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INVESTIGATOR: Eya, J. C.

PERFORMING INSTITUTION:
WEST VIRGINIA STATE UNIVERSITY
PO BOX 1000
INSTITUTE, WEST VIRGINIA 25112

IMPROVING EFFICIENCY OF GROWTH, NUTRIENT UTILIZATION AND MITOCHONDRIAL OXIDATIVE FUNCTION USING ALTERNATIVE FEED INGREDIENTS

NON-TECHNICAL SUMMARY: This project advances genomics and metagenomics-based nutritional approaches in improving productivity and quality of aquaculture in anticipation of climate change. This project will study alternative feed/fish gut microbiome/mitochondrial function/temperature interactions and will generate important information relevant to the development of sustainable, low cost aquaculture feeds. Objectives 1: Determine the interactive effects of various fat sources and temperature on growth performance characteristics, mitochondrial function, and fish gut microbiome in rainbow trout. Sub-objective 1A: Characterize and compare effects of fish versus plant proteins and oils and temperature on growth performance characteristics, mitochondrial function, and fish gut microbiome; Sub-objective 1 B: Evaluate the effects of blending animal and vegetable fats on production efficiency, mitochondrial function, and fish gut microbiome; Sub-objective 1 C: Characterize and compare effects of feeding plant oil versus rendered animal oils and temperature on growth performance characteristics, mitochondrial function, and fish gut microbiome; and Objective 2: Test the ecological resilience of the fish gut microbial community with regard to adaptive energy extraction from alternative feed ingredients. This project will generate new data that will improve understanding of the importance of fish gut microbiome and its role in fish physiology, feed efficiency and growth. Providing an understanding of these issues using aquaculture genomics may lead to commercialization of alternatives resulting in reduced dependence on marine fish resources. Additionally, underrepresented groups will gain hands-on experience in cutting-edge genomics-based research that equips them with skills and knowledge necessary for leadership in STEM-related fields. Moreover, directly incorporating research activities into student education and training and focusing on under-served students will diversify STEM-related workforce.

OBJECTIVES: Objective 1: Determine the interactive effects of various protein/fat sources and temperature on growth performance characteristics, mitochondrial function, and fish gut microbiome in rainbow trout. Sub-objective 1A: Characterize and compare effects of fish versus plant proteins and oils and temperature on growth performance characteristics, mitochondrial function, and fish gut microbiome in rainbow trout; Sub-objective 1 B: Evaluate the effects of blending animal and vegetable fats on production efficiency, mitochondrial function, and fish gut microbiome in rainbow trout; and Objective 2: Test the ecological resilience of the fish gut microbial community with regard to adaptive energy extraction from alternative feed ingredients.

APPROACH: Sub-objective 1A:In order to achieve this objective, a 4x2 factorial study will be conducted comprising of four diets and two temperature regimens for 5 months duration. The four diets will be: Diet 1 = fishmeal-based containing 50% fish oil and 50% plant oil (Camelina oil); Diet 2 = fishmeal-based containing 100% Camelina oil; Diet 3 = plant-based protein (from soybean, lupin and canola meal) containing 50% fish oil and 50% plant oil; and Diet 4 = plant-based containing 100% Camelina oil) and the two temperature regimens will be low (14 oC) and high (20 oC). Each treatment will have 4 replicates.Fish Management: Juvenile rainbow trout will be obtained from the USDA-ARS National Center for Cool and Cold Water Aquaculture and will be used for the study at the WVSU aquaculture wet laboratory. Juvenile rainbow trout weighing approximately 20 ± 5 g will be randomly assigned to 32 162-L aquaria of 26 fish per aquarium. One set of sixteen aquaria will be subjected to 14 oC and the second set of the other sixteen aquaria will be maintained at 20 oC. Each temperature with 16 aquaria will be completely recirculating system with a continuous flow of dechlorinated water at the rate of 1.5L/minutes with partial water exchange in each system. On the first day of the experiment, three fish will be randomly selected from each tank, anaesthetized with MS-222, killed with sharp blow to the head, and frozen at -80 oC, for whole-body composition analyses and for fatty acid profile. An additional three fish from each tank will be randomly selected, killed and tissues collected for biochemical analyses and nuclear- and mitochondrial- encoded gene expression, and intestine will be collected for fish gut microbiota analyses at day 0 (start date for feeding with experimental diets). At the end of the 5 months feeding trial, fish in each aquarium will be individually counted, weighed, and total length measured. Three fish per tank will be randomly selected, killed and frozen for whole-body proximate composition and fatty acid profiles. Additional 3 fish will be randomly selected, killed and tissue samples collected for biochemical and gene expression analyses, and intestine will be collected for gut microbiota analyses. The tissue samples will be flash frozen by immersion in liquid nitrogen and stored at -80 oC. The entire intestinal tract of each of the 3 fish will be removed and immediately frozen −80 °C for gut microbiome analyses. Growth performance characteristics and nutrient efficiencies will be measured described by Eya et al. (2011). The animals will be kept in accordance with the guidelines of the WVSU's Institutional Animal Care and Use Committees.Diet formulation and manufacture, proximate composition and fatty acid profile: (a) Feed manufacture - The ingredients will be ground to less than 0.5 mm in a pin mill, sized with a screener and weighed to produce the batches of each diet. Diet ingredients will be mixed with the vitamin and mineral premixes and additional supplements and extruded. After extrusion, diets will be dried to less than 10% moisture. The dry diets will be top-coated with oil, 50 lbs at a time, in a Kushland cement mixer, bagged and stored in cool dry room. (b) Fish and feed analyses - Proximate composition for feed and whole body moisture, protein and ash will be determined by standard AOAC methods (AOAC 2010). The feeds will be analyzed for amino acid composition. Total lipid in tissues and diets will be extracted and fatty acid methyl esters (FAME) prepared for fatty acid analysis as described by Li & Watkins (2001). The FAME will be quantified using methods described by Twibell et al. (2015).Mitochondrial electron transport system enzyme complex activity: The activities of the respiratory chain enzymes will be determined using the Thermo Fisher Arena 60 automated photometric analyzer system. Isolation and preparation of mitochondrial fractions from liver and muscle of rainbow trout will be performed according to the procedures described by Eya et al. 2010; -with slight modifications for complex I (NADH: Ubiquinone Oxidoreductase), -complex II (Succinate: ubiquinone1 oxidoreductase by Birch-Machin and Turnbull 2001 and Kirby et al. 2007), -complex III (ubiquinol: ferricytochrome c reductase by Jeejeebhoy 2002), complex IV (cytochrome c oxidase by Birch-Machin and Turnbull 2001), -complex V (F1-ATP synthase by Kirby et al. 2007), and Citrate synthase activity by Kirby et al. (2007).Gene expression analyses - The GenomeLab GeXP genetic analysis system (Beckman Coulter Inc.) will be used to simultaneously analyze the expression of 20 genes within a single PCR sample. Genes relevant to the mitochondrial process of oxidative phosphorylation, lipid and amino acid metabolism have been identified and primers will be designed using eXpress Designer software specific for the multiplex analysis. These genes involved in oxidative phosphorylation are of mitochondrial (encoding complex I (nd1), complex III (cytb), complex IV (cox1), complex IV (cox2) and complex V (atp6)), nuclear (peroxisome proliferator activated receptor alpha and beta (pparα and pparβ) and PPAR-γ coactivator-1-alpha (ppargc1α), and uncoupling proteins (UCP) (ucp2α and ucp2β), amino acid metabolism (Cathepsin D (cath d), Cathepsin L (cath l), F-box protein-32 (fbx32), Ubiquitin (ubq) and Lysyl Oxidase (lox), and lipid metabolism ((carnitine palmitoyl transferase, long chain acyl-COA synthase, fatty acid-binding protein (FABP), fatty acid transport protein (FATP) and acyl-COA dehydrogenase).Gut Microbiome Analyses: The effects of the treatments (diet x temperature) on the diversity, stability, and functionality of the trout gut microbiome will be assessed using two approaches: 1. standard ecological diversity metrics based on 16S rRNA gene diversity, and 2. metabolite sampling to directly link diet and microbial functional diversity.Objective1B: - In order to achieve this objective, a 4x2 factorial study will be conducted comprising of four diets and two temperature regimens. The four diets will be: Diet 1 = fishmeal-based containing 25% fish oil and 50% alternative oil mixture from canola, palm and Camelina (1:1:1) and 25% poultry fat; Diet 2 = fishmeal-based containing 25% fish oil and 50% alternative oil mixture and 25% beef tallow; Diet 3 = plant-based protein (from soybean, lupin and canola meal) containing 25% fish oil and 50% alternative oil mixture and 25% pork fat; and Diet 4 = plant-based containing 100% alternative oil mixture and the two temperature regimens will be low (14 oC) and high (20 oC). Feed trial will be for 5 months. Fish management, diet formulation and manufacture, proximate composition and fatty acid profile analyses, growth and nutrient efficiencies determination, mitochondrial electron transport system enzyme complex activity, gene expression analyses, and gut microbiome will be performed as described in objective 1 A.Objective 2: Following the previous experiments, 4 replicate lines of fish will be shifted to one temperature (14°C) and the ecological resilience and adaptive energy extraction capacity of the gut communities will be tested using a reciprocal diet exchange design (Figure 1). From the previous results, the best animal-based diet and the worst plant-based diet in terms of weight gain and feed efficiency will be used because they should represent the greatest adaptive shifts in the gut communities. The metagenomics methods described above will be followed. In addition, alterations of the anaerobic food web will be assessed by measuring SCFAs as described above, and bomb calorimetry will be applied to fecal matter to assess energy extraction efficiency. Sampling of the gut communities will occur simultaneously in each treatment at the beginning and end of the diet shifts. Fish growth will be measured as described above.

PROGRESS: 2020/03 TO 2021/02
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduate and one graduate students had participated in the project and have been trained in all aspects of scientific methods, data collection and analyses. Two graduate students and a Postdoctoral research fellow havebeen recruited for the completion of the project objectives. Because of Covid-19 pandemic, none of the students involved and the PD have participated in any regional or national conferences in the year 2020. How have the results been disseminated to communities of interest?The results from the completed study has been presented to the university community during the public thesis defense of the graduate student that participated in the project. What do you plan to do during the next reporting period to accomplish the goals?Analyses of the samples obtained from the first feeding trial sub-objective 1A(gene expression analyses); sample analyses from subobjective 1B feeding trial and completion of the objective 2 feeding trial and sample analyses.

IMPACT: 2020/03 TO 2021/02
What was accomplished under these goals? Tissues samples from sub-objective 1A have been analyzed for proximate composition, mitochondrial function analyses in terms of mitochondrial biochemistry (mitochondrial complexes enzymatic activities) and gut microbiome and completed. The gene expression analyses for this sub-objectiveis outatanding and the analyses will start next month. The feeding trial forsub-objective 1B has been completed. Fecal materials have been collected for the gut mictobiome analyses. Tissue samples for proximate composition, gene expression and mitochondrial function analyses have been collected. Preparation for the third feeding trial for objective 2 has starteed. The feed formulation and the process of acquiring rainbow trout has commenced.

PUBLICATIONS (not previously reported): 2020/03 TO 2021/02
Type: Theses/Dissertations Status: Other Year Published: 2021 Citation: Abanikannda, M.F. 2020. The effects of plant derived-derived aquafeed and temperature on growth, nutrient utilization efficiency, mitochondrial function and gut microbiobiome in rainbow trout