Caecal Microbiota Composition Research Articles

Peripherally Restricted Activation of Opioid Receptors Influences Anxiety-Related Behaviour and Alters Brain Gene Expression in a Sex-Specific Manner

Although effects of stress-induced anxiety on the gastrointestinal tract and enteric nervous system (ENS) are well studied, how ENS dysfunction impacts behaviour is not well understood. We investigated whether ENS modulation alters anxiety-related behaviour in rats. We used loperamide, a potent μ-opioid receptor agonist that does not cross the blood–brain barrier, to manipulate ENS function and assess changes in behaviour, gut and brain gene expression, and microbiota profile. Sprague Dawley (male/female) rats were acutely dosed with loperamide (subcutaneous) or control solution, and their behavioural phenotype was examined using open field and elevated plus maze tests. Gene expression in the proximal colon, prefrontal cortex, hippocampus, and amygdala was assessed by RNA-seq and caecal microbiota composition determined by shotgun metagenome sequencing. In female rats, loperamide treatment decreased distance moved and frequency of supported rearing, indicating decreased exploratory behaviour and increased anxiety, which was associated with altered hippocampal gene expression. Loperamide altered proximal colon gene expression and microbiome composition in both male and female rats. Our results demonstrate the importance of the ENS for communication between gut and brain for normo-anxious states in female rats and implicate corticotropin-releasing hormone and gamma-aminobutyric acid gene signalling pathways in the hippocampus. This study also sheds light on sexually dimorphic communication between the gut and the brain. Microbiome and colonic gene expression changes likely reflect localised effects of loperamide related to gut dysmotility. These results suggest possible ENS pharmacological targets to alter gut to brain signalling for modulating mood.

Immunoglobulin secretion influences the composition of chicken caecal microbiota

The chicken caecum is colonised by hundreds of different bacterial species. Which of these are targeted by immunoglobulins and how immunoglobulin expression shapes chicken caecal microbiota has been addressed in this study. Using cell sorting followed by sequencing of V3/V4 variable region of 16S rRNA, bacterial species with increased or decreased immunoglobulin coating were determined. Next, we determined also caecal microbiota composition in immunoglobulin knockout chickens. We found that immunoglobulin coating was common and major taxa were coated with immunoglobulins. Similarly, more taxa required immunoglobulin production for caecum colonisation compared to those which became abundant in immunoglobulin-deficient chickens. Taxa with low immunoglobulin coating such as Lactobacillus, Blautia, [Eubacterium] hallii, Megamonas, Fusobacterium and Desulfovibrio all encode S-layer proteins which may reduce interactions with immunoglobulins. Although there were taxa which overgrew in Ig-deficient chickens (e.g. Akkermansia) indicating immunoglobulin production acted to exclude them from the chicken caecum, in most of the cases, immunoglobulin production more likely contributed to fixing the desired microbiota in the chicken caecum.

Chlorella and vegetable oil inclusion in diets for growing rabbits: effects on growth, digestibility, plasma metabolites, and caecal fermentations and microbiota

The inclusion of microalgae in livestock diets has been shown to enhance animal productivity, immune response, and meat quality. However, the role of chlorella (Chlorella vulgaris) in growing rabbit nutrition has been scarcely explored, with available studies focusing on low inclusion levels (<1%) and their effects on rabbit growth and immune response. This study evaluated the growth performance, nutrient digestibility, plasma metabolites, caecal fermentative activity, and caecal microbiota composition of growing rabbits fed diets with different inclusion levels of chlorella and crude fat. A total of 648 mixed-sex Grimaud crossbred rabbits (33 d of age; 841 ± 140 g live weight) were fed 6 experimental diets (96 rabbits per diet for the growth trial) based on a bifactorial design with three dietary inclusion levels of chlorella (0%, 1%, and 2%) and two levels of crude fat (3% and 5%) obtained by the inclusion of soybean oil (1% and 3%, respectively). The trial lasted 38 days until slaughter. From 47 to 51 days of age, 72 rabbits (12 per diet) were submitted to a digestibility trial. At 51 days of age, samples of plasma and caecal content were collected from 36 rabbits (6 rabbit per diet) to analyse plasma metabolites, caecal fermentations, and caecal microbiota. Rabbit live weight at 71 days of age (2 700 g, on average), weight gain (48.8 g/d) and feed conversion ratio (3.27) were unaffected by chlorella inclusion, while feed conversion ratio improved (−5%; P < 0.001) with an increase of crude fat from 3% to 5%. The digestibility of ADF (23.2% vs. 20.9%; P < 0.05) and crude fat (83.8% vs. 85.6%; P < 0.01) improved with the inclusion of chlorella at 2%, as well as the digestibility of crude fat (82.4% vs. 86.9%; P < 0.001) and gross energy (57.3% vs. 58.7%; P < 0.001) with crude fat inclusion at 5%. Plasma non-esterified fatty acids decreased (−19%; P < 0.05) in diets with 5% crude fat. Neither chlorella nor crude fat inclusion levels affected other plasma metabolites, caecal fermentations, or caecal microbiota. Overall, the inclusion of chlorella up to 2% in diets for growing rabbits did not significantly affect diet nutritional value, animal performance, or caecal activity. On the other hand, increasing crude fat to 5% improved the overall feed efficiency.

Essential oils mix effect on chicks ileal and caecal microbiota modulation: a metagenomics sequencing approach.

Microbiota plays a pivotal role in promoting the health and wellbeing of poultry. Essential oils (EOs) serve as an alternative solution for modulating poultry microbiota. This study aimed to investigate, using amplicon sequencing, the effect of a complex and well-defined combination of EOs feed supplement on both ileal and caecal broiler microbiota, within the context of Salmonella and Campylobacter intestinal colonization. For this experiment, 150-day-old Ross chicks were randomly allocated to two groups: T+ (feed supplementation with EO mix 500 g/t) and T- (non-supplemented). At day 7, 30 birds from each group were orally inoculated with 106 CFU/bird of a Salmonella enteritidis and transferred to the second room, forming the following groups: TS+ (30 challenged birds receiving infeed EO mix at 500g/t) and TS- (30 challenged birds receiving a non-supplemented control feed). At day 14, the remaining birds in the first room were orally inoculated with 103 CFU/bird of two strains of Campylobacter jejuni, resulting in the formation of groups T+C+ and T-C+. Birds were sacrificed at day 7, D10, D14, D17, and D21. Ileal and caecal microbiota samples were analyzed using Illumina MiSeq sequencing. At D7 and D14, ileal alpha diversity was higher for treated birds (p <0.05). No significant differences between groups were observed in caecal alpha diversity (p>0.05). The ileal beta diversity exhibited differences between groups at D7 (p < 0.008), D10 (p = 0.029), D14 (p = 0.001) and D17 (p = 0.018), but not at D21 (p = 0.54). For all time points, the analysis indicated that 6 biomarkers were negatively impacted, while 10 biomarkers were positively impacted. Sellimonas and Weissella returned the lowest (negative) and highest (positive) coefficient, respectively. At each time point, treatments influenced caecal microbiota beta diversity (p < 0.001); 31 genera were associated with T+: 10 Ruminoccocaceae genera were alternatively more abundant and less abundant from D7, 7 Lachnospiraceae genera were alternatively more and less abundant from D10, 6 Oscillospiraceae genera were variable depending on the date and 4 Enterobacteriaceae differed from D7. During all the experiment, Campylobacter decreased in treated birds (p < 0.05). This study showed that EO mix modulates ileal and caecal microbiota composition both before and during challenge conditions, increasing alpha diversity, especially in ileum during the early stages of chick life.

Effects of soyhulls with different particle size on the growth performance, blood indices and gut microbiota of yellow feather broilers

ABSTRACT 1. The objective of this study was to determine the effects of soyhulls with different particle sizes on the growth performance, blood indices and gut microbiota of yellow feather broilers. 2. Total of 240 healthy, one-day-old, yellow feather broilers were randomly divided into four groups, with six pen replicates within each group and ten birds per pen. The control group birds were fed the basal diet (Control). For the treatment groups, 5% soyhulls with different particle sizes were included in the basal diet. The particle size geometric mean diameters (d gw) of the soyhulls in the three treatment groups were 299.69 μm (LowPS), 489.85 μm (MediumPS) and 734.83 μm (HighPS) with geometric standard deviation (S gw) 1.75 μm, 1.62 μm and 1.67 μm, respectively. 3. Results showed that the growth performance variables and organ indices were not different among the four groups. The MediumPS group had increased TG, T-CHO, ALT, HDL-C, and GSH-PX levels and decreased T-AOC levels, whereas LowPS and HighPS groups had increased HDL-C and GSH-PX levels (p < 0.05). Microbial diversity analysis showed that the intestinal microbiota of yellow feather broilers mainly included Firmicutes and Bacteroidetes. Inclusion of 5% soyhulls with different particle size had no effect on alpha diversity indices of caecal microbiota. The HighPS group had significantly higher relative abundance of Firmicutes spp. and lower Bacteroidetes spp. compared with the LowPS and MediumPS group but this was not different from the Control group. The relative abundance of Cyanobacteria spp. was significantly higher in the HighPS group than the other three groups. LEfSe analysis showed that there were more enriched biomarker taxa in the groups with soyhulls than the control group. 4. Overall, the inclusion of soyhulls with different particle sizes had limited effects on growth performance, blood indices and caecal microbiota composition of yellow feather broilers.

Dietary Milk or Isolated Legume Proteins Modulate Intestinal Microbiota Composition in Rats.

Shifts toward increased proteolytic fermentation, such as, for example, in athlete and high-protein weight loss diets, may alter the relative abundance of microbial species in the gut and generate bioactive, potentially deleterious metabolic products. In the current investigation, intestinal (caecal) microbiota composition was studied in rats fed diets differing only in their constituent proteins: milk (casein (CAS), lactalbumin (LA)) or legume (Cicer arietinum, Lupinus angustifolius) protein isolates (chickpea protein isolate (CPI), lupin protein isolate (LI)). ANOSIM and Discriminant Analysis showed significant (p < 0.05) differences at both family and genus levels in both microbiota composition and functionality as a consequence of feeding the different proteins. Differences were also significant (p < 0.05) for predicted functionality parameters as determined by PICRUSt analysis. LA induced a generally healthier microbiota composition than CAS, and higher amounts of Methanobrevibacter spp. and Methanogenic_PWY were found in the LI group. LEfSe analysis of bacterial composition and functional activities revealed a number of groups/functions able to explain the different effects found with milk and legume protein isolates. In conclusion, the mostly beneficial modulation of intestinal microbiota generally found with legume-based diets is likely to be due, at least in part, to their constituent proteins.

The Order of Limiting Amino Acids in a Wheat–Sorghum-Based Reduced-Protein Diet for Laying Hens

Understanding the order of limiting amino acids (AA) in reduced-protein (RP) diets for laying hens will facilitate precise feed formulation and ensure that AA requirements are met cost-effectively. The order of the first three limiting AAs—lysine (Lys), methionine (Met), and threonine (Thr)—has been well established in RP laying hen diets. This study aimed to determine the priority order of eight additional limiting AAs (critically important AAs) when formulating wheat–sorghum-based RP diets for laying hens: tryptophan (Trp), valine (Val), isoleucine (Ile), arginine (Arg), leucine (Leu), histidine (His), phenylalanine (Phe), and glycineequivalent (Gly). A total of 330 Hy-Line Brown laying hens were randomly assigned to 11 dietary treatments (30 replicates of individual birds per treatment) from 20 to 39 weeks of age (WOA). Treatments were a standard-protein (17.24% CP) diet as the control (SP); a reduced-protein (15.00% CP) diet with sufficient levels of Lys, Met, and Thr but insufficient levels of the eight experimental essential AA (RP); a reduced-protein diet with sufficient levels of all essential AAs (RP-EAA); and eight subsequent dietary treatments of the RP-EAA diet with one of the experimental essential AAs removed: Trp (RP-EAA-Trp), Val (RP-EAA-Val), Ile (RP-EAA-Ile), Arg (RP-EAA-Arg), Leu (RP-EAA-Leu), His (RP-EAA-His), Phe (RP-EAA-Phe), and Gly (RP-EAA-Gly). Eggs were collected and weighed daily, and feed intake and feed conversion ratio (FCR) were calculated weekly. External and internal egg quality was measured at 29 and 39 WOA. Nutrient digestibility, serum uric acid concentration, caecal microbiota composition, and tibia parameters were measured at 40 WOA. Overall, hens fed the RP-EAA-Val, RP-EAA-Ile, and RP diets presented significantly lower egg mass compared to hens fed the SP, RP-EAA-His, and RP-EAA-Gly diets (p &lt; 0.001). Hens fed the RP diet and RP-EAA-Val diet had a higher FCR compared to those offered the RP-EAA, RP-EAA-Leu, RP-EAA-Phe, and RP-EAA-Gly diets (p = 0.046). Lower protein intake and excretion were observed in hens offered the RP diets compared to hens fed the SP diet (p = 0.001 and 0.018, respectively). Based on the egg mass, Ile may be considered the fourth and Val the fifth limiting AA, after Lys, Met, and Thr, in laying hens fed wheat–sorghum-based RP diets during peak lay. However, if ranked based on FCR, Val may be considered the fourth limiting AA, followed by Trp, Ile, Arg, and His as the co-fifth limiting AAs. Leu, Phe, and Gly may be considered as non-essential AAs for laying hens fed RP diets.

Effects of rutin supplementation on intestinal morphology, antioxidant capacity, immunity, and gut microbiota of laying hens fed a diet containing stored soybean meal

Protein oxidation of soybean meal (SBM) during storage may have adverse effects on the intestinal health of laying hens. Moreover, rutin has anti-inflammatory and antioxidant properties which might be used as a feed additive to mitigate the intestinal damage caused by oxidised protein of SBM. This study aimed to investigate the effects of rutin supplementation on intestinal morphology, antioxidant capacity, immunity and caecal microbiota in laying hens fed a diet containing stored SBM. A total of 384 Hy-Line Brown laying hens (220 days) were randomly allocated into four groups with eight replicates of 12 laying hens each according to a 2 × 2 factorial arrangement with 2 types of SBM (FSM: SBM was stored in the cold storage warehouses at −20 °C for 45 days, and was considered as fresh and control SBM; RTSM: SBM was stored in room temperature warehouse (15 °C to 25 °C), average temperature was 20 °C for 45 days) and 2 levels of rutin (0 and 500 mg/kg). The results showed that the RTSM diet decreased the ileal glutathione peroxidase (GSH-Px) activity, the jejunal superoxide dismutase 2 (SOD2) and ileal NAD(P)H: quinone oxidoreductase 1 (NQO1) mRNA expression levels (p < 0.05), and tended to decrease the jejunal superoxide dismutase 1 (SOD1), ileal glutathione peroxidase 1 (GPX1) and increase the jejunal interleukin-1β (IL-1β) and interleukin-4 (IL-4) mRNA expression levels. Dietary rutin decreased the jejunal crypt depth (CD) (p < 0.05), increased the jejunal total antioxidant capacity (T-AOC) and GSH-Px activities (p < 0.05), decreased the content of interferon-γ (IFN-γ) in the jejunum (p < 0.05), and significantly reduced levels of immunoglobulin G (IgG), IL-1β, tumour necrosis factor-α (TNF-α), IFN-γ, and IL-4 in the ileal mucosa (p < 0.05). Dietary rutin increased SOD2, nuclear factor E2-related factor 2 (Nrf2) and NQO1 mRNA expression levels in the jejunum and GPX1, Nrf2 and NQO1 mRNA expression levels in the ileum, and decreased nuclear factor-κB (NF-κB), IL-1β, IFN-γ and IL-4 mRNA expression in the jejunum and NF-κB, IL-1β, TNF-α, IFN-γ and IL-4 mRNA expressions in the ileum. What’s more, dietary rutin changed the caecal microbiota. PCoA analysis indicated significant structural differences among four groups (p < 0.05), and SBM × Rutin interactions were found in Actinobacteriota and unclassifiedk_norank_d_ Bacteria at the phylum level (p < 0.05). These results suggested that RTSM had slight adverse effects on the intestinal health, and dietary rutin improved intestinal morphology, exerted antioxidant and anti-inflammatory effects via Nrf2 and NF-κB signal pathways, and changed the composition of caecal microbiota. Highlights The RTSM diet has adverse effects on the intestinal health of laying hens compared with the FSM diet. Dietary rutin improved the intestinal health by increasing the intestinal morphology, antioxidant capacity and anti-inflammatory effects. Dietary soybean meal and rutin changed the composition of caecal microbiota.

Negative consequences of reduced protein diets supplemented with synthetic amino acids for performance, intestinal barrier function, and caecal microbiota composition of broiler chickens

The consequences of feeding broiler chickens with reduced protein (RP) diets for gut health and barrier function are not well understood. This study was performed to elucidate the effect of reducing dietary protein and source of protein on gut health and performance parameters. Four experimental diets included 2 control diets with standard protein levels either containing meat and bone meal (CMBM) or an all-vegetable diet (CVEG), a medium RP diet (17.5% in growers and 16.5% in finisher), and a severe RP diet (15.6% in grower and 14.6% in finisher). Off-sex Ross 308 birds were assigned to each of the 4 diets and performance measurements were taken from d 7 to 42 post-hatch. Each diet was replicated 8 times (10 birds per replicate). A challenge study was conducted on additional 96 broilers (24 birds per diet) from d 13 to 21. Half of the birds in each dietary treatment were challenged by dexamethasone (DEX) to induce a leaky gut. Feeding birds with RP diets decreased weight gain (P < 0.0001) and increased feed conversion ratio (P < 0.0001) from d 7 to 42 compared with control diets. There was no difference between CVEG and CMBM control diets for any parameter. The diet containing 15.6% protein increased (P < 0.05) intestinal permeability independent of the DEX challenge. Gene expression of claudin-3 was downregulated (P < 0.05) in birds fed 15.6% protein. There was a significant interaction between diet and DEX (P < 0.05) and both RP diets (17.5% and 15.6%) downregulated claudin-2 expression in DEX-challenged birds. The overall composition of the caecal microbiota was affected in birds fed 15.6% protein having a significantly lower richness of microbiota in both sham and DEX-injected birds. Proteobacteria was the main phylum driving the differences in birds fed 15.6% protein. At the family level, Bifidobacteriaceae, Unclassified Bifidobacteriales, Enterococcaceae, Enterobacteriaceae, and Lachnospiraceae were the main taxa in birds fed 15.6% protein. Despite supplementation of synthetic amino acids, severe reduction of dietary protein compromised performance and intestinal health parameters in broilers, evidenced by differential mRNA expression of tight junction proteins, higher permeability, and changes in caecal microbiota composition.

Slowed gastrointestinal transit is associated with an altered caecal microbiota in an aged rat model.

Gastrointestinal (GI) motility is largely dependent upon activity within the enteric nervous system (ENS) and is an important part of the digestive process. Dysfunction of the ENS can impair GI motility as is seen in the case of constipation where gut transit time is prolonged. Animal models mimicking symptoms of constipation have been developed by way of pharmacological manipulations. Studies have reported an association between altered GI motility and gut microbial population. Little is known about the changes in gut microbiota profile resulting specifically from pharmacologically induced slowed GI motility in rats. Moreover, the relationship between gut microbiota and altered intestinal motility is based on studies using faecal samples, which are easier to obtain but do not accurately reflect the intestinal microbiome. The aim of this study was to examine how delayed GI transit due to opioid receptor agonism in the ENS modifies caecal microbiota composition. Differences in caecal microbial composition of loperamide-treated or control male Sprague Dawley rats were determined by 16S rRNA gene amplicon sequencing. The results revealed that significant differences were observed at both genus and family level between treatment groups. Bacteroides were relatively abundant in the loperamide-induced slowed GI transit group, compared to controls. Richness and diversity of the bacterial communities was significantly lower in the loperamide-treated group compared to the control group. Understanding the link between specific microbial species and varying transit times is crucial to design interventions targeting the microbiome and to treat intestinal motility disorders.

Effects of Different-Sized Cages on the Production Performance, Serum Parameters, and Caecal Microbiota Composition of Laying Hens.

The effects of four different-sized cages-huge (HC), large (LC), medium (MC), and small (SC) cages-on the productive performance, serum biochemical indices, and caecal microbiota composition of Roman laying hens were investigated. At 44 weeks of age, a total of 450 hens were selected and allocated to the four groups, with six replicates each. Equal stocking density (0.054 m2 per bird) was maintained among the four groups throughout the experiment, and number of birds/cage changed for each treatment. After 2 weeks of preliminary trial, the formal experiment was performed from 46 to 60 weeks of age. The laying rate and feed conversion ratio (FCR) were determined daily, antibody titres were measured every 3 weeks, and serum biochemical parameters and caecal microbiota composition were analysed at 60 weeks of age. Compared to HC and SC, the higher laying rate and lower FCR in MC and LC indicated positive effects on egg production and feed efficiency, while SC showed the highest body weight gain (p &lt; 0.05). With increasing cage size, the serum triglycerides (TG) and total cholesterol (T-CH) levels were reduced, and serum glutathione peroxidase (GSH-Px) activity improved, where birds raised in HCs had the lowest serum TG and T-CH and the highest GSH-Px activity. Twenty-nine different phyla and 301 different genera were detected in the caecal microbiota of birds in the four groups. Methanobrevibacter was significantly higher in the SC than in the other groups (p &lt; 0.05). Faecalibacterium was most abundant in the MC compared with the other groups (p &lt; 0.05) and was significantly positively correlated with serum GSH-Px concentration (R = 0.214, p = 0.0017). Lactobacillus was significantly less abundant in the LC and MC than in the HC and SC groups (p &lt; 0.05) and was significantly positively correlated with body weight (R = 0.350, p = 0.0009) but negatively correlated with laying rate and FCR. In conclusion, MC were superior to HC and LC in improving feed conversion efficiency and caecal microflora composition compared to the SC. An appropriate increase in cage size is beneficial to laying hen production and health.

Microbiome-metabolome analysis reveals alterations in the composition and metabolism of caecal microbiota and metabolites with dietary Enteromorpha polysaccharide and Yeast glycoprotein in chickens.

The intestinal microbiome is responsible for the fermentation of complex carbohydrates and orchestrates the immune system through gut microbiota-derived metabolites. In our previous study, we reported that supplementation of Enteromorpha polysaccharide (EP) and yeast glycoprotein (YG) in combination synergistically improved antioxidant activities, serum lipid profile, and fatty acid metabolism in chicken. However, the mechanism of action of these polysaccharides remains elusive. The present study used an integrated 16S-rRNA sequencing technology and untargeted metabolomics technique to reveal the mechanism of action of EP+YG supplementation in broiler chickens fed basal diet or diets supplemented with EP+YG (200mg/kg EP + 200mg/kg YG). The results showed that EP+YG supplementation altered the overall structure of caecal microbiota as evidenced by β diversities analysis. Besides, EP+YG supplementation changed the microbiota composition by altering the community profile at the phylum and genus levels. Furthermore, Spearman correlation analysis indicated a significant correlation between altered microbiota genera vs serum cytokine levels and microbiota genera vs volatile fatty acids production. Predicted functional analysis showed that EP+YG supplementation significantly enriched amino acid metabolism, nucleotide metabolism, glycan biosynthesis and metabolism, energy metabolism, and carbohydrate metabolism. Metabolomics analysis confirmed that EP+YG supplementation modulates a myriad of caecal metabolites by increasing some metabolites, including pyruvic acid, pyridoxine, spermidine, spermine, and dopamine, and decreasing metabolites related to lipid metabolisms such as malonic acid, oleic acid, and docosahexaenoic acid. The quantitative enrichment analysis results further showed that glycolysis/gluconeogenesis, citric acid cycle, tyrosine metabolism, glycine, serine, and threonine metabolism, and cysteine and methionine metabolism were the most important enriched pathways identified with enrichment ratio >11, whereas, fatty acid biosynthesis and biosynthesis of unsaturated fatty acids pathways were suppressed. Together, the 16S-rRNA and untargeted metabolomics results uncovered that EP+YG supplementation modulates intestinal microbiota and their metabolites, thereby influencing the important metabolism pathways, suggesting a potential feed additive.

Comparing a single dose of xylanase to a double dose or cocktail of non-starch polysaccharide-degrading enzymes in broiler chicken diets

This study compared supplementation with a single dose of xylanase to a double dose of xylanase or a non-starch polysaccharide (NSP) degrading enzyme cocktail (NSP-ase cocktail) on productive performance, nutrient utilisation and the gastrointestinal environment in broilers fed commercial diets. Cobb 500 broilers (n=1,080) were fed 12 dietary treatments; four Australian commercial diets (based on wheat-barley, wheat-maize, wheat-sorghum or wheat only) with three different enzyme treatments (single dose of xylanase (16,000 BXU/kg), double dose of xylanase (32,000 BXU/kg) or NSP-ase cocktail (xylanase, β-glucanase, cellulase, pectinase, mannanase, galactanase, arabinofuranosidase). There were 108 pens, nine replicates per dietary treatment, with 10 birds per pen. Performance (total pen body weight, feed intake and feed conversion ratio corrected for mortality) was determined at d 0-35. On d 35, one male and one female were weighed individually and used to determine breast meat, thigh and drumstick weight, dry matter (DM) contents from the gizzard, jejunum and ileum, ileal protein, energy, starch and dry matter digestibility, ileal viscosity and xylo-oligosaccharide (XOS) concentration, caecal microbiota and short chain fatty acid (SCFA) composition. The double dose of xylanase and NSP-ase cocktail had no effect on bird performance, meat yield, ileal viscosity, ileal starch, energy or DM digestibility or digesta DM content. The double xylanase dose and NSP-ase cocktail increased protein digestibility in birds fed the wheat-sorghum based diet (P=0.041) and increased caecal concentration of butyric acid in birds fed the wheat-maize based diet (P=0.040), and propionic, valeric and lactic acid and Bifidobacteria and Enterobacteria spp. in birds fed the wheat-based diet (P&lt;0.05). The double xylanase dose increased XOS production, particularly in birds fed the wheat-barley based diets (P&lt;0.05). The lack of performance effects observed when feeding the double xylanase dose or NSP-ase cocktail suggested that the current recommended xylanase dose (16,000 BXU/kg) is sufficient.

Caecal microbiota composition of experimental inbred MHC-B lines infected with IBV differs according to genetics and vaccination

Interactions between the gut microbiota and the immune system may be involved in vaccine and infection responses. In the present study, we studied the interactions between caecal microbiota composition and parameters describing the immune response in six experimental inbred chicken lines harboring different MHC haplotypes. Animals were challenge-infected with the infectious bronchitis virus (IBV), and half of them were previously vaccinated against this pathogen. We explored to what extent the gut microbiota composition and the genetic line could be related to the immune response, evaluated through flow cytometry. To do so, we characterized the caecal bacterial communities with a 16S rRNA gene amplicon sequencing approach performed one week after the IBV infectious challenge. We observed significant effects of both the vaccination and the genetic line on the microbiota after the challenge infection with IBV, with a lower bacterial richness in vaccinated chickens. We also observed dissimilar caecal community profiles among the different lines, and between the vaccinated and non-vaccinated animals. The effect of vaccination was similar in all the lines, with a reduced abundance of OTU from the Ruminococcacea UCG-014 and Faecalibacterium genera, and an increased abundance of OTU from the Eisenbergiella genus. The main association between the caecal microbiota and the immune phenotypes involved TCRϒδ expression on TCRϒδ+ T cells. This phenotype was negatively associated with OTU from the Escherichia-Shigella genus that were also less abundant in the lines with the highest responses to the vaccine. We proved that the caecal microbiota composition is associated with the IBV vaccine response level in inbred chicken lines, and that the TCRϒδ+ T cells (judged by TCRϒδ expression) may be an important component involved in this interaction, especially with bacteria from the Escherichia-Shigella genus. We hypothesized that bacteria from the Escherichia-Shigella genus increased the systemic level of bacterial lipid antigens, which subsequently mitigated poultry γδ T cells.

Gut microbiota-derived metabolites contribute negatively to hindgut barrier function development at the early weaning goat model.

Early weaning induces intestinal injury, leading to a series of long-term symptoms such as inflammation, malabsorption and diarrhea. In this study, we hypothesized that microbes and their metabolites modulate the host's inflammatory response to early weaning stress in a goat model. A total of 18 female Tibetan goat kids (n = 9) were weaned from their mothers at 28 d (D28) and 60 d (D60) postpartum. D60 and D28 groups were fed the same solid diet ad libitum from weaning to 75 d of age. The colonic epithelium was subject to RNA-sequencing, the caecal digesta metabolomics were assessed by liquid chromatography–tandem mass spectrometry (LC-MS/MS), and the caecal microbiota composition was analysed by 16S ribosomal RNA gene sequencing. We found that early weaning substantially increased the colonic pro-apoptotic gene expression of B-cell lymphoma associated X (Bax), caspase-9, and caspase-3, and decreased the expression of zonula occludens-1 (ZO-1) and claudin-1 (P < 0.01). In addition, a significant Bacteroides acidifaciens enrichment was observed in the hindgut of early-weaned goats (P < 0.01), which negatively correlated with lysophosphatidylcholine products. Similarly, the chemokine signaling, IL-17 signaling, and peroxisome proliferators-activated receptor (PPAR) signaling pathways were upregulated in the colonic mucosa of the early-weaned goats. By applying caecal microbiota transplantation from goats to defaunated C57/6J mice, we confirmed that caecal microbiota of D28 goat kids increased the relative abundance of B. acidifaciens and significantly up-regulated the genes of Bax, G protein–coupled receptor (GPR) 109A, GPR 43, fatty acid binding protein 6, nuclear receptor subfamily 1 group H member 3, angiotensin converting enzyme 2, and IL-6 expression (P < 0.05), and decreased ZO-1, and claudin-1 protein expression in the mice jejunum and colon (P < 0.001). These results proposed that the hindgut microbiota and metabolites mediate the barrier function weakening during early weaning, and the relative abundance of B. acidifaciens was negatively correlated with the hindgut barrier gene expression. This study demonstrates how weaning stress can affect key host–microbe interaction regulators in the hindgut, in a lysophosphatidylcholine dependent and independent manner. Furthermore, based on our mice data, these results are transferable to other mammal species.

Differences in caecal microbiota composition and Salmonella carriage between experimentally infected inbred lines of chickens

BackgroundSalmonella Enteritidis (SE) is one of the major causes of human foodborne intoxication resulting from consumption of contaminated poultry products. Genetic selection of animals that are more resistant to Salmonella carriage and modulation of the gut microbiota are two promising ways to decrease individual Salmonella carriage. The aims of this study were to identify the main genetic and microbial factors that control the level of Salmonella carriage in chickens (Gallus gallus) under controlled experimental conditions. Two-hundred and forty animals from the White Leghorn inbred lines N and 61 were infected by SE at 7 days of age. After infection, animals were kept in isolators to reduce recontamination of birds by Salmonella. Caecal contents were sampled at 12 days post-infection and used for DNA extraction. Microbiota DNA was used to measure individual counts of SE by digital PCR and to determine the bacterial taxonomic composition, using a 16S rRNA gene high-throughput sequencing approach.ResultsOur results confirmed that the N line is more resistant to Salmonella carriage than the 61 line, and that intra-line variability is higher for the 61 line. Furthermore, the 16S analysis showed strong significant differences in microbiota taxonomic composition between the two lines. Among the 617 operational taxonomic units (OTU) observed, more than 390 were differentially abundant between the two lines. Furthermore, within the 61 line, we found a difference in the microbiota taxonomic composition between the high and low Salmonella carriers, with 39 differentially abundant OTU. Using metagenome functional prediction based on 16S data, several metabolic pathways that are potentially associated to microbiota taxonomic differences (e.g. short chain fatty acids pathways) were identified between high and low carriers.ConclusionsOverall, our findings demonstrate that the caecal microbiota composition differs between genetic lines of chickens. This could be one of the reasons why the investigated lines differed in Salmonella carriage levels under experimental infection conditions.

Dietary Supplementation throughout Life with Non-Digestible Oligosaccharides and/or n-3 Poly-Unsaturated Fatty Acids in Healthy Mice Modulates the Gut-Immune System-Brain Axis.

The composition and activity of the intestinal microbial community structures can be beneficially modulated by nutritional components such as non-digestible oligosaccharides and omega-3 poly-unsaturated fatty acids (n-3 PUFAs). These components affect immune function, brain development and behaviour. We investigated the additive effect of a dietary combination of scGOS:lcFOS and n-3 PUFAs on caecal content microbial community structures and development of the immune system, brain and behaviour from day of birth to early adulthood in healthy mice. Male BALB/cByJ mice received a control or enriched diet with a combination of scGOS:lcFOS (9:1) and 6% tuna oil (n-3 PUFAs) or individually scGOS:lcFOS (9:1) or 6% tuna oil (n-3 PUFAs). Behaviour, caecal content microbiota composition, short-chain fatty acid levels, brain monoamine levels, enterochromaffin cells and immune parameters in the mesenteric lymph nodes (MLN) and spleen were assessed. Caecal content microbial community structures displayed differences between the control and dietary groups, and between the dietary groups. Compared to control diet, the scGOS:lcFOS and combination diets increased caecal saccharolytic fermentation activity. The diets enhanced the number of enterochromaffin cells. The combination diet had no effects on the immune cells. Although the dietary effect on behaviour was limited, serotonin and serotonin metabolite levels in the amygdala were increased in the combination diet group. The combination and individual interventions affected caecal content microbial profiles, but had limited effects on behaviour and the immune system. No apparent additive effect was observed when scGOS:lcFOS and n-3 PUFAs were combined. The results suggest that scGOS:lcFOS and n-3 PUFAs together create a balance—the best of both in a healthy host.

Dietary soluble non-starch polysaccharide level and xylanase influence the gastrointestinal environment and nutrient utilisation in laying hens

ABSTRACT 1. The objective of this study was to examine the influence of dietary soluble non-starch polysaccharide (sNSP) level and xylanase supplementation on productive performance, viscosity and pH along the gastrointestinal tract in laying hens. Excreta moisture content, ileal and caecal microbiota and short-chain fatty acid (SCFA) composition and apparent total tract nutrient utilisation were measured. 2. Hyline Brown laying hens (n = 144) were housed individually at 25 weeks of age and allocated to one of the four wheat-based dietary treatments in a 2 × 2 factorial arrangement, consisting of two levels of sNSP (High 13.40 g/kg or Low 11.22 g/kg), with or without xylanase (0 or 12,000 BXU/kg). Birds were fed the dietary treatments for 56 d. 3. Increasing dietary sNSP increased jejunum viscosity, degradability of total NSP, total tract flow of insoluble arabinose, and succinic acid concentration in the caeca (P < 0.05). Feeding high sNSP decreased excreta moisture content, total tract energy retention and free oligosaccharide, total tract flow of soluble and insoluble galactose and insoluble rhamnose and fucose, and ileal acetic and lactic acid concentrations (P < 0.05), and tended to reduce egg production (P = 0.058). 4. Supplementation with xylanase resulted in reduced jejunum and ileum viscosity, caecal pH, excreta moisture, flow of soluble arabinose and glucose and insoluble arabinose and xylose, caecal concentration of Lactobacillus sp. and isobutyric and succinic acid, and ileal concentration of Bacillus sp. and total anaerobic bacteria (P < 0.05). Xylanase application also increased energy retention and insoluble and total NSP degradation, and caecal abundance of Bifidobacteria sp. and valeric acid (P < 0.05). 5. These results reiterated the ability of xylanase to improve nutrient digestibility and reduce excreta moisture content in laying hens, and highlighted the importance of considering dietary sNSP level in laying hen diets.

Fructose, glucose and fat interrelationships with metabolic pathway regulation and effects on the gut microbiota.

The purpose of this 30-day feeding study was to elucidate the changes, correlations, and mechanisms caused by the replacement of the starch content of the AIN-93G diet (St) with glucose (G), fructose (F) or lard (L) in body and organ weights, metabolic changes and caecal microbiota composition in rats (Wistar, SPF). The body weight gain of rats on the F diet was 12% less (P = 0.12) than in the St group. Rats on the L diet consumed 18.6% less feed, 31% more energy and gained 58.4% more than the animals on the St diet, indicating that, in addition to higher energy intake, better feed utilisation is a key factor in the obesogenic effect of diets of high nutrient and energy density. The G, F and L diets significantly increased the lipid content of the liver (St: 7.01 ± 1.48; G: 14.53 ± 8.77; F: 16.73 ± 8.77; L: 19.86 ± 4.92% of DM), suggesting that lipid accumulation in the liver is not a fructose-specific process. Relative to the St control, specific glucose effects were the decreasing serum glucagon (-41%) concentrations and glucagon/leptin ratio and the increasing serum leptin concentrations (+26%); specific fructose effects were the increased weights of the kidney, spleen, epididymal fat and the decreased weight of retroperitoneal fat and the lower immune response, as well as the increased insulin (+26%), glucagon (+26%) and decreased leptin (-25%) levels. This suggests a mild insulin resistance and catabolic metabolism in F rats. Specific lard effects were the decreased insulin (-9.14%) and increased glucagon (+40.44%) and leptin (+44.92%) levels. Relative to St, all diets increased the operational taxonomic units of the phylum Bacteroidetes. G and L decreased, while F increased the proportion of Firmicutes. F and L diets decreased the proportions of Actinobacteria, Proteobacteria and Verrucomicrobia. Correlation and centrality analyses were conducted to ascertain the positive and negative correlations and relative weights of the 32 parameters studied in the metabolic network. These correlations and the underlying potential mechanisms are discussed.

Age-associated changes in caecal microbiome and their apparent correlations with growth performances of layer pullets.

The microbiome in gastrointestinal tracts play an important role in regulating nutrient utilization and absorption, gut immune function, and host growth or development. This study was conducted to investigate the composition and dynamic distribution of caecal microbiota in pullets during the first 16 weeks. Growth performance, immune organs index, and intestinal morphology of pullets were analyzed at 3, 6, 12 and 16 weeks of age. The caecal contents were collected for microbiota analysis by 16S rRNA gene sequencing method. With advancing ages in pullets, the gradually increased average daily feed intake (ADFI), feed conversion ratio (FCR) and intestinal villus height, but the gradually decreased organs index of thymus and bursa were determined. Meanwhile, more abundant caecal bacterial communities were determined from pullets at 12 and 16 weeks of age than those at 3 and 6 weeks of age. Furthermore, the dominant microflora of pullets from different weeks of age were analyzed by using LEfSe: The higher abundance of Blautia, Prevotella, Alistipes, and Eggerthella were found at 6 weeks; Anaerostipes, Oscillospira, Enterococcus and Methanobrevibacter were determined at 12 weeks; and the higher abundance of Parabacteroides, Anaerofustis, Lactobacillus and Butyricimonas were determined at 16 weeks. Further functional predicted analysis by PICRUSt revealed that the endocrine system and carbohydrate metabolism were significantly developed at 3 weeks. The development of the immune system was predicted to be mainly during 6 to 12 weeks, while cardiovascular diseases and circulatory system were during 12 to 16 weeks. In addition, the significantly negative correlation between Bacteroides and villus height, the significantly negative correlation between growth parameters (ADFI and FCR) and Bacteroides, Oscillospira and Alistipes; and the significantly positive relations between growth parameters (ADFI and FCR) and Bilophila, Lactobacillus, Rikenella and Anaerofustis were determined by using Pearson analyses. In conclusion, our data demonstrated that growth performance and intestinal morphology correlate well with caecal microbiota, which could provide new insights to establish or develop nutritional strategies to manage the intestinal health or development of laying pullets.