Objective 1. Identify and analyze gene and gene product interactions that promote protein utilization efficiency and growth in broilers by characterizing the physiological mechanisms underlying stabilization of the chicken gut and improving understanding of nutrient uptake and processing in the cells of the gastrointestinal tract. Objective 2. 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. Objective 3. Develop novel alternative strategies to the use of antimicrobial growth promoters in broilers to eliminate consequences of host reactivity to pathogens and to improve health, nutrient utilization efficiency and growth rate.
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. This project’s focus is to minimize challenges to gut health that both compromise absorption and redirect dietary nutrients from growth to supporting immune function and tissue repair. Objective 1 will identify mechanisms involved in efficient nutrient uptake and processing by cells of the gut. Functional genomics of amino acid transporters will be defined as influenced by nutritional state, pathogen presence, and broiler genotype. Additionally, metabolic interactions between the cellular urea cycle functionality, the nitric oxide cascade and mitochondrial energetics will be characterized according to diet and proinflammatory status. Objective 2 defines the role of the microflora in regulating nutrient uptake and utilization in the gut of growing broilers through metagenomic and metabolomic based modeling strategies, with the additional goal of determining how the broiler gut can better be stabilized by commensal microbes against pathogen influences. Objective 3 addresses alternative strategies to the use of antimicrobials to achieve improved health, nutrient utilization and growth of the broiler. The project will generate validated biomarkers of cell function and stress capable of predicting the health status of the broiler, and a battery of immortalized broiler intestinal cell lines from heritage and high-performance lineages to facilitate rapid assessment of the potential for intervention strategies prior to in vivo modeling.
This is the final report for the Project 8045-31000-106-00D which will end July 25, 2017. New NP101 PrePlan titled “Novel Integrated Nutrition and Health Strategies to Improve Production Efficiencies in Poultry” is being reviewed and established by OSQR. Substantial accomplishments related to each objective were made over the 29 months of the project. Relative to Objective 1, the development and validation of the new technologies and instrumentation, immunological detection reagents, and kinetic enzyme assay methods was accomplished during this year including reporter assays for the measurement intestinal xanthine oxidase, a nonradioactive assay for intestinal nitric oxide synthase activity, mitochondrial ATP synthase activity, and optimization of the chemilumenescent burst activity of immune cells, all in the chicken. The progress directly ties to the objective requirements surrounding the need to define the genomic attributes of the efficiency of nutrient use for growth and gut health. Two critical studies were carried out. The first compared the expression of genes involved in processing and uptake of nutrients, and nutrient sensing mechanisms between fast growing (Ross broilers) and slow growing, heritage meat chickens (ACRBC birds). Birds were fed three diets, each one containing a different level of protein (16%, 22%, or 30%). Tissue samples were taken at several time points between hatch and 4 weeks of age for RNA extraction. By two weeks of age, the average live body weight of Ross broilers was four times that of the ACRBC birds. In the second study, a critical early proinflammatory response pathway was detailed in the chicken wherein for the first time the gut villi epithelial cells of coccidia-infected chickens were shown to generate aberrant chemical modification of intracellular proteins called tyrosine nitrations as early as day 1 after infection. These nitrations were also present in coccidia proteins before infiltrating immune cells were observed in the affected regions. Coordinated with epithelial cell increases in both nitric oxide synthase and xanthine oxidase, generators of the nitrating reactants, the data suggest a new mechanism through which chickens may combat this parasite infection.Substantial understanding was obtained of the nature of complicating factors that exist to generating chicken intestinal epithelial cell lines. Various factors were tested in cell culture conditions and the following observations were made: (a) short-term outgrowth of proliferating epithelial cells from spheroids were obtained from cells scraped from the villus-crypt layer and refined by differential density centrifugation; cells retained epithelial-like morphology up to 4 days before the division arrested and fibroblasts overpopulated the cultures; post-hatch harvest cells were >95% apoptotic. (b) The better cultures were obtained from late-stage embryonic day-18 gut tissue. (c) Duodenal cells were more robust in culture after isolation than cells derived from the ileum. (d) Trypsin and EDTA were toxic to the cells. Hormones with proliferative / anti-apoptotic character were beneficial to the health of the cultures including epithelial growth factor (EGF). (e) Successful insertion of a plasmid containing elements to facilitate the over-expression of red fluorescent protein (RFP-plasmid insertion marker) and telomerase (hTERT immortalization factor) were successful; however, even though transformed cells displayed RFP, the presence of the added telomerase did not override the apoptotic character of the cells for long-term culture proliferation. Relative to Objective 2, the effects of feeding various bovine colostrum preparations to chickens as an alternative to antimicrobial growth promoters were summarized and correlated to the metabolite profile of the gut. Compared to a normal grower diet for broiler chickens, supplementing the diet with normal bovine colostrum or a hyperimmune colostrum containing antibodies against E. coli J-5 bacterin endotoxin, diet containing the colostrum preparations (increased feed efficiency 9%, altered bacterial populations where colostrum was associated with prevalence of Lactobacillaceae, Streptococcaceae, Lactobacillales, and Bacilli, and the normal diet associated with Clostridiales, Clostridia, and poulations of Methanobacteria; significant differences in the effects of the colostrum were evident as a function of the specific section of the gut studied. Colostrum in the chicken feed favored a metabolite profile consistent with aspects of anti-inflammatory character with particular regard to the presence of sphingolipids, eicosanoids, and polyunsaaturated fatty acids. Relative to Objective 3, experiments were conducted in growing Ross broilers to investigate the potential impact of novel forms of vitamin E to stabilize the health of the gut in the effort to develop alternatives to antibiotics to maintain high efficiency of nutrient use towards growth. Two replications of a feeding trial were constructed to identify the tissue distribution of four different forms of vitamin E in the chicken and the impact of increasing tissue levels of gamma- and delta- tocopherols on gut health and mitigation of gut inflammation. In contrast to a mammalian model (bovine), the chicken was observed to be very capable of taking up and distributing both gamma- and delta-tocopherol to both the liver and skeletal muscle. The highest tissue concentrations of gamma- and delta-tocopherol were observed in the duodenum and ileum, respectively. Gut tissue levels of nitrated proteins, a biomarker for proinflammatory stress, were lower in gamma- and delta-tocopherol-fed chickens and suggest a more dynamic role for these vitamin E forms as anti-inflammatory molecules in the gut than originally considered. The data suggest that measures taken to decrease gut inflammation may have an impact on the efficiency of food use by chickens for growth. Additionally, a study was carried out to determine whether lowering the amount of crude protein could result in more rapid clearance of a significant chicken gut parasite (Eimeria) by limiting the amount of protein to the host and thus also limiting the access of these nutrients to the parasite. Normal protein 22% and low protein diets 16% were fed to the chicks beginning at day of infection (chicks age 21 days) through day 7 post infection. The results indicate that lower the crude protein content in feed does not have a significant effect on the course of coccidiosis infection. However, it is also apparent that lowering the protein content did not have a further negative effect on decreased weight gain. The critical SY vacancy was filled with a scientist who will assume the project lead on investigating microbiome correlates to a healthy gut health in association with the development of strategies to maintain sound feed efficiency in broilers that function as alternatives to antimicrobial growth promoters.
1. Adding of bovine colostrum to poultry diets changes gut microbe populations. As the poultry industry moves away from the use of antimicrobials to improve bird performance, new feed additives are being sought whose use will maintain gut health while not sacrificing parameters such as weight gain and feed conversion. The ARS researchers at Beltsville, Maryland, in collaboration with researchers at the Advanced Vaccine Center and the Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, demonstrated that the feeding of diets containing lyophilized normal bovine colostrum or colostrum derived from cows immunized against a modified Gram negative bacterial endotoxin for as little as seven days altered the proportion and varieties of Gram positive and Gram negative bacteria that were associated with the different regions of the gut. Chickens fed with colostrum preparations had a 9% improvement in feed efficiency a more diverse distribution of bacterial types, an increase in gut health.
Bakst, M.R., Welch, G.R., Fetterer, R.H., Miska, K.B. 2016. Impact of broiler egg storage on the relative expression of selected blastoderm genes associated with apoptosis, oxidative stress, and fatty acid metabolism. Poultry Science. 95(6):1411-1417. doi: 10.3382/ps/pew038.
Miska, K.B., Fetterer, R.H. 2017. The mRNA expression of amino acid and sugar transporters, aminopeptidase, as well as the di- and tri-peptide transporter PepT1 in the intestines of Eimeria infected broiler chickens. Poultry Science. 1;96(2):465-473. https://doi.org/10.3382/ps/pew303.
Su, S., Dwyer, D., Miska, K.B., Fetterer, R.H., Jenkins, M.C., Wong, E.A. 2017. Expression of avian beta-defensins in the intestine of Eimeria-challenged chickens. Poultry Science. doi: 10.3382/ps/pew468.