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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

2019 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 on Objective 1. We investigated the effects of sample size and ashing temperature and duration on ash measurement of both algal and non-algal samples. The justification for the study is that many reported methods on ash analysis by dry ashing vary greatly in their conditions, making results uncomparable. We found that for most biomass, both temperature and duration affected ash measurement, and for algae having higher ash content, sample size was also a determining factor. For comparable results, a standardized method of ashing was determined. Significant gains were made in Objective 2, despite a delay in filling the vacant scientist position. Fish from a selected ARS strain and a commercial strain were tested at late term life stages. Commercial strain fish were tested at 100 and 500 grams and were analyzed for protein and amino acid retention. Findings to date from this research demonstrate improved growth and protein retention when dietary formulations are designed to match the stage of growth. The primary goal for Objective 3.A was to determine the differential effects of water quality in raceways (1st, 3rd, or 5th use water) on vitamin and mineral concentrations in rainbow trout reared under commercial hatchery conditions. Water quality declines as water passes through serial reuse raceways, which can lead to stress. Our hypothesis was that the increase in stress would cause lower whole-body levels of vitamins and minerals and possibly deplete trout of many of the essential nutrients, leading to overall poorer health and growth. Final diets containing optimally predicted concentrations of vitamins D and E, and diets with lower and higher concentrations, were formulated and tested in a final feeding trial. Vitamin D and E tissue concentrations for trout in 3rd and 5th use water fed the highest dietary supplemented levels were similar to the control (1st use water). No effects on either growth or other measures of health performance were observed though. In support of Sub-objective 4A, feeding trials were run with ARS-selected trout and a non-selected commercial strain as an experimental control. Fish were fed either a fishmeal control or plant protein-based feed. Microbiota samples were taken from the mucosa and digesta from the distal intestine at three months (prior to enteritis) and at seven months (post enteritis development). Immunological status (cellular and humoral) was evaluated for all groups at both time points pre- and post-stress, Microbiota populations were similar in both strains on the fishmeal diet, but differed for both strains when fed the plant-based feed. These differences on the plant-based feed became more extreme as the fish grew and were especially notable as enteritis began occurring in the non-selected commercial strain. Fish that did not develop enteritis maintained distinct differences in the species of bacteria that they harbored in their intestine. In contrast, disease related bacteria became more prominent in fish that developed enteritis. Physiological differences were also evident as measured by pH, transporter gene expression, proteomics, and transport of amino acids as measured in the plasma of the portal artery. In support of Sub-objective 4B, to test the efficiency of fecal transplantation to modulate host microbiota populations, microbial populations from fecal material were analyzed. Differences between juvenile and adult fish were observed. To test the ability to shift microbiome populations, fecal material from adult fish (>500 grams) was mixed with feed and given to first feeding fry. The transplanted fish showed microbial populations more like adult fish than juvenile non-transplanted fish. These microbial population changes were sustained throughout the sampling period. In support of Objective 5, sub-groups of families from the plant protein selected line of ARS trout were reared from 5 to 250 grams on a complete plant-based feed that did not contain any fishmeal or fish oil. Multiple families were generated from specific crosses between fish with high and low omega-3 fatty acid content in muscle. These fish represent three generations of selection. The fish have been muscle-biopsied and the samples are being analyzed to determine total fatty acid ratios. Mechanisms behind the trait, as revealed by transcriptomic and proteomic analysis of high and low trait responders, reveal the effect is partially due to differential regulation of transportation of fatty acids.

1. Extruded trout feeds benefit trout industry. Extruded aquaculture feeds are more economical and improve water quality compared to conventional expanded feeds. Feed costs make up between 60-80% of total production costs in aquaculture production. Some rainbow trout producers use feeds produced with heat expansion technology. ARS researchers in Aberdeen, Idaho, compared expanded versus extruded feeds under conditions that closely mimic commercial trout production conditions. Use of extruded feeds improved feed conversion, protein retention, and water quality. The use of extruded feeds will greatly benefit the trout industry by increasing productivity and enhancing water quality.

2. Large scale commercial use of trout selected for plant protein diets. Fishmeal is a finite protein source that is in short supply and is increasing in cost. Sustainable production for aquaculture requires the development of feeds formulated with alternative protein sources, of which plant proteins are the most highly recommended. ARS researchers in Aberdeen, Idaho, have selected rainbow trout for increased growth on aquaculture feeds that have completely replaced fishmeal in the diet with plant protein. Non-selected trout develop an intestinal condition termed enteritis when reared on the plant-based feed. However, the selected fish do not develop enteritis and demonstrate improved performance on the plant-based feed compared to conventional commercial trout strains fed fishmeal diets. In 2019 the third largest rainbow trout producer in the U.S. stocked 1 million of these selected fish in production net pens for commercial production on a sustainable feed.

3. ARS trout germplasm selected by a U.S. producer for use in commercial egg sales and production. Most rainbow trout farmers do not manage their own broodstock, but instead purchase eggs for production from outside sources. The second largest commercial egg retailer in U.S. obtained germplasm from ARS trout selected by ARS researchers in Aberdeen, Idaho, for growth and utilization of plant protein feed and is now selling eggs from these lines. The company is expressly marketing eggs from the ARS line as hardier, and have demonstrated their improved growth rate under different environmental conditions, compared to eggs supplied by other egg vendors in the U.S. and abroad. In addition, the company is the second largest commercial producer of rainbow trout and uses ARS germplasm almost exclusively in their production farms.

4. Development of improved methods for the removal of anti-nutritional factors from soybeans. Soybeans are an important oilseed source, providing edible oil, defatted protein meals and related products to the food and feed industries. However, soybeans contain trypsin inhibitors (TI), which are antinutritional and can cause digestive and metabolic diseases and retard growth in animals. It is vitally important to have an analytical method that can accurately measure TI levels in soybean products. The current method approved by American Oil Chemists Society (Method Ba 12-75) and American Association of Cereal Chemists International (Method 22-40.01) has been noted to have problems. Therefore, ARS researchers in Aberdeen, Idaho, developed two improved methods that when compared with the standard method, give more accurate results with less variation and reduced reagent usage. The two methods can be used for measuring TI levels not only in soy products but also in many other TI-containing products.

Review Publications
Cleveland, B.M., Leeds, T.D., Picklo, M., Brentesen, C., Frost, J., Biga, P. 2019. Supplementing rainbow trout (Oncorhynchus mykiss) broodstock diets with choline and methionine improves growth in offspring. Journal of the World Aquaculture Society. 50(3):1-16.
Welker, T.L., Overturf, K.E., Abernathy, J.W. 2019. Effect of aeration and oxygenation on growth and survival of rainbow trout in a commercial serial-pass, flow-through raceway system. Aquaculture Report. 14:1-9.
Liu, K. 2019. Effects of sample size, dry ashing temperature and duration on determination of ash content in algae and other biomass. Algal Research. 40:101486.
Liu, K. 2019. Soybean trypsin inhibitor assay: the sequence effect of adding reagents, factors involved, and mechanistic explanations. Journal of the American Oil Chemists' Society. 96:619-633.
Liu, K. 2019. Soybean trypsin inhibitor assay: further improvement of the standard method approved and reapproved by American Oil Chemists’ Society and American Association of Cereal Chemists International. Journal of the American Oil Chemists' Society. 96:635–645.