Project : USDA ARS

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

Location: Small Grains and Potato Germplasm Research

2024 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
This is the final report for project 2050-21310-006-000D, "Improving Nutrient Utilization to Increase the Production Efficiency and Sustainability of Rainbow Trout Aquaculture," undergoing NP 106 OSQR review. A new project plan titled "Innovations in Feed Science, Farming Practices, and Broodstock Management to Improve Sustainability of Salmonid Aquaculture" will be established once certified by OSQR. In support of Sub-objective 1A, ARS researchers in Aberdeen, Idaho, developed soy protein ingredients with encapsulated oils by mixing extracted soy proteins with vegetable oils in an aqueous solution, followed by spray drying, and other mechanical means. The encapsulated products contained about 65% protein and 20% oil, with improved oxidative stability and had an encapsulation efficiency of around 80%. The process was applicable to other protein sources and scalable to a semi-pilot level. This development addresses the increasing demand for high-energy trout feed (15-25% oil content) desired by the industry. Encapsulated oils in plant protein products extend ingredient shelf-life and prevent oil leakage and oxidative rancidity in high-oil feeds. Over the project's life, different oil-to-protein ratios and spray drying techniques were tested to maximize encapsulated protein content and oil encapsulation efficiency. The process was optimized in the laboratory and scaled up for larger production. At larger scale, the product contained approximately 65% protein and 20% oil with an 84% encapsulation efficiency. The encapsulated product showed minimal changes after addition to a test diet and extrusion processing. For Sub-objective 1B, ARS researchers developed an improved method for accurately measuring acid insoluble ash (AIA), a siliceous compound found in all fish feeds. Digestibility studies were done in rainbow trout and hybrid striped bass comparing AIA as an internal marker to yttrium oxide (the traditional marker for digestibility studies). The goal was to develop an economical method of measuring digestibility of aquaculture feeds. Nine diets were used for testing by mixing three reference diets with three protein ingredients of different sources. Results found that the AIA method proved more variable than the standard method using yttrium. In other Objective 1 research, ARS researchers conducted a collaborative study for measuring a quality index, protein solubility index (PSI) for plant-based fish feed ingredients. This research involved 16 laboratories from 10 countries that measured PSI of 12 selected samples plus a blind duplicate. The study demonstrated robust performance of the proposed AOCS (American Oil Chemists’ Society) method. The results of the study were presented to the AOCS Uniform Methods Committee for approval. Upon approval, this method will serve as a unified index for protein quality, replacing previous indexes. Previously, several existing AIA measurement methods were evaluated, showing substantial variation in accuracy and precision. An improved method was developed that was simpler and more accurate. In one study, four diets with different protein sources were spiked with an ash providing component, Celite, and yttrium before being fed to trout. The new method was less time-consuming and easier to master but showed greater variability compared to yttrium oxide as a marker. In support of Sub-objective 2A (Experiment 3 – Dietary Cork Addition), ARS researchers completed an evaluation of pellet characteristics for floating, cork-supplemented feeds. While previous research using semi-purified diets showed that floating pellets were possible with acceptable characteristics, analysis of diets made for this sub-objective revealed that practical commercial formulations for trout feeds with acceptable pellet characteristics (durability, water stability, sinking rate, and shear strength) were not feasible and still resulted in feces with undesirable characteristics. Formulation modifications continued for further pellet testing. Over the previous four years, early studies determined that poor feed pellet characteristics, fecal durability, and growth performance in rainbow trout fed diets produced by expansion-compression pelleting were due to incomplete starch gelatinization compared to extruded feeds. The primary goal was to identify formulations and processing methods to improve feed characteristics through increased starch gelatinization while maintaining or improving growth and contributing to better fecal characteristics. Later studies identified, formulated, and extruded feeds containing experimental feed binders. Pellet quality was tested using physical and chemical characteristics to identify the six best pellet/binder diet combinations. These diets were used in digestibility and fecal quality trials. After analysis, guar gum sodium alginate and gum Arabic binders were added to diets at different concentrations and evaluated for their effects on fecal stability. Progress on Sub-objective 2B, focused on findings that demonstrated the specific interaction between feed extrusion conditions and starch content. Starch source or composition, whether wheat or barley, did not significantly affect the degree of gelatinization as long as minimum extruder conditions were met. A minimum of 10% starch in the feed mash was required, of which at least 90% would be gelatinized, significantly improving starch digestibility, feed pellet physical characteristics, fecal durability, and fecal particle size. Previous progress during the prior four years involved testing feed formulations by varying extruder conditions to determine optimal and suboptimal conditions for starch gelatinization. Optimal conditions for each setting were determined, and feeds were manufactured using four different overall parameters to achieve the desired gelatinization status. Digestibility trials were initiated, and studies on how extrusion conditions affected starch gelatinization in different wheat and barley varieties with varying amylose levels began. In additional support of Sub-objective 1A, previously determined formulated diets, containing either fishmeal or plant meal as protein sources, were prepared and fed to ARS selected and unselected rainbow trout strains. Feed intake and growth parameters were monitored as the fish grew. Results showed that selected fish performed better on both diets and significantly better on the plant-based diet compared to the non-selected strain. Also in support of Sub-objective 3B included the evaluation of offspring from crosses generated from the previous generation of rainbow trout selected for biosynthesis and conversion of alpha-linolenic acid to healthy omega-3 fatty acids stored in the muscle. At 300 grams, 20 fish from families designated for broodstock were tagged and moved to broodstock raceways. Previous findings for Sub-objective 3A involved spawning and rearing five separate rainbow trout strains on fishmeal control and plant-meal based feeds for eight months. Significant differences were observed between strains in growth, feed conversion ratio, mortality, and development of enteritis (inflammation of the intestine) on both diets. Two strains were chosen for the next milestone, where fish were acclimated in ARS production tanks and fed a control diet and three experimental diets with increasing levels of soybean meal. Evaluation of parameters showed that increasing soybean meal in the diets increased stress gene expression, reduced growth, and decreased downstream water conditions. A study monitoring water conditions across 14 production facilities was conducted, with samples taken from triplicate sections of all flow-through water uses. Samples included water, microbiome, raceway, feed, and fish tissue. Findings showed significant changes in water quality between water uses in different facilities, related to structural differences such as flow and drop between raceways. Microbiome analysis revealed variations through water and across water use systems within facilities and greater differences between facilities. Other experiments, including testing biochar as a fecal binding agent and for dietary absorption of aflatoxin, were completed. For Sub-objective 3B, two generations of lipid-selected rainbow trout were generated and evaluated for comparative fatty acid levels. Crosses were made using muscle fatty acid information from previous generations. Families were reared on a diet containing no fishmeal or fish oil, with lipid provided by 13% alpha-linolenic acid inclusion. At approximately 300g, muscle biopsies were taken from 20 fish per family, and fish were individually tagged. Muscle samples were analyzed for total fatty acid levels. Selected families were kept for broodstock for the next improved generation. Findings showed significant improvement in selected fish to convert and store specific omega-3 fatty acids in muscle when fed an all plant-meal and plant oil-based feed.

Accomplishments
1. Increasing the dietary tolerable limit of corn distiller’s dried grains in tilapia feeds. Soybean meal is the predominant protein source used in tilapia feeds. However, the price of soybean meal is increasing and cutting into the farmer’s overall profit. Distiller’s dried grains from corn, a product produced in the production of ethanol from grain, is an economical alternative to soybean at approximately half the cost. Incorporation of distiller’s dried grains into tilapia feeds has been traditionally low, at no more than 15-20% of diet. ARS scientists in Hagerman, Idaho, found that addition of distiller’s dried grains up to 45% of the diet is tolerable by tilapia with no effect on growth performance. Furthermore, treatment of the feed with phytase (a natural chemical found in all organisms) increases the overall digestibility of phosphorus by the fish with a corresponding decrease in waste from the tilapia which helps reduce the amount of additional phosphorus going into the environment.

2. Development of a PCR-based assay for assessing biosecurity risk of aquafeed for white spot disease. Farm-raised shrimp accounts for over 50% of global shrimp production, driven by nutritionally balanced feed and effective disease prevention. However, the traditional method for testing viruses in aquafeeds has become controversial as it cannot distinguish between harmful and harmless agents in the feed. White spot syndrome virus (WSSV), the most lethal shrimp disease, causes $1 billion in annual losses worldwide. Positive test results using the current method to detect WSSV can lead to feed refusals and trade barriers, even if the feed is safe. To address this, ARS and University of Arizona researchers developed a novel testing method for WSSV in aquafeeds. Their research showed that while WSSV-added feed tested positive using the current method, it did not cause harm to shrimp or test positive with the new method. This improved testing approach will facilitate disease-free certifications of aquafeeds and support the health of farmed aquatic animals.

3. Examination of The Nutritional Value of Novel Sorghum Protein Concentrates. Globally, the market value for plant agriculture is $29.4 billion in 2020, and could surpass $162 billion by 2030, which would make up 7.7% of the global protein market. While sorghum is essential to U.S. agriculture, there is a need to ensure farmers maintain and expand sorghum acreage. An ARS Nutritionist in Bozeman, Montana, has collaborated with Virginia Tech University to develop a research-scaled plant protein product from sorghum (a sorghum protein concentrate) to enhance value-added product marketing opportunities for United States grown sorghum. The novel sorghum protein concentrate was subjected to a thorough nutritional analysis, evaluation for the presence of antinutritional factors, and an in vivo nutrient digestibility determination. Successful demonstration of the nutrient availability of sorghum protein concentrates provides an alternative ingredient for feed producers and another economic stream for sorghum farmers.

U.S. DEPARTMENT OF AGRICULTURE

Small Grains and Potato Germplasm Research: Aberdeen, ID