Location: Animal Biosciences & Biotechnology Laboratory
2022 Annual Report
Objectives
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.
Approach
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.
Progress Report
This is the final report for the Project 8042-31000-108-000D which will end September 19, 2022. New NP101 PostPlan titled “Nutritional strategies to improve production efficiencies in broiler chickens” is being reviewed and established by OSQR. Objective 1 of the project was focused on evaluation of mechanism of action and functional effects of short chain fatty acids as diet additives on gut cell-microbial interactions in broiler chickens. Studies were carried out to determine the effects of addition of butyrate (short chain fatty acids) on performance, microbiota, and gastrointestinal tract nutrient transporter gene expression of broilers infected with Eimeria maxima, a common gastrointestinal pathogen. Performance data from infected chickens indicated that addition of 0.25% butyrate to the diet resulted in increased body weight gain at 7 days post infection (PI). Microbiota analysis indicated more significant changes in bacterial diversity due to infection rather than butyrate supplementation. Expression pattern of genes involved in nutrient uptake indicated that addition of Short-chain fatty acids (SCFAs) does not change expression of these genes in birds infected with Eimeria. The infection produces a decrease in expression of brush border associated genes, especially at 7-days post infection, however this effect is observed regardless of SCFA supplementation. The majority of differences in gene expression were observed between infected and not-infected birds. We concluded that short chain fatty acid may be a promising feed additive for broiler chickens infected with coccidia, but the downstream cellular mechanism responsible for helping to protect the chicken gut and improve growth performance is still unknown. Experiments involving comparison between slow- and fast-growing broilers and short chain fatty acid treatments were not finished due to covid restrictions and maximum teleworking policy.
The second part of the project was related to assessing low level gut inflammation as a model for compromised growth and nutrient uptake, and determinatingg the potential of selective feeding of natural anti-inflammatory constituents to replace antibiotic growth promoters. To answer the goals of this objective, 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 nitrooxidative 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.
Another study was performed 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. Body weight, feed intake and feed conversion ratio were not affected by AGP or probiotic treatment. Supplementation with AGP or probiotic affected the alpha and beta diversity of gastrointestinal tract bacterial populations. Taxonomic analysis revealed significant (P<0.05) changes in relative abundance of some bacterial genera and species, including Escherichia coli and Clostridium celatum, Lactobacillus, Oscillospira, SMB53, Lactobacillus reuteri, and Ralstonia. LEfSe analysis showed that relative abundance of genus Weissella was decreased in AGP treated birds in all parts of the GIT in comparison to non-AGP treated birds. Predicted function of microbiota was also affected by the treatment. Changes in microbiota function and composition were dependent on the gastrointestinal tract localization and to some extent on the duration of probiotic treatment.
The last objective of the project was focused on assessing, developing, and applying microbiome- and metagenome-based strategies in poultry to define the role of the microbiota in the regulation of nutrient uptake and utilization in the gastrointestinal tract during post-hatch development in broilers. An experiment was conducted using six different meat chicken lines/breeds, of which three were slow-growing and three were fast-growing lines to determine whether microbial population distribution patterns are likely to be associated with the phenotypes of those broiler chickens. In a floor-pen setting, all six lines were fed the same diet and were grown until they reached five weeks of age. The birds were characterized by different patterns of growth, with fast-growing broilers having the highest body weight and the lower feed conversion ratio while slow-growing birds were characterized by slower growth rate. Similar patterns were observed for feed intake. Overall, no differences in alpha and beta diversity in bacterial populations of ileum and cecum were observed between different broiler breeds. However, significant effect of breed or breed by age interaction were detected on bacterial composition at every taxonomic level. These results indicate the possibility of host genetic-specific microbiome interaction that could be involved in some of the performance differences seen between breeds.
Additional studies have been conducted to determine the effects of supplementing broiler diets with pure alpha-tocopherol or each of three levels of mixed isoform tocopherol oil (enriched with gamma- and delta tocopherols) on the response to infection with the gut parasite E. maxima. The first trial focused on the recovery phase of the infection largely quantifying the effects of the various tocopherols on the post-infection compensatory gain and feed efficiency (days ten through 14 PI). The second experiment focused on the time surrounding the onset (day 4 PI) and peak (day 7 PI) of the infection to evaluate the mechanism of action of the various tocopherol treatments to mitigate inflammation. Due to retirement of the lead scientist, analysis of the results has been suspended.
Additionally, 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, and 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. Samples have been analyzed for plasma cholic acid, ileal deoxycholic acid, and expression of genes related to bile acid synthesis in the liver as well as transport in both the liver and ileum. FloraMax-B11 is made of lactic acid producing bacteria, which have bile salt hydrolase activities. The partial results (gene expression and plasma cholic) suggest that the probiotic supplementation did not affect the bile acid neosynthesis as expected; however, the bile acid neosynthesis may decrease over time post-hatch, regardless of probiotic supplementation.
A study was conducted to evaluate the effects of in ovo supplementation of threonine on growth performance, intestinal integrity, and intestinal 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 euthanized 2.5 hours later to assess gut leakage and collect tissue samples. 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. The partial data of this experiment show that threonine supplementation did not affect the intestinal permeability post-hatch and growth performance. We believe that the dose of threonine needs to be optimized to elicit the expected changes; therefore, we have decided to repeat the experiment with an optimal dose of threonine. A collaboration with Penn State University, Department of Animal Science, University Park, Pennsylvania, has generated data suggesting that a strain of corn developed by Penn State researchers that is high in flavonoids (polyphenolic compounds) can ameliorate the effects of necrotic enteritis (NE) in broiler chickens. Eimeria infection in the intestines of broiler chicks predisposes them to infection with Clostridium perfringens which can result in NE. The effect of high flavonoid corn diet in chickens with NE was investigated in the gut microbiome. Changed in the microbiome were predominantly associated with NE, however effects of the diets, as well as interaction between infection and diet were also noted.
Accomplishments
1. Choice of 16s rRNA primers and bacterial reference databases influence the microbiome analysis and data interpretation. Microbiota and the integrity of the gastrointestinal tract play important roles in nutrient absorption, immune system development, and disease resistance. Alterations in microbiome homeostasis may lead to a negative effect on feed efficiency, productivity, and health in chickens. Sequencing of nucleic acids is the most common method used to determine taxonomic composition, diversity and bacterial abundance. The method relies on amplifying and sequencing the 16S ribosomal RNA (rRNA) gene with specific 16S rRNA primers. Microbial diversity in the gastrointestinal tract depends on age, feed, feed additives, animal health, anatomical location and environment. The variability in the microbiome could also be affected by the methodology and bioinformatics analysis, including primers specific to hypervariable regions of bacterial 16S rRNA and the reference databases such as Greengenes, SILVA or RDP. ARS researchers at Beltsville, Maryland in collaboration with NEA Bioinformatic group determined that 16S rRNA primer selection as well as the choice of reference database have significant effect on microbiota population’s diversity and taxonomic composition. These results indicate that microbiome data should be interpreted with cautions taking in consideration not only physiological factors but also methodology. The results also indicate the need for standardized methodology for metagenomic analysis in poultry species. Generated data will help development 16S sequencing protocol in poultry species in collaboration with International Microbiome and Multi’omics standards alliance (IMMSA).
Review Publications
Campos, P., Miska, K.B., Kahl, S., Jenkins, M.C., Shao, J.Y., Proszkowiec-Wegla, M.K. 2022. Effects of Eimeria tenella on cecal luminal and mucosal microbiota in broiler chickens. Avian Diseases. 66(1):1-14. https://doi.org/10.1637/21-00068.
Hrabia, A., Miska, K.B., Schreier, L.L., Proszkowiec-Wegla, M.K. 2022. Altered gene expression of selected matrix metalloproteinase system proteins in the broiler chicken gastrointestinal tract during post-hatch development and coccidia infection. Poultry Science. https://doi.org/10.1016/j.psj.2022.101915.
Emami, N.K., Schreier, L.L., Greene, E., Tabler, T., Orlowski, S.K., Anthony, N.B., Proszkowiec-Wegla, M.K., Dridi, S. 2022. Ileal microbial composition in genetically distinct chicken lines reared under normal or high ambient temperatures. BMC Microbiology. https://doi.org/10.1186/s42523-022-00183-y.
Miska, K.B., Schreier, L.L., Kahl, S., Russell, B.A., Qu, Y., Proszkowiec-Wegla, M.K. 2022. Expression of genes associated with nutrient uptake in intestines of chickens with different growth potentials show temporal changes but are not correlated with growth. British Poultry Science. 63(2):179-193. https://doi.org/10.1080/00071668.2021.1966753.
Proszkowiec-Wegla, M.K. 2021. Gastrointestinal anatomy and physiology. Sturkie’s Avian Physiology, 7th edition. p. 485-527. https://doi.org/10.1016/C2019-0-00060-X.
Jenkins, M.C., Parker, C.C., Obrien, C.N., Camp, M.J., Vinyard, B.T., Heeder, C., Proszkowiec-Wegla, M.K. 2021. Metagenomic analysis of 16S Clostridium perfringens amplicons corroborates C. perfringens counts on select agar and C. perfringens PCR analyses of bacteria in broiler farm litter. Avian Diseases. 65:554-558. https://doi.org/10.1637/aviandiseases-D-21-00054.
Campos, P.M., Darwish, N., Shao, J.Y., Proszkowiec-Wegla, M.K. 2022. Choice of microbiota database affects data analysis and interpretation in chicken cecal microbiota. Poultry Science. https://doi.org/10.1016/j.psj.2022.101971.
Miska, K.B., Kahl, S., Schreier, L.L., Russell, B., Kpodo, K.R., Proszkowiec-Wegla, M.K. 2022. Effects of delay of feed post-hatch on the expression of immune related genes and their correlation with components of the gut microbiota. Animals. 12(10):1316. https://doi.org/10.3390/ani12101316.
