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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

2017 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 regard to Objective 1. In one study, hard and soft wheat grains were roller-milled into flour and bran. Protein extraction from both materials and re-solubilized extracts were directly compared using aqueous solutions of varying pH and varying solvent to solid ratios. Results from both hard and soft wheat showed that wheat flour had a protein extractability peak of 80-90% total protein in the alkaline range and another peak of 50-60% in the acidic range. Yet, for bran, only an alkaline peak of 60-70% was observed. The concentrate from flour from the pH 10.75 extraction contained 80-90% protein with 70-80% maximum recovery and had amino acid composition similar to gluten. The concentrate from bran contained 60-70% protein with 50-60% recovery and had amino acid composition different from gluten. Thus, wheat flour protein concentrates could be made by either alkaline extraction or acidic extraction, followed by precipitation at neutral pH. However, concentrates made with the two methods differed in purity and amino acid composition. From bran, only alkaline extraction could be used and protein recovery yield and purity were poorer compared to alkaline extracted protein concentrate from flour. In another study relating to Objective 1, samples of algae, oat grain, oat forage, soymeal and sand were used to investigate factors affecting measurement of total ash by dry ashing and acid insoluble ash (AIA) procedures. AIA is a part of total ash, representing siliceous compounds in herbs and certain food and feed, and serves as a marker in animal nutrition studies. Great variations exist among reports regarding total ash and AIA measurements. Although algae contain silica, there are no previous reports of AIA content for algae. Aside from temperature, dry ashing duration affected ash contents measured. Overnight ashing at 600 degrees Celsius (ºC) is thus recommended. For AIA measurement, there were many factors involved, including ashing conditions and methods to recover AIA. Consequently, significant changes were made to existing methods. The improved methods were less time-consuming, easier to master, and less prone to errors compared to conventional methods. In another study relating to Objective 1, oat forage, a source of leaf protein, was grown and harvested, dried, ground into powder, and solvent extracted for protein. Various factors were investigated, including sample particle size, solvent to solid sample ratio, alkaline solvent concentration, extraction temperature, centrifugation force, and enzyme treatment. The objective was two-fold: to evaluate factors affecting protein extraction and to determine maximum protein extractability under given conditions. All factors tested had significant effects on protein extraction with solvent to solid ratio and alkaline concentration being the most influential. The maximum protein extractability was about 78%, but the conditions that achieved this level required high solvent to solid ratios and a high alkaline concentration. Thus, production of leaf protein concentrate from dried biomass is unlikely to be economically feasible. Progress for Objective 2 has continued but has been limited by a critical vacancy (Research Physiologist). At least one strain of trout has been evaluated for growth rate at 15, 300, and 1,000 grams. Proximate analysis has been performed on samples from these studies and amino acid analysis is currently underway. A second strain of trout has been received at the facility and analysis at different life stages will begin as the fish reach appropriate sizes. Substantial progress was made for Objective 3 to examine vitamin and mineral concentrations for different strains of rainbow trout (Clear Springs and Trout Lodge) and the effect of water source (1st, 3rd, or 5th use water). Samples were collected and examined for differences in whole body mineral (concluded) and vitamin (ongoing) concentrations in rainbow trout. Researchers found that 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 Trout Lodge compared to Clear Springs rainbow trout. Whole body mineral concentrations were significantly higher in rainbow trout in 5th use compared to 1st use water (phosphate, potassium, 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 were unaffected by water source. Little effect of trout strain was observed on rainbow trout mineral concentrations, except for sodium which was higher in Clear Springs compared to Trout Lodge fish. Researchers originally hypothesized 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. This hypothesis is currently being examined. From these data, it does not appear adding extra minerals to feeds would prevent mineral loss or confer 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 for this Objective. Whole body vitamin concentrations are currently being measured and efforts will be focused on this second part of the Objective. Substantial progress was made in Objective 4. Two strains of rainbow trout, the ARS plant-based selected trout and a non-selected domesticated strain, were reared for 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 3 and 7 months. Environmental samples included water and feed, and fish samples included 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 revealed the presence of enteritis in 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 the different 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. In evaluating the effects of transplantation, initial studies progressed in evaluating the ability of antibiotics to strip a host’s intestinal microbiome. It appears that changes in the cocktail of antibiotics used to create gnotobiotic fish and water rearing conditions will require adjustment for this to be successful in rainbow trout. Evaluation of different transplantation methods showed that gavage delivery is the best method to deliver live colonizing microbiota into small fish. Substantial progress was made in Objective 5, where first generation crosses were made in the fall of 2016 using fish with known fillet levels for the nutritional healthy omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Thirty families, from sires and dams with known EPA and DHA muscle fatty acids levels, were generated and the fish from these crosses were reared to 250 grams. Fifteen fish from each family were muscle biopsied and PIT tagged and 10 fish from each family were euthanized and samples were taken from the liver and muscle for fatty acid analysis by gas chromatography-mass spectroscopy (GS-MS) and proteomic and transcriptomic analysis. Protein and RNA have been isolated and libraries prepped for shotgun proteome and RNA-sequencing analysis. Samples for fatty acid analysis have been prepped and fixed and are currently being analyzed by GC-MS.

1. Improved conversion of plant oils to healthy omega-3 fatty acids in fish. Fish oil is increasing in price and decreasing in availability. Through genetic selection ARS scientists have evaluated the ability of certain fish species to biosynthesize and convert plant oils to heart healthy omega-3 fatty acids when reared on feeds free of fish meal and fish oil (i.e. ‘marine free’). Fish reared on the marine free diet showed an increase of 11% increase in omega-3 fatty acids, compared to the previous generation, for this trait in specific crosses. Development of fish strains that do not require fish meal or fish oil, yet have the same health benefits and taste as wild caught species, would significantly improve the sustainability and economics of aquaculture production in the U.S.

2. Improved feed for shrimp. Shrimp is one of the largest globally produced aquaculture species, but commercial losses during rearing can be extremely high. Evaluation of a new formulated shrimp feed containing an additive was performed in ponds at eight separate commercial facilities. Shrimp on the ARS feed had an average survival of 82% versus 40% for standard commercial feed. Use of this feed could potentially double production at commercial shrimp facilities with minimal expense.

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). ARS researchers in Aberdeen, Idaho, recently conducted a study to characterize the ash component in algae. The study is among the 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
Liu, K. 2017. Characterization of ash in algae and other materials by determination of wet acid indigestible ash and microscopic examination. Algal Research. 25:307-321.
Liu, K., Barrows, F. 2017. Wet processing barley grains into concentrates with protein, beta-glucan, and starch. Cereal Chemistry. 94(2):161-169.
Liu, K. 2017. Fractionation of condensed distillers solubles and compositional characterization of its co products. Journal of the American Oil Chemists' Society. 94(1):161-168.
Welker, T.L., Wan, X., Zhou, Y., Yang, Y., Overturf, K.E., Barrows, F., Liu, K. 2017. Effect of dietary tea supplementation on growth performance, fat content, and muscle fatty acid profile of rainbow trout. Aquaculture International. 25(3):1073–1094.