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The microbiota of the gastrointestinal tract influences gut health, which in turn strongly impacts the general health and productivity of laying hens. It is essential to characterise the composition and temporal development of the gut microbiota in healthy layers raised under different management systems, to understand the variations in typical healthy microbiota structure, so that deviations from this might be recognised and correlated with production and health issues when they arise. The present investigation aimed to study the temporal development and phylogenetic composition of the gut microbiota of four commercially raised layer flocks from hatch to end of the production cycle. Non-intrusive faecal sampling was undertaken as a proxy to represent the gut microbiota. Sequencing of 16S rRNA gene amplicons was used to characterise the microbiota. Beta diversity analysis indicated that each faecal microbiota was different across the four flocks and had subtly different temporal development patterns. Despite these inter-flock differences, common patterns of microbiota development were identified. Firmicutes and Proteobacteria were dominant at an early age in all flocks. The microbiota developed gradually during the rearing phase; richness and diversity increased after 42 d of age and then underwent significant changes in composition after the shift to the production farms, with Bacteroidota becoming more dominant in older birds. By developing a more profound knowledge of normal microbiota development in layers, opportunities to harness the microbiota to aid in the management of layer gut health and productivity may be more clearly seen and realised.

Legumes enhance pasture health and soil productivity by fixing atmospheric nitrogen and boosting soil microbiota. We investigated the effects of tropical pasture legumes, including butterfly pea (Clitoria ternatea), seca stylo (Stylosanthes scabra), desmanthus (Desmanthus virgatus), lablab (Lablab purpureus), and Wynn cassia (Chamaecrista rotundifolia), on the soil microbial community and buffel grass (Cenchrus ciliaris) gene expression. Additionally, we explored the impact of a phytogenic bioactive product (PHY) in the coculture system. A pot trial using soil enriched with cow paunch compost included four treatments: monoculture of buffel grass and five legume species with and without PHY supplementation and coculture of buffel grass with each legume species with and without PHY supplementation. Actinobacteriota and Firmicutes were the dominant bacterial phyla. Regardless of PHY application, the coculture of buffel grass with legumes positively influenced microbial composition and diversity. Transcriptomic analysis revealed significant gene expression changes in buffel grass shoots and roots, with each legume uniquely affecting nitrogen metabolism. Lablab and Wynn cassia exhibited similarities in modulating metabolic processes, butterfly pea contributed to mycotoxin detoxification, and desmanthus balanced cell death and growth. Seca stylo enhanced root cell growth and regeneration. These findings offer insights for optimizing legume–grass coculture systems, enhancing soil activity and promoting sustainable agriculture.

Probiotics provided from hatch have a major influence on microbiota development, and together with environmental and bedding microbiota, shape the microbial community of the litter. We investigated the influence of probiotic supplementation and a leaky gut challenge induced using dexamethasone (DEX) on the litter microbial community and litter parameters. The probiotic product was a mix of three Bacillus amyloliquefaciens strains. The litter microbiota were compared to the microbial communities from other gut sections. The litter samples had higher microbial diversity compared to the caecum, gizzard, jejunum, and jejunal mucosa. The high similarity between the litter phylum-level microbiota and gizzard microbiota detected in our study could be a consequence of ingested feed and litter passing through the gizzard. Moreover, the litter microbial community is fundamentally distinct from the intestinal microbiota, as evidenced by the number of genera present in the litter but absent from all the intestinal sections and vice versa. Furthermore, LEfSe analysis identified distinct microbial taxa across different groups, with specific genera associated with different treatments. In terms of litter quality, the birds in the DEX groups had a significantly higher moisture content, indicating successful leaky gut challenge, while probiotic supplementation did not significantly affect the moisture levels. These findings provide comprehensive insights into the distinct microbiota characteristics of litter.

This study explores the impact of a novel phytogenic product containing citric acid, carvacrol, and cinnamaldehyde on buffel grass (Cenchrus ciliaris) seedling emergence. A dilution series of the phytogenic solution revealed a concentration range that promoted seedling emergence, with an optimal concentration of 0.5%. Transcriptomic analysis using RNA-seq was performed to investigate gene expression changes in seedlings under the influence of the phytogenic product. The results revealed that the phytogenic treatment significantly altered the gene expression, with a prevalent boost in transcriptional activity compared to the control. Functional analysis indicated the positive alteration of key metabolic pathways, including the tricarboxylic acid (TCA) cycle, glycolysis, and pentose phosphate pathways. Moreover, pathways related to amino acids, nucleotide biosynthesis, heme biosynthesis, and formyltetrahydrofolate biosynthesis showed substantial modulation. The study provides valuable insights into the molecular mechanisms underlying the phytogenic product’s effects on grass seedling establishment and highlights its ability to promote energy metabolism and essential biosynthetic pathways for plant growth.

Abstract Consumer push towards open and free-range production systems makes biosecurity on farms challenging, leading to increased disease and animal welfare issues. Phytogenic products are increasingly becoming a viable alternative for the use of antibiotics in livestock production. Here we present a study of the effects of commercial phytogenic supplement containing menthol, carvacrol and carvone on intestinal microbiota of layer hens, microbial functional capacity, and intestinal morphology. A total of 40,000 pullets were randomly assigned to two sides of the experimental shed. Growth performance, mortality, egg production and egg quality parameters were recorded throughout the trial period (18–30 weeks of age). Microbial community was investigated using 16S amplicon sequencing and functional difference using metagenomic sequencing. Phytogen supplemented birds had lower mortality and number of dirty eggs, and their microbial communities showed reduced richness. Although phytogen showed the ability to control the range of poultry pathogens, its action was not restricted to pathogenic taxa, and it involved functional remodelling the intestinal community towards increased cofactor production, heterolactic fermentation and salvage and recycling of metabolites. The phytogen did not alter the antimicrobial resistance profile or the number of antibiotic resistance genes. The study indicates that phytogenic supplementation can mimic the action of antibiotics in altering the gut microbiota and be used as their alternative in industry-scale layer production.

The supplementation of antimicrobial growth promoters (AGPs) has been banned in many countries because of the emergence of antimicrobial-resistant pathogens in poultry products and the environment. Probiotics have been broadly studied and demonstrated as a promising AGP substitute. Our study is centred on the effects of a multi-strain Bacillus-based probiotic product on broiler production performance and gut microbial profile in a dexamethasone-induced leaky gut challenge. Two hundred and fifty-six broiler chicks were hatched and randomly assigned into four groups (wheat-soybean meal basal diet (BD) = non-supplemented control (C), BD supplemented with dexamethasone in week 4 (CD), BD containing a probiotic from day one (P), and BD containing a probiotic from day one and supplemented with dexamethasone during challenge week 4 (PD)). The production performance and caecal, gizzard, jejunal lumen and jejunal mucosa swab microbiota were studied by 16S rRNA gene sequencing. The Bacillus probiotic product significantly improved production performance and altered caecal gut microbiota (p ≤ 0.05), but no significant impact on microbiota was observed in other gut sections.

The poultry industry contributes significantly to the global meat industry but faces many production challenges like high-density housing, welfare issues, and pathogenic infections. While antibiotics have commonly been used to treat many of these issues, they are being removed from poultry production globally due to increased microbial resistance. Precision glycans offer a viable alternative to antibiotics by modulating microbial metabolic pathways. In this study, we investigated the effects of precision glycan supplementation on productivity and gut microbiota in broilers. The experiment was conducted in a commercial setting using 32,400 male Ross chickens randomly divided into three sheds with 10,800 birds each. One shed with 12 pen replicates of 900 birds was used as control, while the other two with an equal number of replicates and birds were assigned to precision glycan supplementation. The treatment significantly improved the average daily weight gain and feed conversion ratio, with a significant modification in the abundance of several bacterial taxa in the caecum, ileum, and ileum mucosa microbial communities. There was increased richness and diversity in the caecum, with a reduction in Proteobacteria and an increase in Firmicutes. Richness remained unchanged in the ileum, with an increase in diversity and reduction in pathogenic genera like Clostridium and Escherichia-Shigella. Ileum mucosa showed a lower abundance of mucin degraders and an increased presence of next-generation probiotics. Supplemented birds showed a high level of disease resistance when the farm experienced an outbreak of infectious bronchitis, evidenced by lower mortality. Histological analysis confirmed improvements in the ileum and liver health, where the precision glycan supplementation reduced the area of congested sinusoids compared to the control group in the liver and significantly improved ileum intestinal morphology by increasing crypt depth and surface area. These results collectively suggest that precision glycans offer substantial benefits in poultry production by improving productivity, gut health, and disease resistance.

Pasture dieback is a rapidly expanding decaying pasture syndrome that affects millions of hectares of agricultural land in Queensland, Australia, making it useless for the cattle industry and decimating farmers’ income and welfare. Since the syndrome was first identified in the early 1990s, farmers and agronomists have tried various methods for pasture recovery, including slashing, burning, ploughing and resowing grass, fertilising, destocking, and overstocking. In most cases, after a minimal initial improvement, the grass reverts to dieback within a few weeks. Here, we present an application of potassium humate, a well-known plant growth stimulator, as a possible long-term recovery option. Humate was applied once at the rate of 12 ml per m2. Humate application did not alter the alpha or beta diversity of soil bacterial communities, nor did it change the mineral profile in the soil. However, humate application altered soil microbiota–mineral temporal interactions and introduced subtle changes in the microbial community that could assist pasture recovery. A single humate application increased paddock plant biomass significantly up to 20 weeks post-application. Eleven months after the single application, the paddock was grazed to the ground by the cattle just before the rainfall season. After pasture regrowth, the humate-treated plots significantly improved root morphometric indicators for both grass and dicots and increased the ratio of grass/weeds by 27.6% compared to the water-treated control. While this treatment will not resolve the dieback syndrome, our results invite more research to optimise the use of humate for maximum economic benefit in paddock use under pasture dieback syndrome conditions.

Abstract Sows undergo physiological stress during gestation and lactation, potentially leading to enteric dysbiosis and reduced reproductive potential. Phytogenic additives (PFs) may improve performance via their antioxidant, anti-inflammatory and antimicrobial properties. This study determined whether the provision of a gestation/lactation diet containing PAs would alter the gastrointestinal microbiota of sows and their piglets, and improve performance. Sows received a commercial diet throughout gestation and lactation (CTR; n = 64), a commercial diet throughout gestation and a diet containing PAs in lactation (CTR-PA; n = 63) or a commercial diet containing PAs in gestation and lactation (PA; n = 90). Sows were weighed and backfat recorded after mating and at entry and exit from the farrowing house and piglets were weighed on days 1 and 21 of life. Faecal samples collected from sows at farrowing house entry and piglets at 21 and 35 d were subjected to 16 S rRNA gene amplicon analysis. The addition of PAs to sow diets resulted in more piglets born (P = 0.03), however, it did not improve the number of liveborn piglets (P = 0.14). There were no differences in sow weight, P2 backfat depth or lactation feed intake observed. PAs had no effect on piglet weight or survival to weaning but did alter the faecal microbiota of sows, and this change was observed in piglets at 21 and 35 d. PA supplementation to sows has the potential to increase litter size, while also potentially influencing gastrointestinal tract health of the sow and piglets reared.

Abstract Microbial colonisation of the gastrointestinal tract of newly hatched chicks starts at hatch, seeded from the immediate hatching environment, and quickly results in dense colonisation. The role of ecological factors in gut colonisation has been extensively investigated, as well as the role of micro- and macronutrients in supporting and selecting for bacterial species highly adapted for utilising those nutrients. However, the microbial community contained in poultry feed and its influence on colonisation and maturation of gut microbiota has not been directly addressed. In this study, we compared the microbiota found in poultry feed, with the microbiota of ileum, cecum and excreta, to identify substantial overlap in core microbiotas of the compared groups. We then investigated the microbiota present in raw feedstuffs: meat and bone meal, wheat, corn, canola, barley, soybean, millrun, sorghum, poultry oil, oats, limestone and bloodmeal from four geographically distinct feedstuff suppliers. Each of the feedstuffs had diverse microbial communities. The meat and bone meal and bloodmeal samples had the most complex and distinct microbial populations. There was substantial overlap in the phylogenetic composition found in the grain and seed samples: barley, canola, corn, millrun, oats, sorghum, soybean meal and wheat. Issues related to methodology, viability of microbial communities in the gut and feed, and the implications for biosecurity are discussed.

Pathogen control is a critical issue in the layer industry. Plant-based natural products are firmly replacing the undesirable use of antibiotics in animal production. The poultry industry embraced the opportunity to distance itself from the negative public perception of antibiotic use. In this study, we investigated the effects of a phytogenic product comprising of menthol, carvacrol and carvone on ileum gene expression profile in layers after 16 weeks of continual supplementation. Phytogen supplementation increased endocytosis and autophagy while showing significant predicted cardiovascular protective effects. Statistical comparison with over 100,000 manually curated and comparably reanalysed public datasets suggests that the phytogen effects are highly significantly comparable with transcriptomic effects of clinical antibiotics doxycycline and geldanamycin, and that phytogen can reverse transcriptomic effects of a range of viral diseases and malaria. Our data confirmed the hypothesis that similarly to the original essential oil type antimicrobial constituents of phytogenic products, there may be a range of benefits unrelated to their critical antimicrobial action, contributing to improved bird welfare.

Free-range pullets are reared indoors but the adult hens can go outside which is a mismatch that may reduce adaptation in the laying environment. Rearing enrichments might enhance pullet development and adaptations to subsequent free-range housing with impact on behavior and health measures including gut microbiota. Adult free-range hens vary in range use which may also be associated with microbiota composition. A total of 1,700 Hy-Line Brown® chicks were reared indoors across 16 weeks with three enrichment treatment groups: “control” with standard litter housing, “novelty” with weekly changed novel objects, and “structural” with custom-designed perching structures in the pens. At 15 weeks, 45 pullet cecal contents were sampled before moving 1,386 pullets to the free-range housing system. At 25 weeks, range access commenced, and movements were tracked via radio-frequency identification technology. At 65 weeks, 91 hens were selected based on range use patterns (“indoor”: no ranging; “high outdoor”: daily ranging) across all rearing enrichment groups and cecal contents were collected for microbiota analysis via 16S rRNA amplicon sequencing at V3-V4 regions. The most common bacteria in pullets were unclassified Barnesiellaceae, Prevotella, Blautia and Clostridium and in hens Unclassified, Ruminococcus, unclassified Lachnospiraceae, unclassified Bacteroidales, unclassified Paraprevotellaceae YRC22, and Blautia. The microbial alpha diversity was not significant within the enrichment/ranging groups (pullets: P ≥ 0.17, hen rearing enrichment groups: P ≥ 0.06, hen ranging groups: P ≥ 0.54), but beta diversity significantly varied between these groups (pullets: P ≤ 0.002, hen rearing enrichment groups: P ≤ 0.001, hen ranging groups: P ≤ 0.008). Among the short-chain fatty acids (SCFAs), the propionic acid content was higher (P = 0.03) in the novelty group of pullets than the control group. There were no other significant differences in the SCFA contents between the rearing enrichment groups (all P ≥ 0.10), and the ranging groups (all P ≥ 0.17). Most of the genera identified were more abundant in the indoor than high outdoor hens. Overall, rearing enrichments affected the cecal microbiota diversity of both pullets and adult hens and was able to distinguish hens that remained inside compared with hens that ranging daily for several hours.

Pasture production is vital in cattle farming as it provides animals with food and nutrients. Australia, as a significant global beef producer, has been experiencing pasture dieback, a syndrome of deteriorating grassland that results in the loss of grass and the expansion of weeds. Despite two decades of research and many remediation attempts, there has yet to be a breakthrough in understanding the causes or mechanisms involved. Suggested causes of this phenomenon include soil and plant microbial pathogens, insect infestation, extreme heat stress, radiation, and others. Plants produce a range of phytomolecules with antifungal, antibacterial, antiviral, growth-promoting, and immunostimulant effects to protect themselves from a range of environmental stresses. These products are currently used more in human and veterinary health than in agronomy. In this study, we applied a phytogenic product containing citric acid, carvacrol, and cinnamaldehyde, to investigate its ability to alleviate pasture dieback. The phytogenic liquid-based solution was sprayed twice, one week apart, at 5.4 L per hectare. The soil microbial community was investigated longitudinally to determine long-term effects, and pasture productivity and plant morphometric improvements were explored. The phytogenic liquid significantly improved post-drought recovery of alpha diversity and altered temporal and spatial change in the community. The phytogenic liquid reduced biomarker genera associated with poor and polluted soils and significantly promoted plant and soil beneficial bacteria associated with plant rhizosphere and a range of soil benefits. Phytogenic liquid application produced plant morphology improvements and a consistent enhancement of pasture productivity extending beyond 18 months post-application. Our data show that phytogenic products used in the livestock market as an alternative to antibiotics may also have a beneficial role in agriculture, especially in the light of climate change-related soil maintenance and remediation.

Pasture dieback is a syndrome of unknown cause affecting grasses in Australia, creating significant economic losses to farmers by reducing available livestock feed and paddock carrying capacity. RC3 is a commercial plant growth stimulant tri-sodium salt of trimercapto-S-triazine (TMT) and potassium humate as active ingredients. TMT is commonly used for soil and wastewater remediation by capturing and binding heavy metals, while potassium humate is an organic compound used as a plant growth promoter. We investigated the ability of RC3 to restore soil health and productivity under pasture dieback conditions. RC3 was applied on pasture dieback affected paddock replicate plots once, at a rate of 4 mL/m2, and soil core samples were taken weekly to analyse microbial communities. Plants were collected regularly to measure dry matter and plant morphometrics. Twenty weeks after a single application, dry matter increased in RC3 plots by 900 kg/ha compared to control plots, and at week 48, eleven months after the single application, RC3 plots showed a trend of more grass and dicot species than the control. Morphometric measures suggest minor improvements in dicotyledon plants. Alpha diversity did not change with the application of RC3. Temporal correlation analysis shows that RC3 steadily reduced the presence of genera predominant in poor soils and with extreme environmental conditions over time and prevented the decline of beneficial genera, such as Marmoricola, Actinomadura, Dactylosporangium, and mle1-7.

The application of nanoparticles rose steeply in the last decade, where they have become a common ingredient used in processed human food, improving food properties such as shelf life and appearance. Nanoparticles have also attracted considerable interest to the livestock industry, due to their efficacy in intestinal pathogen control, with the regulatory and consumer driven push for the removal of antibiotic growth promoters. The influence of selenium (Se) nanoparticles was investigated on a diverse and mature broiler caecal microbiota using in vitro culturing and 16S rRNA gene sequencing methods for microbiota characterisation. Caecal microbiota was collected from 4 traditionally grown heritage roosters and grown for 48 h, in the presence and absence of Se nanoparticles, with 2 technical replicates each. The effect of rooster as a biological variable strongly overpowered the effects of nano-Se in the media, resulting in moderate effects on the structure and diversity of the caecal microbial community. However the nanoparticles showed a significant reduction (P

The concept of designer microbiota in chicken is focused on early exposure of the hatchlings to pathogen-free microbiota inoculum, limiting the early access to harmful and pathogenic microorganisms, thus promoting colonisation of the gut with beneficial and natural poultry microbiota. In this study, we controlled colonisation of the intestine in broiler chickens in a large-scale industrial setting via at-hatch administration of a commercial product containing a highly diverse microbiota originating from the chicken caecum. The treatment significantly transformed the microbiota membership in the crop, proventriculus, jejunum and caecum and significantly altered the taxa abundance in the jejunum, jejunum mucosa, and caecum estimated using PERMANOVA and unweighted and weighted UniFrac distances, respectively. The treatment also improved the growth rate in chickens with no significant alteration in feed conversion ratio. A comparison of inoculum product microbiota structure revealed that the inoculum had the highest Shannon diversity index compared to all investigated gut sections, and the number of Observed Species second only to the caecal community. PCoA plots using weighted or unweighted UniFrac placed the inoculum samples together with the samples from the caecal origin.

Poultry production is among the most challenging industries for pathogen control. High animal density and abundance of faecal material demand strict biosecurity measures and continual vigilance in monitoring animal health parameters. Despite this vigilance, dealing with disease outbreaks is a part of farmers’ routines. Phytogenic feed additives comprised of herbs, spices, essential oils, and oleoresins have potent antimicrobial and anti-inflammatory actions. Related studies are gaining substantial interest in human and animal health worldwide. In this study, a commercial blend phytogenic feed additive was supplemented to layers in an industrial free-range production system with 20,000 birds in both control and treatment groups. At the end of the trial, the ileum tissue was sampled for RNAseq transcriptomic analysis to study the host reaction to the supplement. Phytogenic supplement significantly inhibited four cholesterol-related pathways and reduced the Arteriosclerosis disease category towards improved cardiovascular health. The supplemented birds exhibited reduced disease susceptibility for 26 cancer categories with p-values in the range from 5.23 × 10−4 to 1.02 × 10−25. Major metabolic shifts in Lipid metabolism in combination with Carbohydrate metabolism have resulted in a decrease in the Obesity category, altering the ratio of fat and carbohydrate metabolism toward lower fat storage.

This study was conducted to examine the effects of a plant extract mixture, a microencapsulated product composed of eugenol and garlic tincture (PE), on intestinal health in broilers under necrotic enteritis (NE) challenge. A total of 960 d-old mixed-sex Cobb 500 chicks were randomly distributed to 48-floor pens housing 20 birds per pen. Six treatments were applied: UC, unchallenged control; CC, challenged control; PE, challenged group plus PE; AM, challenged group plus antimicrobial (AM); FAP, challenged group plus a full dose of AM with PE; HAP, challenged group plus a half dose of AM with PE in starter, grower and finisher phases. Birds in the challenged groups were inoculated with Eimeria spp. on d 9 and Clostridiumperfringens on d 14. On d 16, the CC group had increased serum fluorescein isothiocyanate dextran (FITC-d), reduced villus surface area, goblet cell number, upregulated CLDN1, JAM2 genes and reduced microbial diversity compared to the UC group (p < 0.05). Birds fed PE had reduced FITC-d, increased goblet cell number and Bifidobacterium compared to the CC group (p < 0.05). Birds fed PE had reduced CLDN5 expression in male birds, and Bacteroides spp. in female birds than CC group (p < 0.05). These findings suggest that PE supplementation mitigates the effect of NE by improving the intestinal health of birds.

Pathogen control is re-emerging as a significant challenge to the health of both humans and animals. The livestock industry is in the process of massively replacing in-feed antibiotics with organic production friendly plant-based products. Nutrigenomics as a science of the effects of food constituents on gene expression is shedding more light on both benefits and detrimental side-effects of feed additive prolonged consumption on the host, indicating the need to understand the feed-host interactions and their influence on the host disease profile. In this study, we investigated the effects of 2% oregano powder supplementation on the liver gene expression in healthy male broilers from the hatch to 6 weeks of age. Deep RNAseq was performed on average 113.3 million paired and quality trimmed sequences per sample and four samples for the control and treatment each. The results demonstrate the severity of oregano effect on liver gene expression with substantial modifications in steroid hormone regulation, fat and carbohydrate metabolism alterations and strong influence on the host disease and function profile. Oregano supplementation was able to interfere with the transcriptional effects of a range of registered drugs and to significantly transcriptionally inhibit a range of cancer disease categories including liver cancer, and to modify fat and carbohydrate metabolism.

Upper limb shows a large number of arterial variations. This case report describes the presence of additional superficial ulnar artery which was used to raise a pedicle flap to cover an arm defect thus avoided using the main vessel of the forearm - radial or ulnar artery. Vascular anomalies occurring in the arm and forearm tend to increase the likelihood of damaging the superficial anomalous arteries during surgery. Superficial ulnar or radial arteries have been described to originate from the upper third of the brachial artery; here we report the origin of the anomalous superficial ulnar artery originating from the brachial artery at the level of elbow with the concomitant presence of normal deep radial and ulnar arteries.