Dietary Phytase- and Lactic Acid-Treated Cereals Caused Greater Taxonomic Adaptations than Functional Adaptations in the Cecal Metagenome of Growing Pigs.

Phosphorus (P) is an essential nutrient for the gut bacteria and the host. Nevertheless, little information exists that indicates to what extent an improved level of P availability in the small intestine leads to functional adaptations in bacterial metabolic pathways in the large intestine. Therefore, we investigated the changes in the taxonomic and functional bacterial metagenome in cecal digesta of growing pigs fed diets containing phytase and/or cereals treated with 2.5% lactic acid (LA) for 19 days (n = 8/diet) using shotgun metagenome sequencing. The phytase supplementation resulted in strikingly distinct bacterial communities, affecting almost all major bacterial families, whereas functional changes were less dramatic among the feeding groups. While phytase treatment decreased predominant Prevotellaceae levels, it seemed that Clostridiaceae, Ruminococcaceae, and Lachnospiraceae filled the opening metabolic niches (P < 0.05). The LA-treated cereals mediated reduced levels of Bacteroidaceae and increased levels of Veillonellaceae, but those results were mainly seen when the cereals were fed as a single treatment (P < 0.05). In association with the taxonomic alterations, phytase caused changes within the major functional pathways corresponding to amino acid metabolism; translation; membrane transport; folding, sorting, and degradation; and energy metabolism, whereas the LA treatment of cereals resulted in decreased enzymatic capacities within the carbohydrate metabolism and energy metabolism pathways (P < 0.05). Metabolic dependencies corresponding to the starch and sucrose metabolism, glycolysis/gluconeogenesis, and citrate cycle pathways were indicated by diet-associated changes in enzymatic capacities related to short-chain fatty acid, methane, vitamin, and bacterial antigen synthesis. Accordingly, the present results support the idea of the importance of the availability of intestinal P for bacterial metabolism. However, the functional profiles were less different than the taxonomic profiles among the dietary treatment results, indicating a certain degree of metabolic plasticity within the cecal metagenome.IMPORTANCE Dietary strategies (e.g., phytase supplementation and lactic acid [LA] treatment of cereals) used to improve the availability of phytate-phosphorus (P) from pig feed reduce the amount of P flowing into the large intestine, whereas LA treatment-induced changes in nutrient fractions alter the substrate being available to the microbiota. In ruminants, lower intestinal P availability compromises the fibrolytic activity of the microbiome. Here, we report that the functional capacities were less dramatically affected than the taxonomic composition by phytase-supplemented and LA-treated cereals. The bacterial community appeared to be partly capable of functionally compensating for the altered flow of P by replacing taxa with higher P needs by those with lower P needs. Therefore, by acting as mucosal immune stimulants, alterations in microbiota-associated molecular patterns (MAMPs) due to the taxonomic shifts may play a greater role for host physiology and health than functional differences caused by differing intestinal P availabilities, which merits further research.

Alterations of the Viable Ileal Microbiota of the Gut Mucosa-Lymph Node Axis in Pigs Fed Phytase and Lactic Acid-Treated Cereals.

The gut-lymph node axis is a critical player in the symbiotic relationship between gut microbiota and the host. However, little is known about the impact of diet-related bacterial shifts in the gut lumen on bacterial translocation into lymph nodes. Here, we (i) characterized changes in the viable microbiota composition along the ileal digesta-mucosa-lymph node axis and (ii) examined the effect of dietary phytase supplementation and lactic acid (LA) soaking of cereals on the bacterial taxonomy along this axis, together with their effect on the mucosal expression of innate immune and barrier function genes in pigs (n = 8/diet). After 18 days on diets, ileal digesta, mucosa, and ileocecal lymph nodes (ICLNs) were collected for RNA isolation and 16S rRNA-based high-resolution community profiling. Bacterial communities were dominated by Lactobacillaceae and Clostridiaceae, with clearly distinguishable profiles at the three sampling sites. Specific bacterial subsampling was indicated by enrichment of the ICLNs with Lactobacillaceae, Lachnospiraceae, Veillonellaceae, and Methanobacteriaceae and less Clostridiaceae, Pasteurellaceae, Helicobacteraceae, and Enterobacteriaceae compared to that of the mucosa. LA treatment of cereals reduced proteolytic taxa in the lumen, including pathobionts like Helicobacteraceae, Campylobacteraceae, and Fusobacteriaceae When combined, phytase- and LA-treated cereals largely increased species richness, while the single treatments reduced Actinobacteria and Bacteroidetes in ICLNs and increased mucosal MUC2 expression. In contrast, phytase reduced mucosal CDH1 expression, indicating altered barrier function with potential effects on bacterial translocation. Overall, both treatments, although often differently, changed the viable microbiome along the digesta-mucosa-lymph node axis in the ileum, probably due to altered substrate availability and microbial-host interactions.IMPORTANCE A host's diet largely determines the gut microbial composition and therefore may influence bacterial translocation into ICLNs. Due to its importance for cell metabolism, the intestinal phosphorus availability, which was modified here by phytase and LA treatment of cereals, affects the intestinal microbiota. Previous studies mainly focused on bacteria in the lumen. The novelty of this work resides mainly in that we report diet-microbe effects along the digesta-mucosa-ICLN axis and linked those effects to mucosal expression of barrier function genes as crucial components for host health. Lymph nodes can serve as reservoir of pathobionts; therefore, present diet-microbiome-host interactions have implications for food safety.

Improvement of Feed Efficiency in Pigs through Microbial Modulation via Fecal Microbiota Transplantation in Sows and Dietary Supplementation of Inulin in Offspring.

As previous studies have demonstrated a link between the porcine intestinal microbiome and feed efficiency (FE), microbiota manipulation may offer a means of improving FE in pigs. A fecal microbiota transplantation procedure (FMTp), using fecal extracts from highly feed-efficient pigs, was performed in pregnant sows (n = 11), with a control group (n = 11) receiving no FMTp. At weaning, offspring were allocated, within sow treatment, to (i) control (n = 67; no dietary supplement) or (ii) inulin (n = 65; 6-week dietary inulin supplementation) treatments. The sow FMTp, alone or in combination with inulin supplementation in offspring, reduced offspring body weight by 8.1 to 10.6 kg at ∼140 days of age, but there was no effect on feed intake. It resulted in better FE, greater bacterial diversity, and higher relative abundances of potentially beneficial bacterial taxa (Fibrobacter and Prevotella) in offspring. Due to the FMTp and/or inulin supplementation, relative abundances of potential pathogens (Chlamydia and Treponema) in the ileum and cecal concentrations of butyric acid were significantly lower. The maternal FMTp led to a greater number of jejunal goblet cells in offspring. Inulin supplementation alone did not affect growth or FE but upregulated duodenal genes linked to glucose and volatile fatty acid homeostasis and increased the mean platelet volume but reduced ileal propionic acid concentrations, granulocyte counts, and serum urea concentrations. Overall, the FMTp in pregnant sows, with or without dietary inulin supplementation in offspring, beneficially modulated offspring intestinal microbiota (albeit mostly low-relative-abundance taxa) and associated physiological parameters. Although FE was improved, the detrimental effect on growth limits the application of this FMTp-inulin strategy in commercial pig production.IMPORTANCE As previous research suggests a link between microbiota and FE, modulation of the intestinal microbiome may be effective in improving FE in pigs. The FMTp in gestating sows, alone or in combination with postweaning dietary inulin supplementation in offspring, achieved improvements in FE and resulted in a higher relative abundance of intestinal bacteria associated with fiber degradation and a lower relative abundance of potential pathogens. However, there was a detrimental effect on growth, although this may not be wholly attributable to microbiota transplantation, as antibiotic and other interventions were also part of the FMT regimen. Therefore, further work with additional control groups is needed to disentangle the effects of each component of the FMTp in order to develop a regimen with practical applications in pig production. Additional research based on findings from this study may also identify specific dietary supplements for the promotion/maintenance of the microbiota transferred via the maternal FMTp, thereby optimizing pig growth and FE.

Functional adaptations in the cecal and colonic metagenomes associated with the consumption of transglycosylated starch in a pig model.

BACKGROUND: Both phylogeny and functional capabilities within the gut microbiota populations are of great importance for influencing host health. As a novel type of resistant starch, transglycosylated starch (TGS) modifies the microbial community and metabolite profiles along the porcine gut, but little is known about the related functional adaptations in key metabolic pathways and their taxonomic identity. RESULTS: Metagenomic sequencing was used to characterize the functional alterations in the cecal and colonic microbiomes of growing pigs fed TGS or control starch (CON) diets for 10 days (n = 8/diet). Bacterial communities were clearly distinguishable at taxonomic and functional level based on the dietary starch, with effects being similar at both gut sites. Cecal and colonic samples from TGS-fed pigs were enriched in Prevotella, Bacteroides, Acidaminoccus and Veillonella, whereas Treponema, Ruminococcus, and Aeromonas declined at both gut sites compared to CON-fed pigs (log2 fold change > ±1; p < 0.001 (q < 0.05)). This was associated with increased enzymatic capacities for amino acid metabolism, galactose, fructose and mannose metabolism, pentose and glucuronate interconversions, citrate cycle and vitamin metabolism for samples from TGS-fed pigs. However, TGS-fed pigs comprised fewer reads for starch and sucrose metabolism and genetic information processing. Changes in key catabolic steps were found to be the result of changes in taxa associated with each type of starch. Functional analysis indicated steps in the breakdown of TGS by the action of α- and ß-galactosidases, which mainly belonged to Bacteroides and Prevotella. Reads mapped to alpha-amylase were less frequent in TGS- compared to CON-fed pigs, with the major source of this gene pool being Bacillus, Aeromonas and Streptococcus. Due to the taxonomic shifts, gene abundances of potent stimulants of the mucosal innate immune response were altered by the starches. The cecal and colonic metagenomes of TGS-fed pigs comprised more reads annotated in lipopolysaccharides biosynthesis, whereas they became depleted of genes for flagellar assembly compared to CON-fed pigs. CONCLUSIONS: Metagenomic sequencing revealed distinct cecal and colonic bacterial communities in CON- and TGS-fed pigs, with strong discrimination among samples by functional capacities related to the respective starch in each pig's diet.

Transglycosylated starch accelerated intestinal transit and enhanced bacterial fermentation in the large intestine using a pig model.

Resistant starch can alter the intestinal nutrient availability and bulk of digesta, thereby modulating the substrate available for microbial metabolic activity along the gastrointestinal tract. This study elucidated the effect of transglycosylated starch (TGS) on the retention of digesta in the upper digestive tract, ileal flow and hindgut disappearance of nutrients, and subsequent bacterial profiles in pigs. Fourteen ileal-cannulated growing pigs were fed either the TGS or control (CON) diet in a complete crossover design. Each period consisted of a 10-d adaptation to the diets, followed by 3-d collection of faeces and ileal digesta. Consumption of TGS decreased the retention of digesta in the stomach and small intestine, and increased ileal DM, starch, Ca and P flow, leading to enhanced starch fermentation in the hindgut compared with CON-fed pigs. TGS increased ileal and faecal total SCFA, especially ileal and faecal acetate and faecal butyrate. Gastric retention time positively correlated to Klebsiella, which benefitted together with Selenomonas, Lactobacillus, Mitsuokella and Coriobacteriaceae from TGS feeding and ileal starch flow. Similar relationships existed in faeces with Coriobacteriaceae, Veillonellaceae and Megasphaera benefitting most, either directly or indirectly via cross-feeding, from TGS residuals in faeces. TGS, in turn, depressed genera within Ruminococcaceae, Clostridiales and Christensenellaceae compared with the CON diet. The present results demonstrated distinct ileal and faecal bacterial community and metabolite profiles in CON- and TGS-fed pigs, which were modulated by the type of starch, intestinal substrate flow and retention of digesta in the upper digestive tract.

Fecal Microbiota Transplant from Highly Feed-Efficient Donors Shows Little Effect on Age-Related Changes in Feed-Efficiency-Associated Fecal Microbiota from Chickens.

Chickens with good or poor feed efficiency (FE) have been shown to differ in their intestinal microbiota composition. This study investigated differences in the fecal bacterial community of highly and poorly feed-efficient chickens at 16 and 29 days posthatch (dph) and evaluated whether a fecal microbiota transplant (FMT) from feed-efficient donors early in life can affect the fecal microbiota in chickens at 16 and 29 dph and chicken FE and nutrient retention at 4 weeks of age. A total of 110 chickens were inoculated with a FMT or a control transplant (CT) on dph 1, 6, and 9 and ranked according to residual feed intake (RFI; the metric for FE) on 30 dph. Fifty-six chickens across both inoculation groups were selected as the extremes in RFI (29 low, 27 high). RFI-related fecal bacterial profiles were discernible at 16 and 29 dph. In particular, Lactobacillus salivarius, Lactobacillus crispatus, and Anaerobacterium operational taxonomic units were associated with low RFI (good FE). Multiple administrations of the FMT only slightly changed the fecal bacterial composition, which was supported by weighted UniFrac analysis, showing similar bacterial communities in the feces of both inoculation groups at 16 and 29 dph. Moreover, the FMT did not change the RFI and nutrient retention of highly and poorly feed-efficient recipients, whereas it tended to increase feed intake and body weight gain in female chickens. This finding suggests that host- and environment-related factors may more strongly affect chicken fecal microbiota and FE than the FMT.IMPORTANCE Modulating the chicken's early microbial colonization using a FMT from highly feed-efficient donor chickens may be a promising tool to establish a more desirable bacterial profile in recipient chickens, thereby improving host FE. Although FE-associated fecal bacterial profiles at 16 and 29 dph could be established, the microbiota composition of a FMT, when administered early in life, may not be a strong factor modulating the fecal microbiota at 2 to 4 weeks of life and reducing the variation in chicken's FE. Nevertheless, the present FMT may have potential benefits for growth performance in female chickens.

Consumption of transglycosylated starch down-regulates expression of mucosal innate immune response genes in the large intestine using a pig model.

Benefits of resistant starch (RS) consumption on host physiology encompass microbial activity-derived attenuation of intestinal inflammation. However, little is known about anti-inflammatory properties of RS of type 4. This study compared the effects of transglycosylated starch (TGS) consumption on the jejunal barrier function and expression of genes related to inflammation, barrier function and the mucosal defence in jejunum, ileum, caecum and colon of pigs. Moreover, interactions of TGS-induced alterations in bacterial metabolites and composition with host mucosal responses were assessed using sparse partial least squares regression and relevance network analysis. Intestinal samples were collected after pigs (n 8/diet; 4 months of age) were fed the experimental diets for 10 d. Consumption of TGS did not modify jejunal barrier function and gene expression. By contrast, TGS down-regulated the caecal expression of zonula occludens-1 and mucin 2 and of genes within the toll-like receptor 4 and NF-κB pro-inflammatory signalling cascade. Relevance networks revealed a microbiome signature on ileal, caecal and colonic mucosal signalling as TGS-derived changes in bacterial genera and fermentation acids, such as propionic acid, correlated with the differently expressed genes in ileum, caecum and colon of pigs. In conclusion, the present findings suggest certain anti-inflammatory capabilities of TGS by down-regulating the expression of pro-inflammatory pathways in the caecal mucosa, which seems to be mediated, at least in part, by TGS-induced changes in microbial action in the large intestine.

Lactic acid treatment of by-products and phosphorus level in the diet modulate bacterial microbiome and the predicted metagenome functions using the rumen simulation technique.

This study used a rumen simulation technique to evaluate the effects of soaking of by-product-rich concentrate (BPC) in 5% lactic acid (LAC; vol/vol) on the rumen microbiota, predicted metagenome, fermentation characteristics, and nutrient degradation without or with supplemented P. The diet was supplemented with 1.6 g of P in the form of monocalcium phosphate per kilogram of dry matter in addition to 284 mg of inorganic P/d per fermentor via artificial saliva. Fermentor fluid was collected for analyses of short-chain fatty acids, fermentation gases, redox potential, and microbiota and feed residues for calculation of nutrient degradation. The microbiota composition was assessed using paired-end Illumina (Illumina Inc., San Diego, CA) MiSeq sequencing of the V3 to V5 region of the 16S rRNA gene. Soaking in LAC reduced the contents of crude protein, neutral and acid detergent fibers, and organic matter fractions as well as ash and P content of the BPC. Both the LAC treatment of BPC and the inorganic P modified the relative bacterial abundances mainly within the predominant orders Bacteroidales and Clostridiales. Supervised DIABLO N-integration networking supported that operational taxonomic units related to BS11, Ruminococcaceae, Christensenellaceae, Eubacterium, and Selenomonas were the most discriminant for the LAC-treated BPC, whereas other operational taxonomic units related to BS11, RFN20, Ruminococcus, and Succiniclasticum were best correlated with the inorganic P supplementation. Integration networking also showed that carbohydrate and pyruvate metabolism, biosynthesis of unsaturated fatty acids, and degradation of several xenobiotics were stimulated by the LAC treatment of BPC. Those data supported the enhanced fermentation activity as indicated by increased total short-chain fatty acid concentration, especially propionate and butyrate, and methane, but decreased ruminal crude protein degradation, with the LAC-treated compared with control-treated BPC. In contrast, despite an increased abundance of imputed functions, such as inositol phosphate metabolism, phosphatidylinositol signaling, and fructose and mannose metabolism, the reduced abundance of the imputed Kyoto Encyclopedia of Genes and Genomes pathway "transcription machinery" as well as the decrease in total short-chain fatty acids and nutrient degradation indicated reduced bacterial metabolic activity with the inorganic P supplementation. In conclusion, soaking of BPC in LAC may favor the proliferation of certain fibrolytic bacterial taxa and stimulate their metabolic activity, whereas the supplemented P to a diet already meeting ruminal P needs may impair ruminal nutrient utilization.

Graded replacement of maize grain with molassed sugar beet pulp modulated ruminal microbial community and fermentation profile in vitro.

BACKGROUND: Molassed sugar beet pulp (Bp) is a viable alternative to grains in cattle nutrition for reducing human edible energy input. Yet little is known about the effects of high inclusion rates of Bp on rumen microbiota. This study used an in vitro approach and the quantitative polymerase chain reaction technique to establish the effects of a graded replacement of maize grain (MG) by Bp on the ruminal microbial community, fermentation profile and nutrient degradation. RESULTS: Six different amounts of Bp (0-400 g kg-1 ), which replaced MG in the diet, were tested using the in vitro semi-continuous rumen simulation technique. The increased inclusion of Bp resulted in greater dietary content and degradation of neutral detergent fibre (P < 0.01). Further, Bp feeding enhanced (P < 0.01) the abundance of genus Prevotella and shifted (P < 0.01) the short-chain fatty acid patterns in favour of acetate and propionate and at the expense of butyrate. A total replacement of MG with Bp resulted in an increased daily methane production (P < 0.01). CONCLUSION: Results suggest positive effects of the replacement of MG by Bp especially in terms of stimulating ruminal acetate and propionate fermentation. However, high replacement rates of Bp resulted in lowered utilization of ammonia and higher ruminal methane production. © 2017 Society of Chemical Industry.

Exploring a Possible Link between the Intestinal Microbiota and Feed Efficiency in Pigs.

Feed efficiency (FE) is critical in pig production for both economic and environmental reasons. As the intestinal microbiota plays an important role in energy harvest, it is likely to influence FE. Therefore, our aim was to characterize the intestinal microbiota of pigs ranked as low, medium, and high residual feed intake ([RFI] a metric for FE), where genetic, nutritional, and management effects were minimized, to explore a possible link between the intestinal microbiota and FE. Eighty-one pigs were ranked according to RFI between weaning and day 126 postweaning, and 32 were selected as the extremes in RFI (12 low, 10 medium, and 10 high). Intestinal microbiota diversity, composition, and predicted functionality were assessed by 16S rRNA gene sequencing. Although no differences in microbial diversity were found, some RFI-associated compositional differences were revealed, principally among members of Firmicutes, predominantly in feces at slaughter (albeit mainly for low-abundance taxa). In particular, microbes associated with a leaner and healthier host (e.g., Christensenellaceae, Oscillibacter, and Cellulosilyticum) were enriched in low RFI (more feed-efficient) pigs. Differences were also observed in the ileum of low RFI pigs; most notably, Nocardiaceae (Rhodococcus) were less abundant. Predictive functional analysis suggested improved metabolic capabilities in these animals, especially within the ileal microbiota. Higher ileal isobutyric acid concentrations were also found in low RFI pigs. Overall, the differences observed within the intestinal microbiota of low RFI pigs compared with that of their high RFI counterparts, albeit relatively subtle, suggest a possible link between the intestinal microbiota and FE in pigs.IMPORTANCE This study is one of the first to show that differences in intestinal microbiota composition, albeit subtle, may partly explain improved feed efficiency (FE) in low residual feed intake (RFI) pigs. One of the main findings is that, although microbial diversity did not differ among animals of varying FE, specific intestinal microbes could potentially be linked with porcine FE. However, as the factors impacting FE are still not fully understood, intestinal microbiota composition may not be a major factor determining differences in FE. Nonetheless, this work has provided a potential set of microbial biomarkers for FE in pigs. Although culturability could be a limiting factor and intervention studies are required, these taxa could potentially be targeted in the future to manipulate the intestinal microbiome so as to improve FE in pigs. If successful, this has the potential to reduce both production costs and the environmental impact of pig production.

Rumen microbial abundance and fermentation profile during severe subacute ruminal acidosis and its modulation by plant derived alkaloids in vitro.

Rumen microbiota have important metabolic functions for the host animal. This study aimed at characterizing changes in rumen microbial abundances and fermentation profiles using a severe subacute ruminal acidosis (SARA) in vitro model, and to evaluate a potential modulatory role of plant derived alkaloids (PDA), containing quaternary benzophenanthridine and protopine alkaloids, of which sanguinarine and chelerythrine were the major bioactive compounds. Induction of severe SARA strongly affected the rumen microbial composition and fermentation variables without suppressing the abundance of total bacteria. Protozoa and fungi were more sensitive to the low ruminal pH condition than bacteria. Induction of severe SARA clearly depressed degradation of fiber (P < 0.001), which came along with a decreased relative abundance of fibrolytic Ruminococcus albus and Fibrobacter succinogenes (P < 0.001). Under severe SARA conditions, the genus Prevotella, Lactobacillus group, Megasphaera elsdenii, and Entodinium spp. (P < 0.001) were more abundant, whereas Ruminobacter amylophilus was less abundant. SARA largely suppressed methane formation (-70%, P < 0.001), although total methanogenic 16S rRNA gene abundance was not affected. According to principal component analysis, Methanobrevibacter spp. correlated to methane concentration. Addition of PDA modulated ruminal fermentation under normal conditions such as enhanced (P < 0.05) concentration of total SCFA, propionate and valerate, and increased (P < 0.05) degradation of crude protein compared with the unsupplemented control diet. Our results indicate strong shifts in the microbial community during severe SARA compared to normal conditions. Supplementation of PDA positively modulates ruminal fermentation under normal ruminal pH conditions.

Processing barley grain with lactic acid and tannic acid ameliorates rumen microbial fermentation and degradation of dietary fibre in vitro.

BACKGROUND: Cereal grains are important ingredients of ruminant diets, but their rapid degradation seriously impairs rumen fermentation and the host's health. The goal of this study was to evaluate whether processing of barley grain with 1 or 5% lactic acid (LA) and 1 or 5% tannic acid (TA), without or with an additional heat treatment (1% LAH or 1% TAH), may affect microbial ruminal abundance, fermentation profile, and nutrient degradation in vitro. RESULTS: Processing with LA lowered (P < 0.05) the concentration of short-chain fatty acids (SCFAs), proportions of branched-chain SCFA, and the acetate-to-propionate ratio. Treatment with 1% TAH and 1% LAH lowered (P < 0.05) gene copy numbers of total protozoa, rumen lipopolysaccharide, and degradation of crude protein, and tended (P = 0.08) to lower the proportion of the genus Prevotella. Treatment of barley grain with 1% LA or 1% LAH stimulated Clostridium cluster XIV. Degradation of fibre was enhanced (P < 0.05) by all LA and TA treatments. CONCLUSION: Chemical and heat treatment of barley grain modulated the ruminal fermentation profile and enhanced fibre degradation; however, processing of grain with LA seems to be superior because this effect was not associated with a concomitant depression in ruminal degradation of organic matter.

Adaptation of the cecal bacterial microbiome of growing pigs in response to resistant starch type 4.

Resistant starch (RS) exacerbates health benefits on the host via modulation of the gut bacterial community. By far, these effects have been less well explored for RS of type 4. This study aimed at gaining a community-wide insight into the impact of enzymatically modified starch (EMS) on the cecal microbiota and hindgut fermentation in growing pigs. Castrated male pigs (n = 12/diet; 29-kg body weight) were fed diets with either 70% EMS or control starch for 10 days. The bacterial profile of each cecal sample was determined by sequencing of the V345 region of the 16S rRNA gene using the Illumina MiSeq platform. EMS diet reduced short-chain fatty acid concentrations in cecum and proximal colon compared to the control diet. Linear discriminant analyses and K means clustering indicated diet-specific cecal community profiles, whereby diversity and species richness were not different among diets. Pigs showed host-specific variation in their most abundant phyla, Firmicutes (55%), Proteobacteria (35%), and Bacteroidetes (10%). The EMS diet decreased abundance of Ruminococcus, Parasutterella, Bilophila, Enterococcus, and Lactobacillus operational taxonomic units (OTU), whereas Meniscus and Actinobacillus OTU were increased compared to those with the control diet (P < 0.05). Quantitative PCR confirmed results for host effect on Enterobacteriaceae and diet effect on members of the Lactobacillus group. The presence of less cecal short-chain fatty acids and the imputed metabolic functions of the cecal microbiome suggested that EMS was less degradable for cecal bacteria than the control starch. The present EMS effects on the bacterial community profiles were different than the previously reported RS effects and can be linked to the chemical structure of EMS.

Dietary calcium concentration and cereals differentially affect mineral balance and tight junction proteins expression in jejunum of weaned pigs.

Ca plays an essential role in bone development; however, little is known about its effect on intestinal gene expression in juvenile animals. In the present study, thirty-two weaned pigs (9·5 (SEM 0·11) kg) were assigned to four diets that differed in Ca concentration (adequate v. high) and cereal composition (wheat-barley v. maize) to assess the jejunal and colonic gene expression of nutrient transporters, tight junction proteins, cytokines and pathogen-associated molecular patterns, nutrient digestibility, Ca balance and serum acute-phase response. To estimate the impact of mucosal bacteria on colonic gene expression, Spearman's correlations between colonic gene expression and bacterial abundance were computed. Faecal Ca excretion indicated that more Ca was available along the intestinal tract of the pigs fed high Ca diets as compared to the pigs fed adequate Ca diets (P> 0.05). High Ca diets decreased jejunal zonula occludens 1 (ZO1) and occludin (OCLN) expression, up-regulated jejunal expression of toll-like receptor 2 (TLR2) and down-regulated colonic GLUT2 expression as compared to the adequate Ca diets (P< 0.05). Dietary cereal composition up-regulated jejunal TLR2 expression and interacted (P= 0.021) with dietary Ca on colonic IL1B expression; high Ca concentration up-regulated IL1B expression with wheat-barley diets and down-regulated it with maize diets. Spearman's correlations (r> 0·35; P< 0·05) indicated an association between operational taxonomic units assigned to the phyla Bacteroidetes, Firmicutes and Proteobacteria and bacterial metabolites and mucosal gene expression in the colon. The present results indicate that high Ca diets have the potential to modify the jejunal and colonic mucosal gene expression response which, in turn, interacts with the composition of the basal diet and mucosa-associated bacteria in weaned pigs.

Microbiomes of unreactive and pathologically altered ileocecal lymph nodes of slaughter pigs.

Microbe-laden dendritic cells are shifted to ileocecal lymph nodes (ICLNs), where microbes are concentrated and an adequate immune response is triggered. Hence, ICLNs are at a crucial position in immune anatomy and control processes of the local immune system. Pathological alterations in ICLNs, such as reactive hyperplasia, lymphadenitis purulenta, or granulomatosa, can harbor a multitude of pathogens and commensals, posing a potential zoonotic risk in animal production. The aim of this study was to characterize the microbial diversity of unreactive ICLNs of slaughter pigs and to investigate community shifts in reactive ICLNs altered by enlargement, purulence, or granulomatous formations. Pyrosequencing of 16S rRNA gene amplicons from 32 ICLNs yielded 175,313 sequences, clustering into 650 operational taxonomic units (OTUs). OTUs were assigned to 239 genera and 11 phyla. Besides a highly diverse bacterial community in ICLNs, we observed significant shifts in pathologically altered ICLNs. The relative abundances of Cloacibacterium- and Novosphingobium-associated OTUs and the genus Faecalibacterium were significantly higher in unreactive ICLNs than in pathologically altered ICLNs. Enlarged ICLNs harbored significantly more Lactobacillus- and Clostridium-associated sequences. Relative abundances of Mycoplasma, Bacteroides, Veillonella, and Variovorax OTUs were significantly increased in granulomatous ICLNs, whereas abundances of Pseudomonas, Escherichia, and Acinetobacter OTUs were significantly increased in purulent ICLNs (P < 0.05). Correlation-based networks revealed interactions among OTUs in all ICLN groups, and discriminant analyses depicted discrimination in response to pathological alterations. This study is the first community-based survey in ICLNs of livestock animals and will provide a basis to broaden the knowledge of microbe-host interactions in pigs.

Higher body fatness in intrauterine growth retarded juvenile pigs is associated with lower fat and higher carbohydrate oxidation during ad libitum and restricted feeding.

PURPOSE: A thrifty energy metabolism has been suggested in intrauterine growth restricted (IUGR) offspring. We characterized energy metabolism and substrate oxidation patterns in IUGR pigs in response to food restriction (FR) and refeeding (RFD). METHODS: Female pigs with low (L; 1.1 kg; n = 20) or normal birth weight (N; 1.5 kg; n = 24) were fed ad libitum after weaning. Half of L and N pigs were food restricted (R; LR, NR) from days 80 to 100 (57% of ad libitum) and refeed from days 101 to 131, while the remaining pigs were fed ad libitum (control, C). Using indirect calorimetry, carbohydrate and fat oxidation (COX, FOX), energy expenditure (EE) and balance (EB), resting metabolic rate (RMR) [all related to kg body weight(0.62) (BW)] and RQ were determined at 4 days before (day 76) and after (day 83) beginning of FR, 4 days before (day 97) and after (day 104) end of FR and 25 days after beginning of RFD (day 125). Body fat and muscle weights were determined at day 131. RESULTS: In spite of higher relative food intake (FI), BW was lower in L pigs. In L pigs, physical activity was lower at age 76 and 83 days compared to N pigs. IUGR did not affect EE or RMR, but resulted in higher COX and lower FOX, causing greater and earlier onset of fat deposition. During FR, EE and RMR of R pigs dropped below that of C pigs, and BW gain was delayed by 30% irrespective of birth weight. In response to FR, COX decreased and FOX increased. During FR, in LR pigs FOX was ~50% of that in NR pigs. After 4 days, but not 25 days of RFD, EB and fat synthesis were higher in pigs previously subjected to FR, indicating early catch-up fat. In R pigs, BW and the abdominal fat proportion were lower at 131 days. CONCLUSIONS: Differences in food intake and substrate oxidation pattern, but not in EE and RMR, between L and N pigs were reflected in higher body fat proportions but lower body and muscle weights in L pigs. Refeeding following FR was initially associated with increased FI, a more positive EB and a more intense stimulation of fat synthesis which did not persist after 25 days of refeeding.

Variations in colostrum metabolite profiles in association with sow parity.

Information about the full spectrum of metabolites present in porcine colostrum and factors that influence metabolite abundances is still incomplete. Parity number appears to modulate the concentration of single metabolites in colostrum. This study aimed to 1) characterize the metabolome composition and 2) assess the effect of parity on metabolite profiles in porcine colostrum. Sows (n = 20) were divided into three parity groups: A) sows in parity 1 and 2 (n = 8), B) sows in parity 3 and 4 (n = 6), and C) sows in parity 5 and 6 (n = 6). Colostrum was collected within 12 h after parturition. A total of 125 metabolites were identified using targeted reversed-phase high-performance liquid chromatography-tandem mass spectrometry and anion-exchange chromatography-high resolution mass spectrometry. Gas chromatography additionally identified 19 fatty acids (FAs). Across parities, colostrum was rich in creatine and creatinine, 1,3-dioleyl-2-palmitatoylglycerol, 1,3-dipalmitoyl-2-oleoylglycerol, and sialyllactose. Alterations in colostrum concentrations were found for eight metabolites among parity groups (P < 0.05) but the effects were not linear. For instance, colostrum from parity group C comprised 75.4% more valine but 15.7%, 34.1%, and 47.9% less citric, pyruvic, and pyroglutamic acid, respectively, compared to group A (P < 0.05). By contrast, colostrum from parity group B contained 39.5% more spermidine than from group A (P < 0.05). Of the FAs, C18:1, C16:0, and C18:2 n6 were the main FAs across parities. Parity affected four FAs (C18:3n3, C14:1, C17:0ai, and C17:1), including 43.1% less α-linolenic acid (C18:3n3) in colostrum from parity group C compared to groups A and B (P < 0.05). Signature feature ranking identified 1-stearoyl-2-hydroxy-sn-glycero-3-phosphatidylcholine and the secondary bile acid hyodeoxycholic acid as the most discriminative metabolites, showing a higher variable importance in the projection score in colostrum from parity group A than from groups B and C. Overall, results provided a comprehensive overview about the metabolome composition of sow colostrum. The consequences of the changes in colostrum metabolites with increasing parity for the nutrient supply of the piglets should be investigated in the future. The knowledge gained in this study could be used to optimize feeding strategies for sows.

Changing dietary calcium-phosphorus level and cereal source selectively alters abundance of bacteria and metabolites in the upper gastrointestinal tracts of weaned pigs.

Several dietary ingredients may affect the bacterial community structure and metabolism in the porcine gut and may therefore influence animals' health and performance. This study investigated the effects of cereal source and calcium-phosphorus (CaP) level in the diet on bacterial microbiota and metabolites, nutrient intake, and gut environment in weaned pigs. Pigs (n=8/treatment) were fed wheat-barley- or corn-based diets with an adequate or high CaP level for 14 days. Effects on microbiota in the stomach, ileum, and midcolon were assessed using quantitative PCR. Data showed that Enterobacteriaceae, Campylobacter spp., and Helicobacter spp., which all contain highly immune reactive lipopolysaccharide (LPS), were abundant at all gut sites. Diet effects on bacteria and metabolites were moderate and occurred mainly in the upper gut, whereas no effects on bacteria, fermentation products, and LPS could be observed in the colon. Differences in carbohydrate intake with corn versus wheat-barley diets selectively stimulated Bifidobacterium in the stomach and ileum. There was a growth advantage for a few bacterial groups in the stomach and ileum of pigs fed the high versus adequate CaP level (i.e., gastric Enterobacteriaceae and ileal Enterococcus, Bacteroides-Prevotella-Porphyromonas, and Campylobacter). Interestingly, gastrointestinal pH was not affected by dietary CaP level. The present findings demonstrate the stability of the bacterial community and gut environment toward dietary changes even in young pigs. The results on stimulation of gastric and ileal Bifidobacterium by corn diets may be employed in nutritional strategies to support gut health after weaning.

Grain-rich diets differently alter ruminal and colonic abundance of microbial populations and lipopolysaccharide in goats.

High grain feeding has been associated with ruminal pH depression and microbial dysbiosis in cattle. Yet, the impact of high grain feeding on the caprine rumen and hindgut microbial community and lipopolysaccharide (LPS) release is largely unknown. Therefore, the objective was to investigate the effect of increasing dietary levels of barley grain on the microbial composition and LPS concentrations in the rumen and colon of goats. Effects were compared with respect to the responses of ruminal and colonic pH and short-chain fatty acid (SCFA) generation. Growing goats (n = 5-6) were fed diets containing 0, 30, or 60% coarsely ground barley grain for 6 weeks. Ruminal ciliate protozoa were counted with Bürker counting chamber, and quantitative PCR was used to compare bacterial populations. Increasing dietary grain level linearly increased (P < 0.05) ruminal numbers of entodiniomorphids. With the 60% grain diet, there was a reduction in ruminal abundance of the genus Prevotella and Fibrobacter succinogenes, whereas the ruminal abundance of Lactobacillus spp. increased compared to the 0 and 30% grain diets (P < 0.05). In the colon, abundance of the genus Prevotella and F. succinogenes increased (P < 0.05) in goats fed the 60% grain diet compared to those fed the other diets. Colonic abundance of Clostridium cluster I was related to the presence of grain in the diet. Ruminal LPS concentration decreased (P < 0.05) in response to the 60% grain diet, whereas its colonic concentration increased in response to the same diet (P < 0.05). Present results provide first insight on the adaptive response of rumen protozoa and rumen and colonic bacterial populations to increasing dietary levels of grain in goats. Although luminal pH largely affects microbial populations, fermentable substrate flow to the caprine hindgut may have played a greater role for colonic bacterial populations in the present study.

Dietary Probiotics Modulate Gut Barrier and Immune-Related Gene Expression and Histomorphology in Broiler Chickens under Non- and Pathogen-Challenged Conditions: A Meta-Analysis.

Data published in the literature about the favorable effects of dietary probiotics on gut health in broiler chickens are inconsistent. To obtain a more comprehensive understanding, we conducted a meta-analysis to assess the effects of probiotics on the gut barrier and immune-related gene expression, histomorphology, and growth in chickens that were either challenged or non-challenged with pathogens. From the 54 articles published between 2012 and 2022, subsets of data, separately for non-challenged and challenged conditions, for response variables were created. The mean dietary probiotic concentrations ranged from 4.7 to 6.2 and 4.7 to 7.2 log10 colony-forming unit/kg under non-challenged and challenged conditions, respectively. Probiotics increased the expression of genes for mucins and tight junction proteins in the jejunum and ileum at weeks 3 and 6. The stimulatory effect of probiotics on tight junction protein expression was partly stronger in challenged than in non-challenged birds. Meta-regressions also showed an anti-inflammatory effect of probiotics under challenged conditions by modulating the expression of cytokines. Probiotics improved villus height at certain ages in the small intestine while not influencing growth performance. Dietary metabolizable energy, crude protein, and days post-infection modified the effects of probiotics on the observed variables. Overall, meta-regressions support the beneficial effects of probiotics on gut integrity and structure in chickens.