Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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. Artificial Intelligence (AI)/Machine Learning (ML) Machine learning was used to determine and evaluate egg quality and fry morphology. These methods were developed through collaborations with Breeding Insights and annotations of trait evaluation with the University of Idaho. ACCOMPLISHMENTS 01 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. 02 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. 03 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.
Impacts
(N/A)
Publications
- Welker, T.L., Sealey, W.M., Tan, R. 2023. Digestibility of corn distiller⿿s dried grains with solubles (DDGS) in practical feeds with phytase and xylanase treatment fed to Nile Tilapia. North American Journal of Aquaculture. 86(1):154-162. https://doi.org/10.1002/naaq.10325.
- Idenyi, J.N., Abanikannda, M., Huber, D.H., Gannam, A., Sealey, W.M., Eya, J.C. 2023. Genome-wide insights into whole gut microbiota of rainbow trout, Oncorhynchus mykiss, fed plant proteins and camelina oil at different temperature regimens. Journal of the World Aquaculture Society. 55(2). Article e13028. https://doi.org/10.1111/jwas.13028.
- Hong, J., Ortiz, J.G., Sealey, W.M., Small, B.C. 2023. Effects of dietary arachidonic acid supplementation in low fishmeal and fish oil-free diets on growth performance, inflammatory response, gut histology, and non- specific immunity in sub-adult rainbow trout, Oncorhynchus mykiss. Aquaculture. 580. Article 740272. https://doi.org/10.1016/j.aquaculture. 2023.740272.
- Liu, K. 2024. Enzymatic and algebraic methodology to determine the contents of Kunitz and Bowman-Birk inhibitors and their contributions to total trypsin or chymotrypsin inhibition in soybeans. Journal of Agricultural and Food Chemistry. 72(20):11782-11793. https://doi.org/10. 1021/acs.jafc.3c06389.
- Welker, T.L., Barrows, F.T. 2023. Improved fecal particle size profile in rainbow trout fed feeds containing different ratios of animal meal and plant protein concentrates: Effect on nitrogen and phosphorus partitioning. North American Journal of Aquaculture. 86(1):84-94. https://doi.org/10. 1002/naaq.10315.
- Welker, T.L., Overturf, K.E. 2023. Effect of dietary soy protein source on effluent water quality and growth performance of rainbow trout reared in a serial reuse water system. Animals. 13(19). Article 3090. https://doi.org/ 10.3390/ani13193090.
- Robinson, K., Duffield, K.R., Ramirez, J.L., Cohnstaedt, L.W., Ashworth, A. J., Jesudhasan, P., Arsi, K., Morales Ramos, J.A., Rojas, M.G., Crippen, T. L., Shanmugasundaram, R., Vaughan, M.M., Webster, C.D., Sealey, W.M., Purswell, J.L., Oppert, B.S., Neven, L.G., Cook, K.L., Donoghue, A.M. 2024. MINIstock: Model for INsect Inclusion in sustainable agriculture: USDA- ARS's research approach to advancing insect meal development and inclusion in animal diets. Journal of Economic Entomology. 117(4):1199-1209. https:// doi.org/10.1093/jee/toae130.
- Kajbaf, K., Overturf, K.E., Kumar, V. 2024. Integrated alternative approaches to select feed-efficient rainbow trout families to enhance the plant protein utilization. Scientific Reports. 14. Article 3869. https:// doi.org/10.1038/s41598-024-54218-2.
- Habte-Tsion, H.M., Hawkyard, M., Sealey, W.M., Bradshaw, D., Meesala, K., Bouchard, D. 2024. Effects of fishmeal substitution with mealworm meals (Tenebrio molitor and Alphitobius diaperinus) on the growth, physiobiochemical response, digesta microbiome, and immune genes expression of Atlantic salmon. Aquaculture Nutrition. 2024(1). Article 6618117. https://doi.org/10.1155/2024/6618117.
- Owens, C.E., Powell, M.S., Gaylord, T., Conley, Z.B., Sealey, W.M. 2024. Investigation of the suitability of 3 insect meals as protein sources for rainbow trout (Oncorhynchus mykiss). Journal of Economic Entomology. 117(4) :1254-1260. https://doi.org/10.1093/jee/toae037.
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Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. In support of Objective 1, a study was conducted to compare acid insoluble ash (AIA) and yttrium oxide as markers for measuring the digestibility of aquaculture feeds in rainbow trout and hybrid striped bass. The goal was to develop a precise and economical method without expensive compounds and elemental analysis. The study found that AIA was less reliable than yttrium as a marker, with apparent digestibility coefficients varying with diets and AIA levels. In addition, research was performed to develop a reliable method for measuring chymotrypsin inhibitor activity in soy-based feed. A new method was optimized, expressing inhibitory activity in arbitrary units. To standardize units, a conversion factor between arbitrary units and µg chymotrypsin inhibited was determined, allowing results to be expressed in mg chymotrypsin inhibited/g sample. If other methods follow the same standardization, comparison of inhibition results among studies would be possible. Also, in support of Objective 1, encapsulated soy protein ingredients containing oils were developed with high encapsulation efficiency and improved oxidative stability. They contained approximately 65 percent (%) protein and 20% oil. In a trial, the encapsulated protein ingredient was compared to a reference diet without encapsulated oils, and the apparent digestibility coefficients of nutrients were determined. These products extend shelf-life and prevent oil leakage and rancidity in high-energy trout feed. Further research under Objective 1 focused on developing sustainable and economical technologies to produce nutritious feed and food products from local protein sources. The USDA method was developed to prepare soy protein concentrates (SPC) from defatted soymeal, using limited resources and water as a leaching solvent. Collaboration with the Soybean Innovation Lab evaluated the feasibility of producing SPC from mechanically processed soy cake using the USDA method. The resulting SPC had increased protein content, higher digestibility, and improved oxidative stability, making it suitable for fortifying staple dishes and addressing protein gaps in Sub-Saharan Africa. This approach contributes to achieving zero hunger in the region. In support of Objective 2, experimental feed binders (guar gum, sodium alginate, and gum Arabic) were evaluated at different dietary concentrations in a growth trial in fiscal year (FY) 2022. Results of this trial were analyzed at the beginning of FY 2023. For Sub-objective 2B, the best performing combination of feed processing (extruder) conditions and feed ingredient particle size were previously evaluated in trout feed, and the four best performing feed pellet types were tested in a growth trial in rainbow trout (Experiment 1). The data were analyzed in FY 2023, and the experiment was completed. For Experiment 2, research examining the effect of extrusion conditions on starch gelatinization using different wheat and barley varieties containing varying levels of amylose were formulated, and the digestibility trial was completed in early FY 2023. Analysis of samples and data are currently being performed and identification of the four best performing wheat and barley sources based on pellet-characteristics will be completed for use in a growth trial by the end of FY 2023. In Experiment 3, floating, cork-supplemented feeds have been formulated with pellet characteristic evaluation (durability, water stability and durability, sinking rate, and shear strength) currently ongoing. In support of Sub-objective 3A, three strains of rainbow trout were reared on a control and a modified plant-based feed to evaluate strain by diet effects on fecal stability, animal health and water quality effects. The experiment has been completed and the harvested samples are being analyzed. Another experiment was started and is still currently in progress involving the incorporation of biochar in diets at different levels to determine its potential for increasing fecal stability and capacity in binding and removing mycotoxin contamination in feed. In support of Sub-objective 3B, family crosses of rainbow trout with known muscle fatty acid ratios were generated in support of the research effort to evaluate genetic improvement of rainbow trout fillets for storage of healthy omega-3 fatty acid in fillets of fish reared on sustainable feeds that do not contain fishmeal or fish oil. The offspring were reared on a feed containing no fishmeal or fish oil and fish from each of these crosses have been biopsied and muscle samples are set for determination of the fatty acid levels. ACCOMPLISHMENTS 01 Development of experimental feed formulations and manufacturing. ARS nutritionists in Bozeman, Montana, collaborated with private aquaculture producers, feed manufacturers, university and federal aquaculture research programs to test new emerging ingredients for extruded aquafeeds, as an alternative to traditional marine and plant meals and oils in a quest for more sustainable and efficient use of resources. The goal of these novel extruded feed formulations is to use precision formulation to increase inclusion levels of lesser-studied feed ingredients to provide a complete diet with the highest feed conversion rate, providing a healthy and easily digestible diet for aquatic animals that maintains its shape and texture in water and is durable for transport and storage. Ingredients such as processed animal proteins, yeast meals, insect meals, emerging vegetable meals/protein concentrates, microalgae, macroalgae, poultry fat, and DHA-rich algal products have been examined as alternatives to ingredients used in conventional formulations. By producing small amounts of these experimental formulations using the industry standard of extrusion cooking technology for controlled testing and validation in collaborator testing facilities, ARS researchers can help accelerate the adoption of these novel ingredients as commercially viable alternatives. 02 Improvement in hatchery effluent water quality through fish feed formulation. Dietary formulation can greatly impact water quality in fish hatcheries. ARS scientists in Hagerman, Idaho, in collaboration with the U.S. Fish and Wildlife Service, and University of Idaho, discovered that ammonia levels in effluent can be significantly reduced by lowering the protein content from 41% to 35% in rainbow trout diets without affecting growth performance. They also determined that phosphorus bound as phytate-P in plant protein sources, normally unavailable to rainbow trout, could be utilized by rainbow trout after treatment with phytase enzyme. With phytase treatment of plant-based feed, rainbow trout were able to meet physiological requirements for phosphorus (negating the need for addition of phosphate) with the added benefit of reduced phosphorus discharge in effluent. Without phytase treatment, trout exhibited poorer growth performance and a marked increase in phosphorus output when fed the plant-based diets. Phytase addition reduced water-soluble P waste loads by 43% from the fishmeal- based feeds and 56% from the plant-based feeds. Department of water quality (DEQ) permitting is continually reducing allowable limits of phosphorus release into downstream rivers and streams from production facilities, thereby limiting production and growth potential. This information is being used by feed manufacturers and producers to meet DEQ requirements allowing aquaculture producers to increase production while reducing harmful effluent effects on receiving waters. 03 Development of a brand new quality index for soy-based fish feed ingredients and diets and a method to measure it. For better utilization as fish feed, it is important to assess quality of protein products accurately and cost-effectively. For decades, several indirect indices, including nitrogen solubility index (NSI), protein dispersibility index (PDI), urease activity, and protein solubility in 0.2% potassium hydroxide, have been used. However, since these methods were developed some 80 years ago, they require specific equipment that is expensive, outdated, or unavailable. To address these issues, ARS researchers recently developed a new protein quality index, hereby referred to as the protein solubility index and a method to measure it. With easy performance and good repeatability, the new index can serve as a unified index to replace most of the existing indirect indices. This now eliminates confusion of multiple tests and terminologies used currently by relevant industries and scientific communities. 04 Evaluated the effects of insect meal inclusion in fish feeds. The decrease in the availability and the increase in the prices of fishmeal and fish oil have prompted the search for sustainable alternatives for aquaculture feeds. Insects, which are part of the natural diet of fish, leave a small ecological footprint because they have a limited need for arable land, may represent a good candidate. ARS researchers in Bozeman, Montana, collaborated with university scientists, trout producers and insect growers to determine the nutritional value of insects for salmonids. Research data demonstrates the potential of these products and provides feed companies with the information necessary to incorporate these products into commercial feed formulations. The global insect feed market is valued at 1.2 million USD and is expected to reach 3 million by 2030. In a 2020 report on the utilization of feed resources in commercial salmonid feeds, insect meal inclusion levels between 2016-2020 grew to low, but reportable levels (0.4% of the total feed volume) for the first time and inclusion levels of insect meals in aquatic animal feeds are proposed to increase such that aquaculture feed markets are forecasted to become the main market for insect meals by 2030.
Impacts
(N/A)
Publications
- Liu, K. 2022. A new method for determining protein solubility index (PSI) based on extraction with 5 mM alkali hydroxide and its correlation with trypsin inhibitor activity in soybean products. Journal of the American Oil Chemists' Society. 99(10):855⿿871. https://doi.org/10.1002/aocs.12643.
- Gulkirpik, E., Donnelly, A., Nowakunda, K., Liu, K., Andrade Laborde, J.E. 2023. Evaluation of a low-resource soy protein production method and its products. Frontiers in Nutrition. 10. Article 1067621. https://doi.org/10. 3389/fnut.2023.1067621.
- Liu, K. 2023. Chymotrypsin inhibitor assay: Expressing, calculating, and standardizing inhibitory activity in absolute amounts of chymotrypsin inhibited. Sustainable Food Proteins. 1(1):30-44. https://doi.org/10.1002/ sfp2.1004.
- Betiku, O., Yeoman, C., Gaylord, T., Ishaq, S., Duff, G., Sealey, W.M. 2023. Evidence of a divided nutritive function in the rainbow trout (Oncorhynchus mykiss) midgut and hindgut microbiomes by whole shotgun metagenomic approach. Aquaculture Reports. 30. Article 101601. https://doi. org/10.1016/j.aqrep.2023.101601.
- Bare, W., Struhs, E., Mirkouei, A., Overturf, K.E., Small, B. 2023. Engineered biomaterials for reducing phosphorus and nitrogen levels from downstream water of aquaculture facilities. Nature Sustainability. 11(4). Article 1029. https://doi.org/10.3390/pr11041029.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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. 02 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. 03 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. 04 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. 05 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.
Impacts
(N/A)
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 ÿ 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.
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Progress 10/01/20 to 09/30/21
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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. Record of Any Impact of Maximized Teleworking Requirement: The maximized telework requirement had a negative impact on the lab⿿s ability to conduct some planned research as scheduled. Maximized telework has severely hindered direct communication and planning sessions with stakeholders and industry collaborators. However, the maximized telework allowed our group to focus our efforts on analyzing results related to Objectives 1, 2, and 3 and to prepare and revise additional manuscripts related to the project. ACCOMPLISHMENTS 01 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. 02 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.
Impacts
(N/A)
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. https://doi.org/10.1016/j.anifeedsci.2021.114818.
- 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. https://doi.org/10.1016/j. aquaculture.2020.735627.
- 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. https://doi.org/ 10.1016/j.cbpb.2021.110589.
- 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. https://doi.org/10.3390/w12020362.
- 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. https://doi.org/10.1002/aocs.12475.
- 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. https://doi.org/10.1002/aocs. 12459|.
- 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. https://doi.org/10.1002/aocs.12514.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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.
Impacts
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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.
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