Performing Department
Nutritional Sciences
Non Technical Summary
The feed industry now promotes use of dietary organic selenium (Se) and supplementation with higher levels of Se for animals. There are currently no identified biomarkers of High-Se status. We previously determined Se requirements for turkeys fed inorganic Se using traditional and selenoprotein mRNA biomarkers, but did not find good biomarkers for supernutritional Se status. Specifically, we now propose to (1) determine Se regulation of traditional biomarkers of Se status (growth, tissue Se concentration and selenoenzyme activity level) in turkeys fed High-Se diets as inorganic Se, to identify potential biomarkers of High-Se status and to better understand the result of high-Se supplementation; (2) In turkey poults fed Se-deficient to High-Se diets as inorganic Se, determine Se regulation of the complete transcriptome in liver using RNA-Seq, to identify biomarkers of supernutritional-, high- and toxic-Se status, to better understand how the turkey adapts to High-Se status, and to better define safe upper limits of Se supplementation; (3) in turkey poults supplemented with organic Se, (a) determine Se regulation of traditional biomarkers of Se status and (b) determine the Se regulation of the complete transcriptome in liver using RNA-Seq, to better characterize the effects of organic vs. inorganic Se supplementation, and to determine minimum dietary Se requirements and safe upper limits for organic Se supplementation. These studies will help to better evaluate the levels and benefits of organic vs. inorganic Se supplementation. These tools and our continued emphasis on their development and application are essential for improving efficiency of the birds directly (genotype selection) and indirectly (the management system, e.g.,nutrition).
Animal Health Component
40%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
Create and share data and technology to enhance the development and application of genomics, epigenomics, and systems biology in poultry.
Project Methods
The following briefly summarize methods that will be used in these studies. We have published use of all cited methods, except RNA-Seq, previously.D1. Poults- Male one-day-old poults will be donated by Jennie-O Turkey Store, Barron WI, and housed in electrically-heated battery brooder cages with raised wire floors in air-conditioned animal quarters (72oF) at the UW Poultry Research Lab (PRL) as described previously. The animal care and treatment protocol is approved by the University of Wisconsin Institutional Animal Care and Use Committee.D2. Poult diets- The diet will be the diet used previously, including supplemental adequate vitamin E, that promotes reasonable growth. This is a Se-deficient semi-purified diet (7 ng Se/g diet, 30% low-Se torula yeast plus 7% additional crystalline amino acids) matching NRC requirements. The poults will be fed the basal diet supplemented with graded levels of Se for 28 d. The Se content of each diet will be confirmed by neutron activation analysis.D3. Tissue Analysis- Poults will be killed by CO2 overexposure followed by exsanguination. Blood will be collected into heparin or EDTA-containing syringes, and tissues (liver, heart, gizzard, kidney, thigh and breast muscle, pancreas, testes) rinsed in 0.15 M KCl and quick frozen.D4. Enzyme Activity Assays- Plasma glutathione peroxidase (Gpx3) and RBC Gpx1 activity are determined, and Gpx1, Gpx4, and thioredoxin reductase (Txnrd) activities determined in the tissues. Protein concentration of each sample is determined by the method of Lowry et al.D5. Total RNA isolation- Under a fume hood, total RNA from the tissues (50-100 mg tissue, n=4-5/diet group) is isolated using the guanidinium isothiocyanate method with TRIzol Reagent (cat. #15596-026, Invitrogen, Carlsbad, CA), following the manufacturer's protocol.D6. Tissue and diet Se determination- Liver and muscle tissue (on dry ice) and diet samples will be shipped from the University of Wisconsin to the University of Missouri Research Reactor (MURR). Se will be analyzed using neutron activation.D7. qPCR- Quantitative real time polymerase chain reaction (qPCR) will be used to determine the levels of selenoprotein mRNA and of non-selenoprotein mRNA in total RNA isolated from tissues.D8. mRNA sequencing- Isolated total liver RNA will be submitted to the UW Sequencing Center. Briefly, RNA will be subjected to quality control, filtered to select polyA RNA (mRNA), Mg fractionated, tagged-cDNA libraries generated, and then subjected to Illunina HiSeq2500 sequencing (minimum 250M reads/library), to generate the FASTQ data files for each sample. Due to the relatively new turkey draft genome sequence and annotation, paired-end sequencing will be performed as advocated by Dr. Kent Reed.D9. RNA-Seq data analysis- In collaboration with Dr. Kent Reed (U Minn), the resulting FASTQ data files will be mapped to the updated turkey genome annotation, and differential expression due to Se status determined for the entire turkey transcriptome, including subsequent gene ontology (GO) analysis.D10. Se-response curves- Our research generates Se-response curves for various biomarkers that collectively describe the Se status of an organism. We use breakpoint analysis to compare parameters in terms of minimum Se requirement, where the plateau breakpoint is defined as the intersection of a line tangent to the steepest slope and the plateau line. These breakpoints are the minimum dietary Se requirement for that biomarker. These curves will be used to assess Se status and determine Se requirements, and a similar approach will be used for assessment of ULs.D11. Statistics-All analyses, except for RNA-Seq, will be conducted with statistical software available in our lab.