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ARS Home » Pacific West Area » Aberdeen, Idaho » Small Grains and Potato Germplasm Research » Research » Research Project #437676

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

Location: Small Grains and Potato Germplasm Research

2021 Annual Report

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.

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
In support of Sub-objective 1A, research continued with Soy protein ingredients containing encapsulated oils that were developed by mixing soybean proteins with vegetable oils and using coacervation and spray drying, two basic techniques for oil encapsulation. The new protein products made at lab scale were found to contain about 70 percent (%) protein content and about 80 to 90% oil encapsulation efficiency. 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 will not only extend the shelf-life of the ingredients, but it also prevents oil leakage and oxidative rancidity of feed associated with high oil inclusion. In support of Sub-objective 1B, various factors affecting the determination of acid insoluble ash (AIA) were systematically investigated, using 16 samples of algae, grains, forage, soymeal, and sand, thus revising the method for measuring AIA significantly. By a simple definition, AIA is the ash that is insoluble in a dilute hydrochloric acid (HCl) solution, and is a part of the total ash, representing siliceous compounds in herbs and certain food and feed. AIA has been used as a marker in animal nutrition studies, since an apparent digestibility coefficient in fish or other livestock animals is an important nutritional parameter for any new ingredient and feed to be developed. Yet, great variations exist among reports regarding AIA measurements. Moreover, all current methods suffer from long running time, low sensitivity, and low reliability. As a result, unrealistic digestibility values or unfavorable results with the use of AIA as a marker in nutritional studies have been observed. Compared with the existing methods for AIA measurement, the new method we developed is less time consuming, easier to master and less prone to analytical errors. This new development could make AIA the preferred marker for nutritional studies of any feed ingredients and feed. In another study relating to Sub-objective 1, for measuring trypsin inhibitor activity in soybean products, two official methods were compared. One method was recently approved, based on improvements developed at the Aberdeen, Idaho, lab two years ago. It uses half the amount of reagents as in previous methods. Our study demonstrated that each method provided different values for the level of trypsin inhibitor. Because the ratio in mg trypsin inhibited per g sample changed with sample trypsin inhibitor activity values and the specific activity of trypsin used, accurate and direct comparison of the two methods was impossible. However, for a rough estimation, values for the initial method were about 50-55% of values obtained using our newly modified method for most samples. The study timely provides the feed industry with a general guideline regarding comparison of trypsin inhibitor values measured by the two methods. In support of Sub-objective 2A, we 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 was used to identify the six best pellet/binder combinations. These six diets were used to perform digestibility and fecal quality trials, which have been completed with data analysis pending. In support of Sub-objective 2B, experimental feeds were manufactured to examine the best performing combination of feed processing (extruder) conditions in which feed moisture (15-30%), barrel temperature (110-170 C), screw speed (150-300 revolutions per minute), and feed ingredient particle size (400, 600, 800, 1000, and 2000 uM). These diets were used in feed particle quality testing as outlined in Sub-objective 2A. Data analysis has not yet been completed. Based on these forthcoming results, the four best performing feed pellet types will be used in feed digestibility and fecal quality trials with rainbow trout in early fiscal year 2022. In support of Sub-objective 3A, diets were formulated and prepared for the comparison of a fish meal control diet with four diets replacing fish meal with increasing concentrations of soybean meal and soy protein concentrate from 15 to 45%. These diets have been prepared by the project fish nutritionist and shipped to the ARS production testing facility. A group of unselected fish (450 fish per group, 300 g) was moved into 30 tanks at this facility for acclimation. This experiment will begin in July, and the fish strains will be evaluated for their ability to use these plant protein-based feeds in third use production water. Growth, stress, proximate tissue analysis, and water quality parameters will be assessed in this experiment. In another project related to Sub-objective 3A, two ARS selected lines of trout and one unselected line were reared from 5 g on diets replacing fish meal and fish oil with plant meal and plant oil. Fish were then sampled at three and seven month intervals to compare effects of strain on dietary utilization and what physiological and metabolic process are altered. Samples are being collected and analyzed by histological evaluation of the liver and intestine, relative fatty acid ratios in the muscle, enzymatic evaluation of plasmid for lipase and stress factors, and gene expression changes in the liver and muscle. In support of Sub-objective 3B, 50 crosses were generated using the ARS Lipid select line. From each of these crosses, 150 fry were kept and reared on an all plant protein/plant lipid diet. At 250 g, 40 fish from each family were pit tagged and biopsied to determine total fatty acid ratios in the muscle. Fish from families showing improved conversion of plant lipid and biosynthesis to healthy omega-3 fatty acids in the muscles will be selected and used for improvement of this trait.

1. Organic acids and essential oils in fish feeds to reduce antibiotic use. In efforts to minimize antibiotic use, alternatives to antibiotics (ATA) have been examined across animal agriculture, including aquaculture. Two promising feed additive classes are organic acids and phytogenic compounds (essential oils or EO). The mode of action of differing additives is not fully described but has shown varied success as antimicrobial agents based on bacterial type. ARS researchers in Stuttgart, Arkansas, and Hagerman, Idaho, along with collaborators at the Bozeman Fish Technology Center, Bozeman, Montana, have performed feeding trials with hybrid striped bass and rainbow trout to test the efficacy of these feed additives on fish performance and health. Results for rainbow trout indicate both organic acids and essential oils improve growth rates and feed efficiency in rainbow trout compared to fish consuming the control diet.

2. Standardization of trypsin inhibitor activity expression units. Trypsin is a digestive enzyme in humans and animals, breaking down proteins. Trypsin inhibitors are naturally present in legume seeds, such as soybeans, and are antinutritional because of their inhibition on trypsin. Among different methods for measuring trypsin inhibitor activity in various protein products, three units have been used for expressing measured results. This makes comparison of results among studies difficult or impossible. To address the problem, ARS researchers at Aberdeen, Idaho, recently conducted a study using an improved trypsin inhibitor assay method developed earlier in the same USDA lab. The study involved developing new approaches to determine conversion factors between different units and standardize the conversion factors against a reference trypsin. The significance of the study is that when measured trypsin inhibitor activity is expressed in absolute amounts of trypsin inhibited and standardization is applied, comparison of among methods will become possible.

Review Publications
Liu, K., Frost, J.B., Welker, T.L., Barrows, F. 2021. Comparison of new and conventional processing methods for their effects on physical properties of fish feed. Animal Feed Science and Technology. 273. Article 114818.
Watson, A., Napolitano, M., Schock, T., Bowden, J., Frost, J.B., Yost, J., Denson, M. 2020. Evaluation of graded levels of soy oil as a fish oil replacement in high soy protein feeds for juvenile red drum, Sciaenops ocellatus. Aquaculture. 529. Article 735627.
Brezas, A., Kumar, V., Overturf, K.E., Hardy, R. 2021. Dietary amino acid supplementation affects temporal expression of amino acid transporters and metabolic genes in selected and commercial strains of rainbow trout (oncorhynchus mykiss). Aquaculture. 255. Article 110589.
Jahromi, N.B., Fulcher, A., Walker, F., Altland, J.E. 2020. Optimizing substrate available water and coir amendment rate in pine bark substrates. Water. 12(2). Article 362.
Liu, K. 2021. Trypsin inhibitor assay: expressing, calculating and standardizing inhibitor activity in absolute amounts of trypsin or trypsin inhibitors. Journal of the American Oil Chemists' Society. 98:355-373.
Liu, K., Seegers, S., Cao, W., Wanasundara, J., Chen, J., Da Silva, A.E., Ross, C., Franco, A.L., Vrijenhoek, T., Bhowmik, P., Li, Y., Wu, X., Bloomer, S. 2021. An international collaborative study on trypsin inhibitor assay for legumes, cereals and related products. Journal of the American Oil Chemists' Society. 98:375-390.|.
Liu, K., Ruiz, N. 2021. Soybean trypsin inhibitor and urease activities and their correlations as affected by heating method, duration, sample matrix and prior soaking. Journal of the American Oil Chemists' Society.