Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

Gut dysbiosis might underlie the pathogenesis of type 1 diabetes. In mice of the non-obese diabetic (NOD) strain, we found that key features of disease correlated inversely with blood and fecal concentrations of the microbial metabolites acetate and butyrate. We therefore fed NOD mice specialized diets designed to release large amounts of acetate or butyrate after bacterial fermentation in the colon. Each diet provided a high degree of protection from diabetes, even when administered after breakdown of immunotolerance. Feeding mice a combined acetate- and butyrate-yielding diet provided complete protection, which suggested that acetate and butyrate might operate through distinct mechanisms. Acetate markedly decreased the frequency of autoreactive T cells in lymphoid tissues, through effects on B cells and their ability to expand populations of autoreactive T cells. A diet containing butyrate boosted the number and function of regulatory T cells, whereas acetate- and butyrate-yielding diets enhanced gut integrity and decreased serum concentration of diabetogenic cytokines such as IL-21. Medicinal foods or metabolites might represent an effective and natural approach for countering the numerous immunological defects that contribute to T cell-dependent autoimmune diseases.

Erratum: Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

This corrects the article DOI: 10.1038/ni.3713.

Transcriptional network analysis of peripheral blood leukocyte subsets in multiple sclerosis identifies a pathogenic role for a cytotoxicity-associated gene network in myeloid cells.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system affecting predominantly adults. It is a complex disease associated with both environmental and genetic risk factors. Although over 230 risk single-nucleotide polymorphisms have been associated with MS, all are common human variants. The mechanisms by which they increase the risk of MS, however, remain elusive. We hypothesized that a complex genetic phenotype such as MS could be driven by coordinated expression of genes controlled by transcriptional regulatory networks. We, therefore, constructed a gene coexpression network from microarray expression analyses of five purified peripheral blood leukocyte subsets of 76 patients with relapsing remitting MS and 104 healthy controls. These analyses identified a major network (or module) of expressed genes associated with MS that play key roles in cell-mediated cytotoxicity which was downregulated in monocytes of patients with MS. Manipulation of the module gene expression was achieved in vitro through small interfering RNA gene knockdown of identified drivers. In a mouse model, network gene knockdown modulated the autoimmune inflammatory MS model disease-experimental autoimmune encephalomyelitis. This research implicates a cytotoxicity-associated gene network in myeloid cells in the pathogenesis of MS.

Pioneering gut health improvements in piglets with phytogenic feed additives.

This research investigates the effects of phytogenic feed additives (PFAs) on the growth performance, gut microbial community, and microbial metabolic functions in weaned piglets via a combined 16S rRNA gene amplicon and shotgun metagenomics approach. A controlled trial was conducted using 200 pigs to highlight the significant influence of PFAs on gut microbiota dynamics. Notably, the treatment group revealed an increased gut microbiota diversity, as measured with the Shannon and Simpson indices. The increase in diversity is accompanied by an increase in beneficial bacterial taxa, such as Roseburia, Faecalibacterium, and Prevotella, and a decline in potential pathogens like Clostridium sensu stricto 1 and Campylobacter. Shotgun sequencing at the species level confirmed these findings. This modification in microbial profile was coupled with an altered profile of microbial metabolic pathways, suggesting a reconfiguration of microbial function under PFA influence. Significant shifts in overall microbial community structure by week 8 demonstrate PFA treatment's temporal impact. Histomorphological examination unveiled improved gut structure in PFA-treated piglets. The results of this study indicate that the use of PFAs as dietary supplements can be an effective strategy, augmenting gut microbiota diversity, reshaping microbial function, enhancing gut structure, and optimising intestinal health of weaned piglets providing valuable implications for swine production. KEY POINTS: • PFAs significantly diversify the gut microbiota in weaned piglets, aiding balance. • Changes in gut structure due to PFAs indicate improved resistance to weaning stress. • PFAs show potential to ease weaning stress, offering a substitute for antibiotics in piglet diets.

Precision glycan supplementation: A strategy to improve performance and intestinal health of laying hens in high-stress commercial environments.

In the dynamic world of animal production, many challenges arise in disease control, animal welfare and the need to meet antibiotic-free demands. Emerging diseases have a significant impact on the poultry industry. Managing gut microbiota is an important determinant of poultry health and performance. Introducing precision glycans as feed additives adds another dimension to this complex environment. The glycans play pivotal roles in supporting gut health and immunological processes and are likely to limit antibiotic usage while enhancing intestinal well-being and overall poultry performance. This study explores precision glycan product as a feed additive supplemented at a continuous dose of 900 g per tonne of feed, in a free-range production system on a large commercial farm. Forty thousand 17-week-old pullets were randomly allocated to one of two separated sections of the production shed, with individual silos and egg-collecting belts. The flock performance, gut microbiota and its functionality were analysed throughout the laying cycle until 72 weeks of age. The results demonstrated that introducing precision glycans improved a range of performance indicators, including reduced cumulative mortality, especially during a major smothering event, where the birds pile up until they suffocate. There was also significantly increased hen-housed egg production, reduced gut dysbiosis score and undigested feed, increased number of goblet cells and improved feed conversion ratio. Additionally, microbiota analysis revealed significant changes in the composition of the gizzard, ileum content, ileum mucosa, and caecal and cloacal regions. Overall, the findings suggest that precision glycans have the potential to enhance poultry egg production in challenging farming environments.

Phytogen Improves Performance during Spotty Liver Disease by Impeding Bacterial Metabolism and Pathogenicity.

A range of antibiotic alternative products is increasingly studied and manufactured in the current animal agriculture, particularly in the poultry industry. Phytogenic feed additives are known for their remarkable ability to suppress pathogens such as Clostridium spp., Escherichia coli, and Salmonella. Other than enhancing biosecurity, improvements in productivity and performance were also observed. However, clear mechanisms for these improvements were not established. In this study, 20,000 Lohman-Brown layers were provided with phytogenic supplement from 16 to 40 weeks of age, and performance parameters were assessed against the same number of unsupplemented control birds. The performance results showed that the birds with phytogenic supplementation presented consistently reduced mortality, increased rate of lay, and increased average egg weight. Functional analysis through shotgun sequencing of cecal metagenomes confirmed a substantial functional shift in the microbial community, showing that phytogen significantly reduced the range of microbial functions, including the production of essential vitamins, cofactors, energy, and amino acids. Functional data showed that phytogen supplementation induced a phenotypic shift in intestinal bacteria LPS phenotype toward the less pathogenic form. The study corroborates the use of phytogenic products in antibiotic-free poultry production systems. The productivity improvements in the number and weight of eggs produced during Spotty Liver Disease justify further optimizing phytogenic alternatives for use in high-risk open and free-range poultry systems. IMPORTANCE The present study establishes the beneficial effects of the continuous phytogenic supplementation reflected in reduced diarrhea and mortality and higher egg productivity under normal conditions and during a natural outbreak of Spotty Liver Disease. Our data points to the importance of phytogen-driven alteration of microbial pathogenicity and fitness-related functional capabilities revealed on the commercial layer farm. Phytogenic product showed an ability to improve the bird's welfare and sustainability in free-range poultry production systems.

Rapid growth of antimicrobial resistance: the role of agriculture in the problem and the solutions.

The control of infectious diseases has always been a top medical priority. For years during the so-called antibiotic era, we enjoyed prolonged life expectancy and the benefits of superior pathogen control. The devastating failure of the medical system, agriculture and pharmaceutical companies and the general population to appreciate and safeguard these benefits is now leading us into a grim post-antibiotic era. Antimicrobial resistance (AMR) refers to microorganisms becoming resistant to antibiotics that were designed and expected to kill them. Prior to the COVID-19 pandemic, AMR was recognised by the World Health Organization as the central priority area with growing public awareness of the threat AMR now presents. The Review on Antimicrobial Resistance, a project commissioned by the UK government, predicted that the death toll of AMR could be one person every 3 seconds, amounting to 10 million deaths per year by 2050. This review aims to raise awareness of the evergrowing extensiveness of antimicrobial resistance and identify major sources of this adversity, focusing on agriculture's role in this problem and its solutions. KEYPOINTS: • Widespread development of antibiotic resistance is a major global health risk. • Antibiotic resistance is abundant in agricultural produce, soil, food, water, air and probiotics. • New approaches are being developed to control and reduce antimicrobial resistance.

Temporal dynamics of gut microbiota in caged laying hens: a field observation from hatching to end of lay.

Gut health has major implications for the general health of food-producing animals such as the layer birds used in the egg industry. In order to modulate gut microbiota for the benefit of gut health, an understanding of the dynamics and details of the development of gut microbiota is critical. The present study investigated the phylogenetic composition of the gut microbiota of a commercial layer flock raised in cages from hatch to the end of the production cycle. This study also aimed to understand the establishment and development of gut microbiota in layer chickens. Results showed that the faecal microbiota was dominated by phyla Firmicutes and Proteobacteria in the rearing phase, but Bacteroidetes in mid lay and late lay phase. The gut microbiota composition changed significantly during the transfer of the flock from the rearing to the production shed. The richness and diversity of gut microbiota increased after week 6 of the flocks age and stabilized in the mid and late lay phase. The overall dynamics of gut microbiota development was similar to that reported in earlier studies, but the phylogenetic composition at the phylum and family level was different. The production stage of the birds is one of the important factors in the development of gut microbiota. This study has contributed to a better understanding of baseline gut microbiota development over the complete life cycles in layer chickens and will help to develop strategies to improve the gut health. KEY POINTS: • Faecal microbiota of caged hens was dominated by phyla Firmicutes and Proteobacteria in the rearing phase. • The gut microbiota composition changed significantly during the transfer of the flock from the rearing to the production shed. • The richness and diversity of gut microbiota increased after week 6 of the flocks age and stabilized in the mid and late lay phase.

The Gut Microbiota of Laying Hens and Its Manipulation with Prebiotics and Probiotics To Enhance Gut Health and Food Safety.

The microbiota plays a vital role in maintaining gut health and influences the overall performance of chickens. Most gut microbiota-related studies have been performed in broilers, which have different microbial communities compared to those of layers. The normal gut microbiota of laying chickens is dominated by Proteobacteria, Firmicutes, Bacteroidetes, Fusobacteria, and Actinobacteria at the phylum level. The composition of the gut microbiota changes with chicken age, genotype, and production system. The metabolites of gut microbiota, such as short-chain fatty acids, indole, tryptamine, vitamins, and bacteriocins, are involved in host-microbiota cross talk, maintenance of barrier function, and immune homeostasis. Resident gut microbiota members also limit and control the colonization of foodborne pathogens. In-feed supplementations of prebiotics and probiotics strengthen the gut microbiota for improved host performance and colonization resistance to gut pathogens, such as Salmonella and Campylobacter The mechanisms of action of prebiotics and probiotics come through the production of organic acids, activation of the host immune system, and production of antimicrobial agents. Probiotic candidates, including Lactobacillus, Bifidobacterium, Bacillus, Saccharomyces, and Faecalibacterium isolates, have shown promising results toward enhancing food safety and gut health. Additionally, a range of complex carbohydrates, including mannose oligosaccharides, fructo-oligosaccharides, and galacto-oligosaccharides, and inulin are promising candidates for improving gut health. Here, we review the potential roles of prebiotics and probiotics in the reshaping of the gut microbiota of layer chickens to enhance gut health and food safety.

Phytogenic products, used as alternatives to antibiotic growth promoters, modify the intestinal microbiota derived from a range of production systems: an in vitro model.

The removal of antibiotics from the feeds used in the livestock industry has resulted in the use of a wide range of alternative antimicrobial products that aim to deliver the productivity and health benefits that have traditionally been associated with antibiotics. Amongst the most popular alternatives are phytogenic product-based extracts from herbs and spices with known antimicrobial properties. Despite embracing such alternatives, the industry is still largely unaware of modes of action, their overall effects on animal health, and interactions with other feed additives such as probiotics. To address some of these issues, three phytogenic products were selected and their interactions with caecal microbiota of layers, grown under six different production systems, were investigated in vitro. Caecal microbiotas were grown with and without phytogenic products, and the changes in microbiota composition were monitored by sequencing of 16S rRNA gene amplicons. Phytogenic products and production system both significantly influenced microbiota composition. The three phytogenic products all altered the relative abundance of species within the Lactobacillus genus, by promoting the growth of some and inhibiting other Lactobacillus species. There were also significant alterations in the Bacillus genus. This was further investigated by comparing the effects of the phytogenic products on the growth of a commercially used Bacillus-based probiotic. The phytogens affected the probiotic mix differently, with some promoting the growth of Bacillus sp. at lower phytogenic concentrations, and fully suppressing growth at higher concentrations, indicating the importance of finding an optimal concentration that can control pathogens while promoting beneficial bacteria. KEY POINTS: • After removal of antibiotics from animal feed, urgent solutions for pathogen control were needed. • Alternative products entered the market without much knowledge on their effects on animal health. • Probiotic products are used in combination with phytogens despite the possible incompatibility.

Characterisation of the intestinal microbiota of commercially farmed saltwater crocodiles, Crocodylus porosus.

The Australian saltwater crocodile (Crocodylus porosus) industry began commercially in the 1980s, producing skins for export and crocodile meat as a by-product. Industry research has thus far focused on strategies to improve production efficiency. In the current study, we utilised 16S rRNA sequencing to characterise the intestinal microbiome of Australian saltwater crocodiles. Samples were collected from 13 commercially farmed crocodiles from six sample sites along the length of the intestinal tract. The results indicate a similar microbiome composition to that found in the freshwater alligator, with the dominate phyla represented by Firmicutes, primarily Clostridia, and Fusobacteria, which appears to be distinct from mammalian, fish, and other reptile phyla which are generally dominated by Firmicutes and Bacteroidetes. The high abundance of 'pathogenic' bacteria, with no apparent consequence to the host's health, is of great interest and warrants further additional investigation. This will enable expansion of the current understanding of host immune function and how it is modified by host and intestinal microbiome interactions.

Selenium nanoparticles in poultry feed modify gut microbiota and increase abundance of Faecalibacterium prausnitzii.

The poultry industry aims to improve productivity while maintaining the health and welfare of flocks. Pathogen control has been achieved through biosecurity, vaccinations and the use of antibiotics. However, the emergence of antibiotic resistance, in animal and human pathogens, has prompted researchers and chicken growers alike to seek alternative approaches. The use of new and emerging approaches to combat pathogen activity including nanotechnology, in particular, silver nanoparticles (NPs), has been found to not only eradicate pathogenic bacteria but also include issues of toxicity and bioaccumulation effects. Other novel metal nanoparticles could provide this pathogen reducing property with a more tailored and biocompatible nanomaterial for the model used, something our study represents. This study investigated the benefits of nanomaterial delivery mechanisms coupled with important health constituents using selenium as a biocompatible metal to minimise toxicity properties. Selenium NPs were compared to two common forms of bulk selenium macronutrients already used in the poultry industry. An intermediate concentration of selenium nanoparticles (0.9 mg/kg) demonstrated the best performance, improving the gut health by increasing the abundance of beneficial bacteria, such as Lactobacillus and Faecalibacterium, and short-chain fatty acids (SCFAs), in particular butyric acid. SCFAs are metabolites produced by the intestinal tract and are used as an energy source for colonic cells and other important bodily functions. Selenium nanoparticles had no significant effect on live weight gain or abundance of potentially pathogenic bacteria.

Correlations between intestinal innate immune genes and cecal microbiota highlight potential for probiotic development for immune modulation in poultry.

Immune function is influenced by the diversity and stability of the intestinal microbiota. A likely trade-off of immune function for growth has been demonstrated in heavier breeds of poultry that have been genetically selected for growth and feed efficiency traits. We investigated the expression of selected innate immune genes and genes encoding products involved in intestinal barrier function to determine whether function changes could be consistently linked to the phenotypic expression of feed conversion ratio (FCR), a common measure of performance within poultry broiler flocks. In addition, we compared individual cecal microbial composition with innate immune gene expression. Samples were utilised from two replicate trials termed P1E1 and P1E2. High (n = 12) and low (n = 12) performing birds were selected based on their individual FCR data from each replicate and combined for microbiota phylogenetic composition and immune gene expression analysis. Toll-like receptor 1 (TLR1La) and zonula occludens 1 (ZO1) were differentially expressed between high- and low-performing broilers. Several taxa were correlated with FCR; of these, unclassified YS2 and ZO1 were also positively correlated with each other. Interactions between taxa and differentially expressed innate immune genes between P1E1 and P1E2 were much greater compared to relationships between high- and low-performing birds. At the level of phylum, reciprocal correlations between tight junction proteins and Toll-like receptors with Bacteroidetes and Firmicutes were evident, as were correlations at the genus level.

Understanding the mechanisms of zinc bacitracin and avilamycin on animal production: linking gut microbiota and growth performance in chickens.

Unravelling the mechanisms of how antibiotics influence growth performance through changes in gut microbiota can lead to the identification of highly productive microbiota in animal production. Here we investigated the effect of zinc bacitracin and avilamycin on growth performance and caecal microbiota in chickens and analysed associations between individual bacteria and growth performance. Two trials were undertaken; each used 96 individually caged 15-day-old Cobb broilers. Trial 1 had a control group (n = 48) and a zinc bacitracin (50 ppm) treatment group (n = 48). Trial 2 had a control group (n = 48) and an avilamycin (15 ppm) treatment group (n = 48). Chicken growth performance was evaluated over a 10-day period, and caecal microbiota was characterised by sequencing of bacterial 16S rRNA gene amplicons. Avilamycin produced no effect on growth performance and exhibited little significant disturbance of the microbiota structure. However, zinc bacitracin reduced the feed conversion ratio (FCR) in treated birds, changed the composition and increased the diversity of their caecal microbiota by reducing dominant species. Avilamycin only produced minor reductions in the abundance of two microbial taxa, whereas zinc bacitracin produced relatively large shifts in a number of taxa, primarily Lactobacillus species. Also, a number of phylotypes closely related to lactobacilli species were positively or negatively correlated with FCR values, suggesting contrasting effects of Lactobacillus spp. on chicken growth performance. By harnessing such bacteria, it may be possible to develop high-productivity strategies in poultry that rely on the use of probiotics and less on in-feed antibiotics.

Effects of concentration of corn distillers dried grains with solubles and enzyme supplementation on cecal microbiota and performance in broiler chickens.

With the increasing production of ethanol for biofuels, a by-product of corn-based ethanol fermentation, dried distillers grains with solubles (DDGS) is finding its way into the feed of agricultural animals including cattle, pigs, poultry, sheep, goats, aquaculture species and horses. Corn DDGS contains very high levels of non-starch polysaccharides and could be considered a good source of fibre. Despite knowledge of the role of the fibre in modulating intestinal microbiota and consequently influencing health, there is currently little information on the interactions between DDGS and intestinal microbiota. We assessed the changes in the cecal microbiota of broilers feed rations supplemented with DDGS (five concentrations: 0, 6, 12, 18 and 24% w/w) with and without presence of digestive enzymes. DDGS concentration was strongly positively correlated (P = 3.7e-17, r = 0.74) with feed conversion efficiency (FCR), diminishing broiler performance with higher concentrations. Additionally, DDGS concentrations positively correlated with Richness index (P = 1.5e-3, r = 0.5), increasing the number of detectable species in the cecum. Among the most affected genera, Faecalibacterium (P = 0.032, r = -0.34) and Streptococcus (P = 7.9e-3, r = -0.39) were negatively correlated with DDGS, while Turicibacter (P = 2.8e-4, r = 0.52) was positively correlated with the DDGS concentration. Enzymes showed minimal effect on cecal microbiota.

The gastrointestinal tract microbiota of the Japanese quail, Coturnix japonica.

Microbiota in the gastrointestinal tract (GIT) plays an essential role in the health and well-being of the host. With the exception of chickens, this area has been poorly studied within birds. The avian GIT harbours unique microbial communities. Birds require rapid energy bursts to enable energy-intensive flying. The passage time of feed through the avian GIT is only 2-3.5 h, and thus requires the presence of microbiota that is extremely efficient in energy extraction. This investigation has used high-throughput 16S rRNA gene sequencing to explore the GIT microbiota of the flighted bird, the Japanese quail (Coturnix japonica). We are reporting, for the first time, the diversity of bacterial phylotypes inhabiting all major sections of the quail GIT including mouth, esophagus, crop, proventriculus, gizzard, duodenum, ileum, cecum, large intestine and feces. Nine phyla of bacteria were found in the quail GIT; however, their distribution varied significantly between GIT sections. Cecal microbiota was the most highly differentiated from all the other communities and showed highest richness at an OTU level but lowest richness at all other taxonomic levels being comprised of only 15 of total 57 families in the quail GIT. Differences were observed in the presence and absence of specific phylotypes between sexes in most sections.

Comparison of fecal and cecal microbiotas reveals qualitative similarities but quantitative differences.

BACKGROUND: The majority of chicken microbiota studies have used the ceca as a sampling site due to the specific role of ceca in chicken productivity, health and wellbeing. However, sampling from ceca and other gastrointestinal tract sections requires the bird to be sacrificed. In contrast, fecal sampling does not require sacrifice and thus allows the same bird to be sampled repeatedly over time. This is a more meaningful and preferred way of sampling as the same animals can be monitored and tracked for temporal studies. The commonly used practice of selecting a subset of birds at each time-point for sacrifice and sampling introduces added variability due to the known animal to animal variation in microbiota. RESULTS: Cecal samples and fecal samples via cloacal swab were collected from 163 birds across 3 replicate trials. DNA was extracted and 16S rRNA gene sequences amplified and pyrosequenced to determine and compare the phylogenetic profile of the microbiota within each sample. The fecal and cecal samples were investigated to determine to what extent the microbiota found in fecal samples represented the microbiota of the ceca. It was found that 88.55% of all operational taxonomic units (OTUs), containing 99.25% of all sequences, were shared between the two sample types, with OTUs unique for each sample type found to be very rare. There was a positive correlation between cecal and fecal abundance in the shared sequences, however the two communities differed significantly in community structure, represented as either alpha or beta diversity. The microbial populations present within the paired ceca of individual birds were also compared and shown to be similar. CONCLUSIONS: Fecal sample analysis captures a large percentage of the microbial diversity present in the ceca. However, the qualitative similarities in OTU presence are not a good representation of the proportions of OTUs within the microbiota from each sampling site. The fecal microbiota is qualitatively similar to cecal microbiota but quantitatively different. Fecal samples can be effectively used to detect some shifts and responses of cecal microbiota.

Microbiota of the chicken gastrointestinal tract: influence on health, productivity and disease.

Recent advances in the technology available for culture-independent methods for identification and enumeration of environmental bacteria have invigorated interest in the study of the role of chicken intestinal microbiota in health and productivity. Chickens harbour unique and diverse bacterial communities that include human and animal pathogens. Increasing public concern about the use of antibiotics in the poultry industry has influenced the ways in which poultry producers are working towards improving birds' intestinal health. Effective means of antibiotic-independent pathogen control through competitive exclusion and promotion of good protective microbiota are being actively investigated. With the realisation that just about any change in environment influences the highly responsive microbial communities and with the abandonment of the notion that we can isolate and investigate a single species of interest outside of the community, came a flood of studies that have attempted to profile the intestinal microbiota of chickens under numerous conditions. This review aims to address the main issues in investigating chicken microbiota and to summarise the data acquired to date.

Layer chicken microbiota: a comprehensive analysis of spatial and temporal dynamics across all major gut sections.

BACKGROUND: The gut microbiota influences chicken health, welfare, and productivity. A diverse and balanced microbiota has been associated with improved growth, efficient feed utilisation, a well-developed immune system, disease resistance, and stress tolerance in chickens. Previous studies on chicken gut microbiota have predominantly focused on broiler chickens and have usually been limited to one or two sections of the digestive system, under controlled research environments, and often sampled at a single time point. To extend these studies, this investigation examined the microbiota of commercially raised layer chickens across all major gut sections of the digestive system and with regular sampling from rearing to the end of production at 80 weeks. The aim was to build a detailed picture of microbiota development across the entire digestive system of layer chickens and study spatial and temporal dynamics. RESULTS: The taxonomic composition of gut microbiota differed significantly between birds in the rearing and production stages, indicating a shift after laying onset. Similar microbiota compositions were observed between proventriculus and gizzard, as well as between jejunum and ileum, likely due to their anatomical proximity. Lactobacillus dominated the upper gut in pullets and the lower gut in older birds. The oesophagus had a high proportion of Proteobacteria, including opportunistic pathogens such as Gallibacterium. Relative abundance of Gallibacterium increased after peak production in multiple gut sections. Aeriscardovia was enriched in the late-lay phase compared to younger birds in multiple gut sections. Age influenced microbial richness and diversity in different organs. The upper gut showed decreased diversity over time, possibly influenced by dietary changes, while the lower gut, specifically cecum and colon, displayed increased richness as birds matured. However, age-related changes were inconsistent across all organs, suggesting the influence of organ-specific factors in microbiota maturation. CONCLUSION: Addressing a gap in previous research, this study explored the microbiota across all major gut sections and tracked their dynamics from rearing to the end of the production cycle in commercially raised layer chickens. This study provides a comprehensive understanding of microbiota structure and development which help to develop targeted strategies to optimise gut health and overall productivity in poultry production.