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

2016 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
Substantial progress was achieved in Objective 1, for both ingredient development and ingredient evaluation. One sub-objective for ingredient development was to explore the use of value-added rice products as ingredients in fish feed. A high-protein rice flour (HPRF) was produced and characterized for chemical composition, protein extractability and enrichment with chemical and physical means. This is a co-product of a commercialized enzymatic process for making high fructose rice syrup from broken rice. Broken rice was also similarly characterized. Results showed that the enzymatic process led to a reduction in starch content from 84.7% in broken rice to 8.3% in HPRF, and a greater than six fold increase in protein, oil, ash, and phytate P. Protein extractability was less than 3% from HPRF and between 5-68% from rice flour, depending on the pH of the solution and particle size of the flour. Aqueous ethanol leaching slightly increased protein content in HPRF but particle size separation led to little improvement. In conclusion, protein enrichment by chemical and physical methods were generally ineffective for high protein rice flour and only alkaline extraction proved to be effective for raw rice. In another study as part of Objective 1, twelve algae samples of different sources were measured for proximate composition and mineral profile, and subjected to wet acid digestion and microscopic examination. The objective was to characterize chemical nature of algae ash, since algae is known for high ash content and understanding the nature of ash can help mitigate and control ash build up during production, and thus significantly affect the value of algal biomass as fish feed. Results showed that these algae samples varied greatly in proximate composition, with protein ranging 12.27-69.68%, oil 0.81-22.29%, ash 1.91-39.75% and total carbohydrate 21.60-75.32%, all on dry matter basis. When the algae samples were subjected to wet digestion, some portion of ash was indigestible. There was a strong positive correlation between ash content and indigestible ash content (with R2 = 0.9371), whereas the latter correlated positively with contents of Calcium, Iron, Aluminum, Barium, and Chromium in the samples. Based on chemical data and micrographs, we concluded that most of the ash in algae was sand, particularly for those with higher ash content. Several feeding studies also contributed towards progress in Objective 1. Two feeding studies and two digestibility studies were conducted with rainbow trout to evaluate new feed ingredients. The first digestibility study determined the apparent digestibility coefficients for four poultry processing meals, fishmeal, barley protein concentrate from a production facility, and a blend of animal by-products to approximate the composition of fishmeal. Samples are being analyzed in the laboratory. A feeding study was conducted with rainbow trout to evaluate the effect of feeding nine sources of soy protein concentrate. Many of these materials are new products to the market. Results indicated that inclusion of many of the new products in the diet of trout supported growth equivalent to trout fed a fishmeal based diet. Another study was conducted to evaluate de-fatted black soldier fly larvae meal (DFBSFL) as a protein source for juvenile rainbow trout. DFBSFL replaced either fish meal or soy protein concentrate (SPC) with inclusion levels of 5, 15, or 25% of DBSFL. An equal level of protein from fishmeal or SPC was removed and the diets were balanced for lysine, methionine and threonine. The results will be used to seek approval of the ingredient for use in fish feeds. Substantial progress was achieved for project Objective 2 relating to nutrient digestibility for different life stages with the completion of seven feeding studies. Three sizes of trout representing the size range during the production cycle (15, 500, and 1,500 grams per fish) were fed either a plant-based or a fishmeal based feed. The apparent digestibility coefficient’s ADC (the percentage of a nutrient that can be digested by trout) for protein, lipid, dry matter, energy and amino acids were determined. The standard method of fecal collection by hand stripping was compared to sedimentation methods for the 500 gram and 1,500 gram trout. The sedimentation method was used for the 15 gram fish since they were too small for hand stripping. The results of the studies with 500 and 1,500 gram fish showed an over estimation of digestibility when collecting feces by sedimentation. Four experiments with 500 gram trout were conducted to determine if sedimentation methodology could be improved to provide results similar to those obtained by hand stripping. Again in each case ADCs were significantly higher with the sedimentation approach. We hypothesized that the sedimentation approach over-estimates digestibility due to nutrient leaching and loss of fine particles from the feces. Studies are underway to determine if further modification of the sedimentation methodology can result in ADC values similar to those obtained by hand stripping. Substantial progress was made for Objective 3, and the first study in the serial-reuse tank system 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) was completed. Samples were collected to examine differences in whole body mineral and vitamin concentrations in rainbow trout. In addition, samples were taken to assess differences in whole genome expression between the water sources. Growth was generally lower as the number of raceway passes increased. In addition, as the number of water passes increased, a greater reduction in growth of Trout Lodge compared to Clear Springs rainbow trout was observed. Likewise, trout raised in water from multiple passes had higher feed conversion (FCR), and FCR values were higher, especially after more water passes, for the Trout Lodge strain. Proximate compositions did not differ significantly, except Clear Springs trout had higher whole body fat content and ash content was higher for fish in 5th use water, regardless of strain. Mineral concentrations in rainbow trout have been quantified, and the data are being analyzed. Substantial progress was made on Objective 4 as DNA isolated from both the mucosa and digest from the proximal, medial, and distal portions of the intestine was sequenced from both selected and control fish lines reared on both fishmeal and plant protein formulated feeds. This was compared with histological data obtained from the same samples. Revisions were made to microbiome transplant protocol lines of fish for the study. Substantial progress was made in Objective 5 as analysis began on samples isolated from second generational crosses. The fish generated from these crosses represent five standard deviations for the trait. Liver and muscle samples have been isolated from trait representatives and are being processed for proteomic and gene expression analysis.

1. Evaluation of algal sources of omega-3 fatty acids to replace fish oil in trout feeds. Fish oil is derived from wild fisheries such as sardines and menhaden, and has been the traditional source of omega-3 fatty acids for farmed fish. However, the high cost of fish oil is currently limiting the expansion of aquaculture and reducing the healthfulness of farmed fish. ARS researchers in Aberdeen, Idaho, determined the nutrient digestibility, palatability, and functionality of a number of new commercial sources from algae. Digestibility of omega-3 fatty acids was found to be high for all products, with no effect on feed intake and minimal effect on feed manufacturing. Identification of alternative sources of omega-3 fatty acids to fish oil for aquaculture feed will decrease dependence on ocean harvested fish and allow farmed fish to remain a heart- and brain-healthy human food.

2. New feed processing technology results in improved water quality of aquaculture farms. Feeding many modern fish feeds results in fecal waste that is very fine and difficult to settle or remove by filtration in flow-through or recirculating aquaculture farms. Traditional fish feed is held together in a pellet by carbohydrate. ARS researchers in Aberdeen, Idaho developed a new method that does not require carbohydrate. The result of feeding feed produced with this new technology (Patent Application Serial Number 14/479,654) is larger and more durable feces, which can be more easily removed from the water, thus improving water quality by reducing nutrients in the effluent. Adoption of this new processing technology will enhance the sustainability of aquaculture production.


Review Publications
Childress, C.J., Fuller, S.A., Rawles, S.D., Beck, B.H., Gaylord, T.G., Barrows, F., Mcentire, M.E. 2015. Lysine supplementation of commercial fishmeal-free diet in hybrid striped bass Morone chrysops x M. saxatilis affects expression of growth related genes. Aquaculture Nutrition. 22:738-744.
Davidson, J., Barrows, F., Kenney, P., Good, C., Schroyer, K., Summerfelt, S.T. 2016. Effects of feeding a fishmeal-free versus a fishmeal-based diet on post-smolt Atlantic salmon salmo salar performance, water quality, and waste production in recirculation aquaculture systems. Aquacultural Engineering. 74:38-51. doi: 10.1016/j.aquaeng.2016.05.004.
Overturf, K.E., Barrows, F., Hardy, R., Brezas, A., Dumas, A. 2016. The role of energy composition of diet affects muscle programming and fiber recruitment, body composition, and growth trajectory in rainbow trout. Aquaculture. 457:1-14.
Abernathy, J.W., Overturf, K.E. 2016. Comparison of ribosomal RNA removal methods for transcriptome sequencing workflows in teleost fish. Journal of Animal Science and Biotechnology. 27(1):60-65.
Welker, T.L., Overturf, K.E., Barrows, F., Abernathy, J.W., Snyder, S.G., Lapatra, S.E. 2015. Short-term feeding cessation prior to harvest does not affect fillet yield in rainbow trout. Annals of Aquaculture and Research. 2(2):1011.
Villasante, A., Powell, M., Murdoch, G., Overturf, K.E., Cain, K., Wacyk, J., Hardy, R. 2016. Effect of anthocyanidins on myogenic differentiation in induced and non-induced primary myoblasts from rainbow trout (Oncorhynchus mykiss). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 196:102-108.
Villasante, A., Powell, M., Moutou, K., Murdoch, G., Overturf, K.E., Wacyk, J., Hardy, R. 2016. Effect of anthocynanidins in myogenic differentiation and antioxidant defense in primary myogenic cells isolated from rainbow trout (Oncorhynchus mykiss). Aquaculture. 454:81-89.
Liu, K., Mahmood, K. 2015. Nutrient composition and protein extractability of oat forage harvested at different maturity stages as compared to grains. Journal of Agricultural Science. 7(12):50-58.