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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

2020 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
This is the final report for project 2050-21310-005-00D, "Integrating the Development of New Feed Ingredients and Functionality and Genetic Improvement to Enhance Sustainable Production of Rainbow Trout," which has been replaced by project 2050-21310-006-00D, "Improving Nutrient Utilization to Increase the Production Efficiency and Sustainability of Rainbow Trout Aquaculture." For additional information, see the new project report. In this project plan, five objectives were outlined to improve the efficiency of trout production through the development of alternative feeds and genetic improvement of fish. Even with a vacancy for a critical scientist (SY) position significant progress was made for each objective. Testing of ingredients was done prior to development and evaluation of novel sustainable feeds. Testing under laboratory and production conditions was utilized to determine the effects of trout strain and life-stage on protein and amino acid retention, and vitamin and mineral requirements. Analysis of microbiome colonization in the intestine of fish was evaluated according to strain and diet and differential distribution of species of bacteria was correlated with the development of enteritis in un-selected fish reared on a plant protein-based feed. The physiological mechanisms behind the ability to biosynthesize and convert plant lipids to healthy omega-3 fatty acids and deposit these lipids in muscle was investigated and probable physiological mechanisms determined. In support of Objective 1, soybean samples were subjected to pretreatments followed by aqueous extraction. The goal was to develop an aqueous extraction process that uses water instead of hexane as a solvent. With an optimized condition of aqueous extraction, a new soy protein product was produced that is suitable for fish feed. In another study, protein extraction from wheat flour and bran was compared using aqueous solutions of varying pH and solvent to solid ratios. Our findings show that protein concentrates can be made from wheat flour by either alkaline extraction or acidic extraction, but from bran only by alkaline extraction. In another study, we developed an improved enzymatic method for measuring starch gelatinization in both wet and dried food and feed products by adding a unique sample pretreatment which could re-solubilize gelatinized starch fully but solubilize native starch minimally, improving total starch measurement, and simplifying the final calculation. In another study, protein extraction from oat forage was investigated. Several extraction factors had significant effects. The maximum protein extraction was 78%, but the conditions to achieve this were quite difficult. Thus, we concluded that production of leaf protein concentrate from dried biomass is unlikely to be economically feasible. In another study, we investigated effects of sample size, ashing temperature and duration on ash measurement of both algae and non-algae samples. We found that for most biomass both temperature and duration affected ash measurement significantly, and for algae having higher ash content, sample size was also a determining factor. Even with a scientist vacancy for Objective 2, significant gains were still achieved. Fish from a selected ARS strain and a commercial strain were tested at most of the lifestages indicated in the plan. Commercial strain fish were tested at 100 and 500 grams and were analyzed for protein and amino acid retention. The ARS strain of fish was evaluated for growth and nutrient retention at the earliest suggested life stage. Findings from this research definitively demonstrate that improved growth and protein retention can be achieved by modulation of dietary formulations to match the stage of growth. In support of Objective 3, we determined vitamin and mineral concentrations for different strains of rainbow trout and the effect of water source. We found that growth performance was generally lower as the number of water passages increased (first through fifth), and this reduction in growth varied by strain. Whole body mineral concentrations were significantly higher in rainbow trout in higher passaged water systems regardless of strain for certain minerals. We originally hypothesized that physiological stress that accompanies late water passage exposure would cause lower levels of whole-body minerals, which was not observed. It is possible that leaching of minerals from feed and feces into the late passaged water systems mitigated stress-related losses. Vitamins A, D, and E decreased in rainbow trout whole body in higher passaged water. However, the differences in whole body concentrations were only significant for vitamins D and E. From these data, it does not appear that adding extra minerals to feeds would prevent mineral loss or confer physiological benefit to trout grown in late passaged water, and therefore, the feeding trial portion with mineral supplementation for improved feed development was concluded. However, it appears that stress-affected changes from first through fifth use water may be causing declines in whole body vitamins D and E. Final diets containing varied concentrations of vitamins D and E were formulated and tested in a final feeding trial. There was not a positive effect on growth or other measures of health performance. Additional supplementation of vitamins and minerals above the recommended levels in diets of rainbow trout in serial-reuse systems does not appear to be warranted. In Sub-objective 3.B, significant progress was not achieved due to a scientist vacancy. Initial testing was done utilizing dietary ingredients such as guar gum, and fecal material was collected and compared to high soy and standard commercial diets. Initial results point to the potential to increase the solidness of fecal samples, but this needs to be more thoroughly evaluated, as fecal material that is too solid can lead to intestinal problems in the fish. In support of Objective 4 and Sub-objective 4.A, both lines of fish were fed a fishmeal-based feed and complete fishmeal replacement plant protein-based feed. Microbiota population differences were found relating to strain and diet. 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 become more extreme as the fish grew and were especially notable as enteritis began occurring in the non-selected commercial line. It was noted that fish that did not develop enteritis maintained distinct differences in the species of the bacteria that they harbored in their intestine, while in fish that developed enteritis, disease-related bacteria became more prominent. Differences were noted between digesta and mucosa sampling and by intestinal region. Physiological differences were also noted as determined by pH, transporter gene expression, proteomics, and monitored amino acid transport from the portal artery. ARS selected fish reared on the plant-based feed showed a higher efficiency and synchronization in the transport and utilization of amino acid, which leads to decreased protein turnover and greater protein retention efficiency. In support of Sub-objective 4.B, we tested the efficiency of fecal transplantation to modulate host microbiota populations microbial populations from fecal material was analyzed and it was noted that culture and sequencing of fecal material revealed differences between juvenile and adult fish even on the same feed. To then test the ability to shift microbiome populations, fecal material was obtained from adult fish and mixed with feed and given to first feeding fry. Transplantation of fecal material into first feeding fish demonstrated significant changes in the developing population of commensal bacteria as opposed to non-transplanted fish. The transplanted fish showed microbial populations more similar to adult fish than juvenile non-transplanted fish. These microbial population changes were found to be sustained throughout the sampling period. In support of Objective 5, families from the plant protein selected line of ARS trout were reared on a complete plant-based feed that did not contain any fishmeal or fish oil from 5 to 250 grams. Multiple families were generated from specific mixed crosses, as evaluated by the fatty acid profiles of the parents. Biopsy results demonstrated significant differences in fatty acid ratios, both within and between families. Multiple families were generated from specific mixed crosses as evaluated by the fatty acid profiles of the parents. Two generations of selection led to an 11 and 14% increase in the measured trait in selected families. Utilizing fish with extreme phenotypes for the trait, isolation and analysis of liver and muscle tissue by transcriptomic and proteomic analysis led to the identification of molecular markers linked to the trait and an understanding of the physiological mechanism behind the trait.

1. Extruded fish feeds are more economical and improve water quality over feeds produced by heat expansion. Feed costs make up between 60-80% of total production costs in aquaculture production. Some producers were feeding diets produced using heat expansion technology. ARS researchers in Hagerman, Idaho, tested and provided producers with information regarding expanded and extruded feeds. The scientists tested a commercial mash that was processed using both extruded and heat-expansion technologies and fed it to trout under production conditions in ARS-maintained tanks. Research findings showed a decrease in feed conversion and increase in protein retention with extruded feeds compared to expanded feeds, and an improvement in water quality, which should lead to a reduction in disease. Initial savings were almost $2 million for one producer.

2. Early removal of fish feed does not reduce fish processing weight. Normal production practices for most aquaculture farms consist of continued feeding of the fish until the day before harvest. At this point fish are at their heaviest and are consuming the highest percentage of feed. ARS researchers in Hagerman, Idaho, demonstrated that removal of feed up to eight days prior to harvest does not reduce processing weight, resulting in significant cost saving through reduced feed. This change in management practices will benefit rainbow trout producers and other finfish producers by reducing costs while also reducing nutrient effluent into receiving waters.

3. Large scale commercial utilization of trout selected for plant protein utilization. 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 Hagerman, Idaho, have selected rainbow trout for increased growth and utilization 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 same feed, but the selected fish do not develop enteritis and demonstrate improved performance on the plant-based feed than non-selected commercial trout on fishmeal diets. This year, Pacific Seafoods, the third largest rainbow trout producer in the United States stocked one million of these selected fish in production net pens for commercial production on a sustainable feed.

4. U.S. producer uses ARS-selected germplasm for rainbow trout egg sales and production. The majority of rainbow trout farmers do not manage their own broodstock and instead purchase their eggs for production from commercial egg producers. ARS researchers in Hagerman, Idaho, have developed trout selected for growth and utilization of plant protein feeds, which is more sustainable than fishmeal feeds. The second-largest commercial egg retailer in the United States is using rainbow trout germplasm developed by the ARS researchers and is now commercially selling eggs from these lines. The company is expressly marketing eggs from this line as hardier and have demonstrated their improved growth rate under different environmental conditions compared to eggs supplied by other, both U.S. and international, egg vendors. This same company uses ARS germplasm almost exclusively in their production farms.

5. Development of improved methods for the removal of anti-nutritional factors from soybeans. Soybeans are an important oilseed, providing edible oil, defatted protein meals and related products to 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. Therefore, TI levels have been an important quality parameter for soy products. It is vitally important to have an analytical method that can accurately measure TI levels in soybean products. Currently, there is a standard method, approved and reapproved by American Oil Chemists Society and American Association of Cereal Chemists International. Yet, some problems still exist for the official method. Therefore, ARS researchers in Hagerman, Idaho, developed two improved methods. Compared with the standard method, the new methods 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, and as such will be of great value to farmers and feed manufacturers looking to increase production and use of sustainable proteins in animal feeds.

Review Publications
Lee, S., Small, B., Patro, B., Overturf, K.E., Hardy, R. 2019. The dietary lysine requirement for optimum protein retention differs with rainbow trout (Oncorhynchus mykiss Walbaum) strain. Aquaculture. 514.
Zhuang, X., Zhao, C., Liu, K., Rubinelli, P., Ricke, S.C., Atungulu, G.G. 2018. Cereal grain fractions as potential sources of prebiotics: current status, opportunities, and potential applications. In: Ricke, S.C., Atungulu, G.G., Rainwater, C. E., Park, S. H., editors. Food and Feed Safety Systems and Analysis. San Diego, CA: Academic Press. p. 173-191.
Wang, R., Liu, Y., Isham, K., Zhao, W., Wheeler, J., Klassen, N., Hu, Y., Bonman, J.M., Chen, J. 2018. QTL identification and KASP markers development for productive tiller and fertile spikelet numbers in two high yielding hard white spring wheat cultivars. Molecular Breeding. 38:135.
Boyles, R., Marshall, D.S., Bockelman, H.E. 2019. Yield data from the Uniform Southern Soft Red Winter Wheat Nursery emphasize importance of selection location and environment for cultivar development. Crop Science. 59(5):1887–1898.
Wang, R., Gordon, T.C., Hole, D., Zhao, W., Isham, K., Bonman, J.M., Goates, B., Chen, J. 2019. Identification and assessment of two major QTL for dwarf bunt resistance in Winter Wheat Line ‘IDO835’. Journal of Theoretical and Applied Genetics. 132:2755-2766.
Kruse, E.B., Esvelt Klos, K.L., Marshall, J.M., Murray, T.D., Ward, B.P., Carter, A.H. 2019. Evaluating selection of a quantitative trait: snow mold tolerance in winter wheat. Agrosystems, Geosciences & Environment. 2(1):1-8.