Research Project: Improving Nutrient Utilization to Increase the Production Efficiency and Sustainability of Rainbow Trout Aquaculture

Location: Small Grains and Potato Germplasm Research

2022 Annual Report

Objectives
The long-term objective of this research project is to provide stakeholders with products and information that can be used to improve sustainable production of rainbow trout. This will be accomplished by identifying novel ingredients with potential for use in aquaculture feeds, isolating new nutritional compounds and improving methods of isolating relevant dietary components, and verifying that formulations and dietary components are beneficial for fish growth and health with minimal impact on receiving waters. Feeds and improved rearing practices will be evaluated on existing commercial strains of rainbow trout. Traits of interest will be identified, and improved trout strains generated and tested. In addition to improvements in feed and strains, feeding and rearing practices will be developed for incorporation into best management plans. ARS researchers will work closely with stakeholders to ensure rapid dispersion of information to the industry. Specifically, during the next five years we will focus on the following objectives: Objective 1: Develop novel methods for creating and evaluating new ingredients and feeds. • Subobjective 1A: Innovate methods to make alternative protein ingredients containing encapsulated oils. (Liu, Vacant Nutritionist and Welker) • Subobjective 1B: Improve assay methods for acid insoluble ash and make it a reliable marker for digestibility studies. Objective 2: Develop feed formulation and processing technologies that minimize impacts on water quality. • Subobjective 2A: Develop feed formulation strategies that increase the stability of fecal castings (durability and particle size) and feed pellets to improve waste collection and water quality (particularly through addition of naturally occurring binders and modification of processing conditions). • Subobjective 2B: Determine the best performing combination of feed processing conditions, starch characteristics (e.g. amylose:amylopectin ratio, particle size, molecular structure), and diet formulation to enhance fecal and feed stability. Objective 3: Identify factors affecting the utilization of sustainable feeds and develop strategies to improve rainbow trout production systems. • Subobjective 3A: Use genomic methods and physiological assessments to analyze the effects of different formulated feeds and water quality conditions in different strains of rainbow trout. (Overturf and Welker). • Subobjective 3B: Improve rainbow trout to convert plant protein and lipids efficiently for enhanced growth.

Approach
Obj 1: Develop novel methods for creating and evaluating new ingredients and feeds. Research Goal: Generate new methods to increase oil content of trout feed via encapsulation to prevent lipid oxidation, oil leakage, and extend shelf-life. Develop an improved assay method for acid insoluble ash (AIA). Oil encapsulation of vegetable oils will be tested by spray drying and coacervation. Various plant protein dispersions will be prepared by testing mixes of soy or other plant proteins. Analysis of the microencapsulated particles and then the digestibility of ingredients captured within the capsules will measured and evaluated when fed to fish. Using different materials with varying levels of ash and AIA, a newly modified technique will compare the utilization of using AIA against existing techniques in determining digestibility of feed ingredients. If we cannot develop a product with 99% EE then 70% EE will be considered valuable. If AIA is low a commercial form of silica will be added. Obj 2. Develop feed formulation and processing technologies that minimize impacts on water quality. Hypothesis: Feed processing and addition of natural binders to commercial diet formulations will increase the durability and stability of trout feces and feed pellets in water. Strategies that increase the stability of feed material to improve water quality will be tested by evaluating processing conditions, the effectiveness of binders, gelatinization and the addition of additives to improve the flotation of diet and fecal particles. A commercial diet formulation for rainbow trout will be processed by extrusion and expansion pelleting. The pellet types/diets will be tested in growth trials with water quality monitoring. Using the best processing conditions three varieties of wheat & barley will be tested. The effects of grain source, feed processing and addition of cork on pellet and fecal characteristics, digestibility, growth, and water quality will be evaluated. If the tested binders do not provide adequate results additional binders may be tested. Obj 3: Identify factors affecting the utilization of sustainable feeds and develop strategies to improve rainbow trout production systems. Research Goal: Measure effects and interactions of trout strain, feed, and water quality to guide development of management practices to increase production efficiency and to use genetic selection to improve conversion of plant lipids to EPA and DHA. The diets from objectives 1 & 2 will then be tested in multiple strains of trout in water of worsening quality. The effect of diet on fish performance will be evaluated across water conditions and compared to performance for these strains and diets when tested under laboratory conditions. To determine the ability of rainbow trout to biosynthesize and convert plant lipid to EPA and DHA, we will generate & test family crosses generated from individuals with known muscle fatty acid ratios. Offspring will be reared on the complete plant-based feed & phenotypes observed & used for selection of the next generation of broodstock. As more robust analysis methods become available, we will implement these procedures in our analyses.

Progress Report
Substantial progress was made for all project objectives. In support of Sub-objective 1A, soy protein ingredients containing encapsulated oils were developed by mixing soybean proteins with vegetable oils into an aqueous solution and spray drying. In one study, a lab method was developed for making soy protein ingredients containing encapsulated oils. The lab method was then scaled up to produce a relatively large quantity of the new protein ingredient. Analysis of this product showed it contained about 65% protein content and 20% oil. The process led to about 84% oil encapsulation efficiency. In a preliminary trial, the new protein ingredient was used to formulate an experimental diet. The effect of the new protein ingredient on feed extrusion was investigated. In the trout feed industry, there is an increasing demand for high energy trout feed, which has led to incorporating higher amounts of oil into feed, in the range of 15-25%. Development of soy protein products containing encapsulated oils not only extends the shelf-life of the ingredients, but also prevents oil leakage and oxidative rancidity of feed associated with high oil inclusion. For Sub-objective 1B, we demonstrated that an acid insoluble ash (AIA), silica found in all feeds and digestive material, can be used as a feasible and economical replacement for yttrium, a chemical element commonly added to feed and used to determine dietary digestibility. In this study, four diets containing proteins of different sources were spiked with three levels of Celite (a commercial AIA) and 0.1% yttrium, before being fed to trout. Feed and fecal samples were measured for AIA using an improved method developed in our lab. Utilizing our improved method of measuring AIA, we now have a precise and economical method of measuring digestibility of aquaculture feeds without the addition of expensive monitoring compounds and elemental analysis. In support of Sub-objective 2A, we previously identified, formulated, and extruded feeds containing experimental feed binders (guar gum, sodium alginate, and gum Arabic). The pellet quality was tested using physical (durability, water stability and durability, sinking rate, and shear strength) and chemical characteristics (proximate composition, total starch, starch gelatinization) and used to identify the six best pellet/binder combinations. These six diets were then used to perform digestibility and fecal quality trials, which have been completed with data analysis partially completed. Under Sub-objective 2B, experimental feeds were manufactured to examine the best performing combination of feed processing (extruder) conditions and feed ingredient particle size on feed pellet quality as outlined in Sub-objective 2A. We chose the four best-performing feed pellet types from the results for a growth trial using rainbow trout. The trial was completed and data analysis is pending. For Experiment 2, research examining the effect of extrusion conditions on starch gelatinization using different wheat and barley varieties containing varying levels of amylose have been formulated. A digestibility trial using the feeds made with these wheat and barley sources will be conducted this Fall in fiscal year (FY) 2022 or early FY 2023. In support of Sub-objective 3A, a substantial amount of work was accomplished in monitoring water conditions across fourteen production facilities. In four facilities samples were taken across triplicate sections from all flow through water uses (between first use through fourth use, first use through fifth use, and first use through eight use). The samples obtained were for the water, microbiome from water, raceway, feed and fish tissue, and fish tissue for gene expression and histology. This is being compared across different production lines reared at the facilities. In one study, samples were taken for fish fed a standard fishmeal expanded feed and then again when the diet was changed to an extruded feed formulated with higher levels of plant protein. Water quality and tissue taken for gene expression have been processed and analyzed, microbiome samples have been sequenced and this massive data set is still being analyzed. In another study, two lines of rainbow trout, ARS plant protein selected line and an unselected production strain, were reared in third use production water for twelve weeks and provided four diets, a control and three diets formulated to contain varying but increasing levels of soybean meal and soy protein concentrate. Samples were taken and are still being processed but noted differences were recorded between the strains and the diets in regards to mortality, growth, and water quality impact. For Sub-objective 3B, selection for improvement continued in both ARS select trout lines, one line improved for growth and utilization of an all plant protein/fish oil diet and the other line reared on an all plant protein and oil diet and selected for improved biosynthesis and conversion of alpha linolenic to docosahexaenoic (DHA) and eicosapentaenoic (EPA) fatty acids stored in fish muscle. Improved growth and health parameters were evident in the plant protein selected fish line, when compared to control and production strains, and these fish are still being released to the industry through material transfer and cooperative agreements and have been incorporated into the production lines representing greater than 75% of commercially produced rainbow trout. Proteomic and metabolomic studies conducted during the past year on this ARS-selected line has revealed interesting aspects related to the physiological changes these fish have undergone during selection when compared to non-selected strains. Furthermore, immunological changes in fish selected on plant-based feeds, as determined by our group and other research labs, has revealed an improvement in nonspecific pathogen resistance and these initial findings were recently published. The ARS line selected for conversion and storage of healthy omega-3 fatty acids is showing continual improvement with EPA and DHA levels in muscle nearly twice that found in unselected trout reared on the same diet.

Accomplishments
1. Determination of improved nonspecific pathogen resistance in ARS select line of rainbow trout. Feed costs and disease losses are the two largest economic components in production aquaculture. ARS researchers in Hagerman, Idaho, have been selecting fish for enhanced capabilities for growth and utilization of an all plant protein-based feed. These fish are currently used in production by the majority of commercial trout producers in the United States. Further evaluation of this strain also shows that the dietary selection has led to an increased resistance to nonspecific pathogens (viral and bacterial) and this increased immunological response has also been recorded by other international research laboratories performing similar research. Unlike other disease-resistant trout strains that have been selected for resistance to one specific pathogen, the Hagerman ARS strain has the capacity to show enhanced pathogen resistance across the ever-changing broad spectrum of devastating pathogens that trout producers face year to year. As pathogens induce reduced health and growth and mortalities are of serious economic concerns for producers, these fish can help mitigate against these losses.

2. Alternative protein sources can improve fecal stability and nutrient leeching in rainbow trout. Uneaten feed and fish feces release nutrients that cause enrichment of effluent water of flow-through trout hatchery systems and negatively impact receiving waters. One of the primary effects is algal blooms from an increase in dissolved phosphorus that can lead to oxygen deprivation and fish kills in streams and rivers. The replacement of fishmeal (FM) in diets of rainbow trout with plant-based protein sources, such as soybean meal (SBM) and soy protein concentrate (SPC), has compounded this problem since these feeds can reduce fecal stability, increase fecal fine particles, and add nutrients, such as phosphorus, to water. ARS researchers at Hagerman, Idaho, and Bozeman, Montana, determined that feeds comprised of a mixture of poultry by-product meal (PBM), corn protein concentrate (CPC), and SPC with guar gum binder produced more stable feces characterized by larger fecal particles and less fine fecal particles in rainbow trout compared to standard fishmeal-based and commercial feeds. Additionally, the fine fecal particle fractions contained significantly more phosphorus. Researchers concluded that feeding the alternative protein feeds would produce more large fecal particles that would settle out of the water column and could be collected, while feeding traditional fishmeal-based feeds would result in higher fine particles and additional phosphorus contribution to effluent with negative impacts to the environment.

3. Investigation of novel distillers’ products to increase production efficiency of U.S. corn and domestic fuel production. Recent advances in the processing of distillers by-products has dramatically changed the resultant nutrient quality of these products. Modern distillers products thereby may have increased usability and value as alternative feedstuffs for some animals, including fish. Working with stakeholder groups involved in corn production, ethanol production and aquaculture, ARS researchers in Aberdeen, Idaho, have investigated the suitability of commercially available novel fermented corn protein products for their application in rainbow trout feeds. These data demonstrate the potential of these products and provide feed companies with the information necessary to incorporate these products into commercial feed formulations. Identification of a suite of potential feed ingredients increases formulation flexibility and thereby protects aquaculture producers from shifting ingredient prices.

4. Developed new and improved methods for measuring acid insoluble ash. In animal nutrition research, markers are used for determining nutrient digestibility of feed and feed ingredients as well as studying digesta kinetics, rumen protein synthesis, herbage intake and species selection. Acid insoluble ash (AIA) is a part of total ash, representing siliceous compounds in herbs, food, feed, and biomasses, due to the natural presence of siliceous compounds and contaminations with dirt and sand. The current method for AIA determination is rudimentary, time-consuming, energy inefficient, prone to errors, and variable in steps and conditions among reports. ARS researchers at Aberdeen, Idaho, systematically investigated effects of various factors at several functional steps on AIA measurement and developed a new method as well as a significantly improved method that are less time consuming, easier to master, and less prone to analytical errors than previous methods. The improved and standardized methodology should make AIA a much better and more reliable marker for animal nutrition studies. These new and improved AIA measuring methods are important to feed formulators and manufacturers as an economic and reliable assay for evaluation of feeds and feed products.

5. Development of a climate smart resource utilizing sorghum in aquatic feeds. Sorghum’s non-genetically modified organism (GMO) status, as well as its status as a resource-conserving grain, can give sorghum a competitive advantage in the valuable and growing plant-based feed and food production sectors. Through partnering with Virginia Tech University with funds provided by the US Sorghum Check-off Program, ARS researchers in Aberdeen, Idaho, have determined available nutrient content of three sorghum varieties for rainbow trout and hybrid striped bass. These values allowed the researchers to determine appropriate inclusion levels in rainbow trout diets and thereby increase sorghum utilization in aquaculture feeds. Further, evaluation of multiple varieties has led to research aiming to develop methods to produce sorghum protein concentrate products by focusing research on those varieties that are best suited for inclusion in aquaculture feeds. These findings are important to sorghum producers, as it has potential as another lucrative market, and provides aquaculture feed manufacturers another available protein source for feeds.

Review Publications
Fawole, F.J., Labh, S.N., Hossain, M.S., Overturf, K.E., Small, B.C., Welker, T.L., Hardy, R.W., Kumar, V. 2021. Insect (black soldier fly larvae) oil as a potential substitute for fish or soy oil in the fish meal-based diet of juvenile rainbow trout (Oncorhynchus mykiss). Animal Nutrition. 7(4):1360-1370. https://doi.org/10.1016/j.aninu.2021.07.008.
Sealey, W.M., Conley, Z.B., Hinman, B.T., O'Neill, T.J., Bowzer, J., Block, S. 2022. Evaluation of the ability of Pichia guilliermondii to improve growth performance and disease resistance in rainbow trout (Oncorhynchus mykiss). Journal of the World Aquaculture Society. 53(2):411-423. https://doi.org/10.1111/jwas.12872.
Bledsoe, J.W., Ma, J., Cain, K., Bruce, T., Rawles, A.A., Abernathy, J.W., Welker, T.L., Overturf, K.E. 2022. Multi-tissue RNAseq reveals genetic and temporal differences in acute viral (IHN) infection among three selected lines of rainbow trout with varying resistance. Fish and Shellfish Immunology. 124:343-361. https://doi.org/10.1016/j.fsi.2022.03.034.
Woolman, M.J., Liu, K. 2022. Simplified analysis and expanded profiles of avenanthramides in oat grains. Foods. 11(4). Article 560. https://doi.org/10.3390/foods11040560.
Liu, K. 2022. Method development and optimization for measuring chymotrypsin and chymotrypsin inhibitor activities. Journal of Food Science. 87(5):2018–2033. https://doi.org/10.1111/1750-3841.16141.
Welker, T.L., Liu, K., Overturf, K.E., Abernathy, J.W., Barrows, F. 2021. Effect of soy protein products and gum inclusion in feed on fecal particle size profile of rainbow trout. Aquaculture. 1:14-25. https://doi.org/10.3390/aquacj1010003.
Mccann, K.M., Rawles, S.D., Lochmann, R.T., Mcentire, M.E., Sealey, W.M., Gaylord, T., Webster, C.D. 2021. Dietary replacement of fishmeal with commercial protein blends designed for aquafeeds in hybrid striped bass (Morone chrysops × Morone saxatilis): Digestibility, growth, body composition, and nutrient retention. Aquaculture Reports. 21. Article 100903. https://doi.org/10.1016/j.aqrep.2021.100903.
Liu, K. 2022. New and improved methods for measuring acid insoluble ash. Animal Feed Science and Technology. 288. Article 115282. https://doi.org/10.1016/j.anifeedsci.2022.115282.
Romano, N., Fischer, H., Rubio-Benito, M., Overturf, K.E., Sinha, A., Kumar, V. 2022. Different dietary combinations of high/low starch and fat with or without bile acid supplementation on growth, liver histopathology, gene expression and fatty acid composition of largemouth bass, Micropterus salmoides. Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology. 266. Article 111157. https://doi.org/10.1016/j.cbpa.2022.111157.
Zarei, M., Amirkolaie, A.K., Trushenski, J.T., Sealey, W.M., Schwarz, M.H., Ovissipour, R. 2022. Sorghum as a potential valuable aquafeed ingredient: Nutritional quality and digestibility. Agriculture. 12(5). Article 669. https://doi.org/10.3390/agriculture12050669.
Hong, J., Bledsoe, J.W., Overturf, K.E., Lee, S., Iassonova, D., Small, B. 2022. Latitude oil as a sustainable alternative to dietary fish oil in rainbow trout (Oncorhynchus mykiss): Effects on filet fatty acid profiles, intestinal histology, and plasma biochemistry. Frontiers in Sustainable Food Systems. 6. Article 837628. https://doi.org/10.3389/fsufs.2022.837628.