1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
This is the second-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 or valerate (short chain fatty acids) on performance of broilers infected with Eimeria tenella, a common gastrointestinal pathogen. Performance data from infected chickens indicated that addition of 0.25% butyrate to the diet can result in increased body weight gain at 7 days post infection (PI). However, at ten days PI there were no differences between infected birds on normal diet or 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 studies; 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 are in progress. Relative to Objective 1, Sub-objective 1.2., this experiment has not been carried out because preliminary results on birds performance and microbiome composition for experimental diets has been inconclusive. We have encountered issues with birds fed the experimental diets, infected birds performance has only improved at 7 days post-infection with only two species of Eimeria. The planned experiment needs to be repeated to clarify the diet issues. Therefore, this experiment will be performed next fiscal year. Relative to Objective 1, Sub-objective 1.3., a study was carried out to determine the effects of addition of butyrate (short chain fatty acids) 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 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 day 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 chicken gut and improve growth performance is still unknown. Relative to Objective 2, Sub-objective 2.1., due to government shut down in the beginning of this year, an experiment described in this milestone will be performed later this FY (late Summer – early Fall). Relative to Objective 2, Sub-objective 2.2., 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. Relative to Objective 3, Sub-objective 3.1, 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. Every week 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. The birds were characterized by different trajectory pattern of growth, with fast-growing broilers having the highest body weight while slow-growing birds were characterized by slower growth rate in comparison to fast growing lines. Similar patterns were observed for feed intake. The fast-growing broilers were also characterized by lower feed conversion ratio. Bacterial DNA from different parts of chicken gastrointestinal tract has been isolated and sequenced. Obtained sequences are currently analyzed for microbial alpha and beta diversity, and taxonomic composition. Relative to Objective 3, Sub-objective 3.2, two experiments were designed and 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. Currently, samples and data collected from these two experiments are being analyzed. The analysis has stopped due to retirement of SY. Additionally: (1) A study was carried out to determine whether different 16S ribosomal RNA (rRNA) primers will affect taxonomic composition and bacterial community diversity. Sequencing of nucleic acids is the most common method used to determine taxonomic composition and bacterial abundance. The method relies on amplifying and sequencing of the 16S rRNA gene with specific 16S rRNA primers. Bacterial DNA isolated from Ross 708 cecal digesta collected at 1, 3 and 5 weeks of age, and eight different, previously published, primer pairs targeting V1-V3, V3, V4, V3-V4, V4-V5, V3-V5 and V4-V6 variable region of the 16S rRNA were employed. Alpha-diversity indexes such as evenness, richness, number of OTUs, beta diversity analysis and taxonomic composition of bacterial population were affected by different 16S rRNA primers. The results suggest that the choice of 16s rRNA primers can influence microbiome analysis and data interpretation. (2) A study was carried out to compare taxonomic composition of chicken gastrointestinal microbiome using two different reference databases, Greengenes and SILVA. The microbiome community plays an important role in the overall nutrition, health and productivity of broiler chickens. Microbial diversity in the gastrointestinal tract depends on age, feed additives, animal health and environment. The variability in the microbiome could also be affected by the methodology in the bioinformatics analysis, including reference databases. Cecal and ileal digesta as well as epithelial scrapings were collected from Ross 708 broilers at 7, 14, 21, 28, and 35 days of age for bacterial DNA isolation and sequencing of the hypervariable V3-V4 region of bacterial 16S rRNA to determine microbial community. Taxonomic composition comparison between Greengenes and SILVA reference databases revealed differences in diversity of classified microorganisms at all taxonomic levels, low abundant bacteria presence and the level of unclassified bacteria. Our research showed that different reference databases can influence microbiome classification at all levels of taxonomic composition. (3) A study was carried out to determine whether access to feed immediately post-hatch will affect ceca development and function in chickens. Ceca are the primary fermentation chambers in chicken gastrointestinal tract, but also plays a role in absorption of water and electrolytes, and synthesis of vitamins and short-chain fatty acids by microbiota. Immediately after hatch Ross 708 broilers had access to feed or were subjected to 48 h feed delay to mimic industry settings. Delayed access to feed up to 72 h is a normal practice in poultry industry due to uneven hatching, sorting, vaccination and transportation to the farms. As shown previously, delayed access to feed affected body weight and feed intake (growth efficiency), microbiome development, gene expression level of proteins involved in tight junction that are responsible for gut permeability as well as the gastrointestinal tract immune system development, but has no effect on majority of gene related to nutrient transport, mitochondria function and immune system development in ceca.
1. Nutrition-relevant gut cell markers as indicator of growth potential. Gene expression changes between fast and slow growing broilers may reflect differences in uptake of nutrients. A study was designed to compare expression of genes that encode proteins located at either the brush border (BB) or basolateral surface (BL) of the gut epithelium among fast and slow growing broiler strains. ARS scientists at Beltsville, Maryland, used six strains of chicks with different growth capacities, three which were fast growing, two of medium growth capacity, and one breed of slow growing chickens. Expression analysis was performed on 14 genes which encode proteins associated with nutrient processing and uptake. The results indicate that there are strain specific differences in gene expression but most likely they are not associate with growth rate. In most cases, genes encoding BB associated proteins increased in expression over time in the duodenum, jejunum, and ileum, while in the ceca the expression decreased. On the other hand, genes encoding BL associated proteins decreased in expression over time in all gut segments with exception of a sugar transporter which increased in expression in the small intestine. The temporal changes in gene expression were very consistent among bird strains.
Proszkowiec-Wegla, M.K., Schreier, L.L., Miska, K.B., Angel, R., Kahl, S., Russell, B.A. 2018. Effect of early neonatal development and delayed feeding immediately post-hatch on jejunal and ileal calcium and phosphorus transporter genes expression in broiler chickens. Poultry Science. 98:1861-1871. http://dx.doi.org/10.3382/ps/pey546.
Miska, K.B., Fetterer, R.H. 2018. The effect of Eimeria maxima infection on the expression of amino acid and sugar transporters aminopeptidase, as well as the di- and tri-peptide transporter PepT1, is not solely due to decreased feed intake. Poultry Science. 97(5):1712-1721.
Miska, K.B., Fetterer, R.H., Su, S., Jenkins, M.C., Lamont, S., Wong, E. 2018. Differential expression of intestinal nutrient transporters and host defense peptides in E. maxima infected Fayoumi and Ross chickens. Poultry Science. 97:4392-4400.
Miska, K.B., Fetterer, R.H. 2019. Expression of amino acid and sugar transporters, aminopeptidase, and the di- and tri-peptide transporter PepT1, between modern fast growing broilers and broilers not selected for rapid growth. Poultry Science. 98:2272-2280. https://doi.org/10.3382/ps/pey583.