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Dietary Sodium Butyrate Decreases Postweaning Diarrhea by Modulating Intestinal Permeability and Changing the Bacterial Communities in Weaned Piglets1,2,3
- Chang Huang4,7,
- Peixia Song4,7,
- Peixin Fan4,
- Chengli Hou4,
- Phil Thacker5, and
- Xi Ma4,6,*
+Author Affiliations
- ↵*To whom correspondence should be addressed. E-mail: maxi@cau.edu.cn or xi.ma@utsouthwestern.edu.
Abstract
Background: The vast majority of substances used as alternatives to antibiotics produce inconsistent results and rarely equal the effectiveness of in-feed antibiotics.
Objective: This study evaluated the effects of the combined use of sodium butyrate (SB) and reduced antibiotics in a piglet diet in promoting performance and to control weaning diarrhea.
Methods: Piglets weaned at 28 d were randomly assigned to a corn-soybean meal control ration [negative control (NC)]; a similar ration with 50 mg kitasamycin/kg, 20 mg colistin sulfate/kg, and 1000 mg encapsulated SB/kg [reduced antibiotics + SB (ASB)]; or to a ration with 100 mg kitasamycin/kg and 40 mg colistin sulfate/kg [positive control (PC)] for 28 d. Performance, diarrhea incidence, intestinal permeability, and changes in the bacterial communities in the ileum and colon were determined.
Results: Weight gain and the ratio of weight gain to feed intake were significantly greater in the ASB and PC piglets than in the NC piglets (P < 0.05). Diarrhea incidence was lower in the ASB and PC piglets than in the NC piglets (P < 0.05). Urinary lactulose to mannitol ratios were 25% and 30% lower, respectively, whereas jejunal and colonic occludin protein expressions were significantly greater in the ASB and PC piglets compared with the NC piglets (P < 0.05). In the intestinal mucosa, malondialdehyde was lower in the ASB and PC piglets (by 42% and 43%, respectively), whereas tumor necrosis factor α (TNF-α) was 63% lower in the ASB piglets and 59% lower in the PC piglets compared with the NC piglets (P < 0.05). 16S ribosomal RNA gene sequence analysis revealed a higher colonic Shannon index and a lower colonic Simpson index in the ASB and PC piglets than in the NC piglets. In addition, the ASB and PC treatments caused a striking decrease in Lactobacillaceae and a noticeable increase in Clostridiaceae in the ileal and colonic lumen, as well as increases in Ruminococcaceae, Lachnospiraceae, and Bacteroidetes in the colonic lumen.
Conclusion: Collectively, our results support an important role for SB in improving performance and decreasing diarrhea incidence in weaned piglets by modulation of intestinal permeability and the bacterial communities in the ileum and colon.
Keywords:
- sodium butyrate
- antibiotic alternative
- performance
- intestinal permeability
- bacterial communities
- piglet
Introduction
The period after weaning is characterized by a high incidence of intestinal disturbances with diarrhea and decreased performance in piglets (1). Subtherapeutic doses of antibiotics have been proven to increase growth rate, improve feed utilization, and reduce the incidence of postweaning diarrhea (2). However, indiscriminate use of antibiotics has led to an increasing number of antibiotic-resistant pathogens as well as public concern with regard to their cross-transfer to humans (3). As a result, the use of antibiotics in diets fed to livestock has been banned in the European Union since 2006. Many materials have been investigated as alternatives (4), among which some organic acids and their salts incorporated in diets are known to be helpful in overcoming the postweaning syndrome (3).
Butyrate, an SCFA, is physiologically produced by large bowel microbial fermentation of dietary carbohydrates in mammals (5). It is quickly absorbed and can be utilized as a major energy source by epithelial cells in the terminal ileum and large intestine (6), as well as stimulating growth of the small intestinal epithelium (5). The effects of butyrate on carcinogenesis (5), inflammation (7), oxidative stress (8), and intestinal barrier function (9) have been described, particularly its activities in promoting gut health both in vitro (10) and in vivo (11).
Because it is easier to handle in the feed-manufacturing process, butyrate is often used in the form of sodium salt instead of free acid (5). Accumulated studies have been conducted with regard to the effects of sodium butyrate (SB)8 on performance in weaned piglets, including feed intake, weight gain, and feed efficiency. One trial by Piva et al. (12) showed that SB supplementation at 800 mg/kg increased weight gain and feed intake in piglets in the first 2 wk after weaning (P < 0.05). In another feeding trial, weaned piglets fed 1000-mg-SB/kg diets showed improved performance compared with those fed a control diet or 500 mg SB/kg (P < 0.05) (13). However, inconsistent results were obtained in other studies. Performance did not differ between piglets fed a basal diet with SB at 0, 1000, 2000, or 4000 mg/kg in an experiment by Biagi et al. (14). Weber and Kerr (15) reported that there was no effect of dietary SB (500, 1000, 2000, and 4000 mg/kg) on overall feed efficiency. In another trial, dietary SB (1000 mg/kg) significantly decreased diarrhea incidence in weaned piglets (P < 0.05) without improvements in weight gain, feed intake, or feed efficiency (16). A conclusion can be drawn that SB does not consistently provide growth-promoting effects to weaned piglets. Therefore, it would appear that SB cannot be used as the sole alternative to antibiotics in piglet feeding.
Although many other countries are expected to introduce an antibiotic ban in diets fed to livestock, antibiotics are still extensively used in many areas of the world. The entire withdrawal of antibiotics in piglet diets would likely result in considerable production loss in these areas because the vast majority of antibiotic substitutes tested produce inconsistent results and rarely equal the effectiveness of antibiotics (4). An efficient strategy may be to use a partial substitution of antibiotics with other growth-promoting substances, as suggested by a previous study in which supplemental acidifiers appeared to act synergistically with avilamycin (17). However, to our knowledge, few studies have focused on the application of SB inclusion, combined with decreased antibiotics, in piglet feeding.
In the present study, we evaluated the effects of the combined use of SB and reduced antibiotics in promoting performance and to control postweaning diarrhea in piglets. Furthermore, to probe into the possible mechanisms for these effects, some variables with regard to intestinal barrier function and changes in the bacterial communities in the ileum and colon were determined.
Methods
Piglets, diets, and experimental protocol.
All procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of China Agricultural University. The heaviest 90 crossbred (Duroc × Landrace × Large White) weaned piglets (weaned at 28 d) were selected from a pool of 30 litters. After a 3-d adaptation period during which all piglets were fed the same base diet, the pigs (10.24 ± 1.90 kg body weight) were assigned to 1 of 3 treatments (n = 30) that were homogenous for weight and sex. A basal diet was formulated to meet the nutrient requirements of the pigs according to the NRC (18) (Supplemental Table 1). Dietary treatments included a corn-soybean meal control ration without antibiotics [negative control (NC)]; a similar ration with 50 mg kitasamycin/kg, 20 mg colistin sulfate/kg, and 1000 mg encapsulated SB/kg [Lideshi, Inc.; reduced antibiotics + SB (ASB)]; and a ration with 100 mg kitasamycin/kg and 40 mg colistin sulfate/kg [positive control (PC)]. The encapsulated structure in SB was used to deter prompt absorption and metabolism of butyrate in the duodenum and jejunum, which ensured considerable release and absorption of butyrate in the lower intestine portion (19).
During the 28-d experiment, pigs were fed their respective diets and allowed ad libitum access to feed and water. The piglets were individually weighed on day 28, and feed consumption per pen was recorded weekly. The amount of feed wasted was recorded daily.
Fecal consistency was visually assessed at 0900 and 1600 h each day by observers who were blind to treatments with the use of a modification of the method described by Ma et al. (20). Fresh excreta were graded by using the following scale: 0 = solid, 1 = semisolid, 2 = semiliquid, and 3 = liquid. The occurrence of diarrhea was defined as production of grade 2 or 3 feces for 2 continuous days.
Small intestinal permeability.
Small intestinal permeability was assessed on day 28 before weighing by using the lactulose to mannitol differential absorption test (21). Briefly, urine samples of piglets per treatment (n = 6) were collected after 6 h of feed deprivation for baseline urinary sugar measurement. After the samples were obtained, 5 mL lactulose (0.4 g/mL; Sigma) and 5 mL of mannitol (0.2 g/L; Sigma) were administered intragastrically to the piglets. Piglets were feed-deprived for the 6-h study period but were allowed to drink water after 30 min. Urinary lactulose and mannitol concentrations were determined by an enzymatic technique (22). Mannitol excretion was corrected by subtraction of baseline values, and the lactulose to mannitol excretion ratio was calculated as an index of intestinal permeability.
Sample collection.
On day 28, 6 blood samples per treatment were harvested from the jugular vein by using tubes without anticoagulant (Becton Dickinson). Blood samples were allowed to clot at room temperature for 20 min and centrifuged at 1610 × g for 10 min at 4°C. Serum was then removed and stored at −20°C until assay. Intestinal segments (duodenum, jejunum, ileum, and colon); mucosa in the duodenum, jejunum, ileum, and colon; as well as chyme from the ileum and colon were obtained and stored at −80°C until further assay (23).
Morphologic evaluations.
Villus height and crypt depth in the duodenum, jejunum, and ileum were determined as previously described (23). Briefly, these segments were fixed in 4% paraformaldehyde for 24 h and then embedded in paraffin wax. Sections of 4 μm were cut and stained with hematoxylin and eosin. Measurements for villus height and crypt depth were taken by using the Axioskop-2 microscope (Olympus) and the Image Processing System (Visitron Systems).
Protein extraction and immunoblot analysis.
The expression of the protein occludin was determined due to its crucial roles in tight junction structure and paracellular permeability (24). The total protein amount contained in the jejunal and colonic tissue samples was extracted according to the method described by a ProteoJET Total Protein Extraction Kit (Fermentas). A Bicinchoninic Acid Protein Assay Kit (Applygen Technologies) was used to determine the protein concentration. Equal amounts of protein extracts (20 μg) were fractionated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). The membranes were blocked with a 5% skimmed-milk solution at room temperature for 2 h and then incubated with diluted antibodies against occludin (1:200; Santa Cruz) and GAPDH (1:20,000; Sigma). After incubation with HRP-conjugated secondary antibody, signals were visualized by the Odyssey Infrared Imaging System (LI-COR Biosciences). Blot analysis was carried out at 6 replicates/treatment by Quantity One software (BioRad Laboratories), with subsequent calculation of the ratio between the band intensities of occludin and GAPDH.
Antioxidant variables and immune indexes.
Serum samples were thawed and thoroughly mixed immediately before testing. Equal amounts of mucosa in different intestinal segments from the same piglet were blended to form a single sample. The blended samples (n = 6/treatment) were homogenized in PBS (10 mM; pH 7.4) and centrifuged at 3000 × g for 10 min at 4°C. The supernatant was stored at −80°C until further assays.
Antioxidant variables, including superoxide dismutase, glutathione peroxidase, glutathione, malonaldehyde, as well as the cytokines complement 3, IL-1β, IL-2, IFN-γ, and TNF-α in serum and intestinal mucosa were all determined by using assay kits according to the manufacturer’s instructions. All of the assay kits were purchased from the Nanjing Jiancheng Bioengineering Institute.
Composition and diversity of the bacterial communities.
DNA in each ileal and colonic chyme sample was extracted according to the manufacturer’s protocol (Bocai Biology). The V3 and V4 regions of the 16S ribosomal DNA gene were chosen for PCR. The primers were 338F (5′ACTCCTACGGGAGGCAGCA-3′) and 806R (5′GGACTACHVGGGTWTCTAAT-3′). The procedure to obtain amplicons was previously described, with modifications (25). The products were examined on a 2% (wt:vol) agarose gel. The amplicons from 6 replicates/treatment were blended in equimolar ratios on the basis of concentration. The blended samples were sent out for MiSeq Next-Generation Sequencing System on an Illumina MiSeq PE300 platform at the Majorbio Bio-Pharm Technology Company. Only sequences >50 bp were used for phylotype analysis at the 0.03 operational taxonomic unit level.
Statistical analysis.
Differences in diarrhea incidence between the 3 treatments were tested by the chi-square contingency test. All other data were analyzed by using SAS version 9.1. The results are presented as means ± SEMs. One-factor ANOVA followed by Tukey’s multiple-range tests were used for equal variances. Kruskal-Wallis 1-factor ANOVA was performed to compare means with unequal variances. For all statistical analyses, P values <0.05 were considered significant.
Results
Performance and diarrhea incidence.
Compared with the NC piglets, weight gain was significantly greater in the ASB and PC piglets (P < 0.05) with feed intake unchanged, resulting in an improved gain to feed ratio in the ASB and PC piglets (P < 0.05) (Table 1). Diarrhea incidence was significantly lower in the ASB and PC piglets than in the NC piglets (P < 0.05) (Table 1). There were no significant differences in performance or diarrhea incidence between the ASB and PC piglets.
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TABLE 1
Performance and diarrhea incidence in weaned piglets fed corn-soybean meal without antibiotics (NC), corn-soybean meal with reduced antibiotics and sodium butyrate (ASB), or corn-soybean meal with antibiotics (PC) for 28 d1
Small intestinal morphology.
Villus height in the jejunum and crypt depth in the duodenum and jejunum were lower in the ASB piglets than in the NC and PC piglets (P < 0.05). The ASB piglets had lower crypt depth in the ileum than did the NC piglets (P < 0.05), whereas the value for the PC piglets was intermediate to values for the NC and ASB piglets (Table 2).
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TABLE 2
Small intestinal morphology of weaned piglets fed corn-soybean meal without antibiotics (NC), corn-soybean meal with reduced antibiotics and sodium butyrate (ASB), or corn-soybean meal with antibiotics (PC) for 28 d1
Villus height in the duodenum and ileum was greater in the PC piglets than in the NC and ASB piglets (P < 0.05). Villus height to crypt depth ratio in the ileum in the PC piglets was also greater than in the NC piglets (P < 0.05), whereas the ratio for the ASB piglets was intermediate to that for the NC and PC piglets (Table 2).
Small intestinal permeability and occludin abundance.
Small intestinal permeability was lower in the ASB and PC piglets as indicated by the decreased urinary lactulose to mannitol ratios compared with the NC piglets (P < 0.05) (Figure 1). The expression of the intestinal tight junction protein occludin in the jejunum and colon was significantly higher in the ASB and PC piglets than in the NC piglets (P < 0.05) (Figure 2). These variables in the ASB piglets were comparable to those in the PC piglets.
FIGURE 1
Small intestinal permeability in weaned piglets fed corn-soybean meal without antibiotics (NC treatment), corn-soybean meal with reduced antibiotics and encapsulated sodium butyrate (ASB treatment), or corn-soybean meal with antibiotics (PC treatment) for 28 d. Values are means ± SEMs, n = 6. Labeled means without a common letter differ, P < 0.05. ASB, reduced antibiotics + sodium butyrate; NC, negative control; PC, positive control.
FIGURE 2
Immunoblot analysis of occludin protein abundance in the jejunal (A) and colonic (B) tissue of weaned piglets fed corn-soybean meal without antibiotics (NC treatment), corn-soybean meal with reduced antibiotics and encapsulated sodium butyrate (ASB treatment), or corn-soybean meal with antibiotics (PC treatment) for 28 d. Representative Western blots are shown. Values are means ± SEMs, n = 6. Labeled means without a common letter differ, P < 0.05. ASB, reduced antibiotics + sodium butyrate; NC, negative control; PC, positive control.
Antioxidant variables.
The serum content of superoxide dismutase in the ASB piglets was significantly higher compared with that in the PC piglets (P < 0.05), whereas the value for the NC piglets was at an intermediate level. Activities of glutathione peroxidase in the intestinal mucosa were increased in the ASB piglets compared with the NC and PC piglets (P < 0.05). The ASB piglets had decreased malondialdehyde in the serum compared with the NC piglets (P < 0.05), whereas the value for the PC piglets was intermediate to that of the NC and ASB treatments. The malondialdehyde concentration in the intestinal mucosa in the ASB piglets was comparable to that in the PC piglets, both of which were significantly lower than in the NC piglets (P < 0.05) (Table 3).
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TABLE 3
Antioxidant variables in serum and intestinal mucosa of weaned piglets fed corn-soybean meal without antibiotics (NC), corn-soybean meal with reduced antibiotics and sodium butyrate (ASB), or corn-soybean meal with antibiotics (PC) for 28 d1
Immune indexes.
Serum IFN-γ was higher in the ASB piglets compared with the NC piglets (P < 0.05), whereas the value for the PC piglets was at an intermediate level. The PC piglets had a lower concentration of TNF-α in serum than did the NC piglets (P < 0.05), whereas the value for the ASB piglets was intermediate to those for the NC and PC piglets. In the intestinal mucosa, TNF-α concentrations in the ASB and PC piglets were lower than in the NC piglets (P < 0.05), although no significant difference was observed between the ASB and PC piglets (Table 4).
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TABLE 4
Immune indexes in serum and intestinal mucosa of weaned piglets fed corn-soybean meal without antibiotics (NC), corn-soybean meal with reduced antibiotics and sodium butyrate (ASB), or corn-soybean meal with antibiotics (PC) for 28 d1
Bacterial composition and diversity.
The ileal lumen harbored decreased bacterial diversity with the ASB and PC treatments, as indicated by the decreased Shannon index and increased Simpson index, whereas the bacterial community in the colonic lumen became more diverse (Table 5). A noticeable increase was observed in Bacteroidetes by the ASB and PC treatments, from 0.7% in the NC piglets to 3.4% in the ASB piglets and 10.3% in the PC piglets (data not shown). The majority of classifiable sequences belonged to Lactobacillaceae (63.8%), Streptococcaceae (18.3%), Pasteurellaceae (7.2%), and Enterobacteriaceae (6.3%) in the ileal lumen, whereas the colonic lumen was dominated by Lactobacillaceae (64.6%), Streptococcaceae (10.1%), Ruminococcaceae (7.7%), Lachnospiraceae (6.0%), and Neisseriaceae (4.0%) in the NC piglets (Figure 3). The decreases in Lactobacillaceae abundance with exposure to the ASB and PC were striking: from 63.8% in the ileal lumen of the NC piglets to 6.8% with the ASB treatment and 8.7% with the PC treatment; the values for the colonic lumen were 64.6%, 44.4%, and 4.4%, respectively. Nonabundant Clostridiaceae became dominant in the ileal lumen, with large increases from 0.3% in the NC piglets to 83.2% in the ASB piglets and 35.0% in the PC piglets (Figure 3). In addition, noticeable increases in Clostridiaceae abundance were also observed in the colonic lumen with the ASB (1.6%) and PC (10.9%) treatments compared with the NC piglets (0.3%) (Figure 3). In addition, certain bacterial families decreased with the ASB and PC treatments, such as Pasteurellaceae and Enterobacteriaceae in the ileal lumen, whereas Ruminococcaceae and Lachnospiraceae were increased in the colonic lumen with the ASB and PC treatments (Figure 3).
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TABLE 5
Bacterial community diversity in the ileal and colonic chyme of weaned piglets fed corn-soybean meal without antibiotics (NC), corn-soybean meal with reduced antibiotics and sodium butyrate (ASB), or corn-soybean meal with antibiotics (PC) for 28 d1
FIGURE 3
Family-level distribution of luminal bacteria in the ileal and colonic chyme of weaned piglets fed corn-soybean meal without antibiotics (NC treatment), corn-soybean meal with reduced antibiotics and encapsulated sodium butyrate (ASB treatment), or corn-soybean meal with antibiotics (PC treatment) for 28 d. ASB, reduced antibiotics + sodium butyrate; NC, negative control; PC, positive control.
Discussion
Kitasamycin is an antibiotic growth promoter that exhibits activity mainly against gram-positive micro-organisms (26), whereas colistin sulfate is a decapeptide with antibacterial activity mainly against gram-negative micro-organisms (27). In the present study, the combined use of SB and reduced antibiotics was introduced given the inconsistent effects of SB fed alone and the still widespread use of antibiotics in many countries. Alterations with regard to intestinal barrier function may explain the improved performance and decreased diarrhea incidence by the ASB and PC treatments.
We first validated that the ASB treatment improved performance in weaned piglets with effects comparable to those for the PC treatment. This observation is similar to that in a previous study (17), which reported that a combination of acidifiers and avilamycin gave better performance-promoting effects than did acidifiers or avilamycin fed alone in piglet diets, although the mechanisms were not well determined. The decreased diarrhea incidences in the ASB and PC piglets were at least partly due to the redefinition of the bacterial communities by the antibiotics. In addition, SB was speculated to influence the intestinal microflora (14) and bacteria virulence (5) in weaned piglets. Our experiment revealed an effective strategy to control postweaning problems with reduced use of antibiotics.
The decreased digestion and absorption of nutrients, fluids, and electrolytes due to villous atrophy and crypt hypertrophy as a result of early weaning may contribute to diarrhea (28). In the present study, the improved villus height in the duodenum and ileum in the PC piglets may partly explain the improved performance and lower diarrhea incidence by the PC treatment. An increase in crypt depth can be used as a predictor of increased crypt cell production rate and overall stimulation of cell turnover in the small intestine, which are generally associated with reduced digestive and absorptive capacity (29). The decreased crypt depth in the duodenum, jejunum, and ileum in the ASB piglets was in agreement with the improved performance.
Increased intestinal secretion or losses of fluid and electrolytes may be considered as a final common pathway of diarrhea production, and many factors interact in subtle ways in the development of diarrhea (28). Increased intestinal permeability is characterized by downregulation of fluid and electrolyte absorption, which leads to fluid and electrolyte accumulation in the bowel (24). In our study, the decreased small intestinal permeability by the ASB treatment may help to confer a decrease in diarrhea incidence. The intercellular tight junction protein complexes play an important role in the mucosal barrier against translocation of intraluminal toxins, antigens, and enteric flora from the lumen into subepithelial tissues and systemic blood circulation (30). Note that the higher expression of occludin in jejunal tissue by the ASB treatment was consistent with the decreased small intestinal permeability observed. The ASB and PC treatments might also decrease paracellular permeability in the hindgut as indicated by the increased expression of occludin in colonic tissue. Previous studies showed that absorption in the large intestine is also involved in the pathophysiology of diarrhea, and a loss of fluid and electrolytes in the small intestine will only cause diarrhea when remedial reabsorption by the large intestine partly or completely fails (31). Collectively, the ASB and PC treatments decreased diarrhea incidence by modulating permeability in both the small intestine and hindgut.
Mucosal oxidative stress has been shown to be sufficient to induce diarrhea and to play an important role during the progression of weaning diarrhea (32). It is likely that the ASB triggered a localized form of attenuation in antioxidant status that was not reflected in the extraintestinal environment due to minor changes in serum antioxidant variables. This discrepancy could be explained by the negligible finding of butyrate in the peripheral blood as a result of rapid metabolism in the gut wall and/or in the liver (5). Glutathione peroxidase is implicated in the protection of gastrointestinal mucosal cells against damage from various insults (32). Malondialdehyde concentrations in tissues and blood are generally used as biomarkers of endogenous lipid peroxidation and free radical-induced damage (33). The increase in glutathione peroxidase in the intestinal mucosa could mainly be attributed to SB, as suggested by the absence of an increase in intestinal mucosal glutathione peroxidase content in the PC piglets, whereas antibiotics are mainly responsible for malondialdehyde decreases in intestinal mucosa. Decreased oxidative stress may lead to reduced damage to the intestinal mucosal barrier, which subsequently leads to reduced intestinal permeability.
Some cytokines are thought to be critical in the predisposition to and exacerbation of some gastrointestinal dysfunctions (34). TNF-α is a central mediator of intestinal inflammatory diseases (34) and has been shown to play a role in the control of intestinal permeability (35). T cell–derived TNF-α inhibits phosphorylation of the myosin light-chain mediated by the myosin light-chain kinase, leading to alternatively disrupted tight junction stability and dysregulation of occludin expression (36). Therefore, the increased occludin expression in the ASB and PC piglets could be partly attributed to decreased TNF-α in the intestinal mucosa. Moreover, TNF-α mediates mast cell degranulation along with some other mediators. TNF-α is, in turn, rapidly released by mast cells after degranulation as are some other inflammatory mediators (24). Mast cell degranulation and the consequent histamine and prostaglandin release profoundly influence intestinal epithelial barrier function (28). The 6-fold decrease in intestinal mucosal TNF-α may indicate an amelioration of mast cell degranulation in the ASB and PC piglets. More research is needed to explain the unexpected increase in serum IFN-γ, whereas the IFN-γ concentration in the intestinal mucosa was not affected by the ASB and PC treatments.
An understanding of how the ASB and PC treatments affect the intestinal bacterial communities may help to reveal the linkages between their changes and improvements in performance and diarrhea incidence. Higher bacterial diversity may be associated with an improved competitive defense against pathogens and decreased vulnerability to environmental perturbations, such as shifts in gut microbial communities, changes in piglet diet, or overt pathogenic challenge (37). Shiga toxin–producing Escherichia coli elicits fluid and electrolyte accumulation in the bowel by disrupting the usual balance of intestinal absorption and secretion toward net secretion (38). The increased bacterial diversity in the colonic lumen and decreased Enterobacteriaceae (mainly Shigella) in both the ileal and colonic lumen may help to decrease diarrhea incidence in ASB and PC piglets.
The colonic lumen harbored a higher number of bacteria associated with degrading complex carbohydrates than did the ileal lumen, which was consistent with previous observations (23, 39). The intestinal bacterial communities may participate in modulation of piglet performance due to the energy derived from hindgut bacterial fermentation. Bacteroidetes is well known for its role in polysaccharide degradation (40). Ruminococcaceae has been consistently found in the hindgut of pigs, and an increase in Ruminococcaceae may improve feed conversion in piglets due to its cellulose-degrading capacity (39). Lachnospiraceae is known to produce an array of bacteriocins and butyrate, and many isolates have shown promise as probiotics (41). In our study, the increases in the relative abundance of Bacteroidetes, Ruminococcaceae, and Lachnospiraceae in the colonic lumen of ASB and PC piglets could help the host obtain more energy from complex polysaccharides that are resistant to the action of digestive enzymes. Whereas the decrease in Lactobacillaceae populations in the ileal lumen may reduce energy losses, previous studies showed that Lactobacilli is the main contributor to microbial bile salt hydrolase activity in the small intestine, which results in impaired lipid absorption by the host animal and consequent dietary energy losses (42). In addition, we propose a hypothesis that the ASB and PC treatments improved the ratio of weight gain to feed via inhibition of the normal microbiota, leading to increased nutrient utilization and a reduction in the maintenance costs of the gastrointestinal system (43). By contrast, an undesirable effect may be the substantially increased Clostridiaceae population in ASB and PC piglets. The persistence of Clostridiaceae in the whole intestinal bacterial communities in ASB and PC piglets and their effect on piglet growth cannot be explained because the possible benefits from intestinal colonization by nonpathogenic Clostridiaceae have not been explored (44).
In summary, the present study showed that the ASB significantly improved weight gain and reduced diarrhea incidence in weaned piglets (P < 0.05). These changes were accompanied by decreased small intestinal permeability, increased intestinal occludin expression, and improvements in some antioxidant variables and immune indexes, as well as considerable alterations in intestinal bacterial communities.
Acknowledgments
We thank Katrina Mohror, South Dakota State University, for excellent assistance in editing this manuscript. C Huang, PS, and XM designed and conducted the research; C Huang, PF, C Hou, and XM analyzed the data; C Huang and XM wrote the manuscript; PT critically reviewed the manuscript; and XM had primary responsibility for final content. All authors read and approved the final manuscript.
Footnotes
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↵1 Supported in part by the National Basic Research Program of China (973 Program, 2013CB117301), the National Natural Science Foundation of China (31528018, 31272448, and 31472101), the National Department Public Benefit Research Foundation (201403047), and Beijing Nova program (xx2013055).
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↵2 Author disclosures: C Huang, P Song, P Fan, C Hou, P Thacker, and X Ma, no conflicts of interest.
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↵3 Supplemental Table 1 is available from the “Online Supporting Material” link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org.
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↵7 These authors contributed equally to this work.
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↵8 Abbreviations used: ASB, reduced antibiotics + sodium butyrate; NC, negative control; PC, positive control; SB, sodium butyrate.
- Manuscript received: June 3, 2015.
- Initial review completed: June 23, 2015.
- Revision accepted: September 29, 2015.