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

Location: Small Grains and Potato Germplasm Research

2023 Annual Report

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

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

Progress Report
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
1. 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.

2. 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.

3. 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.

4. 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.

Review 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.