1. Introduction {#sec1}
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It is well known that early weaning is an effective way to maximize the whole herd production ([@bib18]). However, early weaning is often associated with marked changes in the histology and biochemistry of the gastrointestinal tract of piglets, which resulted in digestive upset, diarrhea and retardation of growth performance ([@bib10], [@bib24]). Antibiotics have been widely used as growth promoters and for therapeutic treatment of gastrointestinal disease in newly weaned piglets ([@bib5]). However, the abuse of antibiotics in animal feeds not only leads to antibiotic residues in animal products and the environment, but also results in bacterial resistant to antibiotic and inhibition of innate immune function ([@bib12], [@bib14]). Therefore, it is necessary to develop effective, safe, economical and environment-friendly alternatives to in-feed antibiotics. Previous studies have shown that organic acids, probiotics and essential oils could be used as substitutes for in-feed antibiotics for their benefits in decreasing the load of pathogenic bacteria in the gut ([@bib3], [@bib37]).

Some researchers have proposed that organic acid could improve growth performance and has antibacterial action primarily via decreasing pH values of the stomach and gut digesta, modulating microbial population, improving nutrients digestion, and other possible mechanisms ([@bib31]). Benzoic acid, as a kind of organic acid, was authorized to be used in pigs at the dose of 0.5% to 1.0% by European Union in 2003. Previous studies indicated that benzoic acid could improve growth performance and nutrient digestibility, and maintain intestinal microflora balance of weaned piglets ([@bib17]). The term probiotics has been defined as live microbial cell preparations or microbial cell components feed supplements, which beneficially improve growth performance by maintaining intestinal microbial balance and stimulating immune response of animals ([@bib20]). *Bacillus* coagulans, as a kind of probiotic, not only has all the characteristics of lactic acid bacteria, but also has strong resistance to acid, high temperature, high pressure, and easy storage properties ([@bib9]). Previous studies reported that *Bacillus* coagulans could improve growth performance, maintain intestinal microflora balance and improve immune response of animals ([@bib41], [@bib22]). Oregano oils, which are essential oils obtained from oregano plant by a steam distillation process, are comprised of more than 20 ingredients; of these ingredients, carvacrol and thymol are the most abundant ([@bib35]). It has been demonstrated that oregano oil could improve growth performance by improving antioxidant capacity and decreasing the number of pathogenic bacteria in the gut of pigs ([@bib7], [@bib40]).

Based on their positive effects, benzoic acid, *Bacillus* coagulans and oregano oils are considered to be good potential alternatives for in-feed antibiotic growth promoter. However, the experimental results of these additives have varied widely and the effect of a single additive is limited. In recent years, organic acids, probiotics and essential oils combined supplementation in animal diets have attracted wide research interest due to their potential 'synergistic' and 'additive' benefits on growth performance ([@bib4], [@bib16]). However, little information is available on the effects of benzoic acid, *Bacillus* coagulans and oregano oils combined supplementation on growth performance, immune status and intestinal health of weaned piglets.

Therefore, the objective of this study was to determine the effects of benzoic acid, *Bacillus* coagulans and oregano oils combined supplementation on growth performance, immune status, intestinal barrier integrity and microflora of weaned piglets, and to evaluate their potential as an alternative to antibiotics growth promoters.

2. Materials and methods {#sec2}
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The experiment was carried out in accordance with the Chinese guidelines for animal welfare and experimental protocol, and was approved by Sichuan Agricultural University Institutional Animal Care and Use Committee (Approval number: CD-SYXK-2017-015).

2.1. Benzoic acid, *Bacillus* coagulans and oregano oil products {#sec2.1}
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Benzoic acid (VevoVitall) was purchased from DSM (China) Company Limited (purity, 99.9%); *Bacillus* coagulans was provided by Sanzheng Group (Kunming, China) at a density of 5 × 10^9^ CFU/g; Oregano oil (free-flowing powder) was provided by Kemin Industries (Zhuhai, China), the major active components in the oregano oil were carvacrol and thymol (contained a minimum of 22 g/kg carvacrol and a minimum of 11 g/kg thymol). Defatted rice bran and silica were used as carriers.

2.2. Animals, diets and experimental design {#sec2.2}
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A total of 25 weaned piglets \[Duroc × (Landrace × Yorkshire)\] with initial body weight (7.65 ± 0.38 kg) were weaned at 21 d of age and randomly assigned to 5 treatments (*n* = 5 piglets per treatment) based on their initial body weight. The dietary treatment groups were as follows: 1) corn-soybean basal diet (negative control, NC); 2) NC + colistin sulfate at 20 g/t + bacitracin zinc at 40 g/t (positive control, PC); 3) NC + benzoic acid at 3,000 g/t + *Bacillus* coagulans at 400 g/t (AB); 4) NC + benzoic acid at 3,000 g/t + oregano oil at 400 g/t (AO); 5) NC + benzoic acid at 3,000 g/t + *Bacillus* coagulans at 400 g/t + oregano oil at 400 g/t (ABO). The pigs were individually housed in metabolic cages (1.5 m × 0.7 m × 1.0 m) with woven wire flooring in a temperature-controlled nursery room (25 to 28 °C) during a 26-d experimental period. All pigs had ad libitum access to feed and water. The basal diet ([Table 1](#tbl1){ref-type="table"}) was formulated to meet or exceed the nutrient requirements recommended by the National Research Council ([@bib32]). The experimental diets consisted of corresponding additive products replaced equivalent maize in the basal diet.Table 1Ingredients composition and nutrients level of the basal diet (%, as-fed basis).Table 1IngredientsContentNutrient level[1](#tbl1fn1){ref-type="table-fn"}ContentMaize29.80Digestible energy, MJ/kg14.72Extruded maize29.85Crude protein19.13Fish meal4.50Calcium0.75Whey powder6.00Total phosphorus0.56Sucrose3.00Available phosphorus0.37Soybean meal10.00Lysine1.30Soybean protein concentrate6.40Methionine0.41Extruded soybean6.70Threonine0.79Soybean oil1.70Tryptophan0.22*L*-lysine-HCl (78%)0.26*L*-threonine (98%)0.02*DL*-methionine (98%)0.09*L*-tryptophan (98%)0.01Choline chloride0.15Salt0.20Limestone0.60Dicalcium phosphate0.37Vitamin premix[2](#tbl1fn2){ref-type="table-fn"}0.05Mineral premix[3](#tbl1fn3){ref-type="table-fn"}0.30Total100.00[^1][^2][^3]

2.3. Growth performance and diarrhea incidence {#sec2.3}
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The pigs were weighed individually at the start and the end of experimental period and the feed intake was recorded on daily basis. These values were used to calculate average daily body weight gain (ADG), average daily feed intake (ADFI) and the ratio of feed to gain (F:G). The feces of all pigs were observed daily throughout the study. Fecal consistency was scored as follows: 0, normal; 1, pasty; 2, semiliquid; and 3, liquid. Pigs with daily fecal consistency scores ≥2 were considered diarrhea ([@bib39]). Diarrhea incidence (%) = Number of diarrheal piglets × Diarrhea days/(Number of piglets × Test days) × 100.

2.4. Sample collection {#sec2.4}
----------------------

Blood samples were collected from pigs via anterior vein into 10-mL vacuum tubes without anticoagulant on d 27 following an overnight fast. Samples were centrifuged at 3,500 × *g* for 10 min at 4 °C, and the serum samples were harvested and stored at −20 °C until further assay. After bled, all pigs were anesthetized by electric shock and then euthanized by exsanguinations. The small intestine was dissected from the mesentery and immediately placed on ice, approximately 20 cm of jejunal tissue sample was removed from the middle portion of jejunum and flushed with ice-cold saline to recover mucosa. Mucosa of jejunum was collected by scraping using a sterile glass microscope slide at 4 °C, and then the samples were rapidly frozen in liquid nitrogen and stored at −80 °C until analyses. At the same time, approximately 3 g of the digesta from the cecum were sampled into sterile tubes separately and immediately immersed in liquid nitrogen, then stored at −80 °C for microbial population determination.

2.5. Disaccharidase activities analysis {#sec2.5}
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Frozen jejunal mucosa scrapings were weighed, thawed and homogenized (5 min) in 9 times the volume (wt/vol) of ice-cold physiologic saline. The mixtures were then centrifuged at 3,500 × *g* for 10 min at 4 °C, the supernatant was collected for disaccharidase activities examination. The total protein concentration of supernatant was determined by using the bicinchoninic acid (BCA) Protein Assay kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer\'s instructions. The activities of disaccharidases (maltase and sucrase) in the jejunal mucosal supernatant were measured by using Disaccharidase Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China) combined with a UV-VIS Spectrophotometer (UV1100, MAPADA, Shanghai, China) according to the manufacturer\'s instructions. The activities of disaccharidases were presented as U/mg protein. One unit (U) of disaccharidase activity was defined as the amount of the enzyme that can hydrolyse 1μmol of substrate per minute under the standard assay conditions.

2.6. Serum and jejunal mucosal cytokines concentrations, and jejunal mucosal secretory immunoglobulin A concentration {#sec2.6}
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In brief, 100 mg of jejunal mucosa was homogenized (5 min) in nine times the volume (wt/vol) of ice-cold PBS solution. The mixtures were then centrifuged at 3,500 × *g* for 15 min at 4 °C, the supernatants were collected and stored at −80 °C until further assay. The jejunal mucosal supernatant and serum were used to detect the tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10) and secretory immunoglobulin A (sIgA) concentrations with commercially available pig ELISA kits from Xinle Co. Ltd. (Shanghai, China) according to the manufacturer\'s instructions.

2.7. Total RNA extraction and quantitative real-time PCR (qPCR) {#sec2.7}
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Total RNA was extracted from jejunal mucosa using TRIzol reagent (TaKaRa Biotechnology Co, Ltd, Dalian, China) following the manufacturer\'s instructions. The concentration and purity of RNA were analyzed spectrophotometrically (Beckman Coulter DU800; Beckman Coulter Inc.), considering the ideal absorbance ratio (1.8 ≤ A260/280 ≤ 2.0). The integrity of RNA was checked by electrophoresis on a 1.5% agarose gel. The RNA samples were reverse transcribed into cDNA using the PrimeScript RT reagent kit (TaKaRa Biotechnology Co, Ltd, Dalian, China) according to the manufacturer\'s instructions. The synthesis involved two steps: 37 °C for 15 min and 85 °C for 5 s. The cDNA was diluted 3-fold and used as a PCR template to evaluate gene expression. The primers were synthesized commercially by TaKaRa Biotechnology Co, Ltd. (Dalian, China), which are listed in [Table 2](#tbl2){ref-type="table"}. All qPCR reactions were done in triplicate on a QuanStudio 6 Flex Real-Time PCR System (Applied Biosystems), using SYBR Premix Ex Taq II (Tli RNaseH Plus; TaKaRa Biotechnology Co, Ltd, Dalian, China). Negative controls were performed in which water was substituted for complementary DNA. The amplification was performed in a final volume of 10 mL. This solution consisted of 5 mL SYBR Premix Ex Taq II (Tli RNaseH Plus), 0.2 mL ROX Reference Dye II, 0.4 mL forward primer, 0.4 mL reverse primer, 1 mL cDNA and 3 mL diethylpyrocarbonate-treated water. The cycling conditions were as follows pre-denaturation at 95 °C for 30 s and 40 cycles of denaturation at 95 °C for 5 s, annealing at the anneal temperatures ([Table 2](#tbl2){ref-type="table"}) for 30 s and extension at 72 °C for 60 s. A dissociation curve was constructed at the end of the reaction to ensure that only one amplification was formed. Target and house-keeping gene amplification efficiencies were calculated according to the specific gene standard curves that were generated from 10-fold serial dilutions, quantifying 6 concentrations. After verification that the primers amplified with an efficiency close to 100%, the results were analyzed using the 2^−ΔΔCt^ method, with porcine glyceraldehyde-3-phosphate dehydrogenase (*GAPDH*) gene as the housekeeping gene ([@bib27]).Table 2Primer sequences used for quantitative real-time PCR.Table 2PrimerPrimer sequence (5′ --3′)Anneal temperature, °CProduct length, bpGenBank accession No.*GAPDH*F: TGAAGGTCGGAGTGAACGGAT\
R: CACTTTGCCAGAGTTAAAAGCA55.7114[NM_001206359.1](ncbi-n:NM_001206359.1){#intref0010}*SGLT1*F: AGAAGGGCCCCAAAATGACC\
R: TGTTCACTACTGTCCGCCAC5996[NM_001164021.1](ncbi-n:NM_001164021.1){#intref0015}*GLUT2*F: TGGAATCAGCCAACCTGTTT\
R: ACAAGTCCCACCGACATGA55.7156[NM_001097417.1](ncbi-n:NM_001097417.1){#intref0020}*PepT1*F: GCCAAAGTCGTCAAGTGC\
R: GGTCAAACAAAGCCCAGA63.3100[NM214347](ncbi-n:NM214347){#intref0025}Claudin-1F: GCCACAGCAAGGTATGGTAAC\
R: AGTAGGGCACCTCCCAGAAG59.0140[FJ873109.1](ncbi-n:FJ873109.1){#intref0030}OccludinF: CTACTCGTCCAACGGGAAAG\
R: ACGCCTCCAAGTTACCACTG59.0158[NM_001163647.2](ncbi-n:NM_001163647.2){#intref0035}*ZO-1*F: CAGCCCCCGTACATGGAGA\
R: GCGCAGACGGTGTTCATAGTT59.0114[XM_005659811](ncbi-n:XM_005659811){#intref0040}Mucin 1F: GTGCCGCTGCCCACAACCTG\
R: AGCCGGGTACCCCAGACCCA59.0141[XM_001926883.4](ncbi-n:XM_001926883.4){#intref0045}Mucin 2F: GGTCATGCTGGAGCTGGACAGT\
R: TGCCTCCTCGGGGTCGTCAC59.0181[XM_003122394.1](ncbi-n:XM_003122394.1){#intref0050}[^4]

2.8. DNA extraction and measurement of intestinal bacterial populations by qPCR {#sec2.8}
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Bacterial DNA in the cecal digesta was extracted using the Stool DNA Kit (Omega Bio-Tek, Doraville, CA, USA) according to the manufacturer\'s instruction. All primers and probes ([Table 3](#tbl3){ref-type="table"}) were commercially synthesized from TaKaRa Biotechnology Co, Ltd. (Dalian, China). Briefly, the number of total bacteria was analyzed by qPCR using SYBR Premix Ex Taq reagents (TaKaRa Biotechnology Co, Lt., Dalian, China) and CFX-96 Real-Time PCR Detection System (BioRad Laboratories, Richmond, CA), and the numbers of *Bacillus, Lactobacillus, Escherichia coli* and *Bifidobacterium* were analyzed by qPCR using PrimerScript TM PCR kit (Perfect Real Time; TaKaRa Biotechnology Co, Ltd, Dalian, China) and CFX-96 Real--Time PCR Detection System (Bio-Rad Laboratories, Richmond, CA) as previously described ([@bib11]). For the quantification of bacteria in the test samples, specific standard curves were generated by constructing standard plasmids as presented by [@bib11]. In addition, bacterial copies were transformed (log~10~) before statistical analysis.Table 3Primer and probe sequences used for quantitative real-time PCR.Table 3ItemPrimer and probe sequence (5' --3′)Anneal temperature, °CProduct length, bpTotal bacteriaF: ACTCCTACGGGAGGCAGCAG\
R: ATTACCGCGGCTGCTGG57.9200*Lactobacillus*F: GAGGCAGCAGTAGGGAATCTTC\
R: CAACAGTTACTCTGACACCCGTTCTTC\
P: AAGAAGGGTTTCGGCTCGTAAAACTCTGTT53.0126*Bifidobacterium*F: CGCGTCCGGTGTGAAAG\
R: CTTCCCGATATCTACACATTCCA\
P: ATTCCACCGTTACACCGGAA57.9121*Bacillus*F: GCAACGAGCGCAACCCTTGA\
R: TCATCCCCACCTTCCTCCGGT\
P: CGGTTTGTCACCGGCAGTCACCT53.092*Escherichia coli*F: CATGCCGCGTGTATGAAGAA\
R: CGGGTAACGTCAATGAGCAAA\
P: AGGTATTAACTTTACTCCCTTCCTC53.096

2.9. Statistical analysis {#sec2.9}
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The data related to growth performance, disaccharidase activities, immune status, gene expression and intestinal bacteria were checked for normal distribution using the Shapiro--Wilk normality test of SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). Then one-way ANOVA test was used to compare the difference of the normal distributed data among groups, followed by Duncan\'s multiple-range tests. Individual piglet served as the experimental unit. Results were presented as means ± standard errors (SE). Moreover, the Chi-square test was used to analyze diarrhea incidence. Differences were considered as significant at *P* \< 0.05.

3. Results {#sec3}
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3.1. Growth performance and diarrhea incidence {#sec3.1}
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The results of growth performance and diarrhea incidence are shown in [Table 4](#tbl4){ref-type="table"}. Compared with NC group, pigs fed AB diet had greater final BW and ADG and lower F:G ratio (*P* \< 0.05). However, no significant difference was observed on the ADFI and diarrhea incidence among treatments (*p* \> 0.05).Table 4Effects of dietary benzoic acid, *Bacillus* coagulans and oregano oil combined supplementation on growth performance and diarrhea incidence of weaned piglets.Table 4ItemNCPCABAOABOInitial BW, kg7.61 ± 0.167.63 ± 0.187.70 ± 0.167.64 ± 0.227.66 ± 0.20Final BW, kg14.20 ± 0.77^a^15.32 ± 0.39^ab^16.42 ± 0.62^b^16.04 ± 0.77^ab^15.96 ± 0.81^ab^ADFI, g436.59 ± 29.65472.76 ± 23.25515.14 ± 35.24515.85 ± 38.76522.17 ± 43.56ADG, g253.46 ± 25.84^a^295.77 ± 12.36^ab^335.38 ± 19.38^b^323.08 ± 25.19^ab^319.23 ± 28.82^ab^F:G1.76 ± 0.09^b^1.60 ± 0.04^ab^1.54 ± 0.05^a^1.60 ± 0.05^ab^1.64 ± 0.07^ab^Diarrhea incidence, %9.238.466.153.085.38[^5][^6]

3.2. Disaccharidase activities and nutrient transporter related gene expression in jejunal mucosa {#sec3.2}
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As shown in [Table 5](#tbl5){ref-type="table"}, no significant difference was observed on the activities of jejunal mucosal maltase and sucrase among treatments (*P* \> 0.05). Compared with NC group, dietary AB increased the mRNA abundance of SGLT1 in jejunal mucosa (*P* \< 0.05).Table 5Effects of dietary benzoic acid, *Bacillus* coagulans and oregano oil combined supplementation on disaccharidase activities and nutrient transporter mRNA abundance in jejunal mucosa of weaned piglets.Table 5ItemNCPCABAOABOMaltase, U/mgprot187.79 ± 18.48197.84 ± 8.70210.44 ± 11.03207.15 ± 24.03211.96 ± 28.95Sucrase, U/mgprot153.98 ± 43.55173.35 ± 13.21214.62 ± 33.59166.29 ± 28.64176.45 ± 31.90*SGLT1*1.00 ± 0.17^a^1.14 ± 0.27^ab^1.69 ± 0.27^b^1.31 ± 0.20^ab^1.61 ± 0.20^ab^*GLUT2*1.00 ± 0.151.22 ± 0.331.65 ± 0.161.35 ± 0.311.42 ± 0.27*PepT1*1.00 ± 0.211.22 ± 0.331.52 ± 0.121.29 ± 0.261.50 ± 0.25[^7][^8]

3.3. Serum and jejunal mucosal cytokines concentrations, and jejunal mucosal sIgA concentration {#sec3.3}
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As shown in [Table 6](#tbl6){ref-type="table"}, compared with NC group, dietary AB and AO decreased serum TNF-α concentration (*P* \< 0.05). Meanwhile, lower serum TNF-α and IL-1β concentrations were observed in ABO group compared with NC group (*P* \< 0.05). Furthermore, compared with NC and PC groups, dietary ABO decreased IL-1β concentration in jejunal mucosa (*P* \< 0.05). However, no significant difference was observed on the concentrations of jejunal mucosal TNF-α, IL-6, IL-10 and sIgA among treatments (*P* \> 0.05).Table 6Effects of dietary benzoic acid, *Bacillus* coagulans and oregano oil combined supplementation on serum and jejunal mucosal cytokines concentrations and jejunal mucosal sIgA concentration of weaned piglets.Table 6ItemNCPCABAOABOSerumTNF-α, ng/L203.49 ± 2.89^b^192.20 ± 8.48^ab^174.93 ± 12.78^a^169.44 ± 12.41^a^168.45 ± 8.27^a^IL-1β, ng/L11.30 ± 0.83^b^10.00 ± 0.59^ab^9.66 ± 0.52^ab^9.44 ± 0.72^ab^8.23 ± 0.42^a^IL-6, ng/L67.56 ± 4.6260.44 ± 4.2559.09 ± 3.5359.43 ± 4.5156.74 ± 3.25IL-10, pg/mL190.65 ± 17.02207.18 ± 8.27218.56 ± 4.71200.32 ± 6.07204.51 ± 12.12Jejunal mucosaTNF-α, ng/L96.67 ± 5.6592.34 ± 10.5382.49 ± 12.3983.81 ± 11.6670.68 ± 9.84IL-1β, ng/L14.11 ± 1.46^b^13.58 ± 1.22^b^11.85 ± 1.31^ab^12.08 ± 1.39^ab^8.51 ± 0.52^a^IL-6, ng/L59.52 ± 7.7758.40 ± 5.8255.12 ± 7.1754.04 ± 7.0650.54 ± 7.57IL-10, pg/mL163.08 ± 18.02181.53 ± 17.93186.32 ± 15.74173.42 ± 19.22170.44 ± 19.37sIgA, μg/mL46.30 ± 2.3955.31 ± 5.3861.68 ± 11.3855.04 ± 6.6659.71 ± 2.98[^9][^10]

3.4. Barrier integrity related gene expression in jejunal mucosa {#sec3.4}
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As shown in [Table 7](#tbl7){ref-type="table"}, compared with NC group, dietary AB increased the mRNA abundance of claudin-1, occludin and mucin2 in jejunal mucosa (*P* \< 0.05). Meanwhile, higher occludin mRNA abundance was observed in ABO group compared with NC group (*P* \< 0.05).Table 7Effects of dietary benzoic acid, *Bacillus* coagulans and oregano oil combined supplementation on barrier integrity related gene expression in jejunal mucosa of weaned piglets.Table 7ItemNCPCABAOABOClaudin-11.00 ± 0.15^a^1.41 ± 0.20^ab^1.70 ± 0.31^b^1.39 ± 0.25^ab^1.53 ± 0.22^ab^Occludin1.00 ± 0.14^a^1.18 ± 0.16^ab^1.52 ± 0.16^b^1.36 ± 0.15^ab^1.54 ± 0.14^b^*ZO-1*1.00 ± 0.170.80 ± 0.111.25 ± 0.291.17 ± 0.081.19 ± 0.18Mucin11.00 ± 0.051.24 ± 0.071.27 ± 0.131.21 ± 0.191.25 ± 0.15Mucin21.00 ± 0.15^a^1.22 ± 0.09^ab^1.41 ± 0.11^b^1.19 ± 0.12^ab^1.11 ± 0.07^ab^[^11][^12]

3.5. Bacteria populations in cecal digesta {#sec3.5}
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The results of intestinal bacteria populations are shown in [Table 8](#tbl8){ref-type="table"}. Compared with NC group, dietary AB increased *Bifidobacterium* and *Bacillus* populations in cecal digesta (*P* \< 0.05); dietary ABO increased *Bifidobacterium* population and decreased *E. coli* population in cecal digesta (*P* \< 0.05). Furthermore, dietary AB and ABO increased *Bacillus* population in cecal digesta compared with PC group (*P* \< 0.05).Table 8Effects of dietary benzoic acid, *Bacillus* coagulans and oregano oil combined supplementation on intestinal bacteria populations in cecal digesta of weaned piglets (log~10~\[copies/g\]).Table 8ItemNCPCABAOABO*Lactobacillus*8.40 ± 0.318.50 ± 0.188.42 ± 0.108.59 ± 0.108.78 ± 0.12*Bacillus*9.84 ± 0.09^ab^9.71 ± 0.05^a^10.16 ± 0.08^c^9.93 ± 0.07^abc^9.99 ± 0.12^bc^*Bifidobacterium*7.88 ± 0.09^a^8.09 ± 0.11^ab^8.19 ± 0.11^b^8.08 ± 0.12^ab^8.24 ± 0.09^b^*Escherichia coli*8.51 ± 0.09^b^8.27 ± 0.23^ab^8.14 ± 0.16^ab^8.17 ± 0.18^ab^7.86 ± 0.16^a^Total bacteria11.29 ± 0.0311.40 ± 0.1111.45 ± 0.1011.39 ± 0.0611.42 ± 0.08[^13][^14]

4. Discussion {#sec4}
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Weaning stress of piglets often causes a wide range of physiological responses, such as impaired intestinal metabolism and function, compromised immunity and decreased antioxidant capacity, which lead to depressed feed utilization efficiency, retarded growth, as well as increased morbidity and mortality ([@bib18]). During last decades, as alternatives to antibiotics, benzoic acid, *Bacillus* coagulans and oregano oil have been used as effective supplements to improve growth performance and decrease diarrhea in piglets. Previous studies have shown that dietary 2,000 mg/kg benzoic acid +100 mg/kg thymol supplementation improved the growth performance and decreased diarrhea in weaned piglets ([@bib21]). [@bib33] reported that benzoic acid and *Bacillus* cereus var. toyoi combined supplementation improved ADG, decreased F:G and diarrhea in weaned piglets. Similarly, the results of the current study revealed that dietary benzoic acid and *Bacillus* coagulans combined supplementation significantly increased ADG and decreased F:G of weaned piglets. This may be due to the better gut characteristics including increased nutrient digestibility, healthy and stable intestinal microflora, and better intestinal barrier integrity.

The nutrient transporters in the small intestine are also very important to the nutrient utilization of animals and human. In the piglet small intestine, glucose absorption depends on 2 types of transport mechanisms: one is glucose transporter 2 (GLUT2), which serves as a facilitated diffusion system through lipid bilayers, and the other is SGLT1, which mediates Na+/glucose co-transport function as a secondary active transporter ([@bib8]). Oligopeptide transporter 1 mainly transports dipeptides and tripeptides from the digestion of dietary proteins ([@bib13]). In the present study, dietary AB increased the mRNA abundance of SGLT1 in jejunal mucosa of piglets, thus resulting in the better nutrient absorption relative to piglets fed NC diet. The result is consistent with the improvement of growth performance in AB group. This may be associated with the changes in the intestinal barrier integrity.

Weaning stress has been reported to cause remarkable disturbances in intestinal barrier function ([@bib34]). Disturbances in the intestinal barrier, characterized by increased intestinal permeability, results in translocation of luminal bacteria, toxins and antigens into the subepithelial tissues, affecting nutrient absorption ([@bib6]). Therefore, an intact intestinal mucosal barrier is pivotal to ensure adequate provision of dietary nutrients to the whole body. The intestinal barrier can be commonly assessed by many indexes, such as the intestinal tight junction proteins and the mucus gel layer ([@bib29]). Tight junction (TJ) proteins (occludin, claudin and intracellular plaque proteins \[ZO and cingulin\]), participate in tight junction structural integrity via binding to the act in cytoskeleton, are considered as major constituents of tight junctions and important regulators of paracellular permeability ([@bib19]). In addition, intestine mucins, such as mucin1 and mucin2, are secreted by goblet cells, have been demonstrated to provide barrier protection against luminal virus, bacteria, and parasites invasions, thus playing important roles in regulating intestinal inflammation ([@bib30]). The present study showed that dietary AB increased the mRNA abundance of claudin-1, occludin and mucin2, and dietary ABO increased occludin mRNA level in jejunal mucosa of weaned piglets. These results indicated that dietary AB and ABO could ameliorate the weaning-associated intestinal damage by improving intestinal barrier integrity in weaned piglets.

As the crucial component of specific immunological responses, sIgA serves as the first line of defense in protecting the intestinal epithelium from enteric toxins and pathogenic microorganisms ([@bib38]). However, in the current study, dietary treatments did not affect on sIgA concentration in jejunal mucosa. A possible reason is that dietary benzoic acid, *Bacillus* coagulans and oregano oil combined supplementation are unable to stimulate sIgA secretion of the jejunal mucosa under normal feeding conditions. Furthermore, cytokine concentration is also widely used to assess the response to pathogens and antigens, both in experimental animal models and in human, as it can affect immune regulation and disease resistance ([@bib36]). Previous studies have demonstrated that dietary benzoic acid, probiotics or essential oils supplementation enhanced immune response in early weaned pigs by modulating the production of inflammatory cytokines (IL-1β, IL-6, TNF-α and IL-10) ([@bib28], [@bib1], [@bib25]). Similar with previous studies, the current study also reported that dietary AB and AO supplementation decreased serum TNF-α concentration, dietary ABO decreased serum TNF-α and IL-1β concentrations. As indicated above, dietary AB, AO or ABO could modulate the immune status of weaned piglets by reducing the production of pro-inflammatory cytokines. Besides the important roles in immunity, cytokines also have important physiological and pathological effects on the intestinal barrier integrity. Pro-inflammatory cytokines such as TNF-α and IL-1β increase intestinal permeability by inducing disruption of tight junctions, whereas anti-inflammatory cytokines, including IL-10 and TGF-β, tend to protect the epithelial integrity ([@bib2]). In the present study, consistent with the decreased IL-1β concentration in jejunal mucosa, increased occludin mRNA abundance in ABO group was observed. However, no difference was observed on IL-10 concentration among treatments. These results indicated that dietary ABO may improve intestinal integrity partially by reducing pro-inflammatory stimulus, rather than enhancing the anti-inflammatory response.

Intestinal microflora is crucial for maturation of the immune system and development of normal intestinal morphology. [@bib23] indicated that a healthy and stable microflora prevents the development of intestinal diseases and results in improved performance. Benzoic acid, *Bacillus* coagulans and oregano oil play an important role in stabilizing the intestinal ecosystem of animals, including increasing the number of health-promoting bacterial species (such as *Lactobacilli, Bacillus* and *Bifidobacteria*) and decreasing the number of potential pathogenic bacterial species (such as *E. coli*) ([@bib15], [@bib26], [@bib42]). Our results demonstrated that dietary benzoic acid, *Bacillus* coagulans and oregano oil combined supplementation can improve intestinal microecological balance, which was evidenced by the improvement in *Bifidobacterium* or *Bacillus* populations, and the decrease in *E. coli* population of cecal digesta. Thus, dietary benzoic acid and *Bacillus* coagulans combined supplementation improve the intestinal microflora could also be an important reason that it could improve the growth performance of weaned piglets.

5. Conclusions {#sec5}
==============

In conclusion, dietary AB supplementation could improve growth performance and intestinal barrier integrity of piglets when fed antibiotic-free diets, which was possibly associated with the improvement of immune status and intestinal microflora. Dietary ABO supplementation is also beneficial to improve immune status and intestinal barrier integrity and microflora of piglets.

Conflict of interest
====================

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.

The authors would like to thank to Jingbo Liu, Yumei Zhao and Lingyan Jia for their collaboration during the experiment. The present study was supported by the 10.13039/501100012166National Key Research and Development Program of China (2018YFD0500605) and the Science and Technology Support Program of Sichuan Province (2014NZ0043, 2016NZ0006).

Peer review under responsibility of Chinese Association of Animal Science and Veterinary Medicine.

[^1]: Nutrients levels were calculated values.

[^2]: The vitamin premix provided the following per kilogram of diets: vitamin A, 9,000 IU; vitamin D~3~, 3,000 IU; vitamin E, 20 IU; vitamin K~3~, 3.0 mg; vitamin B~1~, 1.5 mg; vitamin B~2~, 4.0 mg; vitamin B~6~, 3.0 mg; vitamin B~12~, 0.2 mg; niacin, 30 mg; pantothenic, 15 mg; folic acid, 0.75 mg; biotin, 0.1 mg.

[^3]: The mineral premix provided the following per kilogram of diets: Fe, 100 mg; Cu, 150 mg; Mn, 20 mg; Zn, 100 mg; I, 0.3 mg; Se, 0.3 mg.

[^4]: *GAPDH* = glyceraldehyde 3-phosphate dehydrogenase; *SGLT1* = sodium-glucose cotransporter 1; *GLUT2* = glucose transporter type 2; *PepT1* = oligopeptide transporter 1; *ZO-1* = zonula occludens 1.

[^5]: NC = negative control, a corn-soybean basal diet; PC = positive control, NC + colistin sulfate at 20 g/t + bacitracin zinc at 40 g/t; AB = NC + benzoic acid at 3,000 g/t + *Bacillus* coagulans at 400 g/t; AO = NC + benzoic acid at 3,000 g/t + oregano oil at 400 g/t; ABO = NC + benzoic acid at 3,000 g/t + *Bacillus* coagulans at 400 g/t + oregano oil at 400 g/t; BW = body weight; ADFI = average daily feed intake; ADG = average daily gain; F:G = the ratio of feed intake to gain.

[^6]: ^a,\ b^ Within a row, values with different letter superscripts mean significant difference (*P* \< 0.05).

[^7]: NC = negative control, a corn-soybean basal diet; PC = positive control, NC + colistin sulfate at 20 g/t + bacitracin zinc at 40 g/t; AB = NC + benzoic acid at 3,000 g/t + *Bacillus* coagulans at 400 g/t; AO = NC + benzoic acid at 3,000 g/t + oregano oil at 400 g/t; ABO = NC + benzoic acid at 3,000 g/t + *Bacillus* coagulans at 400 g/t + oregano oil at 400 g/t; *SGLT1* = sodium-glucose cotransporter1; *GLUT2* = glucose transporter type 2; *PepT1* = oligopeptide transporter 1.

[^8]: ^a,\ b^ Within a row, values with different letter superscripts mean significant difference (*P* \< 0.05).

[^9]: NC = negative control, a corn-soybean basal diet; PC = positive control, NC + colistin sulfate at 20 g/t + bacitracin zinc at 40 g/t; AB = NC + benzoic acid at 3,000 g/t + Bacillus coagulans at 400 g/t; AO = NC + benzoic acid at 3,000 g/t + oregano oil at 400 g/t; ABO = NC + benzoic acid at 3,000 g/t + Bacillus coagulans at 400 g/t + oregano oil at 400 g/t; TNF-α = tumor necrosis factor-α; IL-1β = interleukin-1β; IL-6 = interleukin-6; IL-10 = interleukin-10; sIgA = secretion immunoglobulin A.

[^10]: a, b Within a row, values with different letter superscripts mean significant difference (P \< 0.05).

[^11]: NC = negative control, a corn-soybean basal diet; PC = positive control, NC + colistin sulfate at 20 g/t + bacitracin zinc at 40 g/t; AB = NC + benzoic acid at 3,000 g/t + *Bacillus* coagulans at 400 g/t; AO = NC + benzoic acid at 3,000 g/t + oregano oil at 400 g/t; ABO = NC + benzoic acid at 3,000 g/t + *Bacillus* coagulans at 400 g/t + oregano oil at 400 g/t; *ZO-1* = zona occludens 1.

[^12]: ^a,\ b^ Within a row, values with different letter superscripts mean significant difference (*P* \< 0.05).

[^13]: NC = negative control, a corn-soybean basal diet; PC = positive control, NC + colistin sulfate at 20 g/t + bacitracin zinc at 40 g/t; AB = NC + benzoic acid at 3,000 g/t + *Bacillus* coagulans at 400 g/t; AO = NC + benzoic acid at 3,000 g/t + oregano oil at 400 g/t; ABO = NC + benzoic acid at 3,000 g/t + *Bacillus* coagulans at 400 g/t + oregano oil at 400 g/t.

[^14]: ^a,\ b,\ c^ Within a row, values with different letter superscripts mean significant difference (*P* \< 0.05).
