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 third-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 10 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 is in progress. Relative to Objective 1, Sub-objective 1.2., this experiment has not been carried out because preliminary results on bird’s performance and microbiome composition for experimental diets have been inconclusive. We have encountered issues with birds fed the experimental diets; infected bird’s performance was 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 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 (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 non-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, 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 and were supplemented with AGP for 35 days or probiotic for 3, 10 and 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 collect for gene expression and metagenome analysis. Collected data and samples are currently analyzed. 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. 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 were analyzed for microbial alpha and beta diversity, taxonomic composition and predicted function of bacterial community. Data are in preparation for manuscript. Expression of genes related to nutrient transport and uptake was also measured between the fast and slow growing birds. We found that age of birds had an effect on expression of these genes, however their expression did not correlate with growth capacity of the birds. Over time the expression of genes associated with amino acid transporters located at the brush border of the gut epithelium increased, but those associated with the basolateral surface of the gut epithelium tended to decrease with time. 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 an ARS scientist. Additionally: (1) A study was carried out to determine whether access to feed immediately post-hatch will affect mycobiome development in gastrointestinal tract of broiler chickens. Immediately after hatch broiler chicks 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. Microflora of gastrointestinal tract is comprised of bacterial, fungi and viral populations. We have shown previously that delayed access to feed affects body weight and feed intake (growth efficiency), microbiome development, and gene expression level of proteins involved in tight junction that are responsible for gut permeability, as well as the gastrointestinal tract immune system development. In this study, we confirmed that delayed access to feed post-hatch also has impact on fungi population in chickens. (2) Studies were carried out in which birds were infected with Eimeria maxima, E. acervulina, or E. tenella and samples from the intestine and liver were sampled during several time points after infection (between 0 and 14 days post infection). Gene and protein expression of mTOR, which is associated with nutrient sensing, was determined. Surprisingly, expression at both mRNA and protein level depended on the species of Eimeria used in infection. Infection with E. maxima caused little to no change in expression, while E. acervulina and E. tenella infection caused an increase in expression of mTOR. This data suggests that different species of Eimeria have different effects on the chicken. Upregulation in nutrient sensing mechanism leads to increased cell division, and may have a role in recovery from coccidiosis.
5. Record of Any Impact of Maximized Teleworking Requirement:
The experiments in Milestone 2 were planned for spring this year, but because of maximum teleworking policy they were postponed until the policy is lifted.
Payne, J.A., Proszkowiec-Wegla, M.K., Ellestad, L.L. 2019. Delayed access to feed alters expression of genes associated with carbohydrate and amino acid utilization in newly hatched broiler chicks. American Journal of Physiology - Regulatory Integrative & Comparative Physiology. https://doi.org/10.1152/ajpregu.00117.2019.
Payne, J.A., Proszkowiec-Wegla, M.K., Ellestad, L.L. 2020. Delayed access to feed alters gene expression associated with hormone signaling, cellular differentiation, and protein metabolism in muscle of newly hatch chicks. General and Comparative Endocrinology. https://doi.org/10.1016/j.ygcen.2020.113445.