IMPROVING FISH NUTRITION AND PRODUCTION BY UPGRADING PLANT-BASED MEALS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010795
• Project Start Date: Oct 1, 2016
• Project End Date: May 21, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007

Performing Department
Natural Resource Management

Non Technical Summary
Aquaculture is the fastest growing segment of US agriculture and is projected to be the major agricultural growth industry well into the future. Not only are fish and shellfish product supplies necessary to accommodate the population growth, but per capita consumption is increasing as well. Further, the sustainability of wild capture marine fishes for food fish and fish meal markets is at risk with over 87% of stocks fully or overexploited, resulting in a situation where the only production growth that can occur is in aquaculture. The U.S. is a major consumer of wild capture and aquaculture products, importing about 90% of fish and fishery products, but the U.S. is a minor producer.The U.S. seafood trade deficit has recently grown to over $11 billion annually and total U.S. aquaculture production is about $1 billion annually. A large factor that hampers growth of the domestic aquaculture industry is the availability of low cost, sustainable feeds for food fishes. Fish meal is a high quality feed ingredient commonly found in aquaculture diets, however, expansion of the aquaculture industry has placed an increased demand on diminishing marine fish meal resources. Numerous alternatives have been explored and most are either plant products or co-products, and processed animal byproducts. Direct replacement of fish meal with plant sources often results in decreased growth and survival.Given the reduced availability and high cost of marine-derived fish meal, it will become increasingly important to maximize the use of sustainable plant products and co-products to economize fish production. Further, because of the anti nutritional issues involved in the use of plant feedstuffs in fish feeds it is necessary to conduct further research to mitigate these. Low-cost fish-meal replacements will dramatically improve the yield and profitability of aquaculture production of a wide range of aquatic species, thereby allowing the U.S. to continue expansion of its domestic aquaculture industry. Hence, the impetus for this research is to create more sustainable, high quality and economically viable protein ingredients to counter the consistently increasing price trends for fixed supplies of fish meal that constrain the aquaculture industry.

Animal Health Component

60%

Research Effort Categories

Basic

10%

Applied

60%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
302	0810	1010	100%

Knowledge Area
302 - Nutrient Utilization in Animals;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

fish meal

plant protein

bio-processing

enzyme

microbial conversion

thermomechanical

Goals / Objectives
The overarching aim of this research is to assess and resolve issues surrounding the nutritional quality of oilseeds and cereal grains and their coproducts - such as soybean, corn, wheat, carinata, and canola - for use in fish feed applications. The primary objectives of this project includes: 1) determination of nutritional composition and deficiencies that exist in seed meals; 2) evaluate processes - thermomechanical, chemical, enzymatic,and microbial - that may be used to upgrade the nutritional quality of meals; and 3) test palatability, digestibility, and growth performance of promising plant-based products in commercially important finfish diets.Primary targets to be sought in fish meal replacement ingredients include a high protein concentration (65-75%), a balanced essential amino acid profile (relative to fish meal) and high total sulfur amino acid content,low or tolerant levels of a variety of anti-nutritional factors (e.g., fiber, protease inhibitors, phytate, isoflavones, saponins, β-conglycinine, glucosinolates associated with a particular seed meal), and low polluting. The products must be palatable (gustatory and olifactory appeal), highly digestible (e.g., >90% for protein, energy, and amino acids) for feed conversion, and functional for transport and feed manufacturing. Potentially, some test ingredients may also provide innate support for health, stress reduction, and disease resistance.

Project Methods
Processing: Initial processing methods used to modify plant seed meals include combinations of crushing / hull removal,followed by oil extraction via cold pressing and / or solvent extraction. Crushing and oil removal is typically accomplished first because of the value for food or biofuel applications. Residual meals are commonly fed as is to food animals or further processed to improve animal utilization of the meal. Upgrading nutritional content for animal feeding is most commonly done singly or by a combination of thermomechanical, chemical, enzyme, and / or microbial conversion steps. The stepwise approach to be used in this project will follow the current industry practices and will focus on de-hulled, de-oiled meal streams or existing coproducts such as distiller's grains, enzyme treatment, and/or microbial conversion or fermentation.The selection and testing of specific processing approaches, and methods therein, will be done in collaboration with engineering (thermomechanical and chemical processes) and industrial microbiology (strain selection/conversion methods) researchers in the SDSU departments of Agricultural Engineering and Biology and Microbiology. The primary efforts associated with the project director's lab will be testing experimental ingredients and providing feedback to processing.Ingredient evaluation: A three-tiered approach is used to assess the performance of fish fed test ingredients resulting from processing, namely: 1) palatability, 2) protein and energy digestibility, and 3) growth performance. The first two trial types provide process feedback and down-selection of ingredients for subsequent trials - successful ingredients are transitioned into longer term performance validation. Fishes of commercial interest will beused in these trials and typically include rainbow trout and hybrid striped bass or tilapia. These species represent cold and warm water culture applications.Palatability trials will be used to assess the acceptance of processed ingredients. Naïve fish (10-20 g) that have had no previous exposure to plant-based ingredients will be used for these trials. As in all trials, ingredients will be analyzed for proximate composition, amino acids, carbohydrates and potential ANFs prior to diet formulation. Formulationswill bebased on a standard reference diet (standardization provides a means of comparing results among experiments) and test ingredient inclusion is set at 40% of the reference diet (diet manufacturing follows the protocol outlined below for feeding performance trials). Stocking rateswill be20 fish per tank (110-L tanks connected in parallel to a closed-loop recirculating aquaculture system [RAS]) and at least of four tanks (experimental units)will beusedfor each treatment. Following an acclimation period, fish are fed known quantities in three feedings per day. Waste feed will beaccounted for to determine consumption, which is measured daily for a two-week period to provide the palatability assessment.Apparent digestibility coefficients (ADC) of protein, energy, and amino acids of test ingredients derived from processing will be completed using standard methods. The ADCs of reference diets and test ingredient ADCs will be quantified by trace marker methods. Ingredient testing for the measurement of ADCs will use a reference diet formulated to meet species requirements. Each test dietwill becomposed of a 70% fish meal-based reference diet and 30% of the test ingredient, while the tracer concentrations are held constant among diets (diet manufacturing follows the protocol outlined below for feeding performance trials). Separate lots of fishwill beheld in a closed-loop RAS containing six, 750 L tanks. Depending on fish age/size, 100 to 200 fish are held in each tank and fed test diets to satiation for 10 days. Fishwill belightly anesthetized, ventsdried, and then palpated to extrude contents of the distal intestine. Collected digesta is freeze-dried and ground prior to analyses. Analytical methods and calculations for nutrient ADCs follow standard methods.Feeding trials will expand upon palatability and digestibility trial results by determining effects on growth performance and health. Trial diets will be formulated according to known nutrient requirements of the species. Treatment diets will be formulated on an isonitrogenous and isocaloric basis for direct performance comparisons with an FM (+) and base meal (-) control diets. Trace mineral and vitamin premixes will be included at fixed levels in all diets, including controls, to ensure that these nutrient requirements are met or exceeded. Because protein is the general variable of interest (but also peptides and amino acids), fish oil will be used to provide equivalent fatty acid contents in all diets. Ingredientswill be?separately milled then combined according to formulation and blended. Pellets for feeding trials will be produced using a single screw, cooking extrusion system, finished to a final moisture content with a drier-cooler system, and then lipid coated to the target level. Extruded feeds will be analyzed for proximate composition using standard methods.Feeding trial durations will vary by fish size and growth response but typically will range from 90 to 120 days each. Starting sizes are estimated to be 15-20 g for each species. A minimum replication of four experimental units per treatment will be used in all feeding trials. Trials will be conducted in 110-L circular tanks (20-25 fish/tank) or 187-L semi-square tanks (35-40 fish/tank) connected in parallel to a closed-loop RAS. Water quality (e.g., dissolved oxygen, pH, temperature, ammonia, and nitrite) will be monitored every other day.Feed rations will be fed in proportion to tank biomass and split into 3 to 5 daily feedings, depending on fish size. All feed dispensed and fish mortalities will be recorded daily for each tank. Growth performance will be determined by total mass measurements taken at specified intervals; rations will be adjusted in accordance with gains to allow satiation feeding and to reduce waste streams. Consumption will be assessed twice weekly from collections of uneaten pellets in individual tank replicates to estimate feed and protein efficiency.At the end of a feeding trial, sub-samples of fish from each replicate tank will be euthanized and necropsied to quantify muscle ratio, hepatosomatic index, viscerosomatic index, and fillet composition.. Survival, weight gain, growth rate, health indices, and feed conversion will be compared among treatment groups. Additional methods (e.g., sub-acute enteritis, gut microflora analysis, immunological testing) may be applied to assess fish responses and determine mechanisms if appropriate to the design and execution of specific studies.

Progress 10/01/16 to 05/21/20

Outputs
Target Audience:There are several audiences targeted in this line of research including oilseed producers and processors, aquaculture feed manufacturers, food fish producers, and ultimately consumers. Producers and processors will benefit from diversification of plant meal markets, increased demand for upgraded meals, and the creation of multiple product streams with higher value than base meals. The rapidly expanding aquaculture industry will benefit from the sustainable supply of high quality plant protein streams (singly or co-blended), and in the replacement of expensive marine-derived protein for aquaculture. This research also has the potential to economically provide enhanced protein feeds for other livestock that have lower non-nutritional carbohydrate tolerances. Further, feed manufacturers and food fish producers will benefit from stabilized ingredient supply and cost. Consumers will benefit from the increased availability of food fish, which provide a healthy source of dietary protein and lipid. Additional audiences may include the polymer and chemical industries, as co-process streams that evolve from ingredient processing could be high value pre- or probiotic supplements. This research will also lend itself to the management and sustainability of fixed supplies of marine-derived protein. Small-bodied schooling species (e.g., menhaden, herring, anchovy) and processing offal compose the majority of the fish meal commodity. Market competition and pricing have increased significantly for this resource as a result of demand for aquaculture, traditional livestock, and companion animals feed ingredients. Hence, reducing exploitation of this resource by displacing fish meal in animal feeds should enhance sustainability of these stocks and reduce impacts on marine food webs. The PD mentored three undergraduate student researchers who were involved with experiments including processing plant meals, whiteleg shrimp culture, and testing the ideal protein concept for the assessment of essential amino acid requirements. Two of these students received undergraduate awards to support research activities. These mentored research activities provided students with an opportunity to enrich their educational experience, accelerate their development as young professionals, and encourage them to pursue graduate education. Five additional undergraduate students were mentored on agency and industry internships. The PD supervised one post-doctoral researcher and three graduate student researchers (2 Ph.D., 1 M.S.) this year in investigations on seed meal processing, nutritional properties, feeding performance, processing effects and chemical characteristics of co-blended plant meals, feeding trial performance of novel soybean varieties, and the effect of plant-based diets on performance of hybrid striped bass and rainbow trout. The PD provided presentations in two workshops and several society conferences. Changes/Problems:Experienced some problems with bioreactors (i.e., water availability, contamination) used for microbial conversions, but we believe those problems have been resolved at this point. The COVID-19 pandemic has slowed research due to access limitations for some research personnel and contract lab analyses, and supply chain disruptions on materials and supplies. What opportunities for training and professional development has the project provided?planned and conducted experiments, completed data analyses, prepared articles and presentations, and attended meetings to present results. How have the results been disseminated to communities of interest?Results of studies are being published in peer-reviewed journals and delivered through presentations that target the aquaculture industry (see Section 4, Products and 5, Other Products) and relayed to state/regional lay outlets for commodity producers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: (90% accomplished) Most anti-nutritional factors (ANFs) in seed meals (GLS, fiber, starches) under study have been determined. Current research is focused on processing methods (Obj. 2) to reduce or remove ANFs and improve nutritional components (e.g., peptides, amino acids) and fish utilization (nutrient digestibility and absorption) (Obj. 3). Objective 2: Evaluate processes - thermomechanical, chemical, enzymatic hydrolysis, and microbial conversion processes - that may be used to upgrade the nutritional quality of meals (80% accomplished) Carinata meal (CM): The effect of soluble enzymes (alpha-galactosidase, pectinase, and phytase) were tested on the composition of single- and double-washed CM (SCM, DCM). The SCM and DCM contained 55.8 and 60.4% crude protein (CP), and 8.9 and 10.2% crude fiber (CF), respectively. Alpha-galactosidase had no effect on the CP of SCM (55.9%) or DCM (58.9%). However, CF levels were increased to 15.5% and 16.4%, respectively. Pectinase slightly increased the CP (58.1%) of SCM, but slightly decreased the DCM CP (58.1%). Pectinase slightly increased the CF of SCM (11.4%) and DCM (12.0%). Neither enzyme was as effective as cellulase in improving CP content of SCM (63.5%) and DCM (64.5%), and reducing the CF to 6.8% and 7.7%, respectively. However, use of cellulase in combination with pectinase may improve access of cellulase to cellulose and reduce CF. Therefore, combinations of these enzymes are being tested for applications in salmonid diets. SCM was prepared and then fermented with various microbes (Aureobasidium pullulans [2311-1 and 58522], Mucor circinelloides, Neurospora crassa, Pichia kudriavzevii, Paecilomyces variotti, Rhizopus microspores var. oligosporus, Trichoderma reesei) at a solid loading rate (SLR) of 50%. Temperature and pH were optimized for each microbe and then incubated for 168 h. The slurry was centrifuged and the dried solids analyzed for CP, fiber fractions (CF, ADF and NDF), and GLS. Heat sterilization and incubation of SCM for 168 h without microbes decreased total CP content by 3%. Fermentation increased CP titers by 2-5%, with N. crassa yielding the highest CP. Solids loss was negligible for all fermented meals. Fiber (CF, ADF, NDF) contents of SCM were higher than for the raw meal due to removal of soluble fractions during the wash step. Heat sterilization followed by 168 h incubation, with or without microbes, resulted in slight reduction of CF and NDF, and a significant reduction in ADF and GLS contents. Significant reductions in ADF and NDF levels were achieved due to fermentation, but only a slight decrease in CF was observed. N. crassa produced the highest reduction in CF, ADF, and NDF from 10.2, 42.7 and 27.9 in the control to 5.4, 23.8 and 12.3, respectively. Washing and fermenting CM reduced sinigrin in the raw meal (45.2 µmoles/g) by >89%. Objective 3: Test palatability, digestibility, and growth performance of promising plant-based products in commercially important finfish diets (80% accomplished) Effects of converted (microbial) CM (CCM) (10 and 30% inclusion levels) and DCM without conversion (30% inclusion level) on apparent digestibility coefficients (ADCs) and availability of dietary protein were determined in Hybrid Striped Bass. There were no significant differences in protein CP ADCs among diet treatments. However, the CP ADC of the 30% CCM diet ranked much lower and resulted in reduced leucine and phenylalanine ADCs. Arginine, isoleucine, and valine ADCs were only lower than for the reference and 30% DCM diets, and histidine, methionine, and threonine ADCs were lower. The average total amino acid (AA) ADC of the 30% CCM diet was only lower than for the 30% DCM diet. Highest CCM or DCM (30%) increased serum free tryptophan and valine. The reference and 30% WCM diets resulted in the highest serum free total essential AA (EAAs), but only free total EAAs from 30% WCM were elevated continuously over a longer period. Highest CCM or DCM (30%) increased muscle free histidine but reduced leucine and phenylalanine. Only 30% DCM increased muscle free lysine and valine. All inclusions of CCM or DCM increased muscle free methionine. The 30% CCM and DCM maintained elevated muscle free total EAAs over a longer period. Muscle free EAA to lysine ratios showed that only histidine concentrations were adequate for muscle synthesis over the 36-hour trial period. A low-antigen soybean variety (TN; low P34 protein, trypsin inhibitors, and lectin) was investigated as a CP source in two Rainbow Trout (RBT) feeding trials. Both TN and a conventional variety (DV) were solvent extracted (SE) or extruded (EX) and the four meals (i.e., TNSE, TNEX, DVSE and DVEX) were analyzed for composition. The SE (DV=53.6%, TN=48.8%) contained more CP than EX (DV=45.6%, TN=39.4%) meals due to oil removal. Generally, DV meals contained more CP than TN counterparts. The AA composition trend followed that of CP. The trend of lipid and nitrogen free extracts (NFE) was opposite to that of CP. Trypsin inhibitors (units/g) in DVSE (11,402), TNSE (10,925) and DVEX (9,213) were not substantially different. However, TNEX (1,904) contained much lower trypsin inhibitors, due to a rise in temperature of the extruder barrel. The ADCs of the test meals were determined using an indirect method (chromic oxide marker, 0.75%), with the test ingredient and the reference diet (45% CP and 17% lipid) composing 30% and 70% of the test diets, respectively. In addition to the TN and DV meals, a commercial soy product was used as an additional treatment. Following analyses, there were no significant differences detected in CP ADCs among test meals. A feeding experiment to market size evaluated the performance of RBT fed diets containing 20% SE, EX, TN or DV soybean meals, and control and reference commercial diets. The RBT (~24 g) were grown for 155 days. No differences in palatability was observed among the dietary treatments for the first 10 days of feeding. At the end of the study, weight gain and survival of RBT was unaffected by dietary treatment. The feed conversion ratio (FCR) of TNEX and DVSE were higher than for other dietary treatments. Organosomatic indices, body condition (Fulton's K), and fillet yields were also similar among dietary treatments. Histology of the distal gut did not reveal significant differences among treatments. Considerable size variation occurred because the fish (477-535g) had reached sexual maturity and the male:female ratio was dissimilar among replicates. In a second shorter (63 days) growth trial using larger (~60g each) all female RBT, growth metrics of RBT fed the commercial diet were lower than for RBT fed the reference, DVEX and TNEX diets. Consumption of the commercial diet was similar to that of TNSE diet but lower than for other diets. FCR and survival were unaffected by dietary treatments. In additional experiments, SCM was double washed or hydrolyzed using either cellulase, Ronozyme, pectinase or xylanase. Digestibility diets were made that included a reference diet and six test diets in which 30% of the reference diet was replaced with either SCM, DCM or one of the hydrolyzed meals. Chromic oxide was included as an inert marker at 0.75% of the diets. Feces were collected from the RBT (300-500g). However, composition data of the meals, diets and feces plus chromic oxide in diets and feces was delayed due to the COVID-19 lockdown. A growth trial using the processed meal(s) with the best composition and the highest ADCs will be conducted when ADC and composition data are completed. Accomplishing long-term objectives will generate higher value replacement ingredients for fish meal in aquaculture and other food animal diets in a rapidly expanding food animal market. Oilseed producers and processors will benefit due to increased demand and value of sustainable, modified plant-based protein ingredients for aquafeeds.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Brown, M.L., and T. Kasiga. 2020. Modified Brassica carinata seed meal utilization in Hybrid Striped Bass Morone chrysops x M. saxatilis. Aquaculture America 2020. Feb. 9-12. Honolulu, HI.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Landsman, A., B. St-Pierre, M. Rosales-Leija, M. Brown, and W. Gibbons. 2019. Impact of aquaculture practices on intestinal bacterial profiles of Pacific Whiteleg Shrimp Litopenaeus vannamei. Aquaculture America Triennial Conference. March 7-11. New Orleans, LA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kasiga, T., and M.L. Brown. 2019. Effect of carinata Brassica Carinata on trypsin activity, protein and amino acid digestibility and bioavailability in Hybrid Striped Bass Morone chrysops x M. saxatilis. Aquaculture America Triennial Conference. March 7-11. New Orleans, LA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bruce, T.J., L.P. Oliver, B.C. Small, R.W. Hardy, M.L. Brown, S.R. Craig, and K.D. Cain. 2019. Evaluation of fishmeal replacement with soy protein sources on the growth response of Burbot Lota lota. Aquaculture America Triennial Conference. March 7-11. New Orleans, LA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Kasiga, T., and M.L. Brown. 2020. Carinata Brassica carinata utilization in aquaculture. Carinata Biomaterials Summit. Feb. 26-28. Tampa, FL
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kasiga, T., and M.L. Brown. 2019. Re-thinking the ideal protein concept. Aquaculture America Triennial Conference. March 7-11. New Orleans, LA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kasiga, T., and M.L. Brown. 2019. Carinata Brassica carinata utilization in Hybrid Striped bass Morone chrysops X M. saxatilis. Carinata Biomaterials Summit. March 5-7. Quincy, FL.
• Type: Book Chapters Status: Published Year Published: 2020 Citation: Brown, M.L. 2020. Fullfat soybeans in aquaculture. Pages 36 to 48 in R.C. Thaler, editor. Fullfat Soya Handbook, third edition. American Soybean Association, St. Louis, Missouri.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Kasiga, T., B.W. White, T.J. Bruce, and M.L. Brown. 2020. Effect of fish meal replacement with Carinata Brassica carinata in low animal protein diets of rainbow trout Oncorhynchus mykiss (Walbaum) on trypsin activity, protein and amino acid digestibility and bioavailability. Aquaculture Research 51:2134-2149.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ortman, J., S.M. Sinn, W.R. Gibbons, M.L. Brown, J.M. DeRouchey, B. St-Pierre, M. Saqui-Salces, and C.L. Levesque. 2020. Comparative analysis of the ileal bacterial composition of post-weaned pigs fed different high-quality protein sources. Animal: An International Journal of Animal Bioscience. doi:10.1017/S1751731120000014.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Kasiga, T., and M. L. Brown. 2020. Estimation of the protein requirement for juvenile yellow perch Perca flavescens. Aquaculture Nutrition DOI: 10.1111/anu.13059
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Brown, M.L., T. Kasiga, D.E. Spengler, and J.A. Clapper. 2019. Reproductive cycle of Northern Largemouth Bass Micropterus salmoides salmoides. Journal of Experimental Zoology Part A: Ecological Genetics and Physiology DOI:10.1002/jez.2323.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kasiga, T., A.L. Fey, M.A. Berhow, T.J. Bruce, and M.L. Brown. 2019. Growth, feeding and thyroxine-related responses of hybrid striped (sunshine) bass Morone chrysops X M. saxatilis fed de-oiled carinata (Brassica carinata) meal. Aquaculture Nutrition 10.1111/anu.12971.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Landsman, A., B. St-Pierre, M. Rosales-Leija, M.L. Brown, and W. Gibbons. 2019. Investigation of the potential effects of host genetics and probiotic treatment on the gut bacterial community composition of aquaculture-raised Pacific Whiteleg Shrimp, Litopenaeus vannamei. Microorganisms DOI./10.3390/microorganisms7080217.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kasiga, T., and M. L. Brown. 2019. Replacement of fishmeal with carinata Brassica carinata seed meal in low animal protein diets of rainbow trout Oncorhynchus mykiss. Aquaculture Nutrition DOI: 10.1111/anu.12914.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Voorhees, J.M., M.E. Barnes, S. Chipps, and M.L. Brown. 2019. Effects of exercise and bioprocessed soybean meal diets during of Rainbow Trout rearing. Open Biology Journal. DOI: 10.2174/1874196701907010001
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Von Eschen, A., M.L. Brown, and K. Rosentrater. 2019. Influence of amino acid supplementations in juvenile Yellow Perch fed plant protein combinations. Open Journal of Animal Sciences. DOI 10.4236/ojas.2019.92016.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Von Eschen, A., M.L. Brown, and K. Rosentrater. 2019. Fermented soybean meal as a protein source in diets for Yellow Perch (Perca flavescens). Journal of Fisheries Sciences. 13(4): 014-020.
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Koep, E.T., T.J Bruce, W.R. Gibbons, and M.L. Brown. In review. Competitive microbes as a preventative treatment for diseases in aquaculture production. Journal of Aquatic Animal Health.
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Bruce, T.J., T. Kasiga, B.M. White, J.A. Clapper, and M.L. Brown. In review. Acute stress response in rainbow trout Oncorhynchus mykiss fed alternative soybean ingredients. Journal of World Aquaculture Society.
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Kasiga, T., B.M. White, and M.L. Brown. In review. Effects of carinata Brassica carinata in hybrid striped bass Morone chrysops x M. saxatilis diets on enzyme activity, digestibility and bioavailability. Journal of Aquaculture.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kasiga, T., and M.L. Brown. 2019. Nutritional utilization of modified carinata Brassica carinata meals in hybrid striped (sunshine) bass Morone chrysops x M. saxatilis diets. North American Journal of Aquaculture. DOI:10.1002/naaq.10105.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Landsman, A., B. St-Pierre, M. Rosales-Leija, M.L. Brown, and W. Gibbons. 2019. Impact of aquaculture practices on intestinal bacterial profiles of Pacific whiteleg shrimp Litopenaeus vannamei. Microorganisms. DOI/10.3390/microorganisms7040093.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Voorhees, J.M., M.E. Barnes, S. Chipps, and M.L. Brown. 2019. Bioprocessed soybean meal replacement of fish meal in adult rainbow trout (Oncorhynchus mykiss) diets. Cogent Food & Agriculture. DOI 10.1080/23311932.2019.1579482.
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Kasiga, T., B. Karki, J. Croat, J. Kaur, W.G. Gibbons, and M.L. Brown. In review. Process effects on carinata Brassica carinata and camelina Camelina sativa seed meal composition, and palatability in fishes. Journal of Animal Feed Science and Technology.
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Gilham, A., M. Brown, and G. Jordan. In press. Proposed modification of the standard weight (Ws) equation and standard length categories for Arctic Grayling (Thymallus arcticus). North American Journal of Fisheries Management.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:There are several audiences targeted in this line of research including oilseed producers and processors, aquaculture feed manufacturers, food fish producers, and ultimately consumers. Producers and processors will benefit from diversification of plant meal markets, increased demand for upgraded meals, and the creation of multiple product streams with higher value than base meals. The rapidly expanding aquaculture industry will benefit from the sustainable supply of high quality plant protein streams (singly or co-blended), and in the replacement of expensive marine-derived protein for aquaculture. This research also has the potential to economically provide enhanced protein feeds for other livestock that have lower non-nutritional carbohydrate tolerances. Further, feed manufacturers and food fish producers will benefit from stabilized ingredient supply and cost. Consumers will benefit from the increased availability of food fish, which provide a healthy source of dietary protein and lipid. Additional audiences may include the polymer and chemical industries, as co-process streams that evolve from ingredient processing could be high value pre- or probiotic supplements. This research will also lend itself to the management and sustainability of fixed supplies of marine-derived protein. Small-bodied schooling species (e.g., menhaden, herring, anchovy species) and processing offal compose the majority of the fish meal commodity. Market competition and pricing have increased significantly for this resource as a result of demand for aquaculture, traditional livestock, and companion animals feed ingredients. Hence, reducing exploitation of this resource by displacing fish meal in animal feeds should enhance sustainability of these stocks and reduce impacts on marine food webs. The PD mentored three undergraduate student researchers who were involved with experiments including processing plant meals, whiteleg shrimp culture, and testing the ideal protein concept for the assessment of essential amino acid requirements. Two of these students received undergraduate awards to support research activities. These mentored research activities are providing students with an opportunity to enrich their educational experience, accelerate their development as young professionals, and encourage them to pursue graduate education. Five additional undergraduate students were mentored on agency and industry internships. The PD supervised one post doctoral researcher and three graduate student researchers (2 Ph.D., 1 M.S.) this year in investigations on seed meal processing, nutritional properties, feeding performance, processing effects and chemical characteristics of co-blended plant meals, feeding trial performance of novel soybean varieties, and the effect of plant-based diets on performance of Hybrid Striped Bass and Rainbow Trout. The PD provided presentations in two workshops and several society conferences. Changes/Problems:Experienced some problems with bioreactors (i.e., water availability, contamination) used for microbial conversions, but we believe those problems have been resolved at this point. What opportunities for training and professional development has the project provided?The PD, post doctoral researcher (T. Kasiga, Ph.D., Wildlife and Fisheries Sciences) graduate students (T. Schaeffer, Ph.D. candidate, Wildlife and Fisheries Sciences; M. Hama-Salh, Ph.D. candidate, Wildlife and Fisheries Sciences; B. Vanderbush, M.S., Wildlife and Fisheries Sciences), and Wildlife and Fisheries Sciences undergraduate students (A. Muehler, M. Murren, J. Cisneros) jointly planned and conducted experiments, completed data analyses, prepared articles and presentations, and attended meetings to present results. How have the results been disseminated to communities of interest?Results of studies are being published in peer-reviewed journals and delivered through presentations that target the aquaculture industry (see Section 4, Products and 5, Other Products) and relayed to state/regional lay outlets for commodity producers. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Determination of nutritional composition and deficiencies that exist in seed meals Compositional analyses of process-modified oilseed meals will be ongoing to enable comparisons with base and previously modified meals. Objective 2: Evaluate process effects - thermomechanical, chemical, enzymatic hydrolysis, and microbial conversion processes - that may be used to upgrade the nutritional quality of meals A primary task is to evaluate the performance of enzymes and washing in pretreatment steps to upgrade carinata, barley and soy/barley co-blends to optimize microbial conversion and maximize protein content, minimize protein loss in solubles, and improve protein utilization and energy digestibility in fish. Objective 3: Test palatability, digestibility, and growth performance of promising plant-based products in commercially important finfish diets We will focus on conducting fish performance trials using test ingredients as processes yield improved meal compositions. We will also continue to investigate the potential value of co-blended bioprocessed plant meals in practical feed formulations as individual seed meals are compositionally optimized.

Impacts
What was accomplished under these goals? Objective 1: Determination of nutritional composition and deficiencies that exist in seed meals (90% accomplished). Most of the anti-nutritional factors (ANFs) in seed meals (glucosinolates, fiber, starches) have been determined for various meals under study. Current research is focused on processing methods (Obj. 2) to reduce or remove ANFs and improve nutritional components (e.g., peptides, amino acids) and fish utilization (nutrient digestibility and absorption) Objective 2: Evaluate processes - thermomechanical, chemical, enzymatic hydrolysis, and microbial conversion processes - that may be used to upgrade the nutritional quality of meals (50% accomplished). Carinata meal (CM): The effect of soluble enzymes (alpha-galactosidase, pectinase and phytase) were tested on the composition of single- and double-washed CM (SCM, DCM). The SCM and DCM contained 55.8 and 60.4% crude protein (CP), and 8.9 and 10.2% crude fiber (CF), respectively. Alpha-galactosidase had no effect on the CP of SCM (55.9%) or DCM (58.9%). However, CF levels were increased to 15.5% and 16.4%, respectively. Pectinase slightly increased the CP (58.1%) of SCM, but slightly decreased the DCM CP (58.1%). Pectinase slightly increased the CF of SCM (11.4%) and DCM (12.0%). Neither enzyme was as effective as cellulase in improving CP content of SCM (63.5%) and DCM (64.5%), and reducing the CF to 6.8% and 7.7%, respectively. However, use of cellulase in combination with pectinase may improve access of cellulase to cellulose and reduce CF. Therefore, combinations of these enzymes are being tested. SCM was prepared and then fermented with various microbes (Aureobasidium pullulans 2311-1, A. pullulans 58522, Mucor circinelloides, Neurospora crassa, Pichia kudriavzevii, Paecilomyces variotti, Rhizopus microspores var. oligosporus, Trichoderma reesei at a solid loading rate (SLR) of 50%. The pH and temperature were optimized for each microbe and then incubated for 168 h. The slurry was centrifuged and solids were dried for analysis of CP, fiber fractions (crude, ADF and NDF), and glucosinolates (GLS). Heat sterilization and incubation of SCM for 168 h without microbes decreased the total CP content by 3%. Fermentation increased CP titers by 2-5%, with N.crassa yielding the highest CP. Solids loss was negligible for all fermented meals. The fiber (crude, ADF, NDF) contents of the SCM were higher than for the raw meal due to removal of soluble fractions during the wash step. Heat sterilization followed by 168 h incubation with or without microbes resulted in slight reduction of crude fiber and NDF, and a significant reduction in ADF and GLS contents. Significant reduction in the ADF and NDF levels was achieved due to fungal fermentation, but only a slight decrease in crude fiber was observed. N. crassa produced the highest reduction in fibers. Crude fiber, ADF, and NDF for control vs N.crassa fermented meal were 10.2 vs 5.4, 42.7 vs 23.8 and 27.9 vs 12.3%, respectively. Washing and fermenting CM reduced sinigrin from 45.2 µmoles/g in the raw meal to <5 µmoles/g in the fermented meals. Heat sterilization followed by 168 h of incubation with or without microbial fermentation reduced the GLS further to 0.18 µmoles/g. Objective 3: Test palatability, digestibility, and growth performance of promising plant-based products in commercially important finfish diets (50% accomplished). Apparent digestibility coefficients (ADCs) and availability of protein in converted (microbial) CM (CCM) and DCM without conversion was fed (two inclusion levels each) to Hybrid Striped Bass. There were no significant differences in protein CP ADCs among diet treatments. However, the CP ADC of the 30% CCM diet ranked much lower and resulted in reduced leucine and phenylalanine ADCs. Arginine, isoleucine, and valine ADCs were only lower than for the reference and 30% DCM diets, and histidine, methionine and threonine ADCs were lower. The average total amino acid (AA) ADC of the 30% CCM diet was only lower than for the 30% DCM diet. Highest CCM or DCM (30%) increased serum free tryptophan and valine. The reference and 30% WCM diets resulted in the highest serum free total essential AA (EAAs), but only free total EAAs from 30% WCM were elevated continuously over a longer period. Highest CCM or DCM (30%) increased muscle free histidine but reduced leucine and phenylalanine. Only 30% DCM increased muscle free lysine and valine. All inclusions of CCM or DCM increased muscle free methionine. The 30% CCM and DCM maintained elevated muscle free total EAAs over a longer period. Muscle free EAA to lysine ratios showed that only histidine concentrations were adequate for muscle synthesis over the 36-hour trial period. A low-antigen soybean variety (TN; low P34 protein, trypsin inhibitors, and lectin) was investigated as CP source in two Rainbow Trout (RBT) feeding trials. Both TN and a conventional variety (DV) were processed by solvent extraction (SE) or extrusion (EX) and the four meals (i.e., TNSE, TNEX, DVSE and DVEX) were analyzed for composition. The SE meals (DV=53.6%, TN=48.8%) contained more CP than EX meals (DV=45.6%, TN=39.4%) due to oil removal. Generally, DV meals contained more CP than TN counterparts. The AA composition trend followed that of CP. The trend of lipid and nitrogen free extracts (NFE) was opposite to that of CP. Trypsin inhibitors (units/g) in DVSE (11,402), TNSE (10,925) and DVEX (9,213) were not substantially different. However, TNEX (1,904) contained a much lower trypsin inhibitors, which was attributed to a rise (heat lability) in temperature of the extruder barrel. The ADCs of the test meals were determined using an indirect method (chromic oxide marker, 0.75%), with the test ingredient and the reference diet (45% crude protein and 17% lipid) composing 30% and 70% of the test diets, respectively. In addition to the TN and DV meals, a commercial soy protein ingredient was used as an additional treatment. Following analyses, there were no significant differences detected in CP ADCs among test meals. A feeding experiment to market size evaluated the performance of RBT fed diets containing 20% SE, EX, TN or DV soybean meals, and control and reference commercial diets. The RBT (~24 g) were grown in a 36-tank RAS for 155 days, providing five replicates per treatment. No differences in palatability was observed among the dietary treatments for first 10 days of feeding. At the end of the study, weight gain and survival of RBT was unaffected by dietary treatment. The feed conversion ratio (FCR) of TNEX and DVSE were higher than for other dietary treatments. Organosomatic indices, body condition (Fulton's K), and fillet yields were also similar among dietary treatments. Histology of the distal gut did not reveal significant differences among treatments. Considerable size variation occurred due to the fish (477-535g) had reached sexual maturity and the male:female ratio was dissimilar among replicates. In a second trial, 15 larger (~60g each, all female) RBT were grown in a 32-tank RAS for 63 days. Five replicate tanks were used for all dietary treatments except the commercial and DVSE diets which had six replicates. At the end of the study, all data was collected as in the first growth and feeding performance trial. Growth metrics of RBT fed the commercial diet were lower than for RBT fed the reference, DVEX and TNEX diets. Consumption of the commercial diet was similar to that of TNSE diet but lower than for other diets. FCR and survival were unaffected by dietary treatments. Accomplishing long-term objectives will generate higher value replacement ingredients for fish meal in aquaculture and other food animal diets. Oilseed producers and processors will benefit due to increased demand for and value of sustainable, modified plant-based protein ingredients for aquafeeds. Plant-based feeds will serve distributors and aquaculture producers in a rapidly expanding food animal market.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Kasiga, T. 2018. Nutritional Value of modified novel oilseed meals in teleost diets. Ph.D. dissertation, South Dakota State University, Brookings, SD.
• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Voorhees, J. 2018. Impacts of salmonid rearing performance of dietary bio-processed plant-based protein and water velocity. M.S. thesis, South Dakota State University, Brookings, SD.
• Type: Book Chapters Status: Submitted Year Published: 2019 Citation: Brown, M.L. Soybeans in finfish feeds, with special reference to fullfat applications. Pages xx to xx in R.C. Thaler, editor. Fullfat Soya Handbook, third edition. American Soybean Association, St. Louis, Missouri.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Kasiga, T., and M.L. Brown. 2019. Estimation of the protein requirement for juvenile Yellow Perch Perca flavescens. Presented at the Aquaculture America 2019 Triennial Conference, New Orleans, March 7-11
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Kasiga, T., and M.L. Brown. 2019. Effect of Brassica carinata on trypsin activity, protein and amino acid digestibility and bioavailability in Hybrid Striped Bass Morone chrysops x M. saxatilis. Presented at the Aquaculture America 2019 Triennial Conference, New Orleans, March 7-11.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Kasiga, T., and M.L. Brown. 2019. Re-thinking the ideal protein concept. Presented at the Aquaculture America 2019 Triennial Conference, New Orleans, March 7-11. (Platform paper)
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Kasiga, T., and M.L. Brown. 2019. Brassica carinata utilization in Hybrid Striped bass Morone chrysops X M. saxatilis. Presented at the Carinata Biomaterials Summit 2019, Quincy, FL, March 5-7th, 2019.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Brown, M.L. and T. Kasiga. 2018. Effect of Brassica carinata on trypsin activity, protein and amino acid digestibility and bioavailability in Rainbow Trout Oncorhynchus mykiss. 148th Annual Conference of the American Fisheries Society, Atlantic City, NJ, August 19th-23rd. (Poster)
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Brown, M.L. and T. Kasiga. 2018. Performance of Rainbow Trout Oncorhynchus mykiss fed diets containing Brassica carinata meals. 148th Annual Conference of the American Fisheries Society, Atlantic City, NJ, August 19th-23rd. (Poster)
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Kasiga, T., and M.L. Brown. 2018. Nutritional evaluation of modified carinata meals in finfish. Presented at American Oil Chemists Society Annual Conference and Exposition, Minneapolis, MN, May 6th-May 9th. (Platform paper)
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Brown, M.L., T. Kasiga, D.E. Spengler, and J.A. Clapper. 2019. Reproductive cycle of Northern Largemouth Bass Micropterus salmoides salmoides. Journal of Experimental Zoology Part A: Ecological Genetics and Physiology DOI:10.1002/jez.2323.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kasiga, T., A.L. Fey, M.A. Berhow, T.J. Bruce, and M.L. Brown. 2019. Growth, feeding and thyroxine-related responses of hybrid striped (sunshine) bass Morone chrysops X M. saxatilis fed de-oiled carinata (Brassica carinata) meal. Aquaculture Nutrition 10.1111/anu.12971.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kasiga, T., and M. L. Brown. 2019. Replacement of fishmeal with Brassica carinata seed meal in low animal protein diets of rainbow trout Oncorhynchus mykiss. Aquaculture Nutrition DOI: 10.1111/anu.12914.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kasiga, T., and M.L. Brown. 2019. Nutritional utilization of modified Brassica carinata meals in hybrid striped (sunshine) bass Morone chrysops x M. saxatilis diets. North American Journal of Aquaculture. DOI:10.1002/naaq.10105.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Von Eschen, A., M.L. Brown, and K. Rosentrater. 2019. Influence of amino acid supplementations in juvenile Yellow Perch fed plant protein combinations. Open Journal of Animal Sciences. DOI 10.4236/ojas.2019.92016.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Kasiga, T. and M.L. Brown. Process effects on Brassica carinata and Camelina sativa seed meal compositions and palatability in Rainbow Trout Oncorhynchus mykiss. Journal of Animal Feed Science and Technology.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Kasiga, T. and M.L. Brown. Effect of fish meal replacement with Brassica carinata in low animal protein diets of rainbow trout Oncorhynchus mykiss (Walbaum) on trypsin activity, protein and amino acid digestibility and bioavailability. Journal of Aquaculture Research.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Kasiga, T. and M.L. Brown. Effects of high inclusions of processed Brassica carinata meals in hybrid striped bass Morone chrysops X M. saxatilis diets on enzyme activity, digestibility and bioavailability. Journal of Aquaculture.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:There are several audiences targeted in this line of research including oilseed producers and processors, aquaculture feed manufacturers, food fish producers, and ultimately consumers.Producers and processors will benefit from diversification of plant meal markets, increased demand for upgraded meals, and the creation of multiple product streams with higher value than base meals.The rapidly expanding aquaculture industry will benefit from the sustainable supply of high quality protein streams (singly or co-blended), and in the replacement of expensive marine-derived protein for aquaculture.This research also has the potential to economically provide enhanced protein feeds for other livestock that have lower non-nutritional carbohydrate tolerances. Further, feed manufacturers and food fish producers will benefit from stabilized ingredient supply and cost. Consumers will benefit from the increased availability of food fish, which provide a healthy source of dietary protein and lipid. Additional audiences may include the polymer and chemical industries, as co-process streams that evolve from ingredient processing could be feedstocks for production of a variety of high value products. This research will also lend itself to the management and sustainability of fixed supplies of marine-derived protein. Small-bodied schooling species (e.g., menhaden, herring, anchovy species) compose the majority of the fish meal commodity. Market competition and pricing have increased significantly for this resource as a result of demand for aquaculture, traditional livestock, and companion animals feed ingredients. Hence, reducing exploitation on this resource by displacing fish meal in animal feeds should enhance sustainability of these stocks and reduce impacts on marine food webs. The PD mentored four undergraduate student researchers who were involved with experiments including glucosinolate tolerance in hybrid striped bass, microbial conversions of co-blended meals, white-legged shrimp culture, and testing the ideal protein concept for the assessment of essential amino acid requirements. Three of these students received undergraduate awards to support research activities. These mentored research activities are providing students with an opportunity to enrich their educational experience, accelerate their development as young professionals, and encourage them to pursue graduate education. Five additional undergraduate students were mentored on agency and industry internships. The PD supervised graduate student researchers (3 Ph.D., 2 M.S.) this year in investigations on seed meal processing, nutritional properties, and feeding performance, processing effects and chemical characteristics of co-blended plant meals, feeding trial performance of novel soybean varieties, and the effect of plant-based diets on exercise performance of two trout species. The PD provided aquaculture presentations in two workshops and several society conferences.? Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PD, graduate students (T. Kasiga, Ph.D., Wildlife and Fisheries Sciences; T. Schaeffer, Ph.D., Wildlife and Fisheries Sciences; M. Hama-Salh, Ph.D., Wildlife and Fisheries Sciences; J. Voorhees, M.S., Wildlife and Fisheries Sciences), and Wildlife and Fisheries Sciences undergraduate students (A. Fey, A. Muehler, M. Murren, J. Cisneros) jointly planned and conducted experiments, completed data analyses, prepared articles and presentations and attended meetings to present results. How have the results been disseminated to communities of interest?Results of studies are being published in peer-reviewed journals and delivered through presentations that target the aquaculture industry (see Section 4, Products and 5, Other Products) and relayed to state/regional lay outlets for commodity producers (e.g., SD Soybean Leader). What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Determination of nutritional composition and deficiencies that exist in seed meals Compositional analyses of process-modified oilseed meals will be ongoing to enable comparisons with base and previously modified meals. Objective 2: Evaluate processes - thermomechanical, chemical, enzymatic hydrolysis, and microbial conversion processes - that may be used to upgrade the nutritional quality of meals A primary task is to evaluate the performance of enzymes and washing in pretreatment steps to upgrade carinata and barley to optimize microbial conversion and maximize protein content, minimize protein loss in solubles, and improve protein and energy digestibility in fish. Objective 3: Test palatability, digestibility, and growth performance of promising plant-based products in commercially important finfish diets We will focus on conducting fish performance trials using test ingredients as processes yield improved meal compositions. We will also continue to investigate the potential value of co-blended bioprocessed plant meals in practical feed formulations as individual seed meals are compositionally optimized.

Impacts
What was accomplished under these goals? Objective 1: Determination of nutritional composition and deficiencies that exist in seed meals (50% accomplished). Two feeding experiments were conducted to assess the maximum GLS and sinapine concentrations that could be tolerated by HSB. Cold-pressed carinata meal (CPCM) containing 61.2 µmoles of GLS and 6.07 mg of sinapine g-1 of meal was used. Diets in experiment 1 were formulated to provide ≤5.0 µmoles of GLS g-1 of feed ingredients by including ≤8.16% of CPCM in diets. Analyzed GLS and sinapine concentrations in diets were ≤2.71 µmoles and ≤0.181mg g-1, respectively. Dietary GLS breakdown products were undetected. These dietary concentrations of GLS and sinapine did not reduce feed consumption, utilization, or growth, resulting in similar concentrations of thyroxines and consequently no effect on deiodinase enzymes. In experiment 2, diets were formulated to provide 0, 6.12, 12.24 and 18.36 µmoles of GLS g-1 of feed ingredients by including 0, 10, 20 and 30% of CPCM in diets, respectively. Dietary GLS concentrations were 0 to 9.52 µmoles of GLS g-1, respectively. Sinapine concentrations were 0 to 0.75 mg g-1 of diet, respectively. Dietary concentrations of GLS ≥5.58 µmoles of GLS and ≥0.54 mg g-1 of sinapine reduced feed consumption, utilization, and growth. Sinapate esters are undergo oxidation during oil extraction and form complexes with amino acids and proteins in meals, reducing their nutritional value. Results demonstrate the necessity of processing carinata meals to reduce GLS and/or their breakdown products, as well as sinapines and other antinutrients. This is because our findings show that palatability and resulting consumption rates, not impairment of thyroid metabolism, are likely to be the main limitation of unprocessed carinata meals. Objective 2: Evaluate processes - thermomechanical, chemical, enzymatic hydrolysis, and microbial conversion processes - that may be used to upgrade the nutritional quality of meals (30% accomplished). Aerobic conversion (functionally a single wash with microbial metabolism) increased the fiber content of all carinata and camelina meals by >47%. Processing methods that include wash steps reduced glucosinolates titers by >70%, and have enabled increased inclusions (up to 30% of diet) of carinata meals. However, increased replacement of fishmeal will require further processing of carinata meals to reduce fiber to increase growth performance and feed efficiency. Trials are underway using various food grade carbohydrases, in combination with or without aerobic conversion, to lower crude fiber and increase protein. Fish performance trials (Obj. 3) have showed that double washing carinata improved consumption and performance. Therefore, we evaluated the effects of solid loading rate (SLR), mixing time, and double washing carinata on meal composition. Carinata meal was washed in a larger fermenter without pH or temperature adjustments. The slurry was centrifuged and solids were dried, providing the base meal for further processing. The single-washed meal was re-washed, centrifuged, and dried. Mass balance was conducted to determine the percentage of solids, where ~70 to 80% solids were recovered after the second wash. Mixing time did not have any significant impact on the protein level of the dried solids, which averaged 46% for most SLRs. Although protein titers were not significantly impacted by the SLRs, the protein titer for the low SLR (10%) was slightly higher (49.34%) than the average of 46% protein. The percent protein in the supernatant solids averaged 27%. To reduce fiber, carbohydrases tested so far include Ronozyme VP (blend of hemicellulase and pectinase) and cellulase. The two carbohydrases were applied at 2 mg of protein of enzyme per gram of raw unwashed carinata meal and incubated at 500C. The pH was not adjusted for Ronozyme VP and it was adjusted to 5 for cellulase. Ronozyme VP and cellulase reduced the crude fiber content of carinata meal from 9 to 5.8% and 7%, respectively. Ronozyme VP did not fully dissolve in water due to the low (4.5%) protein content compared to cellulase (45%). Soluble pectinases and xylanases are being evaluated in combination with cellulase, with a target of reducing the crude fiber content of washed carinata meals from ~11 to 3%. Objective 3: Test palatability, digestibility, and growth performance of promising plant-based products in commercially important finfish diets (40% accomplished). Palatability of carinata and camelina meals was tested in RBT and HSB and feed acceptance was improved by solvent extraction (carinata, up to 24% improvement; camelina, 28% up to improvement). Extrusion improved the palatability of camelina (15%) and aerobic conversion improved the palatability of carinata test meals (12 to 17%). Protein digestibility (ADC) was generally higher in carinata, 10.4% on average, than camelina meals for both RBT and HSB. Aerobic conversion reduced the protein digestibility of some carinata and camelina meals but the effect was more pronounced in carinata meals. In a RBT growth trial we determined how much FM could be replaced with aerobically converted carinata meals (ACCM). We replaced 25, 50 and 75% of FM in a reference diet containing 20% FM as the sole animal meal. Diets (44% protein, 17% lipid) were balanced for nutrients and fiber. Replacement of more than 25% FM by ACCM resulted in reduced growth, partly due to reduced feed consumption of 9 to 15% of total feed fed. Condition factors also decreased up to 12% with increased FM replacement. Feed conversion ratio (FCR) had an inverse relationship with consumption. Survival was 100%. Results showed that more than 25% of FM cannot be replaced by ACCM in low FM/animal (20%) diets of RBT. Improved utilization of ACCM may occur with more animal meal inclusion or additional processing of ACCM. We conducted a parallel study to determine apparent digestibility coefficients (ADCs) of ACCM. Replacement of FM with ACCM did not alter trypsin activity or protein ADCs. Replacement of more than 25% FM reduced ADCs of arginine, histidine, isoleucine, leucine, phenylalanine, threonine, valine, and tyrosine. Lower ADCs of arginine, histidine, isoleucine, leucine, phenylalanine, threonine, valine, and tyrosine in ACCM diets were not reflected in serum. Serum from RBT fed 75% ACCM had lower lysine concentrations. ACCM diets had reduced essential amino acids (EAA) peak concentrations and resulted in a slower release of EAAs in serum. Ratios of EAAs to lysine showed that tryptophan was the most limiting EAA. However, isoleucine, leucine, methionine, and phenylalanine were inadequate for muscle synthesis, mainly 3 to 9 hours postprandial. We determined the maximum inclusion of ACCM or double-washed carinata meal for HSB, without AC (WCM) in low (20%) animal protein diets. ACCM was included at 10 and 30%, and WCM at only 30% of the diets (44% protein, 12 lipid). After a 106-day growth period, we observed that HSB fed 30% WCM had a similar weight gain to HSB fed the FM reference diet and 30% ACCM, but better than HSB fed 10% ACCM. High (30%) amounts of ACCM or WCM improved feed consumption by >10% of total feed fed. FCR of WCM was 14% better than that of ACCM. Hematocrit (Hk) and hemoglobin (Hb) contents of HSB were improved by ACCM but not WCM. These results showed that the second wash in WCM improved feed utilization more than ACCM. However, the extra wash step in WCM likely reduced the iron content of WCM resulting in lower Hk and Hb. Accomplishing these objectives will generate higher value replacement ingredients for fish meal in aquaculture and other food animal diets. Oilseed producers and processors will benefit due to increased demand for and value of sustainable, modified plant-based protein ingredients for aquafeeds. Plant-based feeds will serve distributors and aquaculture producers in a rapidly expanding food animal market.

Publications

• Type: Book Chapters Status: Accepted Year Published: 2018 Citation: Brown, M.L. Soybeans in finfish feeds, with special reference to fullfat applications. Pages xx to xx in R.C. Thaler, editor. Fullfat soya handbook, third edition. American Soybean Association, St. Louis, Missouri.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Kasiga, T., and M.L. Brown. Effects of carinata Brassica Carinata on trypsin activity, protein and amino acid digestibility and bioavailability in Rainbow Trout Oncorhynchus mykiss. American Fisheries Society, August 19th-23rd, 2018, Atlantic City, NJ.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Kasiga, T., and M.L. Brown. Performance of Rainbow Trout Oncorhynchus mykiss fed diets containing Carinata Brassica carinata meals. American Fisheries Society, August 19th-23rd, 2018, Atlantic City, NJ.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Kasiga, T., and M.L. Brown. Nutritional evaluation of modified carinata meals in finfish. American Oil Chemists Society Annual Meeting and Exposition, May 6th-May 9th, 2018, Minneapolis, MN.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Roberts, B.R., B. Karki , J. Zahler , M. Brown, and W. Gibbons. Fungal fermentation of de-hulled ground barley to increase protein levels. American Oil Chemists Society Annual Meeting and Exposition, May 6th-May 9th, 2018, Minneapolis, MN.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Zahler J., B. Karki , M. Brown, and W. Gibbons. Optimization of submerged fungal incubation process for production of guar protein hydrolysate. American Oil Chemists Society Annual Meeting and Exposition, May 6th-May 9th, 2018, Minneapolis, MN.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Voorhees, J., M. Brown, M. Barnes, and S. Chipps. Inclusion of bio-processed soybean meal and the effects that exercise has on Brown Trout (Salmo trutta). Dakota Chapter American Fisheries Society, Brookings, South Dakota, February 26th  28th, 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Voorhees, J.M., M.L. Brown, S.R. Chipps, and M.E. Barnes. 2018. Impacts on Rainbow Trout (Onchorhynchus mykiss) rearing performance of dietary bioprocessed plant-based protein and water velocity. Dakota Chapter American Fisheries Society, Brookings, South Dakota.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Brown, M., B. White, B. Karki, and W. Gibbons. Performance of Rainbow Trout Oncorhyncus mykiss fed converted soy products. Aquaculture America, Las Vegas, NV, February 19-22, 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Brown, M., J. Vorhees, M. Barnes, and S. Chipps. Impacts on Rainbow Trout Oncorhyncus mykiss rearing performance of dietary bio-processed plant-based protein converted soy products. Aquaculture America, Las Vegas, NV, February 19-22, 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Bruce, T.J., and M.L. Brown. Immunomodulation in freshwater species fed bioprocessed soybean meal. Aquaculture America, Las Vegas, NV, February 19-22, 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Bruce, T.J., R.D. Neiger, and M.L. Brown. Gastrointestinal microbiota and immunological responses of Rainbow Trout Oncorhynchus mykiss fed modified soybean meal ingredients. Aquaculture America, Las Vegas, NV, February 19-22, 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Kasiga, T., B White, and M. Brown. Nutritional value of modified carinata meal as protein sources for hybrid striped bass Morone chrysops x M. saxatilis. Aquaculture America, Las Vegas, NV, February 19-22, 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Kasiga, T., B. Karki, W. Gibbons, and M. Brown. 2018. Utilization of modified carinata meals in finfish diets. Carinata Biomaterials Summit 2018, Panama City, FL, February 18th-20th.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Koepke, J., C. Levesque, W. Gibbons, M. Brown, and R. Kaushik. Evaluation of a bio-processed soybean meal on nursery pig performance and immune status. American Society of Animal Science, Omaha, NE, March 13-17, 2017.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Voorhees, J.M., M.E. Barnes, S. Chipps, and M.L. Brown. July 2018. Rearing performance of juvenile Brown Trout (Salmo trutta) subjected to exercise and dietary bioprocessed soybean meal. Open Journal of Animal Sciences 8:303-328. DOI: 10.4236/ojas.2018.83023.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Voorhees, J.M., M.E. Barnes, S. Chipps, and M.L. Brown. 2018. Direct substitution of fishmeal with bioprocessed soybean meal in brown trout diets. Journal of Fisheries and Aquaculture Development DOI: 10.29011/2577-1493.100043.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhang, J., X. Wang, Y. Lu, S.J. Bhussal, Q. Song, P.B. Cregan, Y. Yen, M.L. Brown, and G. Jiang. March 2018. Genome-wide scan for seed composition provides insights into soybean quality improvement and the impacts of domestication and breeding. Molecular Plant 11:460-472.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Koepke, J., C. Levesque, R. Kaushik, W. Gibbons, and M. Brown. 2017. Evaluation of a bio-processed soybean meal on nursery pig performance and immune status. Journal of Animal Science Doi:10.2527/jas2017.1679.
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Bruce, T.J., Sindelar, S.C., Schulz, D.A., Gibbons, W.R., and Brown, M.L. In Review Characterization of innate immune parameters in yellow perch (Perca flavescens) fed bioprocessed soy-based diets supplemented with minerals and polysaccharides. Aquaculture International.
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Bruce, T.J., S. C. Sindelar, D.A. Schulz, M.L. Brown, and W.R. Gibbons. In review. Evaluation of innate immunity and growth performance in yellow perch fed soy-based diets containing commercial exopolysaccharide. Journal of Aquatic Animal Health.
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Bruce, T.J., S. Das, R.D. Neiger, and M.L. Brown. In review. Immunomodulation in Rainbow Trout Oncorhyncus mykiss fed bioprocessed soy diets and challenged with Flavobacterium psychrophilium. Fish and Shellfish Immunology.
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Bruce, T.J., T. Kasiga, B.M. White, J.A. Clapper, and M.L. Brown. In review. Acute stress response in rainbow trout (Oncorhynchus mykiss) fed alternative soybean ingredients. Journal of the World Aquaculture Society.
• Type: Theses/Dissertations Status: Other Year Published: 2018 Citation: Kasiga, T. 2018. Nutrient-enriched feed ingredients derived from meal streams in oilseed biofuel production. Ph.D. dissertation, South Dakota State University, Brookings.
• Type: Theses/Dissertations Status: Other Year Published: 2018 Citation: Voorhees, J. 2018. Impacts of salmonid rearing performance of dietary bio-processed plant-based protein and water velocity. M.S. thesis, South Dakota State University, Brookings.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Aquafeed manufacturers and finfish producers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PD and graduate students (T. Bruce, Ph.D., Wildlife and Fisheries Sciences; T. Kasiga, Ph.D. , Wildlife and Fisheries Sciences; B. White, M.S., Wildlife and Fisheries Sciences; J. Voorhees, M.S. , Wildlife and Fisheries Sciences) jointly planned and conducted experiments, completed data analyses, prepared articles and presentations and attended meetings to present results. How have the results been disseminated to communities of interest?Results of studies are being published in peer-reviewed journals that target the aquafeed and producer industries (e.g., Journal of the World Aquaculture Society, Aquaculture Research, Journal of Animal Science) and relayed to state/regional lay outlets for commodity producers (e.g., Soybean Leader, Farm Forum, TriState Neighbor). Abstracts for five presentations have been accepted for delivery at Aquaculture America in February 2018. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Determination of nutritional composition and deficiencies that exist in seed meals Compositional analyses of process-modified oilseed meals will be ongoing to enable comparisons with base and previously modified meals. Objective 2: Evaluate processes - thermomechanical, chemical, enzymatic,and microbial - that may be used to upgrade the nutritional quality of meals A primary task is to evaluate the performance of new enzymes in pretreatment steps to upgrade defatted soybean, carinata, barley and canola meals, and further optimize microbial conversion times to minimize protein loss and improve digestibility. We are also examining the effects of processing treatments on residual GLS, and breakdown products, in carinata. Objective 3:Test palatability, digestibility, and growth performance of promising plant-based products in commercially important finfish diets We will focus on conducting fish performance trials using test ingredients as processes yield improved meal compositions. We will also continue to investigate the potential value co-blended bioprocessed plant meals in practical feed formulations as individual seed meals are compositionally optimized.

Impacts
What was accomplished under these goals? Recent research has primarily focused on defatted soybean (SBM), as well as camelina and carinata oilseed meals. ANF targets associated with defatted soybean meal are oligosaccharides, protease inhibitors, and phytic acid. Carinata and camelina oilseed ANF targets include glucosinolates (GLS), phytates, tannins and sinapine, in addition to non-starch polysaccharides and oligosaccharides. Nutritive composition (protein, lipid, amino acids) and ANF analyses were completed on base meals (Objective 1). One task under Objectives 2 and 3 was to assess how nitrogen inclusions may affect bioprocessing to improve nutritional characteristics of SBM for use in aquafeeds. Pretreatment and bioprocessing combined with an economic nitrogen supplementation has the potential to reduce ANFs while simultaneously increasing single-cell protein. Two rainbow trout feeding trials were completed to determine a palatable source and concentration of nitrogen for potential inclusion during bioconversion to mitigate nutrient limitation for Aureobasidium pullulans, the microbe used in bioprocessing. In the first palatability study, three nitrogen sources, ammonium chloride, diammonium phosphate, and urea, were included (1,250 ppm) into diets of rainbow trout (Oncorhynchus mykiss) containing a bioprocessed soybean meal (BSBM) in place of fish meal (FM) and feed consumption was monitored. No significant differences in consumption occurred, however it was noted that the diet containing urea was consumed slightly more than the other nitrogen supplemented diets. Therefore, in a follow-up palatability study, urea was supplemented at incremented (0 to 2,000 ppm) inclusion levels to rainbow trout diets containing BSBM. The study revealed nonsignificant palatability responses when consumption and growth parameters were analyzed among treatments. Hence, the results indicate that up to 2,000 ppm urea can be potentially supplemented to A. pullulans during bioprocessing without any adverse effect on palatability of rainbow trout feeds. In separate feeding trials (Objective 3), six experimental BSBM ingredients were processed and formulated into aquafeeds and fed to rainbow trout and hybrid striped bass (Morone chrysops x M. saxatilis). The performance of these experimental ingredients (BSBM, fractions thereof, and BSBM with enzyme) was tested against a FM control through digestibility and growth trials (105-day). The optimal experimental soy ingredient in each trial was determined based on consumption, growth, feed efficiency, apparent digestibility, and health indices. The experimental ingredient with the highest protein digestibility (ADC-P) when fed to hybrid striped bass was BSBM with enzyme. This ingredient was found to have a significantly higher ADC-P. Rainbow trout growth trials revealed that diets containing BSBM or a washed, base BSBM ingredient provided the best growth and health performance. Further to Objective 3, a 90- day feeding trial using juvenile rainbow trout (27.9 ± 1.4 g; mean ± SE) was completed to evaluate growth, feed efficiency, general health and immunological responses to diets containing several plant-based protein sources and FM. Trout were fed one of four dietary treatments: experimental BSBM, commercial hydrolyzed soy protein concentrate (CSPC), bioprocessed barley protein concentrate (BBPC) and a FM control (FMC). At trial termination, there were no significant differences in relative growth (RG) or specific growth rate (SGR) between fish fed either FMC or BSBM. Fish fed either CSPC or BBPC resulted in significantly lower RG and SGR than the FMC. Significant respiratory burst analysis differences were observed among treatments at day 90, for fish fed either FMC or CSPC diets. Swimming performance experiments were conducted (Objective 3) using two different rainbow trout strains (Erwin x Arlee and Shasta) fed FM replacements. The dietary treatments were either FM or BSBM replacing 60% or 80% of the dietary FM. Flow treatments were the minimal velocity (averaging 3 cm/s for both experiments) required to keep tanks self-cleaning or an elevated level six to ten times that of the minimal velocity. FCR was significantly improved in fish fed diets where 60% of the FM was replaced with BSBM (Table 1). However, SGR was not significantly different among the diets with either strain. There were also no significant differences in weight, length, condition factor, relative fin length, hepatosomatic index, splenosomatic index, or viscerosomatic index among the dietary treatments. None of the dietary treatments were significantly affected by water velocity. However, compared to lower water velocities, higher velocities produced significantly poorer FCR and SGR in Shasta strain rainbow trout. FCR was also significantly higher in the high velocity tanks of the Erwin-Arlee strain, but SGR was not significantly different between the velocities. With the Shasta strain, both the hepatosomatic and viscerosomatic indices were significantly different between the velocity treatments. Fin indices were not significantly different between the velocities in either strain. Up to 80% of the FM component of Shasta strain rainbow trout diets can be replaced with BSBM with no decrease in rearing performance. Also, up to 60% of FM can be replaced in Erwin x Arlee diets. Diets containing BSBM produced similar results to the FM control at both rearing velocities. Process effects on carinata and camelina meals were assessed by subjecting cold-pressed carinata and camelina meals to extrusion, solvent extraction and aerobic conversion or sequential process combinations to determine which process(es) yielded improved composition (Objectives 1 and 2). Carinata meals generally yielded more crude protein than camelina meals for a given process treatment (8 process combinations). Similarly, the fiber contents of carinata seed meals were also generally lower than those resulting from camelina meals. The largest protein increase observed in both carinata and camelina meals was due to hexane-based oil extraction and microbial conversion, but not extrusion. Microbial conversion increased the fiber content of all carinata and camelina meals. However, aerobic conversion reduced GLS of all meals by at least 70% with more reduction observed in camelina meals. Palatability (Objective 3) of carinata and camelina meals was generally improved by solvent extraction and, to a small extent, by aerobic conversion. Protein digestibilities were generally higher in carinata than camelina meals for both rainbow trout and hybrid striped bass but overall protein digestibility was higher in bass than in trout. Microbial conversion lowered the protein digestibility of some carinata and camelina meals but the effect was more pronounced in carinata meals.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Bruce, T.J., and M.L. Brown. 2017. A review of immune system components, cytokines, and immunostimulants in cultured finfish species. Open Journal of Animal Science (Special Issue on Veterinary Vaccines and Immunology) 7:267-288. DOI: 10.4236/ojas.2017.73021.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Bruce, T. J., S.C. Sindelar, J.M. Vorhees, M.L. Brown, and M.E. Barnes. 2017. Performance and immunological responses of rainbow trout (Oncorhynchus mykiss) fed bioprocessed plant-based proteins. Aquaculture Nutrition 23:1160-1168.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: White, B. 2017. An evaluation of nitrogen supplementation and processed soy fractions on the performance of cultured fishes. M.S. Thesis, South Dakota State University, Brookings, SD.
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Bruce, T.J., T. Kasiga, B.M. White, J.A. Clapper, and M.L. Brown. In press. Acute stress response in rainbow trout (Oncorhynchus mykiss) fed alternative soybean ingredients. Journal of the World Aquaculture Society xx:xxx-xxx.
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Bruce, T.J., S. Das, R.D. Neiger, and M.L. Brown. In review. Immunomodulation in Rainbow Trout Oncorhyncus mykiss fed bioprocessed soy diets and challenged with Flavobacterium psychrophilium. Fish and Shellfish Immunology.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Bruce, T.J., R.D. Neiger, and M.L. Brown. 2017. Gut histology, immunology, and the gastrointestinal microflora of rainbow trout (Oncorhynchus mykiss), fed bioprocessed variants of soybean meal. Aquaculture Research DOI: 10.1111/are.13480.