Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209
Performing Department
Agricultural and Environmental Sciences
Non Technical Summary
The aim of the proposed work is to enhance growth, production performance, feed efficiency and profitability of poultry, especially chickens and guinea fowl. An additional goal is to continue developing the guinea fowl as alternative poultry for small scale and limited resource farmers in Tennessee. The optimum requirement for the amino acids methionine and cysteine by the French and Pearl grey guinea fowl are lacking. Also, the minimum amounts of nutrients, especially the essential amino acids that sufficiently drive metabolic processes in poultry are not well established. The requirement of these nutrients for optimum growth and production performance of poultry will be evaluated. The use of direct-fed microbials such as Lactobacillus acidophilus to improve efficiency of nutrient utilization in chickens and guinea fowl will also be evaluated. In completely randomized design, French and Pearl grey guinea fowl and broiler chickens will be assigned to floor pens and subjected to dietary treatments. Dose response experiments with varying dietary levels of each individual nutrient will be conducted from hatch to eight weeks of age. The optimum levels of these nutrients and the effectiveness of direct fed microbials will be determined by growth performance, carcass characteristics, and assay of metabolic indices and gene expression of the experimental birds. Additional cDNA libraries of key organs such as the liver, pancreas and hypothalamus of the guinea fowl will also be constructed and screened to facilitate the identification of economically important metabolic pathways that will guide design of optimum feeding regimens. Findings from this research will be published in the Journal of Poultry Science and optimum levels of these nutrients will be recommended to guinea fowl and traditional poultry producers.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Thelong term goal of this projectis to enhance growth, production performance, feed efficiency and profitability of poultry, especially chickens and guinea fowl. An additional goal is to continue developing the guinea fowl as alternative poultry for small scale and limited resource farmers in Tennessee. Specific objectives are to:Evaluate methionine and cysteine requirement of the French guinea fowl broiler;Evaluate methionine and cysteine requirement of the Pearl Grey guinea fowl;Evaluate lysine homeostasis in performance of guinea fowl and broiler chickens;Construct and screen guinea fowl mRNA libraries to facilitate understanding of key metabolic processes of the guinea fowl and other avian species; andEvaluate the efficacy of direct-fed microbials to further improve efficiency of feed utilization in poultry
Project Methods
To meet objective 1 and 2, evaluation of methionine and cysteine requirement of the French guinea fowl (GF) broilers and Pearl grey guinea fowl, nine diets will be fed to 540 each of French GF broilers and Pearl grey GF from hatch to 8 and 10 weeks of age (WOA), respectively. In a 3x3 factorial arrangement, birds will be fed diets containing 0.40, 0.45 and 0.50% methionine each in combination with 0.35, 0.40 and 0.45% cysteine at hatch to 4 WOA. At 5-8 WOA, the experimental diets will contain 0.35, 0.40, and 0.45% methionine each in combination with 0.35, 0.40 and 0.45% cysteine. The feed will be iso-caloric and iso- nitrogenous containing 3,100 metabolizable energy (ME) Kcal/kg and 23% crude protein (CP) and 3,150 ME Kcal/kg and 21% CP at 0-4 and 5-8 WOA, respectively. Dietary treatments will be replicated three times, fed in marsh form and feed and waterprovided at free choice. Feed consumption and body weight, from which body weight gain (BWG) and feed conversion ratio (FCR) will be calculated, will be measured weekly. Mortality will be recorded as it occurs. At 8 WOA, 20% of the experimental birds will be slaughtered and carcass characteristics which include carcass weight, abdominal fat content and yield of specific carcass components such as breast, thighs and drumsticks will be determined. To meet objective 3, evaluation of lysine homeostasis in performance of GF and broiler chickens, 432 GF and broilers each will be fed diets containing three lysine concentrations during the starter (0-4 WOA) and grower (5-8 WOA) periods. The dietary treatments will comprise 1.425% lysine (125% of NRC recommendation), 1.14% lysine (control) and 0.855% lysine (75% of NRC recommendation). The starter and grower diets will contain 3,100 ME Kcal/Kg,and 23% CP and 3,200 ME Kcal/Kg,and 23% CP, respectively. Feed and water will be provided at free choice. Weekly body weights will be taken until 8 WOA whereas mortality will be recorded as it occurs. Feed consumption and body weight will be measured, and BWG and FCR will be calculated. At8 WOA, 20% of experimental birds will be randomly selected and euthanatized. Tissues will be obtained from the hypothalamus, blood, liver, pancreas, adipose and intestines weighed and snap frozen in liquid nitrogen prior to storage at -80oC until used. These tissues will be used for RNA sequencing and gene expression assays to determine differentially expressed genes that may be influenced by variations in dietary lysine concentrations. To evaluate nutrient sensing and signaling, total RNA will be extracted from 0.3 g tissues from the hypothalamus, pancreas, intestine, adipose tissue and liver using the RNeasy Mini Kit (Qiagen, Valencia, CA). Sequencing RNA libraries will be prepared using Agilent® DNA 1000 Kit and Agilent® High Sensitivity DNA Kit. The Torrent suite and Ion PGM™ system will be used in the RNA sequencing. Key candidate genes will be confirmed using the one-step qPCRand QiaGen's QuantiTect® SYBR® Green PCR Kit. Qualitative data obtained will be presented as fold change in threshold cycle (Ct) relative to an internal control (Chicken 18s ribosomal RNA).To meet objective 4, to facilitate the understanding of key metabolic processes of the GF and other avian species, mRNA libraries of the guinea fowl pancreas, liver, and hypothalamus will be constructed and screened. The pancreas, liver, and hypothalamus will be harvested from adult male GF housed under a 12-hour light/dark cycle and fed a diet comprising of 3,100 kcal ME/kg diet and 23% CP. Total RNA will be isolated from each tissue using Qiagen's RNeasy® Mini Kit. Each sample will be diluted to 50 ng/μl, separated into 50 μl aliquots and immediately frozen at -80°C. The cDNA library will be constructed and sequenced using the Ion touch and Ion Torent Next Generation Sequencer (Life Technologies, Grand Island, NY). The cDNA library will be end-repaired, size selected and then ligated to adapter sequences. The resulting reads will be de-multiplex and reported as separate runs and deposited in the SRA database. Assembly and annotation will be performed on the Blacklight server at Pittsburgh's Super Computing Center (https://biou.psc.edu/blacklight) using Trinity (r2012-08-14) (available at http://trinityrnaseq.sourceforge.net/ ) under the default settings (Struys and Jacobs, 2011, Sauer et al., 2011). Sequences will be annotated using Trinotate (r2013-08-26) (available at http://trinotate.sourceforge.net). Transcript annotation will be performed also using the Trinotate software. The assembled contigs will be submitted to FastAnnotator for comparative annotation and identification of domains and potential enzyme functions. Fast Annotator (available at fastannotator.cgu.edu.tw) will be used to analyze the distribution of gene ontology terms, develop graphical representations of the data set and for enzyme identification. GO terms will be used to identify genes associated with metabolic functions.To meet objective 5, evaluating the efficacy of direct-fed microbials to further improve efficiency of feed utilization in poultry, 690 birds (345 chickens and 345 GF) will be raised for 8weeks using standard rearing procedures. Selected microbes which enhance utilization of nutrients such as phosphorus and nitrogen in-vitro will beconstituted and provided in feed. The probiotics will be supplied 106 to 107 cfu/gm of feed or 107 to 108cfu/ml of water. Body weights and feed consumption of experimental birds will be measured every week and feed and water will be suppliedfree of choice throughout the study. The mode of action of these probiotics will also be evaluated. At 8 WOA, birds will be sacrificed by cervical dislocation. The liver, hypothalamus, kidney, pancreas and intestine samples will be collected, weighed and snap frozen in liquid nitrogen andtransferred into -80°C freezer until analysis. Blood samples will be collected from all experimental birds for evaluation of metabolic profiles associated with feeding the probiotics. Extracted tissue samples will be used for transcriptome and metabolomic studies to understand the interaction between host and probiotic microorganisms. The metabolomic assays will utilize a combination of gas and liquid chromatography (GC and LC), coupled with mass spectrometry (GC-MS; LC-MS; LC-MS/MS), photodiode array (LC-PDA), or flame ionization detectors (GC-FID), to perform a complete examination of volatile and nonvolatile metabolites. The contents of the duodenal loop, ileum, jejunum and proximal colon will also be collected, frozen and later evaluated for the luminal contents resulting from metabolic processes in the gastrointestinal tract and associated organs. Gene expression differences (mean Ct ± standard error) in tissues such as liver, kidney, hypothalamus, pancreas, adipose tissue and intestine among probiotic treatments will be compared by one-way ANOVA. Transcriptome profiling will be performed using the Ion Torrent Genome Machine (Life Technologies, Grand Island, NY).Performance and gene expression data will be subjected to analysis of variance using the General Linear Models (GLM) procedures of SAS® (SAS Institute, 2002). Least significant difference comparisons and the T-test will be utilized, where applicable,to seperate treatment means for main effects when there is a significant F-value. Differences in mortality among dietary treatments will be analyzed using the chi-square method. Significant differences will be denoted by a probability ≤ 0.05.