Skip to main content
ARS Home » Pacific West Area » Aberdeen, Idaho » Small Grains and Potato Germplasm Research » Research » Research Project #428348

Research Project: Integrating the Development of New Feed Ingredients and Functionality and Genetic Improvement to Enhance Sustainable Production of Rainbow Trout

Location: Small Grains and Potato Germplasm Research

2018 Annual Report

Six objectives will be used to improve the efficiency of trout production by the development of alternative feeds and fish better able to use those feeds. Some ingredients are developed in-house and we have both laboratory and pilot scale production capabilities. Digestibility of nutrients from specific ingredients has traditionally been conducted with large fish. The effect of strain and size will be determined. A tank system is available to collect feces both by sedimentation and stripping. The effect of strain and size on protein and amino acid retention will be conducted to determine the need for strain or life stage specific diets. Most trout culture use water from one raceway to another up to 5 times. A 36 tank system located on a commercial farm that receives water from 1st, 3rd, or 5th use will be used to determine the effect of water quality as a stressor on specific mineral and fat soluble vitamins in the tissues. A strain of trout selected to utilize plant-based diets is available and the effect of the gut microflora communities will be characterized. Fish oil has been the source of the heart/brain healthy fatty acids, EPA/DHA. This source is limited by supply and cost. Variability among trout in a specific strain for their ability to biosynthesize EPA/DHA has been identified. Trout with this trait will be bred to enhance the nutritional quality of the fillet for the benefit of the consumer. Objective 1: Develop and evaluate new ingredients and ingredient processing methods to increase nutritional and economic value. 1.A: Develop an improved soybean processing method to simultaneously separate protein and oil and remove anti-nutrients. 1.B: Development of alternative methods for the concentration of protein from wheat, barley, and oats. 1.C: Determine the nutritional and economic value of new and modified ingredients. Objective 2: Determine whether stage specific and strain specific diets are needed by evaluating nutrient digestibility at key life stages with different strains of rainbow trout. 2.A: Determination of the effect of fish size and strain on nutrient digestibility. 2.B: Evaluate the effect of fish size and strain on protein and amino acid retention efficiency. Objective 3: Improve performance of rainbow trout in serial-reuse raceway systems by improving water quality, particularly through modifications to feed formulations, and testing of fish strains. 3.A: Performance of rainbow trout in a serial-reuse system is improved by feeding diets formulated to mitigate stress. 3.B: Develop feed formulation strategies that prevent diarrhea in trout to facilitate waste management. Objective 4: Determine the genetic, physiological, and gut microflora components for improved utilization of plant-based feeds by rainbow trout. 4.A: Isolation and identification of trout microbiota and evaluation of its role in enhanced tolerance to utilization of plant-based feeds. 4.B: Determine the effect of transplantation of microbiota from selected fish. Objective 5: Develop lines of rainbow trout with enhanced abilities to biosynthesize EPA and DHA from plant oils and deposit these nutrients in muscle tissue.

Objective 1: 1.A: An improved aqueous processing method that results in high oil and protein recovery and removal of anti-nutrients will reduce the diarrheic effect of soybeans for trout. Experiments to optimize pretreatment and extraction conditions will be conducted. 1.B: Improved processing methods will increase the nutritional and economic value of protein concentrates from wheat, barley and oats. Trials will be conducted with wheat to optimize starting material and processing conditions to concentrate to 70% protein, and remove the binding effect. This effect of wheat gluten limits inclusion level in extruded feeds. Protein concentrates of barley and oats will be produced using another aqueous fractionation method that features alkaline extraction, centrifugation, and acid precipitation of supernatant. 1.C: A seven phase program will evaluate the nutritional value of alternative ingredients. Complete nutrient and anti-nutrient analysis, fry screening trials, effect on feed intake and extrusion, nutrient digestibility, growth trials, and effect on fecal size will be conducted. Objective 2: 2.A: Nutrient digestibility is affected by either fish size or strain or both. The ADC’s for major nutrients and amino acids will be determined with four unique strains of trout at three sizes (15, 500, 1500 g, 12 trials). 2.B: Nutrient retention efficiency is affected by fish size or strain or both. The same strains and fish size will be used as in 2.A in 12 week growth studies to evaluate protein and amino acid retention. Four diets varying in protein (40/45%) and lipid (20/25%) will be fed. Objective 3: 3.A: Improved diets containing elevated levels of stress-affected minerals and fat soluble vitamins will improve performance of rainbow trout raised in serial-reuse water. The effect of water source (1st, 3rd, & 5th use) as a stressor in three strains of rainbow trout on tissue concentrations of fat soluble vitamins and minerals will be determined. 3.B: Specific combinations of ingredients and prebiotics affect intestinal inflammation and the consistency of rainbow trout feces. To improve waste management dietary factors that affect fecal particle size will be determined. Objective 4: 4.A: Intestinal microflora community structure in rainbow trout is affected by diet and host genotype. Microbial communities will be identified in two strains of trout, one susceptible to soy enteritis and the other resistant. 4.B: Transplantation of microbiota from selected trout fed plant-based feed into non-selected trout will reduce intestinal enteritis when fed plant-based feeds. A cross-over experimental design will be used to determine if different microbial communities can protect a trout from soy induced intestinal enteritis. Objective 5: The ability to biosynthesize EPA and DHA in muscle tissue of rainbow trout fed diets containing plant oils can be selectively enhanced. To evaluate the potential to increase the ability of trout to biosynthesize EPA and DHA in their muscle, variation among individuals and families of rainbow trout will be determined. Individuals with known performance values for this trait will then be selectively bred.

Progress Report
Considerable progress was made with regards to Objective 1. In one study, soybean samples were subjected to pretreatments that included heating, extruding, and pressing, followed by aqueous extraction. The objective was to develop and optimize an extraction process that uses water instead of hexane to separate oil and protein from soybeans. With an optimized processing condition, a new soy protein product with a protein content of 65% and fat content of 4% was produced without the use of any organic solvents (hexane and/or ethanol). It has a protein content between that of defatted meal and a protein concentrate. The new soy protein product also has significantly reduced concentrations of both heat labile and heat stable anti-nutritional factors compared to hexane defatted soymeal. Based on chemical analysis of nutrients as well as anti-nutritional factors, we determined that the newly made soybean protein product is suitable for fish feed. In another study relating to Objective 1, an improved method for measuring the degree of starch gelatinization in both wet and dried food and feed products is being developed. Starch gelatinization is important during processing starch-containing foods or feeds. The extent of starch gelatinization not only determines the textural and organoleptic properties of final products, but also affects human and animal nutrient digestibility through changing enzymatic access to glucosidic linkages. Therefore, having a simple and reliable method to measure the degree of starch gelatinization is important for, not only feed quality evaluation, but also for the nutritional and physiological study of fish fed on a feed. Many methods have been described to measure starch gelatinization, but most are applicable only to purified starch. For processed products, starch must first be isolated, requiring methods that are either laborious or error prone. Previously, an enzymatic method was developed to measure gelatinized starch of dried starchy products in situ. It was a major improvement over the existing methods. The enzymatic method was improved for more accurate measurement of gelatinized starch by fine tuning the unique mechanical re-solubilization step. It was found that this step was an important one since it helps resolubilize starch in dried products and thus effectively eliminate the need for starch isolation before the enzymatic hydrolysis for accurate measurement. In another study relating to Objective 1, an improved method for accurately measuring trypsin inhibitor activity in soybean and other products is being developed. The significance of trypsin inhibitors (TI) in soybeans and other legume products lies in their nutritional implications, since their inactivation by heat treatments parallels improvement in nutritive values and growth performance when fed to animals (including fish). Therefore, TI is considered a key anti-nutritional factor and levels of TI activity (TIA) have been an important quality parameter for proteinaceous food and feed products, particularly those containing soy products. There are many approaches available, including some officially approved methods, however problems still exist. In addressing the problems found with current methods for measuring TIA in soy and other products, factors were studied that affect the TIA assay. Based on new findings, an improved method for measuring TIA in soy and other products is being proposed, which is more reliable than the officially approved methods. Progress for Objective 2 has continued but has been limited by a critical scientist vacancy (Research Physiologist). For Sub-objective 2A, at least one strain of trout has been evaluated for growth rate and nutrient digestibility at 15, 300, and 1,000 grams (g). For Sub-objective 2B, proximate analysis has been performed on samples from these studies and amino acid analysis is currently underway. Amino acid retention has been completed for 15g fish and dietary energy levels are shown to affect specific amino acid retention. Furthermore, the ratio of protein and lipid as an energy source was found to be more important to growth than overall dietary energy in these fish. Strain comparisons have not been run, but a second strain of fish will be tested to continue these comparison studies at the smaller fish sizes. Substantial progress was made in Objective 3 to examine vitamin and mineral concentrations for different strains of rainbow trout and the effect of water source (1st, 3rd, or 5th use water). Samples were collected and examined for differences in whole body mineral and vitamin concentrations in rainbow trout. Growth performance was generally lower as the number of raceway passes increased (1st vs. 5th use water), and this reduction in growth was greater for the commercial control line. Whole body mineral concentrations were significantly higher in rainbow trout in 5th use compared to 1st use water (phosphate, potassium, arsenic, and zinc) regardless of strain. Calcium was also higher in trout in 5th use compared to both 1st and 3rd use water. Whole body copper, iron, magnesium, and sulfur exhibited non-significant increases from 1st to 5th use water. Little effect of trout strain was observed on rainbow trout mineral concentrations, except for sodium which was higher in one strain. It was originally hypothesized that physiological stress that can accompany 5th use water exposure would cause lower levels (loss) of whole body minerals, which was not observed. It is possible that leaching of minerals from feed and feces into the 5th use water mitigated hyperosmotic loss and/or facilitated uptake through the gills in trout. Vitamins A, D, and E decreased in rainbow trout whole body in 5th use compared to 1st use water. However, the differences in whole body concentrations were only significant for vitamins D and E. Vitamin C levels in rainbow trout showed high levels of variation and no conclusions could be reached. From these data, it does not appear that adding extra minerals to feeds prevents mineral loss or confers physiological benefit to trout grown in 5th use water, and therefore, the feeding trial portion with mineral supplementation for improved feed development has been concluded. However, it appears that stress-affected changes from 1st to 5th use water may be causing declines in whole body vitamins D and E. Final diets containing optimally predicted concentrations of vitamins D and E plus concentrations ±1 standard deviation lower and higher are being formulated, and the final study will be conducted to conclude the Objective. Substantial progress was made in Sub-objective 4A. In two separate experiments, two strains of rainbow trout, the ARS plant-based selected trout and a non-selected domesticated strain (now done with a commercial strain and selected academic non-commercial strain), were reared for over seven months on an all plant-protein feed and a fishmeal control feed. Intestinal and other relevant tissue and environmental samples were taken from these fish at three and seven months. Environmental samples included water and feed, and fish samples included gill, liver, kidney, spleen, muscle, and mucosa and digesta from the distal, medial, and proximal sections of the intestine for transcriptomic and microbiota analysis, and whole tissue sampling for histology. Histologic examination in both experiments revealed the presence of enteritis (inflammation of the intestine) of non-selected fish reared on the plant-based feed, but not in any of the other groups. Microbiota samples were isolated, processed, and sequenced to assess the microorganisms present in the different strains fed either the fishmeal or plant-meal based feeds. Initial evaluation showed that there was no difference in the microbiota obtained from water samples from different tanks. However, distinct microbial differences were noted between the feeds. Furthermore, within portions of the intestine, distinct differences were found between selected and non-selected fish on the plant based feed whereas no differences were found between the groups reared on the fishmeal feed. Findings in other tissues showed strain and dietary interactions when the expression of immunological and stress genes was evaluated. Relating to Sub-objective 4B, scientists were unable to use antibiotics to eradicate existing microbial populations in fish larger than 15 grams. Residual microbial populations appeared to preferentially repopulate over transplanted microbial populations and live introduction of material did not show beneficial effects. Therefore, the procedure has been modified to provide transplant microbiota at first feeding in trout fry, before establishment of microbiota populations found in larger fish. These trials are currently underway. Substantial progress was made in Objective 5, where second generation crosses were made in the fall of 2016 using fish with known muscle levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Fatty acid analysis of 450 fish from 30 families generated from parents with known liver fatty acid ratios allowed scientists to identify significant differences for the trait across the tested fish. Twelve fish (per group) were tested and categorized as high, medium, and low performing fish with respect to cumulative muscle EPA and DHA levels. Liver and muscle were isolated from all fish and libraries generated for RNA-sequencing and shotgun-proteomic analysis. The information has been obtained from the transcriptomic and proteomic processed libraries and bioinformatics analysis is now underway to discern the gene and protein differences between the groups, which will then be used to determine the specific physiological mechanisms modified between families and individuals that vary for the trait.

1. Benefits of extruded feeds for trout production. While commercial fish feed produced via steam-compressed pelleting has largely been replaced since the late 1980s by dry extruded feeds, most rainbow trout grown in the U.S. are still fed steam-compressed pellets. ARS scientists located in Hagerman, Idaho, examined the effects of feed pellet processing (extrusion versus expansion-steam pelleting) on feed quality, water quality, fecal durability, and growth in rainbow trout. All feeds had similar chemical composition, but extruded feeds had a significantly higher degree of starch gelatinization than the expansion-steam pelleted feed, which led to extruded feeds having much higher water stability, fecal durability, and lower phosphorus discharge. This work is the first to show that extruded feed pellets are, not only more stable in water than pellets made by expansion-steam pelleting, but they also reduce fecal contributions to waste through improved fecal size and durability in water. The use of extruded feeds in commercial rainbow trout culture could improve waste collection and removal, and reduce pollution in downstream receiving waters.

2. Adding value to broken rice. A huge amount of biproduct known as high protein rice flour (HPRF) is produced annually worldwide from broken rice. To increase the value of broken rice and to expand its end uses, a study was conducted by ARS scientists in Aberdeen, Idaho, in which a commercial HPRF sample was analyzed and tested for feasibility of further protein enrichment by both physical (dry) and chemical (wet) methods. Results showed that HPRF has more than seven-fold increase over the feedstock, not only in protein, oil, and ash contents, but also in phytate content. However, when HPRF with such high phytate content is fed to animals (such as farmed fish) in large amounts, an undesirable excess of phosphorus is excreted. Several options were tested to enrich protein and remove phytate, with the conclusion that an aqueous medium having mild acidic to mild alkaline pH was most effective.

3. Identification of ash content in algae. Algae are a valuable source of both protein and lipid for farmed fish. Yet algae are known for having high ash content (some as high as 70% of dry matter). Scientists in Aberdeen, Idaho, recently conducted a study to characterize the ash component in algae. The study is among a very few to document that silica-containing materials are important contributors of the ash component for algae, particularly those with high ash content. Three types of silica materials were identified in algae: cellular structures of non-diatoms, diatom cell walls, and sandy particles of geologic origin. Contamination by diatoms and sandy particulates are the two major contributors to high ash content of algal samples. Several measures were proposed to produce algae with reduced ash content, which will be more suitable for fish feed.

Review Publications
Welker, T.L., Overturf, K.E., Abernathy, J.W. 2018. Effect of water source and trout strain on expression of stress-affected genes in a commercial setting. North American Journal of Aquaculture. 80(3):249-262.
Welker, T.L., Barrows, F., Overturf, K.E., Gaylord, G., Abernathy, J.W. 2018. Optimization of dietary manganese for rainbow trout, Oncorhynchus mykiss, fed a plant-based diet. Journal of the World Aquaculture Society. 49(1):71-82.
Welker, T.L., Overturf, K.E., Snyder, S., Liu, K., Abernathy, J.W., Frost, J.B., Barrows, F. 2018. Effects of feed processing method (extrusion and expansion-compression pelleting) on water quality and growth of rainbow trout in a commercial setting. Journal of Applied Aquaculture. 30(2):97-124.
Nebo, C., Gimbo, R.Y., Kojima, J.T., Overturf, K.E., Dal-Pai-Silva, M., Portella, M.C. 2018. Depletion of stored nutrients during fasting in Nile tilapia (Oreochromis niloticus) juveniles. Journal of Applied Aquaculture. 30(2):157-173.
Smith, A.A., Dumas, A., Yossa, R., Overturf, K.E., Bureau, D.P. 2018. Effects of soybean meal and high protein sunflower meal on growth performance, feed utilization, gut health and gene expression in artic charr (Salvelinus alpinus) at the grow-out stage. Aquaculture Nutrition.
Smith, A.A., Dumas, A., Yossa, R., Overturf, K.E., Bureau, D.P. 2017. Effects of soybean and sunflower meals on the growth, feed utilization, and gene expression in two Canadian strains of juvenile Arctic charr (Salvelinus alpinus). Aquaculture. 481:191-201.
Liu, K. 2018. Composition and phosphorous profile of high protein rice flour and broken rice and effects of further processing. Journal of the American Oil Chemists' Society.