Diet-induced changes in the jejunal microbiota of developing broilers reduce the abundance of Enterococcus hirae and Enterococcus faecium.

Modern broiler breeds allow for high feed efficiency and rapid growth, which come at a cost of increased susceptibility to pathogens and disease. Broiler growth rate, feed efficiency, and health are affected by the composition of the gut microbiota, which in turn is influenced by diet. In this study, we therefore assessed how diet composition can affect the broiler jejunal gut microbiota. A total of 96 broiler chickens were divided into four diet groups: control, coated butyrate supplementation, medium-chain fatty acid supplementation, or a high-fibre low-protein content. Diet groups were sub-divided into age groups (4, 12 and 33 days of age) resulting in groups of 8 broilers per diet per age. The jejunum content was used for metagenomic shotgun sequencing to determine the microbiota taxonomic composition at species level. The composed diets resulted in a total of 104 differentially abundant bacterial species. Most notably were the butyrate-induced changes in the jejunal microbiota of broilers 4 days post-hatch, resulting in the reduced relative abundance of mainly Enterococcus faecium (-1.8 l2fc, Padj = 9.9E-05) and the opportunistic pathogen Enterococcus hirae (-2.9 l2fc, Padj = 2.7E-08), when compared to the control diet. This effect takes place during early broiler development, which is critical for broiler health, thus exemplifying the importance of how diet can influence the microbiota composition in relation to broiler health. Future studies should therefore elucidate how diet can be used to promote a beneficial microbiota in the early stages of broiler development.

Transcriptomic analysis of intestinal organoids, derived from pigs divergent in feed efficiency, and their response to Escherichia coli.

BACKGROUND: There is increasing interest in using intestinal organoids to study complex traits like feed efficiency (FE) and host-microbe interactions. The aim of this study was to investigate differences in the molecular phenotype of organoids derived from pigs divergent for FE as well as their responses to challenge with adherent and invasive Escherichia coli (E. coli). RESULTS: Colon and ileum tissue from low and high FE pigs was used to generate 3D organoids and two dimensional (2D) monolayers of organoid cells for E. coli challenge. Genome-wide gene expression was used to investigate molecular differences between pigs that were phenotypically divergent for FE and to study the difference in gene expression after challenge with E. coli. We showed, (1) minor differences in gene expression of colon organoids from pigs with low and high FE phenotypes, (2) that an E. coli challenge results in a strong innate immune gene response in both colon and ileum organoids, (3) that the immune response seems to be less pronounced in the colon organoids of high FE pigs and (4) a slightly stronger immune response was observed in ileum than in colon organoids. CONCLUSIONS: These findings demonstrate the potential for using organoids to gain insights into complex biological mechanisms such as FE.

Organoids: a promising new in vitro platform in livestock and veterinary research.

Organoids are self-organizing, self-renewing three-dimensional cellular structures that resemble organs in structure and function. They can be derived from adult stem cells, embryonic stem cells, or induced pluripotent stem cells. They contain most of the relevant cell types with a topology and cell-to-cell interactions resembling that of the in vivo tissue. The widespread and increasing adoption of organoid-based technologies in human biomedical research is testament to their enormous potential in basic, translational- and applied-research. In a similar fashion there appear to be ample possibilities for research applications of organoids from livestock and companion animals. Furthermore, organoids as in vitro models offer a great possibility to reduce the use of experimental animals. Here, we provide an overview of studies on organoids in livestock and companion animal species, with focus on the methods developed for organoids from a variety of tissues/organs from various animal species and on the applications in veterinary research. Current limitations, and ongoing research to address these limitations, are discussed. Further, we elaborate on a number of fields of research in animal nutrition, host-microbe interactions, animal breeding and genomics, and animal biotechnology, in which organoids may have great potential as an in vitro research tool.

Dietary strategies can increase cloacal endotoxin levels and modulate the resident microbiota in broiler chickens.

Endotoxins released from poultry feces have been associated with impaired human health. Because endotoxins are released from gram-negative intestinal bacteria, it was hypothesized that dietary strategies may influence endotoxin excretion via modulation of gut microbiota. We therefore tested dietary strategies that could potentially reduce cloacal endotoxin levels in broiler chickens. One-day-old male Ross 308 (N = 1,344) broilers were housed in 48 pens (N = 8 pens/treatment, 28 chickens per pen) and fed 1 of 6 diets for 35 days (d) in a 3-phase feeding program: a basic diet (CON) that served as the reference diet, or basic diet supplemented with butyrate (BUT), inulin (INU), medium-chain fatty acids (MCFA) or Original XPC™LS (XPC), or a high-fiber-low-protein (HF-LP) diet. A significant (P < 0.05) increase in cloacal endotoxin concentration at d 35 was observed in BUT as compared to CON. Analysis of cloacal microbiota showed a trend (P < 0.07) for a higher gram-negative/gram-positive ratio and for a higher relative abundance of gram-negative bacteria at d 35 (P ≤ 0.08) in BUT and HF-LP as compared to CON. A significant (P < 0.05) increase in average daily gain (ADG) and improved feed conversion ratio (P < 0.05) were observed in MCFA during the grower phase (d 14-28), and a significant (P < 0.05) increase in average daily feed intake (ADFI) was observed in MCFA during d 0 to 28. Broilers fed HF-LP had a significantly (P < 0.05) higher FCR and lower ADG throughout the rearing period. No treatment effects were found on footpad dermatitis, but BUT had worst hock burn scores at d 35 (P < 0.01) and MCFA had worst cleanliness scores at d 21 but not at d 35 (treatment*age P < 0.05), while INU had better cleanliness as compared to CON at d 35 (P < 0.05). In conclusion, especially BUT and HF-LP were able to modulate resident microbiota and BUT also increased cloacal endotoxin levels, which was opposite to our hypothesis. The present study indicates that cloacal endotoxin release can be affected by the diet but further study is needed to find dietary treatments that can reduce cloacal endotoxin release.

Dietary supplementation of zinc oxide modulates intestinal functionality during the post-weaning period in clinically healthy piglets.

BACKGROUND: To improve our understanding of host and intestinal microbiome interaction, this research investigated the effects of a high-level zinc oxide in the diet as model intervention on the intestinal microbiome and small intestinal functionality in clinically healthy post-weaning piglets. In study 1, piglets received either a high concentration of zinc (Zn) as zinc oxide (ZnO, Zn, 2,690 mg/kg) or a low Zn concentration (100 mg/kg) in the diet during the post weaning period (d 14-23). The effects on the piglet's small intestinal microbiome and functionality of intestinal tissue were investigated. In study 2, the impact of timing of the dietary zinc intervention was investigated, i.e., between d 0-14 and/or d 14-23 post weaning, and the consecutive effects on the piglet's intestinal functionality, here referring to microbiota composition and diversity and gene expression profiles. RESULTS: Differences in the small intestinal functionality were observed during the post weaning period between piglets receiving a diet with a low or high concentration ZnO content. A shift in the microbiota composition in the small intestine was observed that could be characterized as a non-pathological change, where mainly the commensals inter-changed. In the immediate post weaning period, i.e., d 0-14, the highest number of differentially expressed genes (DEGs) in intestinal tissue were observed between animals receiving a diet with a low or high concentration ZnO content, i.e., 23 DEGs in jejunal tissue and 11 DEGs in ileal tissue. These genes are involved in biological processes related to immunity and inflammatory responses. For example, genes CD59 and REG3G were downregulated in the animals receiving a diet with a high concentration ZnO content compared to low ZnO content in both jejunum and ileum tissue. In the second study, a similar result was obtained regarding the expression of genes in intestinal tissue related to immune pathways when comparing piglets receiving a diet with a high concentration ZnO content compared to low ZnO content. CONCLUSIONS: Supplementing a diet with a pharmaceutical level of Zn as ZnO for clinically healthy post weaning piglets influences various aspects intestinal functionality, in particular in the first two weeks post-weaning. The model intervention increased both the alpha diversity of the intestinal microbiome and the expression of a limited number of genes linked to the local immune system in intestinal tissue. The effects do not seem related to a direct antimicrobial effect of ZnO.

Sanitary Conditions on the Farm Alters Fecal Metabolite Profile in Growing Pigs.

The aim of this study was to use fecal metabolite profiling to evaluate the effects of contrasting sanitary conditions and the associated subclinical health status of pigs. We analyzed fecal metabolite profiles by nuclear magnetic resonance (1H NMR) from pigs aged 14 and 22 weeks. Pigs kept under low and high sanitary conditions differed in fecal metabolites related to the degradation of dietary starch, metabolism of the gut microbiome, and degradation of components of animal (host) origin. The metabolites that differed significantly (FDR < 0.1) were from metabolic processes involved in either maintaining nutrient digestive capacity, including purine metabolism, energy metabolism, bile acid breakdown and recycling, or immune system metabolism. The results show that the fecal metabolite profiles reflect the sanitary conditions under which the pigs are kept. The fecal metabolite profiles closely resembled the profiles of metabolites found in the colon of pigs. Fecal valerate and kynurenic acid could potentially be used as "non-invasive" biomarkers of immune or inflammatory status that could form the basis for monitoring subclinical health status in pigs.

Local intestinal microbiota response and systemic effects of feeding black soldier fly larvae to replace soybean meal in growing pigs.

Black soldier fly (Hermetia illucens; BSF) larvae as dietary protein source have the ability to deliver nutrients and could possess functional properties that positively support animal productivity and health. More knowledge, however, is needed to assess the impact of feeding a BSF based diet on gut and animal health. Sixteen post-weaned male pigs were randomly assigned to two groups and fed for three weeks with iso-caloric and iso-proteinaceous experimental diets prepared with either soybean meal (SBM) as reference protein source or with BSF as single source of dietary protein. At the end of the trial, the pigs were sacrificed to collect relevant digesta, gut tissue and blood samples to study changes induced by the dietary treatments using ~ omics based analyses. Inclusion of BSF in the diet supports the development of the intestinal microbiome that could positively influence intestinal health. By amine metabolite analysis, we identified two metabolites i.e. sarcosine and methionine sulfoxide, in plasma that serve as markers for the ingestion of insect based ingredients. BSF seems to possess functional properties indicated by the appearance of alpha-aminobutyric acid and taurine in blood plasma of pigs that are known to induce health beneficial effects.

Effects of undigested protein-rich ingredients on polarised small intestinal organoid monolayers.

ABSTRACT: Here, we describe the use of monolayers of intestinal epithelial cells derived from intestinal organoids and transcriptomics to investigate the direct effects of dietary protein sources on epithelial function. Mechanically dissociated 3D organoids of mouse duodenum were used to generate a polarized epithelium containing all cell types found in the tissue of origin. The organoid-derived cell monolayers were exposed to 4% (w/v) of 'undigested (non-hydrolysed)-soluble' fraction of protein sources used as feed ingredients [soybean meal (SBM) and casein], or alternative protein sources (spray dried plasma protein, and yellow meal worm), or controls for 6 h prior to RNA isolation and transcriptomics. All protein sources altered expression of unique biological processes in the epithelial cells. Exposure of intestinal organoids to SBM downregulated expression of retinol and retinoid metabolic processes as well as cholesterol and lipid biosynthetic pathways, consistent with the reported hypotriglyceridaemic effect of soy protein in vivo. These findings support the use of intestinal organoids as models to evaluate complex interactions between dietary ingredients and the intestinal epithelium and highlights some unique host effects of alternative protein sources in animal feed and potentially human food. GRAPHICAL ABSTRACT: Schematic representation of the study. 3-dimensional organoids were generated from mouse duodenum (1). The organoids were subsequently dissociated into single cells (2) and grown as 2-dimensional polarised monolayers (3). Polarized monolayers of organoid cells were exposed to different protein sources [CAS, SBM, SDPP, YMW, or medium control (MC)] for 6 h (4) and further processed for imaging (5) gene expression (6), and biochemical assays (7), to investigate the effects of undigested protein sources on the duodenal epithelium.

Amine Metabolism Is Influenced by Dietary Protein Source.

Growth in world population will inevitably leads to increased demand for protein for humans and animals. Protein from insects and blood plasma are being considered as possible alternatives, but more research on their nutritional quality and health effects is needed. Here, we studied the effect of dietary protein source on metabolism and metabolic amine profiles in serum and urine of mice. Groups of mice were fed semi-purified diets containing 300 g/kg of soybean meal, casein, partially delactosed whey powder, spray-dried plasma protein, wheat gluten meal, and yellow mealworm. Feed and water intake as well as body weight gain were measured for 28 days. After 14 and 28 days, serum and urine samples were collected for measurement of a large panel of amine metabolites. MetaboAnalyst 3.0 was used for analysis of the raw metabolic data. Out of 68 targeted amine metabolites, we could detect 54 in urine and 41 in blood serum. Dietary protein sources were found to have profound effects on host metabolism, particularly in systemic amine profiles, considered here as an endophenotype. We recommend serum over urine to screen for the amine metabolic endophenotype based on partial least squares discriminant analysis. We concluded that metabolites like alpha-aminobutyric acid and 1-methylhistidine are sensitive indicators of too much or too little availability of specific amino acids in the different protein diets. Furthermore, we concluded that amine metabolic profiles can be useful for assessing the nutritional quality of different protein sources.

Multi-Level Integration of Environmentally Perturbed Internal Phenotypes Reveals Key Points of Connectivity between Them.

The genotype and external phenotype of organisms are linked by so-called internal phenotypes which are influenced by environmental conditions. In this study, we used five existing -omics datasets representing five different layers of internal phenotypes, which were simultaneously measured in dietarily perturbed mice. We performed 10 pair-wise correlation analyses verified with a null model built from randomized data. Subsequently, the inferred networks were merged and literature mined for co-occurrences of identified linked nodes. Densely connected internal phenotypes emerged. Forty-five nodes have links with all other data-types and we denote them "connectivity hubs." In literature, we found proof of 6% of the 577 connections, suggesting a biological meaning for the observed correlations. The observed connectivities between metabolite and cytokines hubs showed higher numbers of literature hits as compared to the number of literature hits on the connectivities between the microbiota and gene expression internal phenotypes. We conclude that multi-level integrated networks may help to generate hypotheses and to design experiments aiming to further close the gap between genotype and phenotype. We describe and/or hypothesize on the biological relevance of four identified multi-level connectivity hubs.

Dietary protein sources differentially affect microbiota, mTOR activity and transcription of mTOR signaling pathways in the small intestine.

Dietary protein sources can have profound effects on host-microbe interactions in the gut that are critically important for immune resilience. However more knowledge is needed to assess the impact of different protein sources on gut and animal health. Thirty-six wildtype male C57BL/6J mice of 35 d age (n = 6/group; mean ± SEM body weight 21.9 ± 0.25 g) were randomly assigned to groups fed for four weeks with semi synthetic diets prepared with one of the following protein sources containing (300 g/kg as fed basis): soybean meal (SBM), casein, partially delactosed whey powder, spray dried plasma protein, wheat gluten meal and yellow meal worm. At the end of the experiment, mice were sacrificed to collect ileal tissue to acquire gene expression data, and mammalian (mechanistic) target of rapamycin (mTOR) activity, ileal digesta to study changes in microbiota and serum to measure cytokines and chemokines. By genome-wide transcriptome analysis, we identified fourteen high level regulatory genes that are strongly affected in SBM-fed mice compared to the other experimental groups. They mostly related to the mTOR pathway. In addition, an increased (P < 0.05) concentration of granulocyte colony-stimulating factor was observed in serum of SBM-fed mice compared to other dietary groups. Moreover, by 16S rRNA sequencing, we observed that SBM-fed mice had higher (P < 0.05) abundances of Bacteroidales family S24-7, compared to the other dietary groups. We showed that measurements of genome-wide expression and microbiota composition in the mouse ileum reveal divergent responses to diets containing different protein sources, in particular for a diet based on SBM.