Source: WEST VIRGINIA STATE UNIVERSITY submitted to
NUTRIENT UTILIZATION EFFICIENCY AND MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION RESEARCH FOR FINFISHES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
REVISED
Funding Source
Reporting Frequency
Annual
Accession No.
0223920
Grant No.
(N/A)
Project No.
WVAX-MITOCHONDRIAL
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2010
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Project Director
Eya, JO.
Recipient Organization
WEST VIRGINIA STATE UNIVERSITY
PO BOX 1000
INSTITUTE,WV 25112
Performing Department
Agricultural & Environmental Res Station (AERS)
Non Technical Summary
Aquaculture is the most rapidly developing sectors of the food industry in the U.S. and has been greatly influenced by global developments. As feeds represent up to 80% of the variable cost of production, it is imperative to look at dietary, genetic and other production factors that regulate nutrient retention efficiency, growth and development in different life history stages of cultured fish. Because intensive aquaculture heavily relies upon large inputs of ingredients, such as fish meal and fish oil obtained from wild fish, the sustainability and impact of such activities on world fisheries is questioned. Thus, the replacement of fishmeal as the major protein source by protein sources of plant origin and the development and use of high-nutrient-dense, low pollution diets containing lipids of plant-origin are major objectives for the sustainable development of aquaculture. The primary goal of the project is to understand the fundamental genetic, cellular, and biochemical mechanisms responsible for nutrient utilization efficiency in finfish fed diets of different compositions/formulations, especially plant-based and high-nutrient-dense (high-fat) diets, using genomic-based nutritional approach.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
100%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3023711101050%
3023711102025%
3023711104025%
Goals / Objectives
Objectives: (a) Evaluate the influence of dietary protein and lipid (energy) levels on growth performance characteristics and the expression of gene transcripts involved in mitochondrial oxidative phosphorylation in rainbow trout; (b)Determine the effects of high-lipid diets on protein and mitochondrial proteins oxidation in rainbow trout; (3) Characterize effects of feeding plant-based diets on mitochondrial metabolic efficiency and gene expression (mitochondrial function) in rainbow trout; and (4)Assess the association between mitochondrial function and nutrient utilization efficiency in rainbow trout fed plant-based diets Expected Outputs: Expected outcomes and useful results of this project are many and vital for continued expansion of the domestic aquaculture industry, and will allow for production of sustainable diets for rainbow so that reliable, safe, and nutritious cultured fish reaches the U.S. consumer. The relationships among growth, feed utilization efficiency, and mitochondrial function/biochemistry for rainbow trout fed traditional and plant-based diets will be clarified using new genomics-based nutritional technology (nutrigenomics) in combination with traditional measures. Collecting a variety of data (production data, biochemical, and genetic data) will increase the likelihood of identifying mechanisms that currently constrain the use of plant-based diets, and may reveal new directions for research to optimize feed efficiency and production economics, while strengthening the sustainability of rainbow trout production. Additional expected outcomes from this proposal are that of the research studies teaching and training undergraduate and graduate students in aquaculture methods and principles, the scientific method, aquatic biology, and aquaculture nutrition. Since WVSU is the sole institution that offer aquaculture-related courses in West Virginia for minorities and since some of the collaborating institutions are historically black colleges, this project will increase the visibility and expertise of the researchers at these institutions so as to attract high-quality students, especially minority students, who traditionally do not enter the agricultural sciences. This should increase both ethnic and gender diversity of the food and agriculture (aquaculture) base in this country, because these persons will be qualified to pursue career opportunities within the aquacultural and agricultural sciences, thus, increasing the diversity of the Nation's food and agricultural science base. Also, participating federal agencies (USDA-ARS and USFWS) bring additional benefits and resources to our graduate and undergraduate students
Project Methods
Objective 1: The feeding trial for this objective has been completed and the procedures used were as follows: Diets: A 2 X 3 factorial experiment design was used. The two factors evaluated were two protein levels (40 and 45%) and three lipid levels (10, 20 and 30%). Fish Management: Fish management will be according to the procedures described previously by Eya et al. 2010. Tissues samples were collected for the determination of mitochondrial complexes enzyme activities, and mitochondrial gene expression analyses. Mitochondrial enzyme complex activities: Isolation and preparation of mitochondrial fractions will be performed according to the procedures described by Birch-Machin and Turnbull 2001; Jeejeebhoy 2002 and Kirby et al. 2007 and modified by Eya et al. 2010. Gene expression analyses: Sample preparation, RNA isolation, cDNA synthesis, and real-time PCR- Genes associated with mitochondrial function to be analyzed will include Cytochrome C Oxidase, NADH Dehydrogenase Subunit 1, Adenine Nucleotide Translocase, ATPase Subunit 6, ATP Synthase beta-subunit, Uncoupling Protein2A, Peroxisome Proliferator-Activated Receptor 1-alpha, Peroxisome Proliferator-Activated Receptor Gamma Coactivator alpha using quantitative real-time PCR. Objective 2: Protein and mitochondrial protein carbonyl content of tissue samples obtained from the Objective 1 will be determined as described by Levine et al. (1990) and Youngman et al. (1992), with slight modifications. Objective 3: Diets: Two series of diets will be formulated. First series will be three diets formulated to be isoenergetic (DE = 20 MJ/kg) and isonitrogenous. Diet 1 (fishmeal as the primary protein source; 42% protein and 20%); Diet 2 (plant diet without animal protein source; 42% protein and 20% fat) and will contain corn gluten meal and soy-protein concentrate; Diet 3 (50% fishmeal protein and 50% plant protein sources). The second series will consist of four isonitrogenous and isoenegertic diets (42% protein and 20% fat) that will be primarily plant-based: Diet 4 (soy protein concentrate); Diet 5 (canola protein concentrate); Diet 6 (corn gluten meal), and diet 7 (combination of corn gluten meal, soy protein concentrate and canola protein concentrate) in the ratio of 1:1:1. Fish management and mitochondrial enzyme complex activities and Gene expression analyses: This will be performed according to procedures described in objective 1. Objective 4: Diets: The diet from the objective 3 that give the best growth, improved nutrient utilization efficiency and excellent mitochondrial function will be formulated to mimic commercial ranges of ingredients and be comparable to commercial diets used in the commercial production of rainbow trout. Fish management, mitochondrial enzymes activities and protein oxidation, and gene expression analyses: These will be similar to that described in objectives 1 and 2. Statistical Analyses: All experimental data will be subjected to one-way analysis of variance using General Linear Model of Statistical Analysis (SAS) Institute Inc. (1987) and differences between the treatment means will be considered significant at the probability level of 5%.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Fish feed manufacturers, fish breeders, farmers and students. Rainbow trout with improved growth and nutrient retention will benefit the farmers’ bottom line in terms of profitability. Improved nutrient retention efficiency will benefit the environment by reducing pollution from un-retained discharged as effluents. Students used the research and the findings as part of their MSc. thesis, and will graduate with a degree in Biotechnology opening up new career opportunity. Changes/Problems: Two analytical instruments (Leco bomb calorimeter andThermo Fisher Genesys 2 spectrophotometer)developed faults and have been sent out to vendors for repairs. What opportunities for training and professional development has the project provided? Graduate students that are pursuing the MSc. Degree in Biotechnology used the research for their thesis and one of the students is considering pursuing a PhD degree or career in fisheries management/aquaculture How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? The following analyses:nutrient utilization efficiencies (protein efficiency ratio, protein productive value, energy efficiency ratio, energy productive value, lipid productive value, and lipid efficiency ratio), biochemical analyses of mitochondrial respiratory chain enzyme activities, and gene expression levels of selected mitochondrial genes on tissues (liver, intestine and muscle) will completed and results published in peer-reviewed journals.

Impacts
What was accomplished under these goals? A 2 x 3 factorial experiment was conducted to evaluate the effects of rainbow trout families (high FE or low FE) and diets (45/10 or 45/20 or 45/30 percent protein/fat) on the performance, mitochondrial respiratory chain enzyme activities and gene expression levels in the liver, muscle and intestine. The analyses of mitochondrial respiratory chain enzyme activities were performed on tissues (liver, intestine and muscle). Data analyzed were for weight gain, feed efficiency, feed consumption, specific growth rate, body composition, and visceral fat content. The following analyses are ongoing: nutrient utilization efficiencies (protein efficiency ratio, protein productive value, energy efficiency ratio, energy productive value, lipid productive value, and lipid efficiency ratio), biochemical analyses of mitochondrial respiratory chain enzyme activities, and gene expression levels of selected mitochondrial genes on tissues (liver, intestine and muscle)

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Eya, J.C. Nouaga, R.Y., Ashame, M.F., Pomeroy, C.F., Gannam, A.L. 2013. Effects of dietary lipid levels on growth, feed utilization and mitochondrial function in low- and high- feed efficient families of rainbow trout (Oncorhynchus mykiss). Aquaculture 416-417: 119-128.
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Eya, J.C. Nouaga, R.Y., Ashame, M.F., Pomeroy, C.F., Gannam, A.L. 2013. Effects of dietary lipid levels on mitochondrial gene expression in low-and high-feed efficient families of rainbow trout Oncorhynchus mykiss. (Submitted to Journal of Fish Biology and accepted for publication)


Progress 01/01/12 to 09/30/12

Outputs
Target Audience: The target audience includes fish farmers, fish feed manufacturers, and researchers involved rainbow trout nutrition and selective breeding. Another group of target audience are graduate and undergraduate students participating in the project that are using the research experience as learning resource for their professional development. Changes/Problems: Aquaculture feeding trials were terminated early because water temperature issues. Thismay have affected expectedoutcomes What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A 2 x 3 factorial experiment was conducted to determine effect of trout families (designated as low FE and high FE) and diets (40/10 or 40/20 or 40/30 percent crude protein/fat) on the growth performance characteristics, mitochondrial respiratory enzymatic activities and gene expression in the liver, muscle and intestine. Another 2 x 3 factorial experiment was conducted to determine effect of trout families (designated as low FE and high FE) and diets (45/10 or 45/20 or 45/30 percent crude protein/fat) on the growth performance characteristics, mitochondrial respiratory enzymatic activities and gene expression in the liver, muscle and intestine. Growth performance characteristics, mitochondrial respiratory enzymatic activities and gene expression in the liver, muscle and intestine have been completed for diets containing 40/10, 40/20, and 40/30 percent crude protein/fat.

Publications


    Progress 01/01/11 to 12/31/11

    Outputs
    OUTPUTS: Data collected from the six studies involving factorial experiments were subjected to two-way ANOVA, and differences between means were determined by Duncan's New Multiple Range Test. Treatment main and interaction effects were considered significant at P<0.05. At the end of each feeding trial, the following parameters were determined Percent weight gain = [(final wt - initial wt)/initial wt] x100; Feed efficiency = wet wt gain/dry feed fed; Mean feed consumption = total feed consumed (dry wt)/# of fish; Protein efficiency ratio = live wt gain/protein intake; Protein productive value = [(final protein content&#8722;initial protein content)/protein consumed]; Energy productive value = [(final energy content&#8722;initial energy content)/energy consumed]; Lipid efficiency ratio= live wt gain/lipid intake; Condition factor = wt/length3; and Specific growth rate = [(ln final wt - ln initial wt)/# of days] x 100. Also, mitochondrial oxygen consumption rate, mitochondrial complex enzymatic activities, and mitochondrial gene expression levels were determined for liver, muscle and intestine. Results from the project will be presented at Aquaculture America 2012, Las Vegas, Nevada. Feb 20 - Mar 2, 2012. Results from the project had been presented at the Annual International Conference and Exposition of the World Aquaculture Society, Natal Brazil. June 6-10, 2011; 1890 institutions of Association of Research Directors 15th and 16th Biennial Research Symposium at Atlanta, Georgia, Mar 28-Apr 1, 2009 and Apr 9- 13, 2011, respectively. The project impacted 23 students through participation in directed student research. The feeding and analyses of data were incorporated into classroom teaching, especially for two courses (Principles of Aquaculture and Biology of Fishes) taught at the WVSU. Project resources were to purchase equipment such as ABI 7300 rt-PCR System, thermal cycler and refrigerated centrifuge used in teaching and research currently used for the training of undergraduate and graduate students in molecular biology, especially genomics and nutrigenomics. The results from the project provide some evidence to the association of mitochondrial function with the phenotypic expression of feed efficiency in rainbow trout and channel catfish. Results further showed that mitochondrial respiratory chain activity, gene expression, and protein oxidation are linked to nutrient utilization efficiencies in rainbow trout and channel catfish identified as having low or high feed efficiency. The research served as a platform for hands-on training for graduate and undergraduate students in WVSU in genomic-enabled nutritional research to solving aquaculture related problems. Moreover, this project impacted several students through participation in directed student research and the integration of classroom teaching to performing experiments associated with the project. The research laid the foundation for some of minorities and/or undergraduates/graduates that participated in this project to pursue their graduate education in aquaculture and/or biotechnology having worked side-by-side with multidisciplinary research scientists with different areas of expertise. PARTICIPANTS: Project Director designed, implemented, supervised a research associate, graduate and undergraduate student, and performed data analyses. Rainbow trout for studies 1 and 2 feeding trials were generously supplied by the collaborators (Drs. Greg Weber, Caird Rexroad, Beth Cleveland) at the USDA-ARS National Center for Cool and Cold Water Aquaculture. Feed formulation and manufacture was performed by Dr. Ann Gannam of U.S. Fish and Wildlife Service, Abernathy Fish Technology Center. A research associate, a research technician, and two students participated in the study and their duties included feeding, sampling, collecting and analyzing data. TARGET AUDIENCES: The target audience includes fish farmers, fish feed industries and researchers that are involved in selective breeding of aquaculture species. One graduate and an undergraduate student earned credits hours in directed student research. The data collection and analyses were utilized as teaching resource for the students who participated in the experiment as part of their directed student research and acquired credit units toward their major and thesis. PROJECT MODIFICATIONS: The feeding trials for both studies were terminated after 90 days instead of 112 days because of the failure of the water cooling system.

    Impacts
    Findings from this research for the first time in rainbow trout and channel catfish, demonstrated a strong relationship between diet, strain types and mitochondrial function. Furthermore, results showed variations in the expression levels of some mitochondrial encoded genes and mitochondrial complex enzyme activities in different tissues of rainbow trout and the family with high feed efficiency (FE) and better nutrient utilization had higher numerical values for respiratory chain enzyme activities, down-regulation of hepatic complex I ND1 gene and its up-regulation in the intestine and muscle compared to the low FE family. Data from the research showed that in rainbow trout families with high FE, diet containing 42% crude protein (CP)/20% fat was significantly better than those containing either 42% CP/10% fat or 42% CP/30% fat in terms of growth performance characteristics. Thus, the popular belief that high fat diets (above 20% fat) are necessary for efficient production of fish is not supported by this study. Another aspects of the findings in channel catfish showed that diet containing 32% CP from all-plant protein sources is as good as diets (28% and 32% CP) containing fishmeal for optimum growth and improved FE. However, the mitochondrial function, especially the mitochondrial gene expression levels were significantly down-regulated in fish fed diet containing 32% CP from all-plant protein sources in the liver and intestine. Overall, our data indicate that genetic factor plays a role in rainbow trout and channel catfish production and fish selected for improved growth and high FE when fed practical diet exhibited significant genotype x diet interaction. Thus, if "fine-tuning" of dietary formulations is to be considered in the future, selection strategies that account for that interaction should be considered. The observed changes in some of the selected mitochondrial genes in relation to growth and nutrient utilization efficiencies could serve as marker(s) in breeding selection for genetic improvement of rainbow trout and channel catfish and possibly other aquaculture species.

    Publications

    • Eya, J.C., Ashame, M.F. and Pomeroy, C.F. 2011: Association of mitochondrial function with feed efficiency in rainbow trout: Diets and family effects. Aquaculture 321(1-2):71-84.
    • Eya, J.C., Ashame, M.F., Pomeroy, C.F., Manning, B.B. and Brian, B.C. 2011. Genetic variation in feed consumption, growth, nutrient utilization efficiency and mitochondrial function within a farmed population of channel catfish (Ictalurus punctatus). Aquaculture (Pending).
    • Eya, J.C., Ashame, M.F. Pomeroy, C.F, and Gannam, A. L. 2012. Genetic relationships of growth, feed efficiency and mitochondrial function in rainbow trout (Oncorhynchus mykiss) fed diets differing in dietary lipid levels (Manuscript in preparation).
    • Eya, J.C., Ashame, M.F., Manning, B.B. and Brian, B.C. 2012. Effect of strain and diet on growth performance characteristics and relative expression of genes coding for electron transport chain in channel catfish (Manuscript in preparation).