Objective 1: Evaluate the mechanism of action and functional effects of short chain fatty acid (SCFA) preparations as diet supplements on gut cell-microbial interactions in broilers. Sub-objective 1.1:Compare the effects of SCFA-supplementation on growth parameters in healthy and Eimeria-infected broiler chickens. Sub-objective 1.2 Determine the role of short-term and long-term SCFA diet supplementation on microbiome composition and metabolite profiles in modern production, fast-growing broilers (FGB) and slow-growing (SGB), physiologically robust heritage varieties infected with Eimeria species. Sub-objective 1.3:Evaluate the potential for SCFA supplementation to modulate the expression of genes and proteins associated with nutrient uptake in healthy and Eimeria-infected SGB and FGB lineages. Objective 2: Assess low level gut inflammation as a model for compromised growth and nutrient efficiency, and determine the potential for selective feeding of natural anti-inflammatory constituents to replace antibiotics for growth promotion. Sub-objective 2.1: Establish the capacity of gut inflammation and treatment with traditional antimicrobial growth promoters (AGP) to alter weight gain, feed conversion ratios, and changes in microbiome composition and metabolite profiles in broilers. Sub-objective 2.2:Develop and implement strategies to decrease the effects of bacterial toxins on the gut through the inclusion of novel milk- or plant-derived compounds or fats and oils in the diet. Objective 3: Assess, develop, and apply microbiome- and metagenomic-based strategies in poultry to define the role of the microflora in the regulation of nutrient uptake and utilization in the gastrointestinal tract during post-hatch development of broilers. Sub-objective 3.1: Establish which microbial population distribution patterns are likely to associate with SGB versus FGB production phenotypes in poultry. Sub-objective 3.2: Determine the capacity for diet supplementation with plant-derived gamma (')-and delta (d)-tocopherol-enriched mixed tocopherol oils (predominantly non-alpha-tocopherol isoforms) to modify the antioxidant environment of different segments of the gut in regard to modulating the microbiome of the broiler for improved gut integrity.
This project’s focus is to minimize challenges to gut health that compromise growth by constraining absorption of dietary nutrients and/or and redirecting nutrient use from growth to supporting immune function and tissue repair. Understanding the host-microflora interactions that influence gut health is critical for developing strategies to increase the efficient use of ingested nutrients by broilers for meat production in the absence of antimicrobials. Objective is specific to the topic of alternatives to antimicrobials to maintain efficient growth in broiler chickens through the use of novel formulations of short chain fatty acids, essentially nutritional prebiotics, to offset deficits in growth arising from gut parasitic infection with coccidia. Objective 2 addresses one of the purported mechanisms of actions through which antimicrobial growth promoters improve feed efficiency in poultry, namely, modulating gut inflammation. The intent here is to develop nonantimicrobial, non-drug approaches such as isoforms of vitamin E other than the traditional alpha tocopherol to mitigate gut inflammation thus sparing nutrients for growth that otherwise would be used to fight the inflammation or simply be lost through a lack of intestinal absorption. Objective 3 addresses the role of the gut microflora in optimizing the efficiency of nutrient processing in the gut towards better protein deposition. A unique opportunity will be available through the direct comparison of gut microbiome populations derived from modern fast growing broilers versus those obtained from more slow-growing heritage breeds and varieties. In addition the project will be the first to assess directly the impact of the oxidation-reduction environment in the gut on population changes in the microflora.
This is the fourth-year report for the Project 8042-31000-108-00D which started September 20, 2017. Relative to Objective 1, Sub-objective 1.1., a study was carried out to determine the effects of addition of butyrate (short chain fatty acid) on performance of broilers infected with Eimeria maxima, a common gastrointestinal pathogen. Performance data from infected chickens indicated that, while addition of 0.25% butyrate to the diet resulted in increased body weight gain at 7 days post infection (PI), by 10 days PI there were no differences in body weight between infected birds fed the unsupplemented diet or the diet containing butyrate. Even though a positive effect was observed, birds who were fed experimental diets had lower feed intake in comparison to control diet. This phenomenon needs further investigation; therefore, we are following up to explain the effect of butyrate addition on feed intake. Additionally, gastrointestinal samples for morphological studies have been collected, embedded, sectioned, and stained with hematoxylin and eosin. Morphological analysis of the slides is in progress. Relative to Objective 1, Sub-objective 1.2., this experiment required replication and/or modification as preliminary results on bird’s performance and microbiome composition for experimental diets were inconclusive. We have encountered issues with birds fed the experimental diets. The planned experiment needs to be repeated to clarify the diet issues and will be initiated as soon as the maximum teleworking policy is lifted. Relative to Objective 1, Sub-objective 1.3. a study was carried out to determine the effects of addition of butyrate on expression of genes involved in nutrient uptake in fast growing birds infected with Eimeria maxima, a common gastrointestinal pathogen. Expression pattern of genes involved in nutrient uptake indicates that addition of short chain fatty acids (SCFAs) does not change expression of these genes in birds infected with Eimeria. The infection produced a decrease in expression of brush border associated genes, especially at 7 days PI, however this effect was observed regardless of SCFA supplementation. The majority of differences in gene expression were observed between infected and non-infected birds, rather than between control and supplemented diets. We concluded that SCFAs may be a promising feed additive for broiler chickens infected with coccidia, but the downstream cellular mechanism responsible for helping to protect chicken gut and improve growth performance is still unknown. Manuscript has been published. Relative to Objective 2, Sub-objective 2.1, a study was conducted to determine the effect on traditional antimicrobial growth promoters (AGP) and probiotics on body weight gain, feed conversion ratio, changes in gut permeability and microbiome composition in broiler chickens. In a floor-pen setting, newly hatched chicks were fed the same diet (control) and were supplemented with either AGP (for 35 days) or probiotic (for 3, 10 or 35 days) in water. At every sampling point, weight and feed intake of chickens were recorded. Samples of gastrointestinal tract, as well as the content, were collected for gene expression and metagenome analysis. RNA and DNA has been isolated. Metagenome analysis has been performed. Relative to Objective 2, Sub-objective 2.2., a study was conducted to determine if colostrum (derived from cattle) would serve as an alternative to traditional antimicrobial products. An in-depth metabolite profile of the ileal digesta collected from chickens fed a low level of a purified bovine colostrum for 5 days resulted in a metabolite profile significantly enhanced in its basic anti-inflammatory capabilities. Feed efficiency was improved 9% and the presence of tyrosine-nitrated proteins, gut epithelial nitro-oxidative stress biomarkers, significantly decreased compared to control non-supplemented birds. The improvement in anti-inflammatory capacity of the digesta from colostrum-fed birds was evident in an improved balance in omega-3 versus omega-6 fatty acids, decreased glutathione precursor and metabolites, and an increased oligosaccharide content. The data suggest that low-level, short-term use of bovine colostrum could be beneficial in stabilizing the gut of chickens post-hatch and assist in the efficient uptake of nutrients for growth by providing an environment of compounds with known associations with reducing cell inflammatory processes. Additionally: (1) An experiment was conducted to determine the expression of genes related to immune function and test whether any correlation existed between gene expression and microbiome content. For this study, intestinal samples from birds which were either fed immediately post hatch or delayed for 48 hours post hatch were tested. It has been suggested that delayed feeding post hatch causes decreased production parameters and immune function. In the current studies, gene expression analysis showed that in some cases mRNA level of immune-related genes mRNA was significantly affected by delayed access to feed post hatch. Notably, a correlation analysis between gene expression and microbiome content (at the family, genus, and species levels) indicated that expression of immune-related genes, particularly avian defensins (which interact directly with microbial pathogens resulting in their death), were positively correlated with presence of Clostridium perfringens in in birds with delayed access to feed post hatch. To our knowledge, this study is the first to show correlation between microbiota and immune-related gene expression in birds with delayed access to feed early post hatch. (2) A study was carried out to determine the effects of a probiotic (FloraMax-B11) on bile acid neosynthesis and enterohepatic circulation in broiler chickens. Chickens were raised without supplementation or received either FloraMax-B11 in water for 3, 10, or 35 days, Bacitracin methylene disalicylate, an AGP, in the water for 35 days post hatch. Plasma, ileal mucosa scrapings, ileal and cecal contents, and liver samples were collected at hatch and on days 3, 10, 21, and 35 post hatch. Currently, collected samples are being analyzed for plasma cholic acid, ileal and cecal deoxycholic acid, and expression of genes related to bile acid synthesis in the liver as well as transport in both the liver and ileum. In addition, the protein expression of the mitogen-activated protein kinase (MAPK) signaling pathway in the liver will be investigated. FloraMax-B11 is made of lactic acid producing bacteria, which have bile salt hydrolase activities. Therefore, we expect that the supplementation of FloraMax-B11 will increase bile salt deconjugation thereby reducing enterohepatic circulation and increasing bile acid synthesis in broiler chickens. (3) A study was conducted to evaluate the effects of in ovo supplementation of threonine on growth performance, intestinal integrity, and microbiome development post hatch in broiler chickens. Fertile eggs were injected with either a threonine or saline solution into the amniotic fluid at 17.5 days of incubation. On days 0, 3, 11, and 21 post hatch, chickens were orally administered a fluorescein isothiocyanate labelled dextran (FITC-d) solution and tissue samples and gut leakage were evaluated. Feed intake and body weight were recorded to evaluate growth performance. Serum FITC-d, liver and breast glycogen, intestinal tight junctions and MUC2 gene expression, mucosa morphometry, and intestinal microbiome will be assessed. Threonine, a gluconeogenic amino acid, may increase glycogen reserves and reduce muscle catabolism during the late embryonic stage. It has been linked to increased mucin production, which modulates intestinal microbiome. Therefore, we expect that intra-amniotic injection of threonine will increase glycogen reserves and mucin production thereby improving intestinal and normal microbiome development post hatch. (4) In collaboration with Department of Animal Physiology and Endocrinology, Faculty of Animal Science, University of Agriculture, Krakow, Poland, a study was carried out to determine the role of matrix metalloproteinases in gastrointestinal tract development early post-hatch and during coccidiosis infection. Matrix metalloproteinases are involved in normal physiological processes such as development, morphogenesis, and tissue repair. Expression level of metalloproteinases and their inhibitors was determined in ileal samples during post-hatch development, in jejunum, ileum and ceca during Eimeria maxima infection, and in duodenum and jejunum during Eimeria acervulina infection. Expression profile of metalloproteinases and their inhibitors strongly suggest their role in small intestine development and intestinal remodeling during coccidia infection. (5) In collaboration with Penn State University, Department of Animal Science, University Park, Pennsylvania, a study was carried out to determine the effect of feeding high levels of flavonoids to broiler chicks experimentally infected with Eimeria maxima and Clostridium perfringens on the mortality and morbidity of the subsequent necrotic enteritis infection. The birds which were fed with corn soybean meal derived from corn high in flavonoids had significantly decreased mortality from the experimental necrotic enteritis infection. To determine the changes in the microbiota as well in gene expression (immune related, tight junction, and nutrient processing and uptake genes), the jejunum and ileum as well as the gut contents and scrapings were collected. The DNA and RNA is now being extracted from the collected samples. Use of corn products high in flavonoids may lead to new non-antibiotic growth promoter measures in controlling necrotic enteritis in broilers.
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