the second brain for animal nutrition, The Netherlands: Wagenin- gen Academic Press; 2019. pp. 373–383. 5. Gonzalez-Ortiz G, Gomes GA, dos Santos TT, Bedford MR. New strategies influencing gut functionality and animal performance. In: Gonzalez-Ortiz G, Bedford MR, Bach Knudsen KE, Courtin CM, Classen HL editors. The value of fibre—Engaging the second brain for animal nutrition, The Netherlands: Wageningen Academic Press; 2019. pp. 233–254. 6. Liu JB, Cao SC, Liu J, Xie YN, Zhang HF. Effect of probiotics and xylo-oligosaccharide supplementation on nutrient digestibility, intestinal health and noxious gas emission in weanling pigs. Asian-Austral J Anim Sci. 2018. 31(10): 1660–1669. https://doi.org/10.5713/ajas.17.0908 PMID: 29642680 7. Ribeiro T, Cardoso V, Ferreira LMA, Lordelo MMS, Coelho E, Moreira ASP, et al. Xylo-oligosaccharides display a prebiotic activity when used to supplement wheat or corn-based diets for broilers. Poult Sci. 2018. 97: 4330–4341. https://doi.org/10.3382/ps/pey336 PMID: 30101299 8. Wang S, Yang L, Tang X, Cai L, Liu G, Kong X, et al. Dietary supplementation with high-dose Bacillus subtilis or Lactobacillus reuteri modulates cellular and humoral immunities and improves performance in weaned piglets. J Food Agric Environ. 2011. 9(2 part 1), 181–187. 9. De Maesschalck C, Eeckhaut V, Maertens L, De Lange L, Marchal L, Nezer C, et al. Effects of xylo-oli- gosaccharides on broiler chicken performance and microbiota. Appl Environ Microbiol. 2015. 81: 5880–5888. https://doi.org/10.1128/AEM.01616-15 PMID: 26092452 10. Jain I, Kumar V, Satyanarayana T. Xylooligosaccharides: An economical prebiotic from agroresidues and their health benefits. Indian J Expt Biol. 2015. 53(3): 131–142. PMID: 25872243 11. Yang J, Summanen PH, Henning SM, Hsu M, Lam HM, Huang J, et al. Xylooligosaccharide supplemen- tation alters gut bacteria in both healthy and prediabetic adults: a pilot study. Front Physiol. 2015: 6: 216. https://doi.org/10.3389/fphys.2015.00216 PMID: 26300782 12. Bedford MR. The evolution and application of enzymes in the animal feed industry: the role of data inter- pretation. Br Poult Sci. 2018. 59(5): 486–493. https://doi.org/10.1080/00071668.2018.1484074 PMID: 29877713 13. Pastorelli H, Le Floc’h N, Merlot E, Meunier-Salaun MC, van Milgen J, Montagene L. Feed restriction applied after weaning has different effects on pig performance and health depending on the sanitary conditions. J Anim Sci. 2012. 90: 4866–4875. https://doi.org/10.2527/jas.2012-5309 PMID: 22952368 14. Rozeboom DW, Shaw DT, Tempelman RJ, Miguel JC, Pettigrew JE, Connolly A. Effect of mannan oli- gosaccharide and an antimicrobial product in nursery diets on performance of pigs reared on three dif- ferent farms. J Anim Sci. 2005. 83(11): 2637–2644. https://doi.org/10.2527/2005.83112637x PMID: 16230663 15. Markowiak P, Slizewska K. The role of probiotics, prebiotics and synbiotics in animal nutrition. Gut Pathog. 2018. 10: 21. https://doi.org/10.1186/s13099-018-0250-0 PMID: 29930711 16. NRC. Nutrient rewuirements of swine. Washington, D.C.: The National Academies Press. 2012. 17. Heo JM, Kim JC, Hansen CF, Mullan BP, Hampson DJ, Pluske JR. Feeding a diet with decreased pro- tein content reduces indices of protein fermentation and the incidence of postweaning diarrhoea in weaned pigs challenged with an enterotoxigenic strain of Escherichia coli. J Anim Sci. 2009. 87: 2833– 2843. https://doi.org/10.2527/jas.2008-1274 PMID: 19502498 18. AOAC. Official Methods of Analysis, 17th ed. Association of Official Analytical Chemists, Arlington, VA, USA. 1990. 19. Touchette KJ, Carroll JA, Allee GL, Matteri RL, Dyer CJ, Beausang LA, et al. Effect of spray-dried plasma and lipopolysaccharide exposure on weaned pigs: I. Effects on the immune axis of weaned pigs. J Anim Sci. 2002. 80: 494–501. https://doi.org/10.2527/2002.802494x PMID: 11881933 20. Hanshew AS, Mason CJ, Raffa KF, Currie CR. Minimization of chloroplast contamination in 16S rRNA gene pyrosequencing of insect herbivore bacterial communities. J Microbiol Methods. 2013. 95(2): 149–155. https://doi.org/10.1016/j.mimet.2013.08.007 PMID: 23968645 21. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, et al. Introducing mothur: open- source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol. 2009. 75(23): 7537–7541. https://doi.org/10.1128/AEM.01541- 09 PMID: 19801464 22. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat. methods. 2010. 7(5): 335. https://doi. org/10.1038/nmeth.f.303 PMID: 20383131 23. Oksanen J, Guillaume Blanchet RKF, Legendre P, Minchin PR, O’Hara RB, Simpson GL, et al. Pack- age ‘vegan’. R Packag 2019. version 3.4.0. PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 19 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs 24. Paulson NJ, Olson ND, Braccia DJ, Wagner J, Talukder H, Pop M, et al. Package ‘metagenomeSeq’. 2019. 25. Pastorelli H, van Milgen J, Lovatto P, Montagene L. Meta-analysis of feed intake and growth responses of growing pigs after a sanitary challenge. Animal. 2012. 6: 952–961. https://doi.org/10.1017/ S175173111100228X PMID: 22558966 26. Kim JC, Mullan BP, Pluske JR. Impact of the systemic response to stressors and subclinical and clinical infection on intestinal barrier function and growth in pigs. In Pluske JR Pluske JM editors. Manipulating Pig Production XIV. Werribee: Australasian Pig Science Association; 2013. pp. 62–76. 27. Heinritz SN, Weiss E, Eklund M, Aumiller T, Louis S, Rings A, et al. Intestinal microbiota and microbial metabolites are changed in a pig model fed a high-fat/low-fiber or a low-fat/high-fiber diet. PloS One 2016. 11(4): e0154329. 28. Kruse R, Essen-Gustavsson B, Fossum C, Jensen-Waern M. Blood concentration of the cytokines IL- 1beta, IL-6, IL-10, TNF-alpha and IFN-gamma during experimentally induced swine dysentery. Acta Vet Scand. 2008. 50(1): 32. 29. Thanawongnuwech R, Thacker B, Halbur P, Thacker EL. Increased production of pro-inflammatory cytokines following infection with porcine reproductive and respiratory syndrome virus and mycoplasma hyopneumoniae. Clin Diagn Lab Immunol. 2004. 11: 901–908. https://doi.org/10.1128/CDLI.11.5.901- 908.2004 PMID: 15358650 30. Velazquez-Salinas L, Verdugo-Rodrigues A, Rodriguez LL, Borca MV. The role of interleukin 6 during viral infections. Front Microbiol. 2019. 10: 1057. https://doi.org/10.3389/fmicb.2019.01057 PMID: 31134045 31. Bach Knudesen KE. Fibre and nonstarch polysaccharide content and variation in common crops used in broiler diets. Poult Sci. 2019. 93: 2380–2393. 32. Maharjan P, Mayorga M, Hilton K, Weil J, Beitia A, Caldas J, et al. Non-cellulosic polysaccharide con- tent in feed ingredients and ileal and total tract non-cellulosic polysaccharide digestibility in 21-42-day- old broilers fed diets with and without added composite enzymes. Poult Sci. 2019. 98(9): 4048–4057. https://doi.org/10.3382/ps/pez079 PMID: 30968121 33. Bautil A, Verspreet J, Buyse J, Goos P, Bedford MR, Courtin CM. Adaptation of the microbiome towards fibre digestion: effects of age and dietary ingredients. In: Gonzalez-Ortiz G, Bedford MR, Bach Knudsen KE, Courtin CM, Classen HL editors. The value of fibre—Engaging the second brain for animal nutrition, The Netherlands: Wageningen Academic Press; 2019. pp. 199–216. 34. Bedford MR, Apajalahti J. Exposure of a broiler to a xylanase for 35d increases the capacity of cecal microbiome to ferment soluble xylan. Proc. Poultry Science Association 107th Annual Meeting, San Antonio, Texas, US. 2018. E-Suppl 1: 98–99. 35. Marinho MC, Lordelo MM, Cunha LF, Freire JBP. Microbial activity in the gut of pigs: I. Effect of prebiotic and probiotic supplementation. Livest Sci. 2007. 108: 236–239. 36. Pan J, Yin J, Zhang K, Xie P, Ding H, Huang X, et al. Dietary xylo-oligosacchride supplementation alters gut microbial composition and activity in pigs according to age and dose. AMB Expr. 2019. 9: 134. 37. Gonzalez-Ortiz G, Olukosi OA, Jurgens G, Apajalahti J, Bedford MR. Short-chain fatty acids and ceca microbiota profiles in broilers and turkeys in response to diets supplemented with phytase at varying concentrations, with or without xylanase. Poult Sci. 2020. 99: 2068–2077. https://doi.org/10.1016/j.psj. 2019.11.051 PMID: 32241492 38. Van Craeyveld V, Swennen K, Dornex E, van de Wiele T, Marzorati M, Verstraete W, et al. Structurally different wheat-derived arabinoxylooligosaccharides have different prebiotic and fermentation proper- ties in rats. J Nutr. 2008. 138(12): 2348–2355. https://doi.org/10.3945/jn.108.094367 PMID: 19022956 39. Miller TL, Jenesel SE. Enzymology of butyrate formation by Butyrivibrio fibrisolvens. J Bacteriol. 1979. 138(1): 99–104. https://doi.org/10.1128/JB.138.1.99-104.1979 PMID: 35524 40. Qi M, Nelson KE, Daugherty SC, Nelson WC, Hance IR, Morrison M, et al. Novel molecular features of the fibrolytic intestinal bacterium Fibrobactor intestinalis not shared with Fibrobactor succinogenes as determined by suppressive subtractive hybridization. J Bacterol. 2005. 187(11): 3739–3751. 41. Van Gylswyl NO, Hippe H, Rainey FA. Pseudobutyrivibrio ruminis gen. nov., a butyrate-producing bac- terium from the rumen that closely resembles Butyrivibrio fibrisolvens in phenotype. Int J Sys Bacteroil. 1996. 46(2): 559–563. 42. Kim JC, Heo JM, Mullan BP, Pluske JR. Efficacy of a reduced protein diet on clinical expression of post- weaning diarrhoea and life-time performance after experimental challenge with an enterotoxigenic strain of Escherichia coli. Anim Feed Sci Technol. 2011. 170: 222–230. 43. Pieper R, Kroger S, Richter JF, Wang J, Martin L, Bindelle J, et al. Fermentable fibre amileorates fer- mentable protein-induced changes in microbial ecology, but not the mucosal response, in the colon of piglets. J Nutr. 2012. 142: 661–667. https://doi.org/10.3945/jn.111.156190 PMID: 22357743 PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 20 / 21 PLOS ONE Stimbiotic supplementation in weaner pigs 44. Pluske JR, Kim JC, McDonald DE, Pethick DW, Hampson DJ. Non-starch polysaccharides in the diets of young weaned piglets. In Varley MA, Wiseman J, editors. The weaner pig: nutrition and management. 2001. Wallingford: CABI Publishing. pp. 81–112. 45. Manzanilla EG, Nofrarias M, Anguita M, Castillo M, Perez JF, Martin-Orue SM, et al. Effects of butyrate, avilamycin, and a plant extract combination on the intestinal equilibrium of early weaned pigs. J Anim Sci. 2006. 84: 2743–2751. https://doi.org/10.2527/jas.2005-509 PMID: 16971576 46. Adebiyi AO, Olukosi OA. Apparent and standardised ileal amino acid digestibility of wheat distillers dried grains with solubles with or without exogenous protease in broilers and turkeys. Br Poult Sci. 2015. 56(2): 239–246. https://doi.org/10.1080/00071668.2015.1011606 PMID: 25633411 47. Keesing F, Ostfeld RS. Is biodiversity good for your health? Sci. 2015. 349: 235–236. https://doi.org/ 10.1126/science.aac7892 PMID: 26185230 PLOS ONE | https://doi.org/10.1371/journal.pone.0240264 November 10, 2020 21 / 21