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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

2020 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
This report documents progress for project 2050-21310-006-00D, "Improving Nutrient Utilization to Increase the Production Efficiency and Sustainability of Rainbow Trout Aquaculture," which started December 2019 and continues research from project 2050-21310-005-00D, "Integrating the Development of New Feed Ingredients and Functionality and Genetic Improvement to Enhance Sustainable Production of Rainbow Trout." Considerable progress was made regarding Objective 1. In one study, several existing methods for measuring acid insoluble ash (AIA) content in feed ingredients were carefully reviewed and systematically compared, while factors affecting AIA assay methods were identified. The present study paves a way toward full development of a reliable and efficient method for AIA assay, which could make AIA the preferred marker for animal nutritional studies. In another study relating to Objective 1, several types of soybean products were subjected to varying heat treatments for different distinct durations. Changes in both trypsin inhibitor activity and urease activity were carefully monitored. The objective here is two-fold: to delineate the actual relationship between the indexes and to determine if this relationship is affected by types of soy products and heating methods. Over the past several decades, soybean has become the primary protein source for animal feed, but it contains naturally occurring heat-labile antinutritional factors, such as trypsin inhibitors. Having a maximum nutritional value of soybean meal relies on proper heat processing; under processing leaves soybean products with residual trypsin inhibitor activity at a level detrimental to animal growth, while over processing results in destruction of certain essential amino acids, such as lysine, arginine, and cysteine. Historically, the issue of "adequacy" of heat treatment with soybean meal was settled with the measurement of an indirect analyte, urease activity, based on two practical reasons. First, early studies showed a high correlation between trypsin inhibitors and urease activity in defatted soybean meal. Second, it is much easier and cheaper to measure urease activity than trypsin inhibitor activity. However, over the years, the reliability of urease activity as an indirect index for heat treatment of soy products has repeatedly been questioned. The present study seeks answers to such a question and provides much- needed guidelines to food and feed industries for proper assessment of soy products for heat treatment adequacy. In support of Objective 2, we recently determined that the primary cause of poor feed pellet characteristics, fecal durability, and growth performance of rainbow trout fed diets produced by expansion-compression pelleting was due to incomplete starch gelatinization when compared to extruded feeds. For Sub-objective 2B, we have tested the feed formulations by varying the extruder conditions (barrel temperature, moisture, shear force, and residence time) to determine the optimal and sub-optimal conditions for starch gelatinization. Now that these conditions have been refined, we can initiate the feeding and digestibility trials. Significant progress has been made in regards to Sub-objective 3A. Eggs from five strains of rainbow trout that were spawned and fertilized on the same date were obtained from commercial producers and our ARS selected stock. These fish were separated into groups and have concurrently been reared on either a fishmeal control diet or complete plantmeal protein replacement diet for five months. Samples have been taken monthly and final samples are to be taken at the end of eight months. In regards to Sub-objective 3B, the lipid-selected fish are selected and tested during odd numbered years, so these fish are currently growing and will not be mature until the fall of 2020. Family crosses will be generated and analysis and selection of fish from this group will take place in 2021.

1. Novel volumetric quantification method for fecal particle size classification in rainbow trout. Alternative plant protein sources, such as soybean meal, can interfere with digestion due to the presence of anti-nutritional factors that cause a diarrhea-like condition in rainbow trout, resulting in very fine fecal particles that can adversely affect aquaculture systems and the environment. However, affordable and easy-to-implement methods to measure fecal quality do not exist. A team of ARS scientists in Hagerman, Idaho, have developed a simple, volumetric method which relies on visual measurement of settled fecal samples. The method separates feces into three particle size-classes. Independent observations confirmed that the method is reproducible and without observer bias. Due to the ever-increasing addition of plant protein products into trout diets, this method is of high value to researchers and fish producers to evaluate the impacts on fecal and water quality.

2. Fecal particle size of rainbow trout influenced by amount and type of soy protein products and gum inclusion in feed. Replacement of fishmeal with alternative plant proteins, especially soybean meal, can cause a diarrhea-like symptom in rainbow trout (RBT), characterized by very fine fecal particles called fines. These fines do not settle out in raceway effluent for collection and contribute to the pollution of receiving waters. A team of ARS scientists in Hagerman, Idaho, conducted experiments to determine the effect of different soy protein sources on fecal quality and then these results were used to refine practical formulations using the soy-based protein sources. All-soy protein produced feces in RBT with high fecal fines and low levels of large particles, while diets containing fishmeal and other plant protein concentrates produced feces having a balanced particle size distribution. Refinement of the plant protein dietary formulations and addition of a binder demonstrated that fecal particles were larger and could be collected prior to escapement into receiving waters. This is important information for trout growers and feed companies because it shows that crude fiber in some plant proteins causes undesirable fecal particle profiles in RBT, and addition of dietary binders could significantly alleviate this negative effect and improve water quality of effluent.

3. Method to measure trypsin inhibitors in various protein products. Trypsin inhibitors (TI) are naturally occurring proteinaceous substances that are abundant in legume seeds and cereal grains. These inhibitors are antinutritional and/or bioactive and reduce the utilization of plant proteins in aquaculture feeds. As plant proteins are being used to replace fishmeal as the predominant protein source in aquaculture feeds, it is important to have a standard method to measure trypsin inhibitors in various protein products with high sensitivity and precision. ARS researchers at Hagerman, Idaho, recently carried out a collaborative study involving 12 laboratories in four continents: North America, South America, Asia and Europe, to evaluate the performance of a proposed method for determination of trypsin inhibitor activity in soybeans, pulses, grains and their processed products. The proposed method was a significant improvement over the current AOCS (American Association of Cereal Chemistry) method for TI assessment in soy products and is being evaluated as an approved AOCS method.

4. Improved method to measure starch content and gelatinization in wet and dried food and feed products. Starch is an important component of various food and feed products. Starch gelatinization is an important physicochemical process during processing starch-containing foods or feeds. The degree of starch gelatinization (DSG) affects not only physiochemical and sensory properties of starchy products, but also their susceptibility to enzymatic digestion and thus nutritional properties for humans or animals. A simple and reliable method that can accurately measure the total starch content and DSG is important for not only feed quality evaluation but also for the nutritional and physiological study of fish fed on a feed. ARS researchers at Hagerman, Idaho, recently developed an improved method for simpler and more accurate measurement of both total starch and gelatinized starch in situ for wet and dried products. This provides a valuable new tool for food and feed researchers to study the role of starch in food and feed products.

5. Supplying improved rainbow trout germplasm to the largest producers in the United States. Rainbow trout production is the second largest finfish aquaculture production sector in the United States. ARS researchers in Hagerman, Idaho, have led a rainbow trout selection program for the past 20 years. Through agreements and fish disbursement, the ARS researchers released improved germplasm via eggs, milt, and fry to the three largest rainbow trout producers in the United States with the germplasm to be used for production and incorporation into producer broodstock lines. This germplasm will comprise nearly 70% of all rainbow trout produced in the United States.

Review Publications
Liu, K., Liu, Q. 2020. Enzymatic determination of total starch and degree of starch gelatinization in various products. Food Hydrocolloids. 103.
Blaufuss, P., Gaylord, G., Sealey, W., Overturf, K.E., Powell, M. 2020. Selection on a plant-based diet reveals changes in oral tolerance, microbiota and growth in rainbow trout (Oncorhynchus mykiss) when fed a high soy diet. Aquaculture. 525.
Romano, N., Kumar, V., Yang, G., Kaibaf, K., Rubio, M., Overturf, K.E., Brezas, A., Hardy, R. 2020. Bile acid metabolism in fish: disturbances caused by fishmeal alternatives and some mitigating effects from dietary bile inclusions. Aquaculture. 12(3):1792-1817.
Welker, T.L., Overturf, K.E., Barrows, F. 2020. Development and evaluation of a volumetric quantification method for fecal particle size classification in rainbow trout fed different diets. North American Journal of Aquaculture. 82(2):159-168.
Liu, K., Wise, M.L. 2021. Distributions of nutrients and avenanthramides within oat grain and effects on pearled kernel composition. Food Chemistry. 336.