106 Barrows 5366 21310 003 00D PostPlan

Project Plan
NP 106 Aquaculture
August 2009
Old ARS Research Project Number

5366-21310-003-00D
Management Research Unit

5366-05 Small Grains and Potato Germplasm Research Unit
Location
Hagerman and Aberdeen, Idaho (Aberdeen)
Title
Improving Sustainability of Rainbow Trout Production by Integrated Development of Improved
Grains, Feeds, and Trout.
Investigators
Frederic T. Barrows Lead Scientist, Research Physiologist, Fish 1.0
J. Michael Bonman Research Leader 0.05
Vacant Research Physiologist, Fish 1.0
Gongshe Hu Plant Geneticist 1.0
Kehsun Liu Grain and Oilseed Chemist 1.0
Kenneth E. Overturf Research Geneticist, Fish 1.0
Scientific Staff Years

5.05
Planned Duration
60 months
Post-Peer Review Signature Page

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Signatures and Dates Must Be Complete Prior To Distributing this Project Plan to Peer Reviewers
Barrows, 5366-21310-003-00D, Improving Sustainability of Rainbow Trout Production by Integrated

Development of Improved Grains, Feeds, and Trout.
This project plan was revised, as appropriate, according to the peer review recommendations and/or other
insights developed while considering the peer review recommendations. A response to each peer review
recommendation is attached. If recommendations were not adopted, a rationale is provided.
John Michael Bonman___________________ 6/29/2009____

Research Leader Date
This final version of the project plan reflects the best efforts of the research team to consider the
recommendations provided by peer reviewers. The responses to the peer review recommendations are
satisfactory.
John Michael Bonman___________________ 6/29/2009_____

Center, Institute or Lab Director Date
The attached plan for the project identified above was created by a team of credible researchers and
internally reviewed and recognized by the team’s management and National Program Leader to establish
the project’s relevance and dedication to the Agricultural Research Service’s mission and Congressional
mandates. It reflects the best efforts of the research team to consider the recommendations provided by
peer reviewers. The responses to the peer review recommendations are satisfactory. The project plan has
completed a scientific merit peer review in accordance with the Research Title of the 1998 Farm Bill
(PL105-185) and was deemed feasible for implementation. Reasonable consideration was given to each
recommendation for improvement provided by the peer reviewers.
______________________________________ ______________
Area Director (original signature required) Date
September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

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TABLE OF CONTENTS:
Cover Page………………………………………………………………………………………i
Signature Page………………………………………………………………………………....ii
Table of Contents……………..……………………….…….……………………...…………iii
Project Summary…………………………………….…………………………………..…....iv
Objectives………………………………………………………………………….……………1
Need for Research………………………………………………..…………….……………..2
Scientific Background ……………………………….…………………………….…………3
Approaches and Procedures ………………………………………………………………13
Milestones and Outcomes ……………….…………………………………………………26
Literature Cited ……………………………………………………………………………….43
Past Accomplishments ……………..………………………………………………………52
Issues of Concern Statements ……………….……………………………………………67
Appendices ………...…………………………………………………………………………72

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Project Summary:
This project will use parallel, interconnected lines of research to improve the
sustainability and production efficiency of rainbow trout (Figure 1). Alternative ingredients from
multiple sources will be used directly, developed, or modified in order to eliminate or reduce fish
meal and oil in trout diets. Concurrently, trout populations will be screened to identify variation
in important traits and to identify genes specific to the utilization of these feeds and to elucidate
the genetic basis for improvement. A series of digestibility trials will be performed to define
available nutrient levels from currently available and enhanced plant products. Supplementation
needs for specific amino acids and minerals in fish meal-free feeds will be determined.
Biochemical, physiological and genetic responses of fish fed alternative diets will be determined
using genetic and physiological tools. Carbohydrates and fatty acids in grains differ in both type
and abundance from fish meal and oil, and understanding the metabolic and genetic factors
affecting nutrient utilization of trout will improve selection efforts in both trout and grains, and
increase the precision of nutrient supplements, thereby reducing excretory waste. The
anticipated outcomes from the project are: 1) improved feeds for fish farmers, environmental
compliance through reduced nutrient release, identified traits and markers to aid stock
improvement efforts 2) alternate ingredients, reduced costs, price stability, and improved diet
formulations for feed manufacturers; 3) new markets and products for grain farmers; and 4) a
safe, sustainable, nutritious food supply and cleaner environment for U.S. consumers.

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Objectives
The overall goal of this project is to improve the sustainability and production efficiency of
rainbow trout by developing innovative feeds that reduce dependence on marine fishery
resources. This will be accomplished through the integrated development of improved
ingredients and feeds and elucidation of the molecular mechanisms underlying metabolic
processing of these ingredients (Figure 1).
The specific objectives are:

Objective 1: Identify and develop grain lines with desirable traits for either direct or indirect use
in aquafeeds.
Objective 2: Develop mechanical, chemical and biological methods to improve the nutritional
and anti-nutritional profile of grains, by-products and other alternative ingredients.
Objective 3: Determine nutritional value of alternative ingredients (protein, lipid, energy)and

develop practical feed formulations for improved strains of fish.
Objective 4: Determine optimal nutrient supplementation levels for specific life stages of
improved strains of trout.
Objective 5: Use gene expression analyses to advance the understanding of gene targets for
improving nutrition, growth, and development processes under production
conditions.
Objective 6: Identify phenotypic differences in rainbow trout for growth and utilization of plant-
based sustainable diets and determine the genetic variation for the identified
traits.
Need for Research

All commercially important marine fish populations are considered currently fully
exploited or overexploited by USA and international fisheries management agencies, with the
possible exception of the Alaskan fishery. Seafood consumption, however, is increasing due to
the influence of aquaculture production. Over the next 20 years, aquaculture production must
increase by 500% to meet expected demand of fish for human consumption. Fish meal and fish
oil, which are produced from capture fisheries of industrial species not used for direct human
consumption, have been primary ingredients of fish feeds, but there are concerns that supplies
are insufficient to meet the growing demand to produce fish feeds. In fact, limited production of
fish meal and fish oil is predicted to limit industry expansion by 2013 (Tacon 2003). These
products are also costly, variable in quality and availability, and their continued production is
considered by some to be unsustainable. To allow for the continued expansion of aquaculture,
alternate sources of protein and oil must be developed and production efficiencies must be
increased. Developing new diet formulations in which fish meal and fish oil are replaced with
alternative ingredients can reduce fish feed cost and variability and move the aquafeed industry
to a more sustainable foundation. However, some alternative ingredients can create problems
with reduce feed intake lower feed digestibility, cause metabolic alterations or health problems
in fish and reduce growth rates of fish. Plant-derived ingredients, and other alternative sources,
must be modified to improve nutritional quality, reduce levels of anti-nutrients and meet
specifications for aquafeeds by genetic selection of the plant, post-harvest processing, and feed
formulation and processing.

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Evaluation of alternative ingredients and development of complete feeds

A large number of plant derived products are potential substitutes for fish meal in trout
feeds. However, each ingredient has one or more properties that reduce its nutritional value
relative to fish meal. Methods to improve positive attributes and reduce or eliminate possible
anti-nutritional factors are needed. To ensure economical feasibility, processing methods must
be developed that are capable of simultaneously concentrating multiple high value components
from grains and their co-products.
Most experimental diets in which alternative plant ingredients completely replace
fishmeal fall short of supporting optimal fish growth and/or feed conversion efficiencies. Often
nutritional limitations were identified as potential restrictions to performance but frequently
unknown nutritional inadequacies or other unknown limits to ingredient utilization have not yet
be defined. Some formulations have not considered amino acid availabilities of the alternative
ingredients, and this information is critical to optimize growth and nitrogen retention.
Determining the optimum dietary levels of essential amino acids and the optimum ratio of one to
another that is necessary to support optimal growth at the lowest protein levels is essential to
improve sustainability and production efficiency. Formulating feeds to contain an optimum
amino acid profile and establishing dietary amino acid levels on a digestible basis when feeds
contain alternative ingredients will add flexibility and accuracy in feed formulation and help to
maintain high growth performance of fish fed the improved feeds.
If feeds containing little or no fish meal are to be accepted by the fish farming industry,
the factors responsible for growth rate reduction must not only be identified but solutions to
these nutritional problems are required. In nature, trout have evolved as
carnivores/insectivores. Therefore, distinct genetic stocks must be selected to optimize the
growth efficiency of trout fed plant-based feeds. The first step in this process is to identify
genetic variability for commercially relevant traits. The development of molecular tools for
quantifying these traits and selecting improved families is also vital. Recent research has
indicated that rainbow trout strains selected for improved utilization of plant-based feeds
perform better than unselected parental strains. For aquaculture to continue its growth,
domesticated stocks of fish that can utilize the plant-based feeds of the future will be needed.
Relevance to ARS National Plan- This project falls within the following components of the
National Program 106 (Aquaculture): 1) Defining Nutrient Requirements, Nutrient Composition
of Feedstuffs and Expanding Alternative Ingredients 2) Understanding, Improving, and
Effectively Using Animal Genetic and Genomic Resources
This project’s focus on feed formulations and feeding strategies to reduce dependence
on marine fish-based protein and oils in aquaculture diets relates directly to the National
Program component “Defining Nutrient Requirements and Nutrient Composition of Feedstuffs
and Expanding Alternative Ingredients”. The project’s focus on molecular identification of traits
linked to growth, health and feed utilization which will enhance efforts of the National Trout
Broodstock Selection Program, fits under National Program Component Understanding,
Improving, and Effectively Using Animal Genetic and Genomic Resources. Customer input at
the USDA/CSREES/ARS workshop held in 2008 noted a sense of urgency to increasing
production efficiency, which is the primary goal of this project.
Inclusion of this project within the Small Grains and Potato Germplasm Research Unit in
Aberdeen, Idaho, is unique among fish nutrition projects. The unit has an extensive collection of
small grain germplasm (more than 130,000 accessions of diverse origin) and expertise in plant
breeding and genetics. Other CRIS projects within the unit are complementary to the present
project. The Unit is currently analyzing the National Small Grains Collection at Aberdeen and
then using the resulting information in the development of cultivars possessing nutrient
characteristics important for aquafeeds. Researchers in Aberdeen were the first to identify low
phytate strain mutations and are now transferring this trait to more productive and adapted

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barley genotypes. Thus, there exists great potential for modifying plant strains to possess the
requisite biochemical components for the production of nutritionally complete fish feeds.
Potential Benefits- Attaining these objectives will provide economic, environmental, and
health benefits to several sectors of society. Commercial barley and oat producers will benefit
from improved cultivars and new market demand for their raw and value-added products.
Commercial fish producers will benefit from improved production efficiencies accruing from
superior feeds and enhanced genetic strains. The use of feeds with reduced or no fish meal will
enhance the sustainability of aquaculture by lessening dependence on marine products. Feed
manufacturers will benefit from the availability of alternate feed ingredients, knowledge of
specific nutrient requirements and access to proven diet formulations. Consumers will benefit
from an affordable, high quality protein supply that is produced in an environmentally
sustainable manner with reduced nutrient levels (phosphorus) in surface waters from
aquaculture production.
Anticipated products- Four categories of products are expected from this project. First,
improved oat and barley traits and germplasm will be identified. Second, improved ingredients
will be developed for use in trout feeds. Third, feed formulations and nutrient supplement
specifications for non-fish meal feeds will be developed. Fourth, genes and traits involved with
nutrient metabolism and utilization in trout fed non-fish meal feeds will be identified,
characterized, and made available for genetic selection programs for stock improvement.
Customers- Trout farmers, fish feed manufacturers, grain producers and consumers will
all use products from this project and will be essential parts of field testing the alternative diets
developed in this project. Selective breeding programs for rainbow trout will employ genetic
markers for traits associated with metabolism of plant products by trout. Consumers demanding
safe aquaculture products will enjoy healthy products produced in an environmentally
responsible manner.
Scientific Background
Objective 1; Improve quality value of small grains. Beta-glucan (BG) is a dietary
fiber in barley grain that critically influences the value of the grain to the end-user. BG
concentration, for example, directly affects the availability and price of ingredients derived as by-
products from the industrial utilization of this grain. Barley is considered a good source of starch
for ethanol production, but BG reduces flowability of the slurry reducing process efficiencies. A
better understanding of the genetic mechanisms controlling BG metabolism in small grains will
provide the tools needed for stronger grain selection programs.
The mixed linkage (1-3), (1-4)-beta-D-glucan, simply called BG here, is a polymer of
glucose which is deposited as cell wall in cereal grains. The unique structure of this type of BG
makes it soluble and thus the most important dietary fiber for human health. BG mainly exists in
cereal crops including barley, oat, rye, and wheat (Smith and Harris 1999). Barley and oat
grains have the highest BG content making them the two major sources; BG comprises 3-8% of
barley (Holtekjoln et al. 2006) and 3-6% of oat (Asp et al. 1992). In cereal species, BG
accumulates in the cell walls of the developing caryopsis and surrounding maternal tissues and
eventually becomes a major component of the cell wall of the endosperm (Carpita 1996). As a
unique type of fiber, BG has critical impacts on human health, feed value, and industrial
utilization. BG is one of the primary target traits in barley and oat breeding programs. Cultivars
with both high and low contents are important with high contents focused on the human health
markets and low contents for malting, animal feed, and biofuel production. High levels of BG
might interfere with protein and starch separation and recovery by wet methods. Low BG
barley grains may contribute to high efficiency of protein and starch separation for Dr.
Liu’s work proposed in Objective 2.

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The benefits of BG consumption on human health have attracted substantial attention.

Barley BG has a cholesterol-lowering effect (Keenan et al. 2007; Behall et al. 2004; Li et al.
2003; McIntosh et al. 1991; Newman et al. 1989) that may contribute considerably to heart
disease prevention in humans. Recent scientific study also indicates that dietary fiber intake
can reduce the risk of diabetes (Zhang et al. 2006). Therefore, development of healthy food,
specifically BG, fiber-rich food resources, could contribute to reducing these health related
problems.
BG also negatively affects malt and beer quality (Fincher 1992) as well as ethanol yield
for biofuel production. BG content in malt is a concern because it causes difficulty in the filtering
processes during brewing (Bamforth and Barclay 1993) and haze formation (Coote and Kirson
1976). In addition barley is an economical ingredient for animal feeds that is cheaper than other
grains such as corn. However BG content makes it unpopular as a feed ingredient for mono-
gastric animals, including poultry and fish, due to the reduction of metabolizeable energy (ME)
from the grain. (Campell et al. 1989; Jeroch and Dänicke 1995). In order to increase the ME
value of barley, reductions in BG content will be required. The biological effects of BG on seed
quality and other agronomic traits are unclear. Genetic study of BG content has lagged behind
studies of other traits and strengthening those studies will greatly improve the knowledge and
practical utility of the trait. Aquafeed development will also benefit from the industrial utilization
of barley grains due to improved BG contents through the increased availability of economic by-
products. Therefore, we propose to genetically manipulate the BG levels in barley lines to
improve industrial and feed value that will result in the greater availability of economical, high
quality ingredients for aquafeed development.
The most important connection between BG and Aquafeed development is the
availability of appropriate genetic lines for better improvement of BG levels in barley. The
barley lines in the Small Grain Germplasm Collection database were first checked for
their BG profiles. We decided to use the alternative approach of mutant screening
because of the limitation of BG levels and adaptability of the lines in the germplasm
collection. We used a chemical mutagenized method with the cultivar Harrington for
mutant induction. Two mutants were discovered in the M1 generation for the higher and
lower BG contents compared to the parental line. The high BG line was designated M38
and low BG line was designated M351.
Objective 2; Ingredient Enhancement. A limited number of ingredients are currently used in

trout feeds, relative to other domesticated animals, due both a lack of characterization of
existing products and a lack of high protein products available. Grains and bio-fuels by-
products have many positive characteristics, but may not contain enough protein to replace fish
meal or may contain anti-nutrient that limit their inclusion rate. Therefore, physical, chemical or
biological modification of non-traditional aquafeed ingredients may produce new ingredients for
trout feeds and find new uses for underutilized material.
Barley and oats. In addition to the benefits to human nutrition described above, barley
and oats can be potential sources of low-cost proteins with high nutritional value for animal
feeds. However, their protein contents (8-15%) are much lower than levels in fish meal (about
60%). These grains also contain other components, such as starch and phytate, that may limit
their use in aquafeeds. There are several reports of processing barley and oats into a fraction
enriched with protein, beta-gluan (BG), or starch. Basically these methods can be grouped into
two major categories: dry fractionation and wet extraction. Dry methods include 1) pearling
(Yeung and Vasanthan 2001), 2) roller milling (Izydorczyk et al. 2003), 3) milling followed by air
classification (Wu et al. 1994), and 4) milling followed by sieving (Wu et al. 1994). Wet methods
typically involve solvent extraction, screening and precipitation (Cluskey et al. 1973; Wu et al.
1979; Ma 1983) and can include enzymatic treatments (Barrows et al. 2009). BG is generally
extracted from barley and oats with alkali and precipitated by ethanol solution (Bhatty 1995;

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Temelli 1997). A hot water extraction of BG from the grain followed by freeze-thaw of the
extract was reported in a U.S. Patent Application US 2002/0192770 A1 by Morgan (2002). Wet
methods to isolate starch from barley are also reported (Linko et al. 1989; Andersson et al.
2001). However, all reported studies on fractionating barley or oats by dry or wet methods
focused on enrichment of only one (Ma 1983; Bhatty 1995; Temelli 1997) or two nutritional
components (Linko et al. 1989; Wu and Stringfellow 1995). Preliminary work with barley at our
laboratory on simultaneous separation of protein, BG and starch using both dry and wet
methods demonstrated the feasibility of this approach. Additional work is needed to improve the
efficiency of the newly developed methods and to appropriately modify them for pilot scale
production. After both laboratory and pilot scale methods are successfully demonstrated with
barley, research will focus on developing similar methods for oats.
DDGS from grain-based ethanol production. A dramatic increase in fuel ethanol
production in the U.S. has led to a rapid increase in the supply of distillers dried grains with
solubles (DDGS). Marketing of DDGS is critical to the sustainability of ethanol production, but
high fiber and phytate content limit its use for some animal feeds. Additionally, there is
significant variation in the chemical composition and physical properties among varieties of
DDGS (Belyea et al. 2004) and this affects market price. Three approaches are being explored
to improve the quality of DDGS for animal feeds. First, variation in quality is being addressed by
studies designed to improve our understanding of nutrient changes during conversion of corn to
ethanol, and by standardizing analytical methods. In our laboratory, the particle size distribution
of DDGS and its relationship to contents of various nutrients was recently investigated (Liu
2008) while a follow up study identified the effect of chemical and physical properties in the
ground corn on those of the resulting DDGS (Liu 2009). To date, limited information exists
regarding changes in nutrient profiles during the dry grind process (Noureddini et al. 2009) or
standardization of analytical methods for DDGS (Thiex, 2009). Second, the quality of DDGS for
aquafeeds is being improved by further processing to increase protein and simultaneously
decrease fiber and other unwanted constituents (Wu and Stringfellow 1982; Singh et al. 2002;
Srinivasan et al. 2005). We recently investigated, for example, a method of combing sieving
with winnowing to affect compositional changes in DDGS (manuscript submitted). Each
reported method has beneficial attributes but a high quality consistent product is not yet
available; a combination of techniques or alternate methods may be necessary. Third, the
process of raw material selection and the modification of ethanol production methods to produce
modified DDGS with improved nutritional profiles need further evaluation. For example, low-
phytate corn or other crops are being bred (Raboy 2002, Pilu et al. 2003), that, when utilized as
a feedstock for ethanol production, could lead to production of a low-phytate DDGS.
Modification of process steps, such as adding a feedstock pretreatment, has also shown
promise in improving the nutritional profile of DDGS (Singh et al. 2005). In order to meet the
demanding specifications of aquafeeds for a high-protein ingredient, all three approaches will
need to be evaluated and put in practice.
Algae biomass. Marine algae’s are currently being investigated not only as sources of
bio-fuel, but also as a method to remove CO2 from power plant off-gas. A genetically diverse
group of organisms comprising thousands of species are attractive natural sources of nutrients
and bioactive compounds. Historically, algae have been consumed directly as a vegetable
(seaweeds). They are now used as a source of polysaccharides or polyunsaturated fatty acid
oils (Becker 2004; Spolaore et al. 2006). The biomass of algae is generally a powdery product
that is marketed as food supplements. Nevertheless, dried algae have not garnered significant
market share as food items or food substitutes in the U.S. (Becker 2007). The major obstacles
to increased algal consumption are the powdery consistency, dark green color and slightly fishy
smell. There is also concern regarding potential long-term toxicity or other effects on human
health. Nevertheless, there is a growing market for algae biomass in animal nutrition (Becker
2004; Spolaore et al. 2006). The use of algae in feed for farmed fish could be another

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promising application of this resource. In fact, some of the obstacles for human consumption,
such as pigment and odor, turn out to be advantages when algal meals are included in
aquafeeds. The beneficial effects of Porphyra in the diet of red sea bream were described by
Mustafa et al. (1995), for example. The additive appeared to improve body weight gain and
increase triglyceride and protein deposition in the muscle. Another positive effect of algae
inclusion in fish feed is improved resistance of fish to stress or diseases (Kyle 2004).
Traditionally, live (wet) algae have been used in aquaculture for years, but due to the high costs
and difficulties associated with production, transportation or storage, dry algae products have
received more attention.
Co-product of algal bio-fuel production. In order to minimize the potentially deleterious
environmental and agricultural consequences associated with current land-based bio-fuel
feedstock, some algae species are being considered as promising sources of raw material for
future bio-fuel production (Dismukes et al. 2008). Key advantages of algae include prolific
growth rates, the ability to clean up water resources with excess nutrients, and the ability to
grow on lands that are marginal for other agricultural purposes. The potential of algae as
high-yield sources of lipids (25-50%) and fermentable biomass (starch and glycogen, 20-50%)
was documented in research conducted by the National Renewable Energy Lab and its
contractors within the Aquatic Species Program during the 1980s and 1990s (Sheenhan et al.
1998). More importantly, a significant fraction of the residual biomass following lipid and
carbohydrate extraction is protein that is expected to pass through largely unaltered by the mild
conditions used for fermentation to ethanol or those used for lipid extraction for biodiesel
production. Thus, this co-product of bio-fuel production from algae can be directed toward
secondary markets, such as fish feed. Research into the use of algae as a viable bio-energy
feedstock is in its infancy relative to more thoroughly investigated terrestrial feedstock; yet,
significant research efforts are underway (Chisti 2007; Dismukes et al. 2008).
The protein content of algae differs according to species. Some species have protein
concentrations as low as 10% on a dry matter basis (dmb), while others are as high as 65%
(Spolaore et al. 2006). In addition, Rodde et al. (2004) examined the chemical composition of
Palmaria palmate and observed clear seasonal variation in ash (15-27%, dmb), protein (14-
30%) and a low molecular weight carbohydrate, floridoside (3.3-25%). In addition, concerns
have been raised with regard to potentially high levels of nucleic acids, heavy-metals, and
known and unknown toxins in algae biomass (Becker 2004). Therefore, before algae biomass
or its derivative products can be developed into fish feed ingredients, they must be thoroughly
characterized for nutritional value and the presence of possible anti-nutritional factors.
Objective 3; Alternative Ingredients and Practical Feeds. Determination of the nutritional

value of ingredients is a primary consideration in developing alternative feed formulations.
Glencross et al. (2007) recently outlined strategies for evaluating the nutritional value of
ingredients in aquaculture feeds. Some commonly available ingredients, as well as promising
new ingredients, are being evaluated for rainbow trout as well as other commercially produced
fish species in the United States. Lower nutrient digestibility from alternative ingredients has
been identified as a factor that limits inclusion in fish feeds (Gaylord and Gatlin 1996;
McGoogan and Reigh 1996; Cheng and Hardy 2002; Gaylord and Rawles 2005). Knowledge of
nutrient availability for each ingredient is essential for its efficient use in alternative diet
formulations. The importance of having diets balanced on an available energy basis as well as
an available amino acid basis has been well demonstrated across multiple production animal
species (Pomar et al. 1991; Warnants et al. 2001; Yamamoto et al. 2002). Gaylord et al. (2008)
determined the digestibility of protein and energy of 24 common ingredients in extruded diets for
rainbow trout. Apparent amino acid availability coefficients were also determined for these
ingredients (Gaylord et al. 2009). While this information provides the necessary insight needed
to formulate diets with ingredients used in today’s trout feeds, as new ingredients are

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developed, new cultivars of plants produced, or co-products from related industries are
released, it is essential to fully understand the nutrient availabilities and anti-nutrient limitations
of each ingredient.
The need to determine the nutritional value of new ingredients is obvious, but re-
evaluating traditional ingredients over time is also important due to changes that may occur in
the composition of commercially available products. Moreover, changes in growing conditions,
agricultural practices, and processing occur over time and can effect not only the composition of
an ingredient, but also its nutrient availability. One example, of this is lupin meals. Glencross et
al. (2008) determined that considerable variability in nutrient content existed in lupin meals
sampled over a three year period and that this variability altered the digestibility of nutrients and
energy in the product for rainbow trout. Soybean meal is another example where the nutritional
profile has been altered by newly developed processing technologies or selective breeding
(Barrows et al. 2008; Baker and Stein 2009). Selective breeding of cereal grains can also
change the digestible energy (Gaylord et al. 2009) or phosphorus content for rainbow trout
(Overturf et al. 2003).
As previously noted, while it is important to increase the number of alternative
ingredients available for feed formulation, and to fully understand their nutritional value, the next
step in aquafeed development is to evaluate the interactive effects of multiple combinations of
ingredients. Some studies have evaluated single ingredient effects on fish performance and
health (Romarheim et al. 2006), but unknown interactions may occur when novel ingredients are
included in a diet with various other ingredients. Santigosa et al. (2008) noted limitations in
completely replacing fish meal with a combination of plant proteins which they demonstrated
were probably due to reduced digestive enzyme capacity in the gastrointestinal tract of rainbow
trout. However, this study was not able to parse out which feed ingredient, or potential anti-
nutrient, was responsible for the reduced enzymatic capacity and, therefore, whether a single
ingredient or combination of plant ingredients led to the observed results could not be
discriminated. Although the nutritional value of an ingredient combination may be sufficient
based on compositional analysis and digestibility estimates, specific combinations of ingredients
may yield better or worse fish performance in longer term feeding trials for a variety of unknown
factors. Some of the confounding factors may be associated with pellet quality or acceptability
(Hansen and Storebakken 2007; Sorensen et al. 2009), anti-nutrients (Tacon 1997), or
interactions of ingredient components during extrusion that may lower nutritional value
(Erbersdobler and Somoza 2007). Ruohonen et al. (2007) described the need to examine
potential interactions among nutrients to optimize diets for finfish. Evaluating the effect of a
variety of ingredient combinations on fish performance will not only allow identification of
positive and/or negative interactions, but will also help determine the economic value of each
ingredient.
Objective 4; Optimal Supplementation of Practical Diets. Relative to terrestrial species,

basic requirement information for all nutrients is limited for various life stages of rainbow trout.
While knowledge is needed for fatty acid requirements, amino acids and minerals are the most
limiting as new ingredients are included in practical diets.
Minerals - The nutrient composition of most grains and fish meal are very different (NRC
1993), and un-supplemented, fish meal-free diets, may be low in several key minerals. Fish
meals can contain high levels of ash due to the inclusion of bones and as such grains contain
much lower levels of the macro-minerals phosphorus, magnesium and potassium (NRC 1993).
In addition, much of the phosphorus in grains is also unavailable due to phytic acid (Ogino et al.
1979). Diets not containing fish meal need supplementation with phosphorus to ensure
requirements (NRC 1993). It is essential, however, for commercial trout production to minimize
phosphorus discharge and optimize dietary phosphorus levels due to strict regulation of
phosphorus discharge in hatchery effluent. Hence, a great deal of research has focused on

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phosphorus requirements in fish (Cheng et al. 2004; Lellis et al. 2004; Sugiura et al. 2004). In
studies with diets containing fish meal, levels of other minerals, such as magnesium and
potassium, were adequate and not supplemented. However, a strong inter-relationship exists
between phosphorus, magnesium, calcium, potassium and other trace minerals in absorption
and metabolism (Aikawa 1981; Ensminger et al. 1990; Berdanier 1998). As ingredients sources
change balancing dietary minerals is further complicated due to the ability of fish to absorb Ca
through the gills (NRC 1993); thus, calcium hardness of the rearing water may influence the
need for supplemental minerals when diets low in fish meal are fed. Requirements and
utilization of P by rainbow trout is well characterized (Cheng et al. 2004; Sugiura et al. 2004),
but the diets used in those studies contained fish meal that contributed significant quantities of
Mg, Ca, Na and P. When fish meal-free, all-plant protein diets were supplemented with MgO,
NaCl, and KCl and fed to trout during a 15 week study, a significant increase in protein and
energy retention was observed, indicating the importance of these minerals to long term
performance (Barrows et al. submitted).
As discussed above, replacement of fish meal with plant-derived ingredients in the diet
decreases macro-minerals and increases dietary phytate. Both of these changes can affect
trace mineral nutrition of the trout (Apines et al. 2004). Adequate availability of trace minerals is
vital for optimal fish performance (Watanabe et al., 1997) and phytate not only lowers the
availability of phosphorus but it has also been shown to interact directly and indirectly with trace
minerals to reduce availability to animals (Odell 1969; Davies and Olpin 1979; Lo et al. 1981;
Apines et al. 2003). For example, calcium-bound phytate increases chelation of zinc to form co-
precipitates (Anon 1967) that may decrease endogenous zinc re-absorption as well as affect
availability of dietary zinc (Morris 1986). Increasing the phytate level from 1.1 to 2.2% in channel
catfish diets containing 50 mg zinc/kg decreased weight gain, feed efficiency, and zinc content
in the fish (Satoh et al. 1989). With 1.1% phytate in diets, channel catfish require about 200 mg
zinc/kg feed, which is 10 times higher than their dietary requirement for available zinc (Gatlin
and Wilson 1984). Dietary calcium levels have also been shown to affect not only zinc status
(Morris and Ellis 1980), but also interactions of phytate with calcium and zinc can effect growth
and health of trout (Davis and Gatlin 1986). Calcium is known to have antagonistic effects on
copper absorption and utilization (Davis and Mertz 1987). In addition there is a strong
antagonism between zinc and copper, which further complicates balancing diet for these
minerals as ingredients sources and water chemistry changes. Inadequate mineral nutrition can
result in reduced fish health with either a zinc deficiency (Kiron et al. 1993) or unbalanced
dietary levels of zinc which inhibits copper absorption (Knox et al. 1984) resulting in decreased
immune function (Hambidge et al.1986).
One approach to increasing bioavailability of minerals is to feed amino acid chelates or
proteinates. Chelates of zinc and amino acids have been found to increase bioavailability
compared to inorganic sources (Rojas et al. 1995). Experiments conducted to define nutrient
requirements typically estimate the minimum amount of nutrient necessary to maintain normal
growth, survival, tissue saturation, or to maintain some specific metabolic function, and are
usually conducted for periods of time much shorter than a production cycle. While these
minimum requirements are generally adequate to prevent deficiency and death, they may not
prevent marginal deficiencies and or excesses that could result in lower nutrient utilization
efficiencies or predispose fish to infection when fed for extended periods of time (Sealey and
Gatlin 2002a). However, information on the effects of marginal deficiencies or excesses is
lacking because these conditions are difficult to characterize and often overlooked (Lall and
Olivier 1993). Tests of immunocompentance can aide in determinations of optimum nutrient
levels. Alterations in immune response occur earlier than other physiological processes, when
body micronutrient reserves are depleted or become excessive, and they predict long-term risk
of infection and mortality (Blazer 1991; Sealey and Gatlin 2002b). Appropriate tests for
immunocompentance include descriptive and functional assays that examine both the

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nonspecific and specific branches of the immune response in addition to experimental disease
challenges (Blazer 1991; Lall and Olivier 1993).
Objective 5; Gene Expression Analysis. During the past decade molecular tools are
increasingly being used to complement standard hatchery techniques in assessing the
physiological response of agriculture animals. Changes in gene expression in rainbow trout
have been found to correlate with lipid peroxidation during early development (Fontagne et al.
2008), amino acid regulation of mTOR signaling (Seiliez et al. 2008), age and muscle accretion
(Johansen and Overturf 2005; Gahr et al. 2006;), carbohydrate metabolism (Kirchner et al.
2003a, 2003b), protein degradation and incorporation of plant meal protein (Overturf and
Gaylord 2009), and transcription of glucose receptors with inclusion of vegetable oil in salmonid
diets (Menoyo et al. 2006). An enhanced understanding of the genes directly involved with
nutrient utilization and their regulation is essential to optimize fish growth with diets formulated
to have fish meal and fish oil substantially replaced with sustainable products. Transcriptional
regulation is the initial step in the genomic regulation of a physical trait. The use of northern
blots, microarrays and quantitative PCR has provided a wealth of information regarding the
correlation between expression and phenotype. Gene expression studies have identified and
correlated changes in gene expression with meristic and quantitative traits involved with
disease, toxicology, metabolism, growth and health (Morley et al. 2004; Schadt et al. 2005;
Visscher et al. 2008; Andersson 2001) and for use in the detection and development of
molecular markers (Hoffmeyer et al. 2000). While microarrays are invaluable in presenting a
global picture of the transcriptome, quantitative PCR is much more precise in determining
relative and absolute changes for the expression of single genes between individuals and
groups. The use of small gene expression panels is a valuable method for studying the
molecular control of physical traits and for the discovery of candidate genes involved with traits
when the pathways involved in regulation are known. The complete sequence for the genes
involved in metabolism are known for most higher vertebrates and these sequences are also
known for salmonids. The development and use of gene expression panels containing probes
linked to specific metabolic pathways or physiological events in rainbow trout can be a highly
efficient method for determining very minute and specific differences between families and
alterations in nutrient partitioning due to dietary changes (Etherton 2000; Clarke 2004).
Objective 6; Identify Phenotypic Differences; To fully realize the potential of sustainable

aquaculture diets formulated to either reduce or completely replace fish meal and oil it is first
necessary to determine the limits of utilization for material commonly found in plant and animal
by products. Although protein can be isolated, concentrated and refined from plant material the
cost of performing these procedures is relatively substantial and economically limits their
utilization in aquaculture diets. It is necessary to understand how salmonids process nutrients
from different sources, especially in regards to changes in amino acid composition,
carbohydrate levels, and fatty acid composition. Knowledge of how metabolism, protein
turnover, and energy partitioning pathways interact will be of great assistance in developing
diets composed of sustainable plant and animal byproduct materials and will prove beneficial in
the identification of markers for selection programs looking to improve fish health and growth
performances on these diets. The three main metabolic pathways that will be focused on in this
objective deal with the processing of carbohydrates, fatty acids and protein.
Carbohydrate utilization is limited in fish and the necessary energy requirements can be
handled by the catabolism of amino acids (Stone 2003). Several avenues of research have
attempted to elucidate the mechanisms regarding energy partitioning and cellular utilization of
carbohydrate to gain a better understanding of glucose metabolism in salmonids. The first step
in circulating glucose metabolism is uptake by the cell and uses active glucose transporters.
Studies have found that several glycolytic genes and transporters are inducible and activity

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increases when carbohydrate levels are increased in the diet (Panserat et al. 2001; Kirchner et
al. 2003a, 2003b). The expression and activity of several transporters and glucose processing
enzymes have been characterized and quantified but the reason trout ineffectively metabolize
glucose in most cell types has yet to be determined. Therefore, a goal of this project is to define
the nutritional influences sustainable plant meal and animal byproduct diets will have on the key
enzymes of glucose metabolism. This information will enable researchers to better understand
how the carbohydrate material in formulated diets is being processed and its effect on nutrition
partitioning. This, in turn, will allow nutritionists to optimally incorporate carbohydrates and
formulate diets more precisely to meet the nutritional needs of fish.
Fish oil is currently being used at its maximum availability and future growth in
aquaculture will require the development of alternative, sustainable, oil sources. Dietary lipids in
fish can be oxidized to provide energy, incorporated into cell membranes, or deposited in
adipose tissue. To ensure high growth rates in aquaculture species the lipid content and the
fatty acid composition of the diet needs to be balanced to maintain optimal nutritional benefits
for the fish and for the production of a healthy consumer product. Lipid levels have been
increasing in salmonid feeds for energy so that the dietary protein can instead be used for
muscle accretion instead of used to meet energy requirements (Turchini et al. 2009). Too much
lipid can lead to the deposition of undesirable levels in the fish carcass.
Fatty acid metabolism in fish is considered to be essentially an identical process to that
in mammals. In fish, however, lipids are the primary source of energy. As plant oils and animal
fats are incorporated into rainbow trout feeds in place of fish oils, the fatty acid profiles of the
diets will be altered and as a consequence the profiles in the fish will also change (Hardy et al.
1987). This effect is well documented, but alterations in metabolism and health of the animal as
affected by changes in dietary fatty acids are not as well understood. Rainbow trout have a
requirement for n-3 fatty acids that can be supplied by linolenic acid (LNA) to support normal life
functions (NRC 1993), but it is not clear if trout can synthesize adequate quantities of
eicosapentanoic acid (EPA) and docosahexanoic acid (DHA) from this precursor to support the
growth rates attained in commercial production.
The metabolic machinery for elongation and de-saturation of LNA to EPA and DHA has
been demonstrated to be active in rainbow trout (Buzzi et al. 1996; Tocher et al. 2004). Seiliez
et al. (2001) observed increased 6-desaturase-like gene expression that can be nutritionally
modified by either the carbohydrate quantity or fat type. Increased rates of synthesis of n-3
HUFA from LNA would be beneficial in maintaining the HUFA content of fish consuming plant
oils, and therefore the healthfulness of trout fillets for human consumption. The effects of
altered fatty acid profiles on fish health have been demonstrated and reviewed by Turchini et al.
(2009). Menoyo et al. (2006) found that increased levels of vegetable oil in salmonid diets
increased expression of glucose transporters and caused a shift in plasma triglyceride and
glucose levels. Furthermore, Montero et al. (2003) have demonstrated effects on
immunosuppression and stress resistance in fish when 60% of the dietary fish oil is replaced
with plant oils. Therefore, these and other physiological changes in fish due to modifications of
lipids in diet need to be addressed in order to economically meet the needs of the fish, the
growers, and the consumers.
In protein metabolism when diets are formulated with plant and animal byproduct
ingredients there is concern in regards to metabolic and growth regulation (Kimball and
Jefferson 2006). Limitations of amino acids and improper amino acid ratios can greatly diminish
the efficiency of protein utilization and retention. Martin et al. (2003) replaced fishmeal with
soybean meal and observed an increase in protein synthesis and protein consumption, but no
increases in growth were observed. It was then concluded that synthesized protein retention
was reduced while ammonia excretion was increasing. Research has been carried out to
address the idea of an ideal amino acid ratio for growth of rainbow trout and Atlantic salmon
(Green and Hardy 2002; Gaylord et al. 2007; Hevroy et al. 2007). Other work has addressed

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the alterations in the proteome of trout fed increased levels of plant proteins (Martin et al. 2003).
Increased dietary energy levels also have been demonstrated to influence the overall rate of
protein synthesis and protein retention efficiency, but curiously without a concomitant increase
in protein growth rates (Bolliet et al. 2000). However, research involving protein turnover and
muscle accretion in salmonids have been focusing on protein degradation factors which appear
to be sensitive to dietary energy and actively involved in muscle accretion (Salem et al. 2005,
2006, 2007; Overturf and Gaylord 2009). An integrative approach to define the effects dietary
modifications have on growth physiology, metabolic regulation and nutrient portioning in rainbow
trout will be instrumental in optimizing dietary formulation containing protein and oil from
sustainable plant and animal byproducts and in generating improved stocks of fish.
Related Research; This project is closely coordinated with the units CRIS projects 5366-2100-
02800D, “Small Grains Genetics and Germplasm Enhancement” and 5366-21000-025-00D,
“Plant and Seed Seed Chemistry Genetics”. Barley and oat germplasm stocks and breeding
materials are being evaluated for their potential value in fish feeds. Incorporation of these
improved grains in trout feeds may aid in the selection of trout stocks for enhanced utilization of
alternative protein source diets and also provide potential benefits in the area of harmful effluent
reduction and health. This project is also closely coordinated with the research projects of the
NCCCWA unit CRIS’s 1915-31000-001-00D, “Utilizing Genetics for Enhancing Cool and Cold
Water Aquaculture Production”. The goal of that project is to develop improved strains of
rainbow trout for many characteristics. Our work will provide to their project identified traits that
may be selectively improved, markers, and germplasm of fish selected for improved utilization of
grain-based feeds. The USDA Cooperative Agreement 5366-21310-003-02S “Development of
Plant-Based Feeds for Rainbow Trout” is integral to the proposed project plan as the projects
are co-located at the Hagerman Fish Culture Experiment Station for fish culture and laboratory
operations. In addition the USDA project 5341-31410-003-00D “Converting Alaska fish by-
products into value added ingredients and products” is a potential source of alternative nutrients
and ingredients that may be utilized to reduce fish meals from reduction fisheries utilized in
rainbow trout feeds. The USDA Inhouse project 5366-21310-003-04T “Development of
Biologically Enhanced Plant Proteins to Replace Fish Meal in Trout Feed” is cooperative
research between Dr. Barrows and Montana Microbial Products to develop and test Biologically
Enhanced Plant Protein for rainbow trout feeds. A Small Business Grant project number MONK-
20085-02050 “Buhl Ethanol Project: Co-producing ethanol and Barley Protein Using Local
Resources” through CSREES MONK exist to advance the commercialization of a facility for co-
producing ethanol and Barley Protein Concentrate the later of which has been utilized in test
diets for rainbow trout. The HATCH project RI00H-895 “The role of follostatin in muscle growth
of salmonids” relates to the current project in that it is addressing the mechanisms by which
rainbow trout may have improved growth rate and more efficient feed conversion through down
regulation of myostatin by follastatin. The HATCH project WNP00913 “Molecular mechanisms
regulating skeletal muscle growth and differentiation” relates to the current project by
addressing dietary components that might alter satellite cell physiology utilizing rainbow trout
cell lines. The CSREES WN.Z project WNZ-YOUNG “Western Regional Aquaculture Center –
19th Annual Work Plan relates to the current project plan through its proposed goal to study the
effects of feed and water temperature in relation to quality of trout. The USDA ARS in house
project 1930-32000-003-00D “Development of sustainable land-based aquaculture production
systems” is a collaborative project with the current CRIS project through the impact of feeds on
water quality. Specifically, the plant-based feeds developed by the current CRIS project will be
tested for waste solids and nutrients relative to fish meal based feeds for rainbow trout in
recirculating aquaculture systems. The EVANS-ALLEN project WVAX-Aquaculture of the
CSREES-WVAX “Protein replacement in feeds for aquaculture finfish species” relates to the
proposed project in that it will determine the efficacy of a novel recovered protein from

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poultry/municipal waste as an ingredient for rainbow trout and define the nutritional value of the
protein through digestibility studies and feeding trials. The CSREES WVAX project WVAX-EYA-
CBG “Molecular characterization of mitochondrial functions as tool to select for feed efficiency in
finfishes” is related to the current project in identification of strains of trout with superior
mitochondrial function to improve feed efficiencies and identify gene expression changes in the
strains of trout. This project is related to a project at the Eastern Regional Research Center,
CRIS 1935-41000-072-00, “Economic Competitiveness of Renewable Fuels Derived from
Grains and Related Biomass”, which has an objective to develop more efficient processes for
converting hulled and hulless barley to fuel ethanol and improved, BG-free feed co-products.
The approach is to develop BG-degrading enzyme technology to reduce fermentation viscosity
and improve the production of ethanol from barley. This project is also somehow related to a
project at the Central Regional Research Center, CRIS 3620-41440-019-00, Improved Isolation,
Modification, and Functionality of Grain Proteins for New Product Development, which has a
focus on functional properties of isolated seed proteins.
This project is related to that at North Central Agricultural Research Laboratory,
Brookings, SD, CRIS No. 5447-41000-002-00, Improving the Value and Utilization of Ethanol
Manufacturing Co-products”, which has a focus on improving physical and flowability property of
DDGS, led by Kurt Rosentrater. We will utilize Dr. Rosentrater expertise in evaluating physical
properties of DDGS, and he will source appropriate DDGS through his industry contacts.
APPROACHES AND RESEARCH PROCEDURES

Objective 1: Development of improved grain lines. (Lead: Hu; Support: Liu, Obert)
Sub-objective 1A. Determine the effect of BG mutations in barley on performance of major
agronomic and quality traits.
The goal of this sub-objective is develop near iso-genic lines of barley and in order to
determine the effect of the mutation on agronomic and quality traits both in laboratory and field
experiments. Results will provide a solid basis to justify their uses in development of value-add
barley cultivars.
Hypothesis 1A. BG mutation alone will not affect agronomic or quality traits.
Experimental Design: Two mutants of M38 and M351 have been identified from the
chemically-mutagenized barley cultivar of Harrington. M38 has increased BG content by at least
50%, while the BG content of M351 is decreased by 90%. To develop near iso-genic lines, two
mutants will be back-crossed to the parental cultivar of Harrington in the greenhouse and then
self-pollinated twice to obtain a BC1F2 (back crossed F2) generation in both the greenhouse
and in the field at the University of Idaho Research and Extension Center, Aberdeen. Progeny
lines from the BC1F2 generation will be harvested separately and assayed for BG contents for
each of the lines using our modified enzymatic procedure (Hu and Burton 2008). Four
homozygous lines of each mutant and corresponding wild-type will be selected based on their
BG content compared to the mutant and wild type parental lines using the following criteria: 1)
similar plant morphology; 2) similar BG content in grains; 3) the BG contents in selected families
are not segregating in self-pollinated progenies. The selected four homozygous lines for each
mutant and wild-type category of the mutant will be individually pooled to represent the near iso-
genic lines, respectively, for the specific mutant. Since near iso-genic lines of a particular mutant
will have a similar genetic background except for the mutant gene, phenotypic differences
between the lines are theoretically due to the different allelic functions of the mutant gene. The
near iso-genic lines developed will be used for comparison of performance of other traits.
Evaluations of agronomic traits will be conducted both in Aberdeen and Tetonia Idaho locations
providing different environmental conditions due to differences in elevation at the two sites.
Major agronomic traits including yield potential, lodging, and disease resistance will be
evaluated at each location for two years. The field experiments will use a randomized block

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design with at least three replications. Differences between near iso-genic lines in agronomic
traits will be investigated by statistical analyses using the mixed model for which genotype is
the fixed effect while locations and years are random effects. Other quality traits such as
seed weight, total starch, oil, and protein contents will be determined in Dr. Keshun Liu’s lab at
Aberdeen using the routine protocols. To test the utility of using M351 in feed barley
development, an experiment will be conducted to determine if less BGase is needed to treat the
low BG mutant grains. Standard dosage of beta-glucanase utilization is 500 units per gram of
flour (MegaZyme, Ireland) and feed supplement (Yaghobfar et al., 2007) will be used as
controls. Reductions of BGase by 20%, 40%, and 60% will be tested on M351 and its
corresponding wild-type to compare the BG changes. Evaluation of beta-glucanase doses in the
M351 and the wild type will be conducted using our BG measurement protocol (Hu and Burton
2008). Collaborating with Dr. Liu at Aberdeen, two mutants and their near iso-genic wild types
will be tested for differences of protein extraction efficiency. If the BG contents in grains affect
extraction efficiency, this information will guidance useful for feed barley improvement and for
plant-based fish feed development because an efficient protein extraction is the critical
step in Aquafeed development. Results from this study should be useful in development of
barley cultivars for potential feed utilization or BG extraction.
Contingencies: Field experiments may be compromised due to the un-controllable natural
conditions. If this occurs, we extend the study for an additional season. Any pollination failure
may delay the plant materials development. Duplicated cross pollination will be conducted.
Collaborations: Dr. Don Obert will help to setup the field experiments and assist with statistical
analysis of the agronomic traits for evaluation of near iso-genic lines.
Sub-objective 1.B. Genetic mapping of the BG mutations.

Experimental design: Mutants of M38 and M351 will be crossed to wild-type barley lines to
develop mapping populations. Initial crosses have been made of two mutants to the six-row
barley cultivar Steptoe, whose BG content is approximately 6%. The differences in BG content
between the parents should allow clear scoring and identification of the mutant and wild-type
phenotypes in the progenies. The F1 seeds from the crosses will be planted to generate F2
plants. Seeds from F2 plants will be planted to obtain F3 plants that will be harvested
individually. BG content will be measured for each of the 300 to 400 F3 families generated from
each cross. A minimum of 100 F3 homozygous families with either mutant or wild-type BG
levels will be identified. Ten seeds from each homozygous family will be germinated and used
for DNA extraction. Four DNA pools representing the M38 mutant, the M351 mutant, and their
corresponding wild-type backgrounds will be made by DNA extraction from a mix containing an
equal amount of tissue of each homozygous family from the same phenotypic group. Each DNA
pool will be made from a minimum of 40 families. The pooled DNA samples will be analyzed for
polymorphism between the mutant and corresponding wild-type using the powerful DArT
(Diversified DNA Array Technology) service provided from Triticarte Pty Ltd, Australia. Two
parental DNA samples will be used as reference controls in the same DArT analysis. Candidate
loci identified by DArT analysis will be further validated using nearby DNA markers such as
simple sequence repeats (SSRs). Close-linked DNA markers, particularly PCR-based markers,
will be identified during the mapping of the mutant genes in mapping populations. Those
identified markers could be very valuable for marker-assisted selection to track the mutant
alleles in the breeding program.
Contingencies: Polymorphism between the mutant and corresponding wild-type DNA pools
may not be identified due to the genome similarity in the regions that the mutant gene resides.
As a backup, we will cross the mutant of M351 to a more genetically distance Oregon Wolf
Barley Dominant line that has a beta glucan content of about 8.0%, and the M38 will be crossed
to Morex which has a lower BG content of 5.0%. These crosses will be made at the same time
as the crosses with Steptoe. The homozygous families will be identified and will serve as

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backups, and similar DNA pools from those backup crosses will be made and subjected to the
DArT analysis if necessary.
Collaborations: Dr. Eric Jackson, USDA-ARS, Aberdeen, Idaho, will assist in the DArT marker
mapping experiments.
Objective 2: Ingredient Modification (Lead: Liu; Support: Barrows)

Sub-objective 2.A. Simultaneous separation of high value nutrients from barley and oats.
Experimental design: Both laboratory and pilot scale methods will be evaluated for barley and
oats. The objective is to simultaneously enrich protein, BG and starch from these grains. Two
types of fractionation will be evaluated. Dry fractionation methods include dehulling, pearling,
milling and sieving or air classification will be performed (Wu et al. 1994; Yeung and Vasanthan
2001; Izydorczyk et al. 2003). Effects of individual dry fractionation methods and their
combinations on contents and recovery rates of protein, BG and starch in resulting fractions will
be determined. Particle size of each dry fraction will also be measured to find relationships
between particle size and nutrient contents, according to a standard method (ASAE Standards,
2003). For wet extraction, grains are dehulled and milled. This is followed by solvent extraction
for nutrient separations (Cluskey et al. 1973; Ma 1983; Bhatty 1995; Temelli 1997). Different
solvent types and extraction times will be evaluated. Solvent types include water, alkaline, acid,
and organic solvents will be used to extract and/or precipitate protein, BG and starch.
Intermediate and final fractionated products will be analyzed for chemical composition and
recovery rates.
The total nitrogen/protein content will be measured by an AOAC combustion method,
using a protein analyzer (Model FP-528, Leco Corp. St. Joseph, MI). The protein content will
be calculated with a conversion factor of 5.75, a common value for cereal grains and related
products. Crude fat content will be measured according to AOCS Am 5-04 procedure (AOCS
2005) using a fat analyzer (Model XT10, Ankom Technology, Macedon, NY). BG will be
measured according to the Approved Method 32-23 (AACC 2000), using the BG enzymatic
assay kit supplied by Megazyme Intl. (Wicklow, Ireland). Starch will be measured according to
an enzymatic method using a starch test kit (R-Biopharm, Inc., Marshall, MI). The content of
phytate will be measured extracting samples with 0.4M HCl and 0.7M Na2SO4. Phytate P is
then recovered as a ferric precipitate and assayed colorimetrically. Mineral content will be
determined with an inductively coupled plasma-optical emission spectrometer.
During wet extraction, BG tends to form a viscous solution which leads to a problem of
separation. Removing of BG before extraction or minimizing its hydration will be evaluated.
Effects of lipids on both dry and wet fractionation methods will also be investigated. Lipids from
milled grains will be removed with solvent extraction. Defatted flour will undergo the same
fractionation procedures described above and results will be compared with those from full-fat
flours. In addition, using the improved processing methods, comparison of the nutrient profiles
of barley and oat fractions with different BG contents will be made. Barleys containing different
BG levels will be produced in Objective 1. Once the laboratory methods are developed, pilot
scale quantities will produced for in vivo evaluation in Objective 3.
All data will be analyzed using the Analysis of Variance Procedure (JMP, version 5,
SAS, Cary, NC, USA). For wet fractionation methods a factorial treatment design will be used
with solvent type and extraction time as the main effects.. Treatment effects in all statistical
analyses will be considered different when probabilities for a greater F value are less than 0.05.
Contingencies: High levels of BG might interfere with protein and starch separation and
recovery by wet methods. If this occurs, enzymatic treatments with beta-glucanase will be
evaluated. Alternatively, selection of low BG crops, developed in Objective 1, can be used
Collaborations: Drs. Don Obert and Phil Bregitzer, ARS, Aberdeen, ID, will provide a variety of
barley and oat samples of differing composition from select genetic lines; Dr. Victor Raboy,
ARS, Aberdeen, ID, will provide low phytate barleys.

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Sub-objective 2.B. Improve nutritional and economic values of biofuel coproducts.

Experimental design: Two major sources of biofuel co-products, or their feedstock, will be
evaluated for development into ingredients for trout feeds. DDGS and related products (such as
distiller grains or DDGS from cereal grains rather than corn) and algal biomass and/or biofuel
co-products made from algae will be evaluated by a two-phase approach.
Phase 1: A set of analytical methods will be compiled from existing methods and
modified (when necessary) to accurately characterize physical properties and chemical
composition (i.e. nutrients and anti-nutrients) of bio-fuel co-products and algae biomass. The
lipid fractions will be evaluated for fatty acid composition while protein quality will be evaluated
for amino acid profile. Particle size distribution of these products and its relationship with
chemical composition will also be characterized. Analytical methods to be compiled and
modified for suitability of analyzing these materials will include combustion method for total
nitrogen/protein content (AOAC 2002), a solvent extraction method for crude fat content (AOCS
2005), an enzymatic method for total starch (assay kit supplied by Megazyme Intl., Wicklow,
Ireland), a wet extraction and ferric precipitation method for phytate (Liu et al. 2007), an
inductively coupled plasma-optical emission method for minerals (Liu et al. 2007), a GC method
for fatty acid composition (Liu 1994) and a HPLC method for amino acid profiling (Fleming et al.
1992; AOAC 1995).
DDGS evaluation will also include monitoring chemical changes during entire conversion
process (from corn to DDGS) by collecting and analyzing intermediate samples at every key
processing step of the dry grind method from several production plants. Components to be
quantified will include protein, residual starch, crude fat, total carbohydrates, minerals, different
types of phosphorus, fatty acid composition and amino acid composition.
Algae will be produced by collaborators but will be selected based upon compositional
analysis. The levels of nucleic acids and heavy metals (such as Pb and Cd) will be measured
for algae and its derivative products, since some of these products may be produced using CO2
from power plants. Nucleic acid content will be measured according to method of Ding and
Canter (2004). The heavy metals will be measured by an inductively coupled plasma-optical
emission method (Liu et al. 2007).
Phase 2: Processing methods, both mechanical and chemical, will be developed to
fractionate bio-fuel co-products and algae biomass for increasing protein content and reducing
or removing unwanted components. Mechanical methods will include milling, air classification,
sieving and their combination. During sieving, two methods will be evaluated, stacked sieve
method and reverse sieve method (Liu 2009).
Chemical methods will be evaluated with different solvents with varying concentrations,
extraction durations, and temperatures to determine maximal extraction efficiency as follows.
Protein enrichment will first use alkaline extraction followed by acid precipitation. Ethanol will be
used to extract simple sugars and other polar constituents. Effect of lipid on protein enrichment
will also be studied. This will be carried out by first extracting oil with hexane. Biological
methods will include application of enzymes to facilitate nutrient release and anti-nutrient
destruction. Combinations of mechanical, chemical and biological methods will also be
explored to maximize efficiency and control cost. Using similar analytical procedures in Phase
1, all fractions will be evaluated for chemical composition.
Finally, the nutritional value of newly developed protein ingredients will be evaluated in
Objective 3. If fish feeding trials show limited digestibility or bioavailability, processing
conditions will be re-evaluated.
All data will be analyzed as described above in Sub-objective 2a. For wet extraction a
factorial treatment design will again be used with solvent type, extraction time, concentration,
and temperature as the main effects. Second order regression models will be fit to estimate
optimal extraction time, solvent concentration and temperature effects on extraction efficiencies.

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Contingencies: Algae biofuel is just being developed, so its co-products may not be available.
In this case, DDGS and algae biomass will be the primary focus. If high levels of omega-3 fatty
acids are found in algal biomass, it will be evaluated as both a protein and EFA source.
Collaborations: Dr. Kurt Rosentrater, USDA-ARS, Brookings, SD, will source DDGS samples
and analyze physical properties of DDGS, such as density, flowability, etc. Dr. James Levine
(Kent Bio-Energy) and Dr. Alina Kolkowski (Carbon Capture Corporation) will provide algae
biomass or algal meals.
Objective 3; Alternative ingredients and practical feed formulations. (Lead: Barrows;

Support Liu, Overturf)
The research in this objective, and that in Objectives 4, 5, and 6 are being conducted
concurrently to collaborative research with the University of Idaho through the Specific
Cooperative Agreement (SCA) “Improving the Competiveness of Rainbow Trout Production by
the Integrated Development of Improved Grains, Feeds, and Trout”. This new SCA will be
effective in 2009 and active until 2014.
Sub-objective 3.A. Determine the nutritional value of alternative feed ingredients.
Experimental design: A four step process will be used to determine the nutritional value of
alternative ingredients. First, the nutrient and anti-nutrient profile of each alternative feed
ingredient will be determined using standard procedures to determine the moisture, ash, crude
protein, starch, lipid, gross energy and amino acid content. Anti-nutrients to be determined will
be based upon known substances in base material and include, but are not limited to, trypsin
inhibitor activity, stachyose, raffinose, and saponins. Pending ingredients include algal meals,
DDGS, barley protein concentrates, rice protein concentrates, bacterial and yeast proteins.
Second, the effect of the alternative ingredient on feed intake will be determined using standard
laboratory procedures (Stone et al. 2005). Third, the apparent nutrient and energy digestibility,
and amino acid availability will be determined from the ingredients in compounded, extruded
diets. The methodologies employed will include standard established procedures. Alternative
ingredients will be developed from within the project (Objective 2), from commercial sources and
through collaboration with industry developing products. The methods of Cho et al. (1982) and
Bureau et al. (1999) will be used to estimate apparent digestibility coefficients (ADCs). A
complete reference diet meeting or exceeding all known nutritional requirements of trout will be
blended with the test ingredients in a 70:30 ratio (dry-weight basis) to form test diets. In each
digestibility trial a standard fish meal (IFN 5-01-977, Menhaden Special Select, Omega Protein
Inc., Houston TX) will be included as a reference. Dry matter and ash analysis of ingredients,
diets and feces will be performed according to standard methods (AOAC 1995). The
indigestible marker, yttrium oxide, will be included in the diet at 0.10% and content determined
by inductively coupled plasma atomic absorption spectrophotometry (Perkin-Elmer Corporation,
Norwalk, Connecticut, USA) following wet ashing with nitric acid (AOAC 1995). Crude protein
(N x 6.25) will be determined by the Dumas method (AOAC 1995) on a Leco nitrogen analyzer
(FP428, LECO Corporation, St. Joseph, Michigan, USA). Amino acids will be analyzed by high
performance liquid chromatography (HP1100, Agilent Technologies, Wilmington, Delaware,
USA) following hydrolysis (AOAC, 1995) using pre-column o-phthaldehyde derivatization
(Fleming et al. 1992). Lipid will be determined by ether extraction (AOAC 1995). Total energy
will be determined by adiabatic bomb calorimetry (Parr1281, Parr Instrument Company Inc.,
Moline, Illinois, USA). Phytate will be determined using methods of Harland and Oberleas
(1986). Fourth, growth studies will be conducted that focus on defining the utility of the
ingredients which will be presented in sub-objective 3.b.
All experimental feeds will be produced by cooking extrusion (Buhler Inc., DNDL-44,
Minneapolis, MN), a pulse-bed drier (Buhler Inc., FB-50, Minneapolis, MN) and vacuum-coater
for oil application (Pflauer Milling, V100, Ontario CA). Process conditions will be recorded for

Barrows 17
pressure, temperature in each barrel section, feed rate of water and dry material, torque, and
specific mechanical energy, for each diet.
The PROC ANOVA procedure, SAS Software Version 9.2 (SAS institute, Inc., Cary, NC)
will be used to conduct analysis of variance (Ott 1977) in which diet will be defined as a fixed
effect and each tank of fish will be the experimental unit. Differences among treatment means
(n= 3 t/trt) of digestibility coefficients for protein, individual amino acid availability, and mean
amino acid availability within and among test ingredients will be determined using the Tukey-
Kramer procedure for pair-wise comparisons (SAS 2009). Treatment effects in all statistical
analyses in this project will be considered different when probabilities for a greater F value are
less than 0.05.
Contingencies: At any point in the evaluation, if an ingredient is determined to have low
nutritional value it may be dropped from the evaluation. During the project ingredients new to
the market may be identified and included following the same procedures.
Collaborations: Mr. Clifford Bradley (Montana Microbial Products) will collaborate by providing
biologically concentrated plant proteins. Dr. James Levine (Kent Bio-Energy) and Dr. Alina
Kolkowski (Carbon Capture Corporation) will provide algal meals.
Sub-objective 3.B. Development and evaluation of alternative feed formulations.

Hypothesis: Specific ingredient blends of alternative ingredients will improve growth and
nutrient retention of rainbow trout fed fish meal free diets.
Experimental design: Ingredients selected will be based upon nutrient content and digestibility
information developed in Objective 2 and 3a, cost, sustainability, and commercial availability.
Ingredients will include conventional and novel type ingredients. All diets will be formulated on a
digestible nutrient basis and combined with information already determined for commercially
available ingredients in the previous CRIS. Combining these data, diets will be formulated to
have complimentary ingredient combinations to meet the nutritional needs of rainbow trout on a
digestible nutrient basis. Mixture model experimental design will be utilized to formulate diets
to meet formulation goals based on digestible nutrient targets for the diets. Combinations of
ingredients will be based on classification of ingredients for their major nutrient contribution.
A total of 1,375 fish will be randomly distributed among experimental tanks.
Twenty five, fingerling trout (~15 g/f) will be placed into 145-L tanks and fed the experimental
diets for 12 weeks. Feeds will be formulated to contain 45% crude protein and 20% lipid on wet
weight basis to meet all other known nutrient requirements of trout (NRC, 1993). A control diet
based upon fish meal (IFN 5-01-977) and fish oil (Table 2), a commercial fish meal based
reference diet (Silver Cup Trout), and 10 experimental formulations will be utilized in each trial.
Diets will be produced as outlined in Objective 3a, and supplemented with essential
nutrients (i.e. amino acids and minerals) to meet or exceed NRC recommendations
(1993). The ratio of plant to animal ingredients will be held constant within each diet
series within each experiment. Five tanks per diet (treatment) will be randomized with respect
to position in the laboratory. Each tank will be supplied with 4 L/min of untreated, constant
temperature (14.5 C), gravity-fed spring water. Fish will be fed three times per day, six days
per week to apparent satiation for a period of 12 weeks. Fish in each tank will be group-weighed
and counted at the beginning and every 21 days. From the initial population of fish, and from
each tank at the end of the studies, ten fasted fish (72 hours) will be sacrificed and processed
into a puree using a Robot Coupe food processor (Robot Coupe R-2, Ridgefield, MS), and sub-
sampled for proximate and mineral analyses. Methods for determining proximate composition
and methods to determine digestibility of the feeds and statistical analysis of the data were
described previously. Growth performance will be analyzed as a repeated measures, mixed
model. Multivariate analysis of variance will be utilized to assess the effects of diet levels on
tissue storage efficiencies and growth performance (SAS 2009).

Barrows 18
Contingencies: If acceptable growth and feed efficiency of trout is not attained in laboratory
studies, changes in feed formulation will be made. These changes could include deletion of
certain ingredients or the inclusion of fish meal/oil in the formula.
Collaborations: Dr. Hardy (UI) will provide expertise and analytical and fish-rearing
laboratories. Dr. Steven Summerfelt (Freshwater Institute) and Dr. Steven Craig (Low Salinity
Inc.) will provide field testing in recirculation systems, and Dr. Scott LaPatra (Clear Springs
Foods) will provide field testing in flow-through systems. Dr. Steve Rawles, USDA-ARS
Stuttgart National Aquaculture Research Center, is involved in ongoing collaborations across
fish species regarding ingredient combination effects in trout and hybrid striped bass and will
provide expertise in diet formulation, experimental design and multivariate statistical analyses.
Mr. David Brock (Rangen Feeds) and Mr. Chris Nelson (Silver Cup Feeds) will provide advice
on ingredient choice and practical feed formulation and production of pilot scale quantities of
experimental feeds.
Objective 4: Refined mineral supplementation for plant-based diets (Lead;Barrows;

Support; Overturf)
Hypothesis: Supplementation of plant-based diets for trout with specific minerals is necessary
to optimize growth, nutrient retention and feed efficiency.
Experimental design: A series of three feeding trials will be conducted with advanced
fingerling rainbow trout feeding graded levels of K (KCL), Mg (MgO), Ca, and P (Ca2PO4) in a
plant-based diet (Barrows et al., 2008). Eight graded levels of each mineral source will be fed
from 0 to 0.80% of the diet with KCL in 0.10% increments, from 2.25 to 4.0% of the diet with
Ca2PO4 in 0.25% increments, and from 0 to 0.16% of the diet with MgO in 0.02% increments.
Each study will contain an un-supplemented fish meal (IFN 5-01-977) based diet (Table 2) and
the same diet supplemented with the basal level of the mineral being tested in that trial. Basal
supplementation levels of 0.60% KCL, 2.50% Ca2PO4, and 0.06% MgO will be used for the
mineral not being evaluated in that study and added to the fish meal free diet described by
Barrows et al. (2008). A fourth study will be conducted to determine optimal zinc
supplementation level of practical diets with zinc supplied in both inorganic (ZnSO4) and
chelated forms. Both of these sources will be added in 8 graded levels to a diet containing
optimal macro-mineral supplementation levels determined in studies described above. Fish
rearing conditions and procedures, and feed manufacturing process will be as described in
Objectives 3a and 3b. Growth, feed efficiency, protein, energy and mineral retention, liver and
kidney histology, hematocrit and plasma ion balance will be monitored and analyses will be
conducted as previously described. Whole body mineral, liver copper, and plasma Mg, Ca, Cu,
Zn and P, will be determined by inductively coupled plasma atomic absorption
spectrophotometry (Perkin-Elmer Corporation, Norwalk, Connecticut, USA) following wet ashing
with nitric acid (AOAC 1995). Hepatosomatic index (liver weight/body weight), intraperitoneal
fat, and fillet yield will be determined. Proximate composition of the fish will be determined to
assess protein and energy retention. Metabolic pathways for specific minerals that indicate the
optimal metabolic concentrations of the minerals will be assayed for activity. To determine
possible links between performance and gene expression basic metabolic and mineral specific
genes will be evaluated from at least 10 basic regulatory metabolic genes, such as PPAR,
pyruvate carboxylase, acylCoA dehydrogenase, and mineral specific genes such as
metallothionein and zinc transporters for zinc in the liver (Cousins 1979; Liuzzi et al., 2001),
Dose-response curves for growth and physiological responses as affected by specific mineral
deficiencies will be determined using non-linear saturation kinetics modeling (SAS 2009; Mercer
et al. 1989). Slope ratio comparison will be made with Zn sources (Jaramillo et al. 2009).
Nonspecific immune responses will be assessed from blood samples collected at the
beginning and end of the zinc study. Fish will be anesthetized and blood collected from the
caudal vasculature using a heparinized syringe. Total plasma protein and immunoglobulin will

Barrows 19
be measured as describe by Siwicki et al. (1994). Serum lysozyme levels will de determined as
described by Sankaran and Gurnani (1972). After blood collection, fish will be sacrificed and
head kidney and spleen of test animals will be removed. Tissues will be pooled by tank
(experimental unit) and a portion will be used to isolate phagocytic cells for functional assays
according to the methods of Stave et al. (1983). Respiratory burst activity (Secombes 1990),
phagocytosis (Seeley et al. 1990), and macrophage killing (Anderson and Siwicki 1996) will be
determined with minor modifications as described by Sealey and Gatlin (2002b). As previously
described mineral availability has been linked to immune response. This project will also
evaluate the effects of mineral availability on immune response by measuring the expression of
genes that have been demonstrated to immunologically correlate with pathogen dose response
(Overturf et al. 2004). Therefore the remaining tissue will be used to characterize the molecular
expression of immunologically related factors and cytokines including tumor necrosis factor,
interleukin 8, compliment factor C3, hepicidin, and CD-8 as outlined by Overturf et al. (2004).
Contingencies: If no response to a supplemental mineral is observed, that mineral will not be
included in future evaluations with other minerals. If a level other than the basal level is
determined to be optimal, that level will be used in future studies.
Collaborations: Dr. Sealey will coordinate sample collection and laboratory analyses for
measurements of immunocompetence. Dr. Hardy (UI) will collaborate with feed formulation and
experimental design and analyses.
Objective 5: Gene expression analyses (Lead; Overturf; Support; Barrows)

Sub-objective 5.A. Determine how changes in expression of previously tested and defined
metabolic genes regulating fatty acid metabolism and muscle growth correlate with energy
partitioning and growth in rainbow trout.
Hypothesis: Relative expression changes in a panel composed of a limited number of specific
genes will correspond with physiological variation seen in trout utilization of sustainable
formulated diets.
Experimental Design: To first test for changes in gene expression correlative to dietary
changes, actively growing rainbow trout between 40 and 120 grams will be fed a control diet
containing commercial levels of fish meal and fish oil. These fish acclimated to the control diet
will then be used in the following studies. In one study the experimental animals will be split
among six treatment groups with one group receiving the control diet and the other five groups
being pair fed on diets that have the fish meal replaced with 20, 40, 60, 80, and 100% of a blend
of plant meals. The relative level of each plant product will be maintained in each of the diets.
The plant meal product used will consist of a material that is identified as economically feasible
for utilization in aquaculture diets and which has been previously evaluated as a feed product
within this research project. Triplicate tanks of 30 fish will be reared on their respective diet for
16 weeks with active monitoring of average fish growth and feed intake. At the end of the
experiment individual weights will be taken and fish analyzed for muscle and intraperitoneal fat
ratios, and whole body proximates. Feed conversion ratios and percent energy retention will be
calculated for each group. For gene expression analysis, RNA isolated from individual liver and
muscle tissues will be used. Liver samples will be run against a panel of 15 (desaturases,
elongases, etc.) genes involved with fatty acid synthesis and conversion and for genes involved
in pathway substrate shuttling (Table 1). Furthermore, a panel of 11 muscle regulatory and
protein degradation genes (MyoD, MURF1, calpain, cathepsin, etc.) will be run on RNA from
muscle samples to analyze for links between energy partitioning and muscle accretion. A similar
experiment will also be performed using a diet that is fish meal free and will evaluate the effects
of serially replacing fish oil with a soy/flax oil blend that is low in EPA and DHA and high in
linolenic and linoleic acids. This experiment will be set up the same as the serial replacement
fishmeal experiment requiring eighteen tanks per treatment with 30 fish per tank. Another panel
of genes will be included in the analysis of these samples containing genes known to be

Barrows 20
involved with fatty acid metabolism and metabolic regulation, such as lipoprotein lipase, PPAR
(α β, and γ), carnitine palmitoyl transferase, and sterol regulatory binding protein 2 (SREBP2).
Regression analysis will be used to evaluate relationships between inclusion of sustainable
protein or oil product, measured physical differences (i.e. growth rate, muscle ratio, HSI, etc.)
and the expression of genes being evaluated.
Contingencies: There are two contingencies to consider, one problem is that no physical
differences are discernable in the trout on the diets and the second possibility is that none of the
genes show a correlation in expression with the physical parameters being measured in the fish.
Regarding the first potential problem, in prior experiments rainbow trout have been reared on
these diets at this facility and differences have been detected. But if no differences are noted in
this experiment then the experiment would be rerun utilizing diets containing less refined
products. For the second problem, if no gene correlations are noted for the genes involved in
the panels used then the initial number of genes would be expanded to contain other genes
involved in both fatty acid metabolism and muscle growth.
Collaborations: Dr. Ron Hardy and staff of the University of Idaho will collaborate with
experimental setup, sample isolation and testing.
Sub-objective 5.B. Identification of SNP markers for improved growth on plant based diets for
rainbow trout.
Hypothesis- Single-nucleotide polymorphisms can be identified in genes that are found to
change in expression according to physiological changes seen in trout reared on sustainable
plant based diets.
Experimental Design: From prior research and the gene panel study described in the previous
sub-objective, genes found to modulate according to growth or other physiological changes in a
manner significant to the utilization of a sustainable plant or animal by product based feed will
be analyzed for potential single-nucleotide polymorphisms (SNPs). Genes of interest as
determined by linear regression analysis showing a significant correlative change in expression
with growth, food conversion, muscle ratio or other measured physical trait will be sequenced
from 32 different individuals using 20 fish from the rainbow trout strain used in the original
experiments and 12 fish from outside populations. Alignment analysis and identification of single
based polymorphism will be done using the program Sequencher (Gene Codes Co., Ann Arbor,
MI). The identified SNPs will then be evaluated to determine if they are linked to predicted
physiological modifications for which the expression of these genes was originally found to
correlate. For example if changes in Δ6 dehydrogenase expression is found to correlate with the
conversion of linoleic acid to docosohexaenoic acid in fish then SNPs identified for this gene will
be initially evaluated against fish samples with known conversion differences. Basic analysis
will consist of sorting fish from the original experiments into groups according to documented
physical differences and then genotyping the individuals. Correlation analysis will be run to
determine if identified SNPs are significantly linked to the phenotypes.
Contingencies: It is possible that the initial SNPs identified will not be linked to the relative
physical changes that are seen in fish on the different diets. Since a correlative link has already
been established between gene expression and the physical trait, then the best solution would
be to go back and sequence more 5 prime and 3 prime untranscribed regions and identify SNPs
from these areas as these regions are known to play a major role in regulating gene expression.
Collaborations: Shawn Narum, Columbia Intertribal Fish Commission, will collaborate in gene
sequencing, SNP analysis and marker design and genotyping.
Objective 6: Phenotypic differences and genetic variation for identified traits. (Lead;
Overturf; Support; Barrows)

Barrows 21
Sub-objective 6.A. Identify variation in physiological performance of rainbow trout that is

specific to the utilization of plant protein diets, versus fishmeal diets, and determine their genetic
components.
Hypothesis: Differences in growth and other physiological parameters exist between rainbow
trout families when reared on either fish meal and fish oil based diets or sustainable plant and/or
animal by product based diets.
Experimental Design: To determine the potential for improvement of rainbow trout for growth
on plant or animal by-product sustainable diets, 30 maternal half-sib families of fish will be
generated splitting eggs from females and crossing them to two different males using the
following design, dam 1 x sire 1, dam 1 x sire 2, dam 2 x sire 3, dam 2 x sire 4, etc). The
families will then be fed a control feed containing fish meal and fish oil comparable to
commercial diets and two experimental diets; one diet completely replacing fish meal with
protein from sustainable plant or animal by product sources but still containing fish oil equal to
levels used in the control diet and a second diet formulated to replace both fish meal and fish oil
completely with protein and oil from sustainable sources. Individual fish will be tagged and
groups of 6 families communally reared with 30 fish per tank in triplicate and 30 tanks per
treatment. The fifteen fish/family to be fed each diet will be weighed initially and at the end of the
16-week experiment. Proximate analysis will be performed including whole body lipids and
muscle ratios. Samples of plasma, liver and muscle will be taken from all fish. Plasma samples
will be tested for identified growth factors and glucose levels and RNA isolated from the liver
and muscle samples for quantitative expression analysis against fatty acid metabolism and
muscle growth gene panels. Correlation analysis will be run against fish within families and
between families for the genes against growth, IGF-I level, muscle ratio and against individual
fatty acid levels. A factorial ANOVA will be run to determine effects of family, diet and
interactions. Genetic variance components for growth and other physical factors and their
relative heritability will be estimated using REML.
Contingencies: Genetic variation for dietary utilization has been found between salmonid
families when reared on fish meal or plant meal based diets. This study aims to identify factors
that correlate with the genetic and physiological variations detected from previous studies and
use these factors to identify how energy partitioning might be different between the fish on the
diets and families that more efficiently utilize the sustainable based diets. If none of the
variables currently described in this study shows a strong correlation with related genetic or
dietary changes then we propose broadening our gene panels as there are a multitude of genes
described that have been found that are modulating according to the nutrient status of the
animal. Also we will look to identify other physical traits such as muscle firmness, muscle fiber
number or intraperitoneal fat levels for use in comparing dietary effects.
Collaborations: Richard Towner, Genetic Consulting, will collaborate with experimental design
and genetic analysis.
Sub-objective 6.B. Determine if genetic variation exists in rainbow trout for the metabolism of
plant oils to yield increased deposition of DHA and EPA in fish tissue and determine the genes
involved.
Hypothesis: Variation exists in rainbow trout for the conversion of certain plant oils to DHA and
EPA and its deposition in fish tissues.
Experimental Design: For the evaluation of conversion of plant oils and increased deposition
of EPA and DHA in rainbow trout, 30 families of rainbow trout will be communally reared and fed
a diet composed of plant protein and a blend of soy and flax oils. The lipid component of this
feed will be composed of a flax soy blend that is high in 18:3n-3 (α linolenic acid) and 18:2n-6
(linoleic acid) and contains no eicosapentaenoic acid (EPA, 20:5n-3) or docosohexaenoic acid
(DHA, 22:6n-3). For this experiment 6 families of rainbow trout, at 5 fish per family will be
stocked in 140 l tanks to yield six replicate tanks- for a total of 30 tanks. Actively growing fish

Barrows 22
between 20 and 80 grams in size will be individually tagged and fed the diet for a period of 12
weeks. Three fish from each family per tank will be sampled at the completion of the feeding
trial. Initial and final samples will be processed for whole body proximate analysis. Liver and
muscle samples will be collected for proximate, expression and enzymatic analysis. Whole body
lipid and fatty acid analysis will be performed. Fillet samples will be collected and analyzed for
lipid composition and fatty acid profiles. RNA and protein will be isolated from liver samples and
analyzed for expression of elongase, Δ5 and Δ6 desaturase, and acyl CoA dehydrogenase.
Anova analysis will be used to determine significant differences for level and type of lipid. If
differences are determined for the level and type of fatty acid deposited in the fillet and no
correlative change in gene expression is found then, where possible, the enzyme activity will be
tested for the genes listed above.
Contingencies: There is a potential that since these fish have been reared on a fish oil based
diet prior to this experiment that the residual EPA and DHA will be preferentially retained in the
body and this experiment will not be able to adequately detect conversion and deposition
differences. If this happens then the experiment will be repeated and the fish will be started at a
smaller size (less than 5 g) and reared to at least 80 g. This increase in size should dilute out
any residual EPA and DHA originally stored in these fish. Some studies have also shown that
the presence of EPA and DHA appears to be necessary to stimulate certain metabolic
processes, to account for this the experiment can also be run but instead of using a feed without
any fish oil, the diet will be formulated to contain reduced levels (5% or lower) of fish oil. Finally
if variation in fatty acids conversion and deposition are detected but do not correspond with
gene expression or enzyme activity of the genes listed then the list of genes for analysis will be
expanded to include other genes known to be involved with lipid metabolism such at lipoprotein
lipase and TNFα, among others.
Collaborations: Dr. Ron Hardy and staff of the University of Idaho will collaborate with
experimental setup, sample isolation and testing.
Physical and Human Resources:

There are 5.5 SY’s available (listed on title page) for this project and 4 technicians (analytical
laboratory), one fish culture technician and one feed production technician. In addition to the
equipment described in each objective below, complete field plots, green houses, and fish
rearing facilties are available either in ARS facilities (plants) or through current Specific
Cooperative Agreement with University of Idaho.
Objective 1; Laboratory equipment at Aberdeen Molecular Genetics Lab are available for
completion of the proposed experiments and include a variety of centrifuges, PCR thermo-
cycler, ABI3130 genotype analyzer, Gel electrophoresis setups, and Gel-Doc system (Bio-Rad)
for DNA isolation, DNA marker analysis. Plate reader (Bio-Tek), cyclone mill, and multiple-
channel pipettes are available for BG measurements. Two rooms of greenhouse are available
each year for plant material growth. Unlimited field space is available for population planting.
Objective 2; The Grain Chemistry and Utilization Lab located at Aberdeen, ID, has two
separate physical rooms, one is dedicated for chemical analysis and other to laboratory scale
processing. The analytical lab is equipped with a HPLC (Waters), a GC (Aligent), a total
nitrogen/protein analyzer (Leco), a fat analyzer (Ankom Technology), an ash furnace, a
spectrophotometer, a high speed centrifuge, and an evaporator, and three analytical balances,
etc. The processing lab has an electric seed scarifier, a barley pearler, a set of sieves with a
shaker, etc. Production of pilot-scale quantities of the enhanced ingredients will be done at
Feed and Nutrition Laboratory, Bozeman, MT.
Objective 3&4; Analytical laboratory equipment available for completion for the goals
are: LC-MS (Agilent Inc), a GC (Shimadzu), a nitrogen/protein analyzer (Leco), a fat analyzer
(Ankom Technology, Inc), a spectrophotometer, ovens and muffle furnace, centrifuges, rotovap,
analytical balances, etc. Fish rearing space is available through collaboration with UI for tanks

Barrows 23
at the HFCES and include; 42, 475 liter tanks; 128, 144 liter tanks; 64, 144 liter rectangular
troughs; 12, stack incubation units (Heath); 5, 1.2 x 7.6 m raceways; 18, 1.5 m circular tanks;
15, 1.8 m circular tanks; 3, 3.0 m circular tanks.
All feed manufacturing will be done at the Feed and Nutrition Laboratory in Bozeman,
MT, through an Interagency Agreement with the US Fish and Wildlife Service. All necessary
equipment for ingredient preparation, mixing, and pellet formation is available including hammer
mills, pulverizers, mixers, cold extrusion, cooking extrusion (Buhler DNDL-44), driers and
vacuum coaters for oil application. 30, 420 liter fish tanks equipped with excess feed collection
systems are available for 2, 12 week studies per year through an Interagency Agreement.
Objectives 5&6; Fish rearing space is available through a Specific Cooperative
Agreement with UI for tanks at the HFCES (listed above in objectives 3&4) and raceways at the
Idaho Springs facility. The physical resources available include RNA extraction equipment
including a Mixer Mill 301, ABI 3730 and 3130XL genetic analyzers for genotyping, ABI 7900
and ABI 7500 Fast Sequence Detection Systems for analysis of gene expression, a Perkin-
Elmer Scanarray 5000 for analysis of hybridized array slides and a modified Hybaid
hybridization station for performing hybridizations, a Fluidigm EPI Genetic Analysis System for
SNP genotyping, and a Tecan Freedom Evo liquid handling robot for reaction setup. The project
also possesses a Sakure tissue embedding and sectioning station for the preparation of tissues
for in situ staining and a Zeiss digital fluorescent scope for visualization and software for
quantitative analysis.
Project Management and Evaluation - Project team members have a diverse set of expertise,
providing the opportunity to produce significant progress, yet also providing challenges in
communication and collaboration. To maximize collaboration, objectives in this Project Plan,
were designed to work across expertise, integrating skill sets, to attain the goals of each
objective. Figure 1 illustrates the interconnections and dependence of each researcher on
another. Even though researchers for this project are located at three different facilities, this
distance has been not been a detriment to the progress of the past project. For example, a
protein concentrate to be made in Aberdeen ID, using improved grains will be processed into a
complete feed at the laboratory in Bozeman, MT and then fed to trout in a diet study at the
Hagerman ID location. The team also meets in one location on a quarterly basis, to update
each other on progress, and more importantly coordinate upcoming collaborations. Milestones
are reviewed and updated at the Project Meetings. Video-conferencing technologies are also
used to assist in communication during Unit meetings.

Barrows 24
Milestones:
Project Title Improving Sustainability of Rainbow Trout Project No. 106 5366-21310-002-00D
Production by Integrated Development of Improved
Grains, Feeds, and Trout
National Programc Aquaculture
Objective 1 Identify and develop grain lines with desirable traits for either direct or indirect use in aqua feeds.
Subobjective 1a Determine the effect of Beta-glucan mutations on other agronomic and quality traits.
NP Action Plan Componentf Component 3. Defining Nutrient Requirements, Nutrient Composition of Feedstuffs and
Expanding Alternative Ingredients
NP Action Plan Problem 3B: Evaluate the Nutritional Value of Alternative Sources of Protein and Lipid
Statementg
SY Progress/
Hypothesish Teami Months Milestonesj Changesk Productsl
GH 12 Make back-crosses
GH 24 Develop back-cross
populations
GH 36 Identification of near Near-isogenic lines
iso-genic lines available for field
evaluations
GH 48 Compare the near
iso-genic lines in
field and lab
GH 60 Finish field and lab Germplasm
testing useful for further
breeding,
manuscript
Production by Integrated Development of
Improved Grains, Feeds, and Trout
Objective 1 Identify and develop grain lines with desirable traits for either direct or indirect use in aqua feeds.
Subobjective 1b Genetic mapping of the beta-glucan mutations.
Statementg
SY Progress/
GH 12 Finish all crosses
GH 24 Develop F2 populations
GH 36 Advance the mapping Beta-glucan
populations mapping
population
GH 48 DNA isolations for the
populations
GH 60 Mapping out mutant Markers useful for
genes further breeding,
manuscript

Barrows 25
Objective 2 Develop mechanical, chemical and biological methods to improve the nutritional and anti-nutritional
profile of grains, by-products and other alternative ingredients.
Statementg
SY Progress/
Subobjective 2.A. Simultaneous separation of high value nutrients from barley and oats.
KL 12 Fine-tune lab Use of low phytate Barley protein
method for wet barley, low beta- concentrate (70%),
fractionation of glucan barley beta-glucan (60%)
barley and starch (97%)
fractions,
KL 24 Method Oat protein

development for dry concentrate, oat
fractionation of oats beta-glucan and oat
and wet starch
fractionation of oats
KL, FB 36 Pilot scale Pilot amounts of
production of barley protein
and oat products concentrates for
fish trial.
KL, FB 48 Evaluation of
products in
Objective 3a
60
Subobjective 2.B. Improve nutritional and economic values of bio-fuel co-products
12
KL 24 Characterization Enhanced DDGS
and fractionation ingredient
DDGS
KL 36 Characterization If lipid content is Enhanced algal
and fractionation of high, work on both protein and oil
algae biomass. protein and lipid ingredients for fish
ingredients feed
KL, FB 48 Pilot scale-up for Pilot amounts of
fractionating DDGS protein and/or oil
and algae biomass. ingredients for fish
trials in Objective 3
KL, FB 60 Characterization If algal biofuel Enhanced algal bio-
and fractionation of residue is not fuel coproduct
algal biofuel residue available, continue
study algae

Barrows 26
Objective 3 Determine nutritional value of alternative ingredients, protein, lipid, and energy and develop
practical feed formulations for the improved strains of fish.
Subobjective 3a Determine the nutritional value of alternative feed ingredients.
NP Action Plan Componentf Defining Nutrient Requirements, Nutrient Composition of Feedstuffs
and Expanding Alternative Ingredients
NP Action Plan Problem Problem Statement 3B: Evaluate the Nutritional Value of Alternative
Statementg Sources of Protein and Lipid
SY Progress/
Quantification FB, KL 12 Additional Database of
of ingredients’ ingredient
compositional nutritional as well as nutritional profiles
analysis, anti-nutritional updated
palatability, and specifications
nutrient added to ongoing
digestibility of database for trout
alternative feed feed ingredients
ingredients in 24 Digestibility trial
order to performed
estimate their FB, KL 36 Palatability trial
nutritional and performed
economical FB, KL 48 Oat and barley Additional
values
products evaluated ingredient
from 2a information
FB, KL 60 Additional Database of
ingredients’ ingredient
nutritional as well as nutritional profiles,
anti-nutritional inclusion limits and
specifications nutrient
added to ongoing digestibilities
database for trout available for feed
feed ingredients formulation
software

Barrows 27
Objective 3 Determine nutritional value of alternative ingredients, protein, lipid, and energy and develop
practical feed formulations for the improved strains of fish.
Subobjective 3b Development and evaluation of alternative feed formulations.
f
NP Action Plan Component Defining Nutrient Requirements, Nutrient Composition of Feedstuffs
and Expanding Alternative Ingredients
NP Action Plan Problem Problem Statement 3B: Evaluate the Nutritional Value of Alternative
Statementg Sources of Protein and Lipid
SY Progress/
Combinatorial FB, KL 12
effects of 24 Potential ingredient
ingredients on combinations
rainbow trout identified
performance FB, KL 36 Feeding trial
will differ based completed and data
on nutritional analyzed
values
48
determined in
Sub-objective FB, KL 60 Optimized Demonstration trial
3A. commercial with farmers “Field
ingredient mixtures Day”
identified and field
tested

Barrows 28
Objective 4 Refined mineral supplementation for plant-based diets
NP Action Plan Componentf Defining Nutrient Requirements, Nutrient Composition of Feedstuffs and
NP Action Plan Problem Problem Statement 3A: Determine Nutrient Requirements
Statementg
SY Progress/
Addition of FB, KO 12 Complete K trial
minerals to FB, KO 24 Complete Mg trial
plant-based FB, KO 36 Complete P trial A recommended
trout feeds will macro- mineral
improve growth premix defined for
and health. trout fed plant-
based feeds
FB, KO 48 Complete Zn trial A recommended Zn
source and level for
trout fed plant-
based feeds
FB, KO 60 Integrate
information into
Objective 3b, 5, 6

Barrows 29
Objective 5 Use gene expression analyses to advance understanding of gene targets to improve nutrition,
growth, and development processes under production conditions.
Subobjective 5a Determine how changes in expression of previously tested and defined regulatory
metabolic genes regulating fatty acid metabolism and muscle growth correlate with
energy partitioning and growth in rainbow trout.
NP Action Plan Componentf Defining Nutrient Requirements, Nutrient Composition of Feedstuffs and
NP Action Plan Problem Defining Nutrient Requirements, Nutrient Composition of Feedstuffs and
Statementg Expanding Alternative Ingredients
SY Progress/
Relative expression KO, FB 12 Genes for panel Identified genes for
changes in a panel Protein exp started study.
composed of a limited KO, FB 24 Protein exp finished Isolated
number of specific experimental
genes corresponding samples.
with physiological KO, FB 36 Protein samples Manuscript
variation seen in trout analyzed Identified genes
utilization of different Lipid exp started useful in dietary
formulated sustainable analysis.
diets. KO,FB 48 Lipid exp finished Isolated samples
for analysis.
KO, FB 60 Lipid samples Determination of
analyzed and specific regulatory
completed. checkpoints for
growth and protein
turnover that can be
used to enhance
determination of
dietary utilization
and can be used for
selectively
improving identified
traits.

Barrows 30
Objective 5 Use gene expression analyses to advance understanding of gene targets to improve nutrition,
growth, and development processes under production conditions.
Subobjective 5b Identification of SNP markers for improved growth on plant based diets for rainbow
trout.
f
NP Action Plan Component Understanding, Improving, and Effectively Using Animal Genetic and
Genomic Resources
NP Action Plan Problem Develop and Implement Genomic Tools in Genetic Improvement
Statementg Programs
SY Progress/
SNPS can be KO, FB 12 Genes are chosen for Identified genes for
identified in genes SNP analysis study.
that are found to KO, FB 24 Genes are sequenced Sequence of genes
change in and SNPs identified for analysis.
expression KO, FB 36 Genotype SNP to SNP assays.
according to designated fish groups
physiological
KO, FB 48 Evaluate correlation of Production of new
changes seen in
SNP with phenotyped genotyping
trout reared on
markers.
sustainable plant
based diets. KO, FB 60 Evaluate correlation of Markers for
SNP with phenotyped selection,
manuscript

Barrows 31
Objective 6 Identify phenotypic differences in rainbow trout for growth and utilization of plant-based sustainable
diets and determine the genetic variation for identified traits.
Subobjective 6a Identify variation in physiological performance of rainbow trout that is specific to the
utilization of plant protein diets, versus fishmeal diets, and determine their genetic
components.
NP Action Plan Componentf Enhancing animal performance, well-being, and efficiency in diverse
productions systems
NP Action Plan Problem Improve Growth, Nutrient Utilization, and Well-being
Statementg
SY Progress/
That there are KO, FB 12 Generate trout Produced crosses
significant families and diets of trout.
differences KO, FB 24 Rear families for Families of fish of
between tested several months on size suitable for
rainbow trout diets study.
families for
growth and other
KO, FB 48 Take samples and Regulatory
physiological
arrange and metabolic
parameters when
analyze data checkpoints defined
reared on either
for the energy
fish meal and fish
partitioning and
oil based diets or
specific nutrient
sustainable plant
utilization.
and/or animal by
product based
diets. KO, FB 60 Analaysis of data Traits identified that
complete. can be used for
selection for
improvement trout
utilization of plant
components.

Barrows 32
Objective 6 Identify phenotypic differences in rainbow trout for growth and utilization of plant-based sustainable
diets and determine the genetic variation for identified traits.
Subobjective 6b Determine if genetic variation exists in rainbow trout for the ability to process plant oils
with increased deposition of DHA and EPA lipids in fish tissue.
f
NP Action Plan Component Enhancing animal performance, well-being, and efficiency in diverse
productions systems
NP Action Plan Problem Improve Growth, Nutrient Utilization, and Well-being
Statementg
SY Progress/
Variation exists in KO 12 Arrange crosses Crosses of fish
rainbow trout for selected and made.
the conversion KO, FB 24 Generate families Families generated
and deposition of and make diets for study.
lipids from plant KO, FB 36 Feed families on Determination of
oils to omega 3 diets for several variability of lipid
fatty acids. months, take conversion in trout
samples and families.
arrange and
analyze data
KO, FB 48 Write up data Identification of
specific regulatory
lipid checkpoints.
KO 60 Analysis complete Defined traits and
markers linked to
lipid utilization for
use in selection
program.

Barrows 33
Accomplishments from Prior Project Period
Title; The integration of nutritional, genetic and physiological approaches to improve production
efficiency of rainbow trout (Oncorhynchus mykiss).
Investigators; Frederic T. Barrows, J. Michael Bonman, T. Gibson Gaylord, Gongshe Hu,

Keshun Liu, and Kenneth E. Overturf
Summary Impact on Objective 1

Small grains are abundant ingredients for sustainable plant-based Aquafeed development.
Chemical compositions and related grain quality are very important for the grains to be used as
a nutrient source and cost-effective ingredient. Genetic modification of the grain compositions
will greatly influence the value and possibly create better germplasm lines for development of
the aquafeed friendly varieties. One major quality trait in barley grain is the BG that is very
attractive for commercial extraction because of its confirmed health beneficial to human.
Increase of the BG content will improve the market value of barley grains and may enhance the
by-product production from the BG extraction process. The by-product may be used as protein
rich and cost-effective aquafeed ingredient. Low BG grains are desired for animal feed, biofuel,
and fermentation. Significant reduction of barley BG will enhance its utilizations in those areas
and eventually benefit aquafeed development by obtaining cost-effective by products from those
processes.
We have identified several barley mutations with dramatic changes of BG contents in our plant
genetic project.
Description
Those mutations may be used in improvement of barley that will help our project. Those
mutants may be good materials for practical uses or concept-proofing experiments. We need to
experimentally test the mutants. In the current plan, we will continue to advance the genetic
generations for the early identified two mutants and to do various tests. We will develop the near
iso-genic lines for the two mutants so that the comparisons of the mutant and corresponding
wild type alleles are scientifically reliable. Agronomic test will provide evidence for the potential
use them in the breeding. Quality tests including protein, oil, and protein extraction efficiency will
provide evidence for their potential effects on other quality traits or process efficiency. Genetic
mapping of the mutants will contribute to the better understanding of basic science of genetic
mechanisms controlling the beta-glean metabolism and to d help development of molecular
markers used in barley breeding.
Summary of Accomplishments—Impact on Objective 2
Development of laboratory procedures to remove surface materials from cereal grains

layer by layer
For cereal grains, many nutrients are concentrated in the outer layers. A major problem that
hinders cereal research is lack of simple and small-scale methods to abrade grains. We
developed laboratory-scale methods to effectively remove the outer layers of cereal grains and
consequently addressed the problem. The new methods for abrading grains allow grain
scientists to produce experimental materials and evaluate nutrients contents in various grain
layers. The methods have provided valuable means for dry fractionating grains to enrich protein

Barrows 34
and other valuable components at a laboratory scale, and will be used in the current plan.
Based on the same principle, a simple method for measuring wheat hardness using the
inexpensive lab machines was also developed.
1. Liu, K.S. 2007. A modified laboratory method to remove outer layers from cereal grains
using a barley pearler. Cereal Chem. 84(4):399-46.
2. Liu, K.S. 2007. Laboratory methods to remove surface layers from cereal grains using a
seed scarifier and comparison with a barley pearler. Cereal Chem. 84(4):407-414.
3. Liu, K.S. 2008. Measurement of wheat hardness by seed scarifier and barley pearler and
comparison with single-kernel characterization system. Cereal Chem. 85(2):165-173.
4. Liu, K.S. and R.A. Moreau. 2008. Concentrations of functional lipids in abraded fractions of
hulless barley and effect of storage. J. Food Sci. 73(7):C569-C576.
Study on phosphorous and mineral distribution within a barley kernel and effect of low
phytate crop
Using the newly developed grain pearling method, the effect of low phytate (LP) mutation on
contents and distribution patterns for different types of phosphorous and minerals within a
barley seed were investigated. Phytate is the main storage form of phosphorus (P) in grains. It
is known to bind mineral cations. Results showed that there is no difference in mineral
concentrations and distribution patterns between conventional barley variety and low phytic acid
mutants. This implies that there is no direct role of localization of phytic acid synthesis in mineral
distribution within a barley seed and that LP breeding does not lead to reduction in mineral
contents in barley seeds. This study helps clarify controversy on the subject within scientific
community and provided direct evidence to justify LP breeding programs. In the current plan,
low phytate grains will also be investigated.
Liu, K.S., Peterson, K.L., and Raboy, V. 2007. A comparison of the phosphorus and mineral
concentrations in bran and abraded kernel fractions of a normal barley (Hordeum vulgare)
cultivar versus four Low Phytic Acid (lpa) isolines. J. Agric. Food Chem. 55 (11):4453-4460.
Study on factors affecting sieving performance and efficiency

The subject of this study was prompted by a surprising observation during dry fractionation of
barley flour by sieving at our laboratory. It was found that in separating barley flour, when other
conditions were kept same, a reverse sieve process, that is, flour is sifted in a fine to coarse
order by multiple sieving steps with each step using a single sieve, gave a better sieving
efficiency and performance than the conventional stacked sieve method. The objectives of the
study were: (1) to make a systematic comparison between the stacked and reverse sieve
methods for separation of flour, and (2) determine the effects of flour type, milling method,
sieving duration and sieve percussion on sieving performance using both the stacked and
reverse sieving methods. This study showed how the selected variables can affect sieving
efficiency and performance. It was also the first to report increased sieving efficiency of reserve
sieve method over the conventional stacked sieve method. The observed difference between
the two sieving procedures can be explained by the beneficial effect of oversized particles on
reducing sieve blinding by near or sub-sieve sized particles. Furthermore, by using the reverse
sieve procedure, the mass frequency of finer particle classes was increased and the difference
in protein content among sieved fractions was enlarged. Since hundreds of millions of tones of
particulate material are subjected to industrial sieving/screening each year, an understanding of
the factors affecting sieving efficiency and performance has great economic significance. It also
provides guidelines for the current plan with regard to using sieving method to dry fractionate
grains and biofuel co-products.

Barrows 35
Liu, K.S. 2009. Some factors affecting sieving performance and efficiency. Powder
Technology. 193:208-213.
Development of dry processing methods to fractionate barleys for simultaneously

concentrating several valuable components
We explored methods of pearling, milling, followed by sieving, and their combinations to dry
fractionate barleys for producing meals enriched with protein, BG and/or starch. The protein
fraction is intended for use as an ingredient for trout feed. Results show that pearling affected
the efficiency of subsequent milling methods followed by sieving, in term of nutrient enrichment
and recovery rates. The milling method also had significant effects on efficiency of sieving for
nutrient enrichment and recovery rates, as did the genotype. For protein, pearling alone was
the best method to enrich it, but for BG and starch, in order to achieve maximum nutrient
shifting, a combination of pearling and milling followed by sieving was necessary. Therefore,
although dry fractionation is the method of choice for separating barley into fractions with
varying levels of protein, BG and/or starch, selection of a specific single or combined method is
needed for achieving maximum shifts of a particular nutrient. The study provides significant
information to those who use dry fractionation methods to enrich protein, BG and/or starch. It
has impact on the current plan with regard to dry fractionating oats and other grains for
improving the nutrient profile of ingredient fractions for aquafeeds.
Liu, K.S., Barrows, F.T. and Obert, D. 2009. Dry fractionation methods to produce barley
meals varying in protein, BG and starch contents. J. Food Sci. 74(6):C487-C499.
Quality evaluation of dried distillers grains with solubles (DDGS) and impact of raw corn
quality
The research on DDGS is a timely subject. DDGS is a co-product of ethanol production from
corn and other cereal grains. It is a mix of particulate materials and has a protein content as
high as 30%. DDGS is now a major commodity in the world trade, yet there is a lack of uniform
quality parameters and methods to measure them. We investigated and found that particle size
distribution of DDGS is a characteristic property of a particular sample and that some key
components such as protein content vary significantly among fractions with different particle
sizes. The finding promotes better understanding of physical and chemical properties of DDGS,
and will potentially have impact on how DDGS products are evaluated for quality.
Furthermore, we also investigated effects of particle size distribution, compositional and color
properties of ground corn on quality of DDGS. Oftentimes, corn processors believe that ground
corn and DDGS are interrelated in certain quality parameters. Yet, previous research, although
rather limited, has not established this relationship. The study was conducted to define the
relationships between ground corn and DDGS. Results show that the geometric mean diameter
of particles of the whole fraction and mass frequency of individual particle size classes between
ground corn and DDGS varied, but particle size distribution of the two correlated very well (r =
0.807). There also were positive correlations in contents of protein and non-starch
carbohydrate between corn and DDGS. Variation in nutrients and color attributes were larger in
DDGS than in corn. These observations disagree with previous reports and provide scientific
basis to partially support the common belief expressed by processors regarding relationships
between corn and DDGS. This research will assist in defining the interrelated quality attributes
of feedstock that may improve the final product (DDGS) nutrient profile for utilization in
aquafeeds.

Barrows 36
1. Liu. K. S. 2008. Particle size distribution of distillers dried grains with solubles (DDGS) and
relationships to compositional and color properties. BioResource Technology. 99:8421-
8428.
2. Liu. K.S. 2009. Effects of particle size distribution, compositional and color properties of
ground corn on quality of distillers dried grains with solubles (DDGS). BioResource
Technology. 100:4433-4440.
Summary of Impact on Objective 3 and 4.
Taurine Supplementation of Plant-based Trout feeds.

Growth of carnivorous fish fed plant-based diets is usually less than fish fed fish meal based
diets, even when all known nutrient requirements are met. A study was conducted that
identified the sulfur-containing amino acid taurine to be conditionally indispensable to trout when
fed plant based feeds. This amino acid is abundant in fish meal, but virtually absent in plant-
derived ingredients. Supplementation of this amino acid to plant based feeds increased growth
of trout to that equivalent to trout fed fish-meal based feeds. Another study demonstrated that
increased levels of methionine could not replace taurine. Addition of this commercially available
synthetic amino acid to plant-based trout feeds represents a viable approach to increasing
growth rate of rainbow trout. Additional collaborative experiments have demonstrated that
taurine is a conditionally indispensable amino acid in the cobia (Rachycentron canadum), a
marine carnivorous fish species with increasing commercial culture interest. Taurine is now
utilized in commercially produced plant-based diets for rainbow trout and cobia.
Gaylord, T.G., Teague, A.M. and Barrows, F.T. 2006. Taurine supplementation of all-plant
protein diets for rainbow trout (Oncorhynchus mykiss). Journal of the World Aquaculture
Society. 37:509-517.
Gaylord, T.G., Barrows, F.T., Teague, A.M., Johansen, K.A., Overturf, K.E. and Shepherd, B.
2007. Supplementation of taurine and methionine to all-plant protein diets for rainbow
trout (Oncorhynchus mykiss). Aquaculture 269:514-524.
Lunger, A.N., McLean, E., Gaylord, T.G., and Craig, S.R. 2007. Taurine supplementation to
alternative dietary proteins used in fish meal replacement enhances growth of juvenile
cobia (Rachycentron canadum). Aquaculture 271:401-410.
Nutrient digestibility from alternative ingredients

Rainbow trout feeds are currently processed by cooking extrusion to increase the carbohydrate
digestibility of plant starches and to make the feed float to enable visual inspection of feeding
activity, but limited data on the nutrient digestibility from ingredients undergoing extrusion
processing is currently available. All other work determining nutrient digestibility and availability
has used a variety of methods and formulations that may not be directly comparable to current
industry standards. The amino acid availability and nutritive value of common and novel
feedstuffs must be defined to produce cost effective diets for rainbow trout. The database of
amino acid availability in feedstuffs for fish is extremely small. In one experiment, twenty four
ingredients were evaluated for apparent digestibility of nutrients including protein and amino
acid availability, digestible energy and organic matter digestibility. In another experiment
various barley and wheat cultivars that have been bred for improved nutritional value were
assayed for energy and starch digestibility for rainbow trout. A third experiment tested the
effects of a novel oil extraction process from soybeans on the digestibility of nutrients to trout

Barrows 37
from the remaining soybean meal. The information garnered from these experiments has
increased the ability to formulated trout feeds on an available nutrient basis and especially
increased the data available on the apparent amino acid availability of feed ingredients in
extruded feeds. The ability to formulate diets based on digestible nutrient and energy using
commercially applicable processing technology will allow for precision formulation of trout feeds
and limit over or under formulation of trout diets.
Gaylord, T.G., Barrows, F.T., and Rawles, S.D. 2008. Apparent Digestibility of Gross Nutrients
from Feedstuffs in Extruded Feeds for Rainbow Trout, Oncorhynchus mykiss. Journal of
the World Aquaculture Society. 39:827-834.
Gaylord, T.G., Barrows, F.T., Rawles, S.D., Liu, K., Bregitzer, P., Hang, A., Obert, D., and
Morris, C. 2008. Apparent digestibility of nutrients in extruded diets from cultivars of
barley and wheat selected for nutritional quality in rainbow trout (Oncorhynchus mykiss).
Aquaculture Nutrition. On-line early 10.1111/j.1365-2095.2008.00595.x
Gaylord, T.G., Barrows, F.T. and Rawles, S.D. (in press) Apparent Amino Acid Availability from
Feedstuffs in Extruded Diets for Rainbow Trout Oncorhynchus mykiss. Aquaculture
Nutrition.
Evaluation of balancing amino acid profile in plant-protein based diet formulations for
rainbow trout to reduce total dietary protein. Current diets for rainbow trout may be over
formulated with protein to meet individual amino acid requirements. Results from a 12-week
feeding trial demonstrate that when diets are formulated for available amino acids, instead of
crude protein, growth rate can be maintained and total dietary protein can be reduced.
Synthetic amino acids are supplemented to provide a better amino acid balance than currently
suggested in the literature. Supplementing with synthetic lysine, methionine and threonine
reduced total dietary protein by 11% and increase protein retained as growth by 35%. The
impact of this research will be to reduce both feed cost and nitrogenous waste released into the
environment through increased protein retention efficiencies.
Gaylord, T.G. and F.T. Barrows. 2009. Multiple amino acid supplementations to reduce dietary
protein in plant-based rainbow trout (Oncorhynchus mykiss) feeds. Aquaculture.
287:180-184.
Vitamin supplementation of extruded plant-based trout feeds

Vitamin supplementation levels for rainbow trout have been refined for feeds formulated with
fish meal and other animal protein sources that may supply some of the vitamin needs of
rainbow trout. As increasing concentrations of plant proteins are utilized in lieu of fish meals
and other animal protein sources refinements in vitamin supplementation levels are necessary.
This also is the case with feed processing. Most of the vitamin requirements have been
determined in diets utilizing purified ingredients and cold-pelleted feeds. Limited information
has been available on the effects of cooking extrusion on vitamin recoveries in feeds. An
experiment was performed that addressed the cooking extrusion losses in plant-based trout
feeds and the effects of modifying vitamin supplementation levels for extrusion losses and
ingredient interactions. The new vitamin premix formulation was validated for improving trout
performance and fish health in plant-based and fish meal based diets. This modified vitamin
formulation is now commercially available and utilized by the trout feeds industry.

Barrows 38
Barrows, F.T., Gaylord, T.G., Sealey, W.M., Porter, L., and Smith, C.E. 2008. The effect of
vitamin premix in extruded plant-based and fish meal based diets on growth efficiency and
health of rainbow trout, Oncorhynchus mykiss. Aquaculture. 283: 148-155.
Summary of Impact on Objective 5.
Identification of genes involved in metabolic regulation and energy partitioning with

dietary changes.
Genes were identified and assays developed for evaluating the expression of genes involved in
muscle regulation, carbohydrate and lipid utilization, protein turnover and metabolic pathway
signaling. Correlative differences were determined for genes with incorporation of sustainable
plant meal or oils in diets and with with growth and protein accretion. These identified genes will
now make up the panels that will be used to more carefully analyze the metabolic and growth
differences found upon incorporation and blending of different plant and sustainable material in
diets upon the overall physiology of the animal. Corollary gene changes between pathways
were also identified and characterized. Furthermore, enzymatic assays were also developed
and tested to further clarify the extent of the protein activity on identified physiological changes.
Overturf, K. and Gaylord, T.G. 2009 Determination of relative protein degradation activity at
different life stages in rainbow trout (Oncorhynchus mykiss). Comparative Biochemistry and
Physiology Part B. 152:150-160.
Johansen, K., and Overturf, K. 2006 Alterations in expression of genes associated with muscle
metabolism and growth during nutritional restriction and refeeding in rainbow trout. Comparative
Biochemistry and Physiology, Part B 144:119-127.
Summary of Impact on Objective 6.
Objective 6: Identify phenotypic differences in rainbow trout for growth and utilization of plant-
based sustainable diets and determine the genetic variation for identified traits.
Nutrition by Genetic Interaction

The ability to utilize selective breeding of rainbow trout for improved utilization of plant-
based feeds has shown promise. Collaborative research with the USDA-ARS National
Center for Cool and Cold Water Aquaculture and Troutlodge Inc. has shown that within
commercial strains of trout a genotype x diet interaction occurs and that genetic potential
exist for increased utilization of plant-based diets within the Kamloop strain. These
studies were also corroborated at the HFCES where families selected for growth on
plant meal based diets improved for growth and the fastest growing families ranked
separate when reared on fish meal based diets. Furthermore, SNP markers were
identified, developed and characterized, according to genes involved with fish health and
growth. Pierce, L.R., Palti, Y., Silverstein, J.T., Barrows, F.T., Hallerman, E.M., Parsons,
J.E. Family growth response to fishmeal and plant-based diets shows genotype x diet
interaction in rainbow trout (Oncorhynchus mykiss). Aquaculture 278; 37-42. 2008.
Campbell, N., Overturf, K. and Narum, S. 2009 Characterization of 22 novel single

nucleotide polymorphism markers in steelhead and rainbow trout. Molecular Ecology
Resources 9:318-322.

Barrows 39
Overturf, K., LaPatra, S., Towner, R., Campbell, N., Narum, S. 2009. Relationship between
growth and disease resistance in rainbow trout (Oncorhynchus mykiss). Journal of Fish
Diseases. submitted
Metabolic and physiological changes associated with genetic variation and dietary
carbohydrate and lipid changes.
Studies using distinct family crosses of rainbow trout reared on high and low carbohydrate diets
found significant interactions between family, weight gain, plasma glucose levels, and the
expression of glycolytic and energy partitioning genes. For families reared either on a low or
high carbohydrate diet decreased plasma glucose levels were significantly to growth rate for
most but not all families which then was found to correlate with plasma glucose levels.
Gluokinase and PPARβ were also found to correlate with carbohydrate utilization. Also in two
different studies involving families of trout fed diets containing fish oil or completely replaced
with plant oil distinct differences were found for growth, egg development and egg lipid
deposition, and genes involved with lipid conversion. These studies provided the identification of
physiological modification and gene expression regulation that we are using to more precisely
study the metabolic changes and genetic differences that are significant with the incorporation
and blending of sustainable plant and animal byproduct material in aquaculture diets.
Description of how the objectives and accomplishments relate to the current plan.
The accomplishments of the terminating project have built a body of data that have allowed for
plant-based feeds to be formulated for rainbow trout that can support good growth, production
efficiency and health. One limitation is that the costs of the diets are often in excess of current
commercial diet formulations. New technologies and varieties of ingredients may reduce the
cost of ingredients but determination of their nutritive value to rainbow trout will have to be
assessed as well as performance of trout fed these novel ingredients.
The data from the terminating project on the digestibility of nutrients from alternative ingredients
will be built upon by the evaluation of new ingredients being developed for the aquafeed
industry. The current plan will expand upon this database in evaluating combinations of
ingredients that will provide optimized nutrient profiles for trout metabolism and identify potential
unfavorable effects on trout performance.
The refinements in vitamin supplementation of extruded plant-based feeds in the terminating

project has determined modification of vitamin supplemented was necessary to optimize fish
health and production efficiencies relative to fish meal based feeds fro rainbow trout. These
same principles will apply to mineral supplementation needs in plant-based feeds relative to fish
meal based feeds where the endogenous contribution of fish meal to mineral supplies will have
to be accounted for as plant based ingredients are utilized to a greater extent. This is due to the
lower mineral content of plant ingredient relative to fish meals as well as potential interactions
with anti-nutrients such as phytic acid with mineral absorption for the diet.
The identification of genes that are correlatively regulated in response to dietary changes
provides us with the molecular tools necessary to determine how fish physiologically and
metabolically respond to different sustainable ingredient when incorporated into diets
individually, blended and at different levels. From this point our research can better define the
positive and negative effects of certain ingredients and begin to identify genes involved in traits
that might be selectively improved in fish to enhance their utilization of certain sustainable
products.

Barrows 40
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92:61-73.

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digestibility, metabolizable energy, egg production, egg quality and intestinal morphology
of the broiler chicks and layer hens fed hull-less barley based diets. Pakistan Journal of
Biological Sciences 10, 2257-2266.
Yamamoto, T., Shima, T., Furuita, H. and Suzuki, N. 2002. Influence of feeding diets without
fish meal by hand and by self-feeders on feed intake, growth and nutrient utilization of
juvenile rainbow trout (Oncorhynchus mykiss). Aquaculture 214:289
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pearled barley flours as affected by the degree of pearling. J. Agric. Food Chem.
49:331-335.
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Load, and the Risk for Gestational Diabetes Mellitus. Diabetes Care 29:2223-2230.

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Past Accomplishments of Frederic T. Barrows
Education:
Ph.D. Animal Nutrition, Department of Animal Science, Iowa State Univerisity,1988
M.S. Poultry Nutrition, Department of Animal Science, Iowa State Univerisity,1984
B.S. Animal Science, College of Agriculture, Iowa State University,1978.
Experience:
2003-present Research Physiologist, Fish with USDA/ARS, Small Grains and Potato
Germplasm Research Facility, Hagerman, Idaho. Development of plant-
based feeds for commercial trout production.
1988-2003 Research Physiologist, U.S. Fish and Wildlife Service, Fish Technology
Center, Bozeman, Montana. Developed feeds for a variety of fish
species including threatened and endangered species, and fish for
recreational fisheries or aquaculture production.
1982-1988.1 Graduate Research Assistant, Department of Animal Science, Iowa State
University, Ames, Iowa. Developed a series of feed formulation for
various life stages of walleye (stizostedium vitreum). Also developed
culture methods required for intensive culture of walleye with formulated
feeds. Assisted in field collection of fish samples for surveys on the
Missouri river. Assisted with data collection for laying hen and turkey
production trials.
Accomplishments:
Development of fish meal-free trout diets. The goal of a fish meal free diet for carnivorous fish
has been declared impossible by anti-aquaculture groups and that same goal has been
identified as the most pressing issue by US aquaculture stakeholders. Dr. Barrows has lead a
team that developed fish meal free feeds that produce growth and feed efficiency equivalent to
trout fed a fish meal based diet. A complete set of in-vivo apparent digestibility coefficients
(major nutrient classes and amino acids) for both alternative ingredients and common feed
ingredients was developed. This information was not previously available, and now feeds are
balanced on an available amino acid basis. Nutrients present in fish meal, but not found in plant
derived ingredients, were identified and optimized, which include but are not limited to insoitol,
electrolytes, and taurine. Anti-nutrients present in some plant ingredients were identified.
Information was combined and feeds were cost-effectively formulated and tested both in the
laboratory and in commercial settings. Based on this work, a first of its kind Demonstration
Project was conducted at the College of Southern Idaho, which included a field day of
presentations, outreach displays, and an opportunity for farmers to taste trout fed grain based
diets. Development and commercialization of an open-formula vitamin premix for plant-based
feeds. Information on vitamin requirements for fish is incomplete and severely lacking relative to
terrestrial species and an open-formula premix had not been updated for over 20 years. Since
that time new forms of vitamins and new feed processing techniques have become available
and adopted. An open-formula vitamin premix was developed and tested in fish meal and plant-
based diets for rainbow trout. As part of this work, Dr. Barrows was the first to identify the need
to add insoitol to diets without fish meal.
Publications;
1. Barrows, F.T., Gaylord, T.G., Sealey, W.M., Porter, L., Smith, C.E. The effect of vitamin
premix in extruded plant based and fish meal based diets on growth efficiency and
health of rainbow trout, Oncorhynchus mykiss. Aquaculture, 283, 148-155. 2008.

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2. Barrows, F.T., Gaylord, T.G., Stone, D.A.J., and Smith C.E. Effect of protein source and
nutrient density on growth efficiency, histology, and plasma amino acid concentration of
rainbow trout (Oncorhynchus mykiss Walbaum). Aquaculture Research 38:1747-1758.
2007.
3. Barrows, F. T., D. Bellis, A. Krogdahl, J. T. Silverstein, E. M. Herman, W. M. Sealey, M.
B.Rust and D. M. Gatlin III. Report of the Plant Products in Aquafeed Strategic Planning
Workshop: An integrated, interdisciplinary research roadmap for increasing utilization of
plant feedstuffs in diets for carnivorous fish. Reviews in Fisheries Science. 16(4):449-
455. 2008.
4. Barrows, F.T., Gaylord, T.G., Sealey, W.M., Haas, M.J., Stroup, R.L. Processing soybean
meal for biodiesel production; effect of a new processing method on growth performance
of rainbow trout, Oncorhynchus mykiss. Aquaculture, 283, 143-147. 2008.
5. Pierce, L.R., Palti, Y., Silverstein, J.T., Barrows, F.T., Hallerman, E.M., Parsons, J.E.
Family growth response to fishmeal and plant-based diets shows genotype x diet
interaction in rainbow trout (Oncorhynchus mykiss). Aquaculture 278; 37-42. 2008.
6. Barrows, F., Bradley, C., Kearns, R., Wasicek, B., Hardy, R. Protein concentrate from
starch containing grains: composition, method of making, and use thereof. U.S. Patent,
Confirmation # 1423. April 2009.
7. Gaylord, T.G., Barrows, F.T., Rawles, S.D., Liu, K., Bregitzer, P., Hang, A., Obert, D., and
Morris, C. Apparent digestibility of nutrients in extruded diets from cultivars of barley and
wheat selected for nutritional quality in rainbow trout (Oncorhynchus mykiss).
Aquaculture Nutrition. Published on-line early, June 2nd, 2008.
8. Gaylord, T.G., Barrows, F.T., and Rawles, S.D. Apparent Digestibility of Gross Nutrients
from Feedstuffs in Extruded Feeds for Rainbow Trout, Oncorhynchus mykiss. Journal of
the World Aquaculture Society. Accepted October 23rd, 2008.
9. Sealey, W., Barrows, F., Hang, A., Johansen, K., Overturf, K., LaPatra, S. and Hardy, R.
Evaluation of the ability of barley varieties containing different amounts of β-glucan to
alter growth and disease resistance of rainbow trout Oncorhynchus mykiss. Animal Feed
Science and Technology, 141:115-128. 2007.
10. Sealey, W., Barrows, F., Johansen, K., Overturf, K., LaPatra, S., and Hardy, R.
Evaluation of the ability of partially autolyzed yeast and GrobioticTM-A to improve disease
resistance of rainbow trout Oncorhynchus mykiss. North American Journal of
Aquaculture 69:400-406. 2007.
11. Gatlin, D.M. III, Barrows, F. T., Bellis, D., Brown, P., Campen, J., Dabrowski, K.,
Gaylord, T.G., Hardy, R. W., Herman, E., Hu, G., Krogdahl, Ǻ., Nelson, R., Overturf, K.,
Rust, M., Sealey, W., Skonberg, D., Souza, E., Stone, D., Wilson, R. and Wurtele, E.
Aquaculture Research 38: 551-579. 2007.
12. Palti, Y., J.T. Silverstein, H. Wieman, J.G. Phillips, F.T. Barrows, J.E. Parsons.
Evaluation of family growth response to fish meal and gluten-based diets in rainbow trout
(Onchorhyncus mykiss). Aquaculture, 255: 548-556. 2006.
13. Barrows, F.T. and W.A. Lellis. Effect of diet processing method and ingredient
substitution on feed characteristics and survival of larval walleye, Sanders vitreus.
Journal of the World Aquaculture Society. 37(2): 154-160. 2006.
14. Gaylord,T.G., A.M. Teague., F.T. Barrows. Taurine supplementation of all-plant protein
diets for rainbow trout (Onchorhyncus mykiss). Journal of the World Aquaculture Society
37(4):509-517. 2006.
15. Barrows, F.T., D.A.J. Stone, R.W. Hardy. The effects of extrusion conditions on the
nutritional value of soybean meal for rainbow trout (Oncorhynchus mykiss) Aquaculture
265:244-252. 2007.

Barrows 54
16. Liu, K.K.M., F.T. Barrows, R.W. Hardy, F.M. Dong. Body Composition, growth
performance and product quality of rainbow trout (Onchorynchus mykiss) fed diets
containing poultry fat, soybean/corn lecithin, or menhaden oil. Aquaculture. 238:309-328.
2004.
17. Lellis, W.A., F.T. Barrows, and R.W. Hardy. Effects of phase-feeding dietary phosphorus
on survival, growth, and processing characteristics of rainbow trout, Oncorhynchus
mykiss (Walbaum). Aquaculture. 224; 607-616. 2004.
18. Stone, D.A., R.W. Hardy, F.T. Barrows, Z.J. Cheng. Effects of extrusion on nutritional
value of diets containing corn gluten meal and corn distiller’s dried grain for rainbow
trout, Onchorhynchus myskiss. Journal of Applied Aquaculture 17(3):1-20. 2005.
19. Schwertner, M.A., K.K.M. Liu, F.T. Barrows, R.W. Hardy, F.M. Dong. Performance of
Post-juvenile Rainbow Trout Onchorynchus mykiss Fed Diets Manufactured by Different
Processing Techniques. World Aquaculture. 34(2):162-174. 2003.
20. Zhu, S., S. Chen, R.W. Hardy, F.T. Barrows. Digestibility, growth and excretion response
of rainbow trout (Onchorynchus mykiss Walbaum) to feeds of different ingredient size.
Aquaculture Research 32(11):885-893. 2001.

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Past Accomplishments of J. Michael Bonman
Education:
University of Oregon, Biology, BA, 1974

Washington State University, Botany, MS, 1977
Washington State University, Plant Pathology, Ph.D., 1980
Experience:
2002-present Research Leader and Location Coordinator, USDA-ARS, Small Grains

and Potato Germplasm Research Unit
1991-2002 Research Associate and Technical Leader for Fungicide Optimization, E.I.
DuPont de Nemours and Company, Crop Protection, Stine-Haskell Research Center,
P.O. Box 30, Newark, DE 19714.
Responsible for leading a team of biologists who conduct research on optimizing the
attributes of new synthetic chemical “lead areas” for disease control in cereals, grapes,
potatoes, rice, and other crops.
1982-1991 Plant Pathologist, International Rice Research Institute, MC PO Box 3127,

Makati City 1271, Philippines.
Postdoctoral Fellow, Rockefeller Foundation, Pak Chong, Thailand.
Accomplishments:
As a post-doctoral fellow, conducted field studies with downy mildew disease of tropical
maize at the Thai National Corn and Sorghum Research Center. Discovered previously
unknown wild hosts of the downy mildew pathogen and developed a method for scoring
downy mildew resistance on maize seedlings.
While at IRRI, worked with plant breeders to develop improved rice germplasm and
conducted studies to demonstrate the value of partial resistance to rice blast disease in
lowland tropical rice. Results from this work lead to changes in blast-resistance
selection criteria for lowland breeding materials. Conducted studies with breeders and
geneticists on blast-resistance inheritance studies using both traditional and molecular
techniques and developed the first set of near-isogenic lines with single major genes for
blast resistance and a set of recombinant inbred lines used to map quantitative trait loci
for blast resistance. The lines developed in these studies are being widely used in rice
blast research and practical breeding efforts.
With DuPont was responsible for leading a team of biologists who conduct research on
optimizing the attributes of new synthetic chemical “lead areas” for disease control in
cereals, grapes, potatoes, rice, and other crops. Past major accomplishments at DuPont
included: 1) developing domestic and international field-testing capabilities for rice
fungicides; 2) developing fungicide testing methods that lead to the discovery of new rice
fungicides; 3) establishing a model for research cooperation between DuPont colleagues
in the United States and Japan; and 4) leading a multi-disciplinary team that resolved a
serious screen-reproducibility problem.

Barrows 56
Past Accomplishments of Gongshe Hu
Education:
Hebei Agricultural University, BS, 1982

Kansas State University, Genetics, MS, 1990
Kansas State University, Plant Pathology, Ph.D., 1995
Experience:
2005-present Research Geneticist, USDA-ARS, Small Grains and Potato Germplasm

Research Unit, Aberdeen,, Idaho. Development of plant-based feeds for commercial
trout production
1998-2004. Research Associate, Assistant specialist in Plant Gene Expression Center,

Albany, California. For genetic research on genetic dissection of the N gene signal
transduction pathway.
1995-1998. Postdoctoral fellow, Missouri University, Columbia, Missouri.
Accomplishments:
As a post-doctoral fellow in the University of Missouri, conducted genetic study of maize

lesion-mimic mutants. Discovered the genetic and chemical mechanisms for the
dominant lesion phenotype of the mutant Les22. This research is the first example of a
metabolic disorder in plants leading to the cell death phenotype.
While at Plant Gene Expression Center, Using tomato plants bearing the N gene as a
system for the genetic dissection of the N signal pathway, identified signal transduction
pathway components. Leading a small team, first developed a seedling lethal screening
approach by exploiting the temperature sensitivity of the N gene. This approach enabled
high throughput screening of fast neutron mutagenized tomato seeds. Successfully
isolated a recessive mutant sun1-1 that abolished N gene function. Genetic and
biological characterization showed that sun1-1 suppressed several other R genes
including Ve, Bs4, and I1. The mutant also suppressed salicylic acid induction and basal
resistance. Mapped and cloned the gene using positional cloning.
Publications:
1. Hu, G., Liang, G.H., Wassom, C.E. Chemical induction of apomictic seed formation in
maize. Euphytica 56: 97-105. 1991.
2. Hu, G., Kofoid, K.D., Liang, G.H.. An unstable mutation for pigmentation in kernels of
'Calico' sorghum. Hereditas 115: 163-167. 1991.
3. Hu, G., Hulbert, S. Evidence for involvement of gene conversion in meiotic instability of
the Rp1 rust resistance genes of maize. Genome 37: 742-746. 1994.
4. Hu, G., Hulbert, S. Association of a lesion mimic phenotype with certain Rp1 gene
combinations MNL 69:99. 1995.

Barrows 57
5. Hu, G., Hulbert, S. Construction of compound Rp1 rust resistance genes in maize.
Euphytica 87: 45-51. 1996.
6. Hu, G., Richter, T., Hulbert, S., Pryor, T. Disease lesion mimicrycaused by mutations in
the rust resistance gene rp1. Plant cell 8: 1367- 1376. 1996.
7. Hu, G., Webb, C., Hulbert, S. Adult-plant phenotype of the Rp1-DJ compound rust
resistance gene in maize Phytopathology 87: 236-241. 1997.
8. Hulbert, S., Hu, G., Drake, J. Kansas rust resistant sweet corn population A and B.
HortScience 32: 1130-1131. 1997.
9. Hu, G., Yalpani, N., Briggs, S., Johal, G.S. A porphyrin pathway impairment is
responsible for the phenotypic manifestation of a dominant disease lesion-mimic
mutation of maize. Plant Cell 10: 1095-1105. 1998.
10. Johal, G. S., Briggs, S., Gray, J., Hu, G. Methods and compositions for r egulating cell
death and enhancing disease . Patent No. 6455297. 2002.
11. Hu, G., de Hart, A.K.A., Li, Y., Ustach, C., Handley, V., Navarre, R., Hwang , C.F.,
Aegerter, B.J., Williamson, V.M., Baker, B. EDS1 in tomato is required for resistance
mediated by TIR-class R gene and the receptor-like R gene Ve. The Plant Journal 42:
372-391. 2005.
12. Hu, G., Jackson, E.W., and Bonman, J.M. Expansion of PCR-based marker resources in
oat by surveying genome-derived SSR markers from barley and wheat. Crop Science
47: 2004-2012. 2007.
13. Jackson E.W., Obert D.E., Menz M., Hu G., Avant J.B., Chong J., Bonman J.M.
Characterization of and mapping oat crown rust resistance genes using three
assessment methods. Phytopathology 97: 1063-1070. 2007.
14. Barrows, F. T., Bellis, D. , Brown, P., Campen, J., Dabrowski, K., Gatlin III, D.M.,
Gaylord, T.G., Hardy, R. W. , Herman, E. , Hu, G., Krogdahl, Ǻ ., Nelson, R., Overturf,
K., Rust, M., Sealey, W., Skonberg, D., Souza, E., Stone, D., Wilson, R., Wurtele, E.
Expanding the utilization of sustainable plant products in aquafeeds – a review.
Aquaculture Research 38: 551-579. 2007.
15. Jackson, E.W., Obert, D., Hu, G., Menz, M., Bonman, JM. Qualitative and Quantitative
trait loci conditioning resistance to Puccinia coronata races NQMG and LGCG in the oat
(Avena sativa L.) cultivars Ogle and TAM O-301. Theoretical and Applied Genetics 116:
517-527. 2008.
16. Hu, G., Burton, C. Modification of the standard enzymatic protocol to a cost- efficient
format for mixed-linkage (1→3, 1→4)-β-D-glucan measurement. Cereal Chemistry 85:
648-653. 2008.
17. Jackson, E.W., Wise, M., Bonman, J.M.,Obert, D.E., Hu, G. and Peterson. D.M.. QTLs
Affecting -tocotrienol, -tocopherol, and total tocopherol concentrations detected in the
Ogle/TAM O-301 oat mapping population. Crop Science 48:2141-2152. 2008.
18. Hulbert,S., Pryor, T., Hu, G., Richter, T., Drake, J. Genetic fine structure of resistance
loci. In the Gene for gene relationship in plant-parasite interactions. I.R. Cruite, E.B.
Holub, and J. J. Burdon, eds (New York: Cab Internationals). pp 27-43. 1997.
19. Bennetzen, J., Richter, T., Hu, G., Sanmiguel, P., Hong, K., Frederick, R.,

Barrows 58
Hulbert, S. Organization and hyperevolution of rust resistance genes in maize. In

Advances in Molecular Genetics of Plant-Microbe Interactions, ed. Daniels, M.(Kluwer,
Boston), Vol. 3, pp. 261-266. 1994.
20. Johal, G. S., Briggs, S., Gray, J., Hu, G. 2001. Methods for making male-sterile plants.
Patent Application # 20010023501

Barrows 59
Past Accomplishments of Keshun Liu, Research Chemist
Education:
Ph.D. Food Science, Michigan State University, E. Lansing, MI, 1986-1989
M. S. Food Science, Michigan State University, E. Lansing, MI, 1984-1986
B. S. Horticulture, Anhui Agricultural University, China, 1978-1982
Experience:
9/2005-Present. Research chemist, USDA, ARS, Aberdeen, ID. Developing enhanced
protein ingredients of plant origin for use in trout feed.
2003-2005 Adjunct Associate Professor, University of Missouri, Columbia, MO. Conducted
independent research on high moisture extrusion of soy proteins into fibrous meat
analogs.
1992-2002 Sr. Food Scientist/Manager, Monsanto Co. Directed studies that assessed
safety of biotech crops from compositional/nutritional standpoints & led a soyfood
laboratory for researching and evaluating specially soybeans.
1990-1992 Postdoctoral associate, University of Georgia, Griffin, GA. Initiated and
conducted series of basic and applied research on food legume texture as affected by
prolonged storage, with an objective to provide a scientific explanation.
Accomplishments:
Since joining ARS trout-grain team in fall, 2005 (last member to join the team), Dr. Liu has
established a Grain Chemistry and Utilization Laboratory at the Small Grain and Potato
Germplasm Research Unit, ARS, Aberdeen, ID. His major research accomplishments at ARS
included: 1) development of laboratory methods to abrade grains, 2) elucidation of effects of low
phytate barley on distribution of minerals and phytate within kernel, 3) development of methods
for improved sieving efficiency by reverse sieving over the conventional stacked sieve method,
4) development of a dry fractionation method to produce barley meals varying in protein, beta-
glucan and/or starch, and 5) establishment of particle size distribution as a quality parameter of
DDGS and its relationship with physicochemical properties.
At the University of Missouri, Dr. Liu and his colleagues developed a technique to objectively
measure the degree of fiber formation in extruded soy proteins based on fluorescence
polarization spectroscopy. He also carried out experiments that refined the understanding of
protein-protein interactions and fiber formation mechanisms.
At Monsanto Co., Keshun established an analytical and soyfood laboratory, developed new
analytical methods for screening breeding lines, and explored end uses of soybeans. He made
concrete efforts to promote soybean food utilization through research, publications, and
outreach, and became well-known for his expertise on soybeans and soyfoods. He is author/co-
author of more than 35 publications on soy, wrote or edited two reference books: “Soybeans:
Chemistry, Technology and Utilization (1997) and “Soybeans as a functional food (2004)”. He
organized or co-organized two international conferences and over 15 symposia on soy, and was
a frequently invited speaker on the subject to both domestic and international audience.
Publications:
1. Liu. K.S. 2009. Effects of particle size distribution, compositional and color properties of
ground corn on quality of distillers dried grains with solubles (DDGS). BioResource
Technology. 100:4433-4440..

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2. Liu, K.S., Barrows, F.T. and Obert, D. 2009. Dry Fractionation Methods to Produce Barley
MealsVarying in Protein, Beta-Glucan and Starch Contents. J. Food Sci. Accepted for
publication
3. Liu, K.S. 2009. Some factors affecting sieving efficiency and performance. Powder
Technology. 193:208-213.
4. Liu. K. 2008. Particle size distribution of distillers dried grains with solubles (DDGS) and
relationships to compositional and color properties. BioResource Technology. 99:8421-
8428.
5. Liu, K. and F.-H. Hsieh. 2008. Protein-protein interactions during high-moisture extrusion for
fibrous meat analogs and comparison of protein solubility methods using different solvent
systems. J. Agric. Food Chem. 56:2681-2687.
6. Liu, K. 2008. Measurement of wheat hardness by seed scarifier and barley pearler and
comparison with single-kernel characterization system. Cereal Chem. 85(2):165-173.
7. Liu, K.S. and R.A. Moreau. 2008. Concentrations of functional lipids in abraded fractions of
hulless barley and effect of storage. J. Food Sci. 73(7):C569-C576.
8. Liu, K. 2007. A modified laboratory method to remove outer layers from cereal grains using
a barley pearler. Cereal Chem. 84(4):399-406.
9. Liu, K. 2007. Laboratory methods to remove surface layers from cereal grains using a seed
scarifier and comparison with a barley pearler. Cereal Chem. 84(4):407-414.
10. Liu, K., Peterson, K.L., and Raboy, V. 2007. A comparison of the phosphorus and
mineral concentrations in bran and abraded kernel fractions of a normal barley (Hordeum
vulgare) cultivar versus four Low Phytic Acid (lpa) isolines. J. Agric. Food Chem. 55
(11):4453-4460.
11. Liu, K. and Hsieh, F.-H. 2007. Protein-protein interactions in high moisture extruded
analogs and heat-induced soy protein gels. J. Am. Oil Chem. Soc. 84:741-748.
12. Melinda, C. M., K. Liu, Trujillo, W.A. and Dobert, R.C. 2005. Glyphosate-tolerant
soybeans remain compositionally equivalent to conventional soybeans during three years of
field testing. J. Agric. Food Chem. 53 (13): 5331-5335.
13. Yao, G. K. Liu and F. Hsieh. 2004. A new method for characterizing fiber formation in
meat analogs during high moisture extrusion. J. Food Sci. 69: E303-E307.
14. Liu, K. (Ed.) 2004. Soybeans as Functional Food and Ingredients. AOCS Press,
Champaign, IL.
15. Liu, K. 1997, 1999. Soybeans: Chemistry, Technology, and Utilization. Kluwer
Academic Publishers, New York, NY.
16. Ang, C.Y.W., K. Liu, and Y-W. Huang. (Eds.) 1999. Asian Foods: Science and
Technology. Technomic Publishing Co. Inc. Lancaster, Pennsylvania.
17. Liu, K., JY Gai, et al. (Eds), 2002. Proceedings of China & International Soy Conference
and Exhibition, Beijing, China. Chinese Cereals and Oils Society. Beijing, China, Nov. 6-9.
18. Liu, K. 2008. Food use of whole soybeans, Ch. 14. in “Soybeans: Chemistry,
Production, Processing and Utilization”, L.A. Johnson, P.J. White and R. Galloway, eds.,
AOCS Press. Urbana, IL. pp 441-481.
19. Wang, X. and Liu, K. 2005. Extraction with compressed petroleum gases for specialty
oil and meal products INFORM. April issue.

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Past Accomplishments of Kenneth E. Overturf
Education:
Ph.D. Cell and Molecular Biology, University of Nevada-Reno, 1994
BS Biology, Boise State University, 1989
Experience:
2000-present Research Geneticist for USDA/ARS, Small Grains and Potato Germplasm
Research Facility, Aberdeen, Idaho. Conducting research on the heritability
between trout strains and within trout families while analyzing their ability to
utilize different food sources.
1998-2000 Research Associate, Gene Therapy Department, Comprehensive Cancer Center,
University of Alabama at Birmingham, Birmingham, Alabama. Conducted cancer
gene therapy research using engineered viral vectors in live animal systems.
Performing research with animal models for dissecting the genetics behind
certain types of cancer.
1994-1998 Postdoctoral Fellow, Medical and Molecular Genetics Department, Oregon
Health Sciences University, Portland, Oregon. Investigated different inherited
diseases, their prevalence in the population and attempted to isolate the genes
behind their disease status. Investigated certain aspects of different inherited
metabolic diseases and tested different gene replacement strategies for
therapeutic correction. Experimentally generated and tested the first phenotypic
model that was corrected via gene therapy.
1998 Scientific Consultant for Stem Cells, Sunnyvale, California. Performed research
determining the regeneration potential of specific subpopulations of cells residing
within the liver.
1989 Student Fellowship for the Department of Energy, Idaho National Engineering
Laboratory, Idaho Falls, Idaho. Analyzed micro-organisms isolated from
radioactive containment pods and determined their characteristics and identity.
Accomplishments:
Experimentally generated and tested the first phenotypic model that was corrected via gene
therapy. Helped create and test engineered retroviruses for treatment of the disease Hereditary
Tyrosinemia Type I in a mouse model and demonstrated complete correction of disease
phenotype. This was the first animal model ever phenotypically cured through gene therapy
measures. Dr. Overturf has previously performed analyses quantifying the transcriptional
regulation of genes coding for several key enzymes in the glycolytic pathway. Application of
new technologies in Dr. Overturf’s laboratory on gene expression will allow for an even broader
array of gene products to be screened for regulation by altered nutritional status of the trout. Dr.
Overturf has developed and tested many real-time quantitative PCR probes for genes involved
with protein degradation, metabolism, muscle development, and nutrient partitioning.
Publications Ken Overturf:

1. Thorgaard, G.H., Bailey, G.S., Williams, D., Buhler, D.R., Kaattari, S.L., Ristow, S.S.,
Hansen, J.D., Winton, J.R., Bartholomew, J.L., Nagler, J.J., Walsh, P.J., Vijayan, M.M., Devlin,
R.H., Hardy, R.W., Overturf, K.E., Young, W.P., Robison, B.D., Rexroad, C. and Palti, Y. 2002.
Status and opportunities for research in rainbow trout. Comp. Bio. Physiol. B 133:609-646.

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2. Overturf, K., Casten, M., LaPatra, S.L., Rexroad III, C. and Hardy, R.W. 2003. Comparison of
growth performance, immunological response and genetic diversity of five strains of rainbow
trout (Oncorhynchus mykiss). Aquaculture 217(104):93-106.
3. Overturf, K., LaPatra, S.L. and Reynolds, P.N. 2003. The effectiveness of adenoviral vectors
to deliver and express genes in rainbow trout (Oncorhynchus mykiss). Journal of Fish Diseases:
26 (2):91-101.
Overturf, K., Raboy, V., Cheng, Z.J. and Hardy, R.W. 2003. Mineral availability from barley low
phytic acid grains in Rainbow Trout (Oncorhynchus mykiss) diets. J. Aqua. Nutr. 9:239-
246.
Overturf, K., Bullock, D., LaPatra, S. and Hardy, R. 2004. Genetic selection and molecular
analysis of domesticated rainbow trout for enhanced growth on alternative diet sources.
Environ. Bio. Fishes 69: 409-418.
Biga, P.R., Peterson, B.C., Schelling, G.T., Hardy, R.W., Cain, K.D., Overturf, K. and Ott, T.L.
2004. The effects of recombinant bovine somatotropin (rbST) on tissue IGF-I, IGF-I
receptor, and GH mRNA levels in rainbow trout (Oncorhynchus mykiss). Gen. Comp.
Endocrin. 135:324-333.
Biga, P.R., Cain, K.D., Hardy, R.W., Schelling, G.T., Overturf, K. and Ott, T.L. 2004. Growth hormone
differentially regulates muscle myostatin-I and –II and increases circulating cortisol in rainbow
trout (Oncorhynchus mykiss). Gen. Comp. Endocrin. 138:32-41.
Johansen, K.A., and Overturf, K. (2005) Sequence, conservation, and quantitative expression of
rainbow trout Myf5. Comparative Biochemistry and Physiology, Part B 140:533-541.
Powell, M., Overturf, K., Hogge, C. and Johnson, K. 2005. Detection of Renibacterium
salmoninarum in Chinook salmon Oncorhynchus tshawytscha using Quantitative PCR.
Journal of Fish Diseases. 28:615-622.
Biga, P.R., Peterson, B.C., Schelling, G.T., Hardy, R.W., Cain, K.D.,Overturf, K., and Ott, T.L.
2005 Bovine growth hormone treatment increased IGF-I in circulation and induced the
production of a specific immune response in rainbow trout (Oncorhynchus mykiss).
Aquaculture 246:437-445.
Johansen, K., and Overturf, K. 2005 Quantitative expression analysis of genes affecting muscle
growth in rainbow trout (Oncorhynchus mykiss). Marine Biotechnology 7:576-587.
Johansen, K., and Overturf, K. 2006 Alterations in expression of genes associated with muscle
metabolism and growth during nutritional restriction and refeeding in rainbow trout.
Comparative Biochemistry and Physiology, Part B 144:119-127.
Overturf, K., and LaPatra, S. 2006 Quantitative expression of (Walbaum) of immunological

factors in rainbow trout, Oncorhynchus mykiss (Walbaum) after infection with either
Flavobacterium psychrofilum, Aeromonas salmonicida, or infectious haematopoietic
necrosis
virus. Journal of Fish Diseases 29:215-224.
Johansen, K.A., Sealey, W.M. and Overturf, K. 2006 The effects of chronic immune stimulation

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on muscle growth in rainbow trout. Comparative Biochemistry and Physiology - Part B:

Biochemistry and Molecular Biology 144:520-531.
Stone, D., Gaylord, T.G., Johansen, K., Overturf, K., Sealey, W. and Hardy, R. 2007 Evaluation
of the effects of repeated fecal collection by manual stripping on the, plasma cortisol levels,
TNF-α gene expression, and digestibility and availability of nutrients from hydrolyzed
poultry and egg meal by rainbow trout, Oncorhynchus mykiss (Walbaum). Aquaculture
275:250-259.
Gatlin, D.M. III, Barrows, F. T., Bellis, D., Brown, P., Campen, J., Dabrowski, K., Gaylord,
T.G., Hardy, R. W., Herman, E., Hu, G., Krogdahl, Ǻ., Nelson, R., Overturf, K., Rust,
M., Sealey, W., Skonberg, D., Souza, E., Stone, D., Wilson, R. and Wurtele, E. (2007).
Aquaculture Research 38:551-579.
Sealey, W., Barrows, F., Hang, A., Johansen, K., Overturf, K., LaPatra, S. and Hardy, R. 2007
Evaluation of the ability of barley varieties containing different amounts of β-glucan to alter
growth and disease resistance of rainbow trout Oncorhynchus mykiss. Animal Feed
Science
and Technology 141:115-128.
Gaylord, T.G., Barrows, F.T., Teague, A.M., Johansen, K.J., Overturf, K.E., and Shepherd, B.
2007 Supplementation of taurine and methionine to all-plant protein diets for rainbow trout
(Oncorhynchus mykiss). Aquaculture 269:514-524
Overturf, K. and Gaylord, T.G. 2009 Determination of relative protein degradation activity at
different life stages in rainbow trout (Oncorhynchus mykiss). Comparative Biochemistry and
Physiology Part B. 152:150-160.
Campbell, N., Overturf, K. and Narum, S. 2009 Characterization of 22 novel single nucleotide
polymorphism markers in steelhead and rainbow trout. Molecular Ecology Resources
9:318-322.

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Issues of Concern Statement
Animal Care:
This research involves rainbow trout (Oncorhynchus mykiss), and all studies conducted as part
of this project will adhere to policies and conditions approved by the Animal Care & Use
Committee, University of Idaho.
Endangered Species:
No endangered species will be used in this project
Environmental Impact Statement:

All experiments of this project will be conducted in laboratories and raceway facilities of the
University of Idaho and the U.S. Fish and Wildlife Service/BFTC. The research project has been
examined for potential impacts on the environment and has been found to be categorically
excluded under ARS regulations for the National Environmental Policy Act.
Human Study Procedure:

No humans will be subjects of the experiments of this project.
Laboratory Hazards:
This research involves working with hazardous and radioactive materials. All required permits
have been received. All hazardous and radioactive materials are handled with appropriate
protective clothing and used in fume hoods or approved space as required. All wastes are
documented, collected, and disposed of in accordance with established regulations and location
safety plans. No radioactive materials will be used at the HFCES.
Occupational Safety & Health:

The Station participates in a Occupational Safety and Health Program as outlined by the
University of Idaho and the USDA/ARS. Employees have yearly training on OSH issues and
inspections of the facility by both the University of Idaho Occupational Safety and Health officer
as well as USDA/ARS/PWA Safety and Health Manager.
Recombinant DNA Procedures:

No recombinant DNA procedures will be used in this project.
Homeland Security
Intellectual Property Issues

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Appendix
Page Description
47 Figure 1
48 Figure 2
48 Figure 3
Table 1
ARS Collaborators
50 Letter of Collaboration, Jeffery Silverstein, USDA/ARS, National Program 106
51 Letter of Collaboration, Caird Rexroad, USDA/ARS, National Program 106
52 Letter of Collaboration, Harmeet Guraya, USDA/ARS, National Program 306
53 Letter of Collaboration, Kevin Hicks, USDA/ARS, National Program 307
54 Letter of Collaboration, Steve Rawles, USDA/ARS, National Program 106
55 Letter of Collaboration, Chhorn Lim, USDA/ARS, National Program 106
Other Federal Agency Collaborators

56 Letter of Collaboration, Bob Mooth, DOI, US Fish and Wildlife Service
57 Letter of Collaboration, Shawn Narum, CRITFC
University Collaborators
57 Letter of Collaboration, Gary Thorgard, Washington State University
58 Letter of Collaboration, Matt Rise, University of Wisconsin
59 Letter of Collaboration, Ron Hardy, University of Idaho
61 Letter of Collaboration, Wendy Sealey, University of Idaho
Industry and Non-Profit Collaborators

62 Letter of Collaboration, Steve Summerfelt, Freshwater Institute
64 Letter of Collaboration, Richard Towner, GenTec Consulting

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Table 1
Example of a few of the genes currently in use for panel analysis.
Metabolic Muscle growth and Protein turnover

development
Aldolase B Mef2A Calpains 1 and 2
Aspartate aminotransferase Mef2C Calpastatin long and short
Acyl CoA dehydrogenase Murf4 Caspase 3, 8, and 9
Δ5, Δ6 and Δ9 desaturases Myf5 Cathespin L and D
Elongase Myogenin Proteasome 20
Fructose 1,6 bisphosphatase Myostatin 1 and 2
Glucokinase MyoD 1 and 2
Glutamate dehydrogenase Murf1
Glutathione S transferase FoxO
Glutathione peroxidase Conserved edge expressed pr
hexokinase
PPAR α,β,γ
Phosphoglucomutase
Phosphoglycerate kinase
Pyruvate carboxylase
Pyruvate dehydrogenase
Pyruvate kinase
TNFα
Transaldolase

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Table 2 Composition of fish meal control diet

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65 Letter of Collaboration, Chris Nelson, Silver Cup Feeds
66 Letter of Collaboration, Clifford Bradley, Montana Microbial Products

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Figure 1. Integrated development of Improved grains, feeds and trout.

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Figure 2. Growth (% of initial weight) of an unselected strain of rainbow trout (R9) versus a
growth selected strain (House Creek) fed methionine deficient (Met (-)) or methionine replete
(Met (+)) diets.

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Figure 3. Protein retention efficiency of an unselected strain of rainbow trout (R9) versus a
growth selected strain (House Creek) fed methionine deficient (Met (-)) or methionine replete
(Met (+)) diets.

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United States Department of Agriculture

Research, Education and Economics
Agricultural Research Service
June 1st, 2009
Dr. Frederic T. Barrows, Ph.D.

Lead Scientist and Fish Nutritionist
USDA/ARS/SGPGRU
Hagerman Fish Culture Experiment Station
3095F National Fish Hatchery Road
Hagerman, ID 83332
Dear Dr. Barrows:
I would like to confirm my support and willingness to collaborate with you and Dr. Gibson
Gaylord on Objective 3: Determine the nutritional value of alternative ingredients and develop
practical feed formulations for improved strains of fish and Objective 4: Determine optimal
nutrient supplementation levels for specific life stages of fish of the USDA/ARS CRIS project
titled “Improving Sustainability of Rainbow Trout Production by Integrated Development of
Improved Grains, Feeds, and Trout”.
As the goals of your project are closely allied with those of ours,“Integrated Approaches for
Improving the Efficiency and Sustainability of Morone and Other Warm Water Fish
Production”, collaboration in the areas of fish meal/oil replacement and dietary supplementations
will not only help optimize diets containing alternative proteins and oils for carnivorous fish but
will also more efficiently leverage NP106 assets at ARS facilities in Hagerman, Idaho, Bozeman,
Montana, and Stuttgart, Arkansas. To that end, I look forward to determining digestibility
estimates, helping formulate practical diets, designing feeding trials, and applying multivariate
statistics in research to evaluate novel protein and oil substitutes in diets for both trout and hybrid
striped bass.
Sincerely,
Steven D. Rawles, Ph.D.

Fish Nutritionist
Harry K. Dupree - Stuttgart National Aquaculture Research Center P.O. Box 1050 • 2955 Highway 130 East • Stuttgart, AR
72160-1050 Phone: (870) 673-4483 • Fax: (870) 673-7710 • E-mail: [email protected]

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Barrows, 5366-21310-003-00D, Improving Sustainability of Rainbow Trout Production by Integrated

John Michael Bonman___________________ 6/29/2009____

John Michael Bonman___________________ 6/29/2009_____

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Need for Research………………………………………………..…………….……………..2

Scientific Background ……………………………….…………………………….…………3

Approaches and Procedures ………………………………………………………………13

Milestones and Outcomes ……………….…………………………………………………26

Literature Cited ……………………………………………………………………………….43

Past Accomplishments ……………..………………………………………………………52

Issues of Concern Statements ……………….……………………………………………67

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

The specific objectives are:

Objective 3: Determine nutritional value of alternative ingredients (protein, lipid, energy)and

Need for Research

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Evaluation of alternative ingredients and development of complete feeds

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

The benefits of BG consumption on human health have attracted substantial attention.

Objective 2; Ingredient Enhancement. A limited number of ingredients are currently used in

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Objective 3; Alternative Ingredients and Practical Feeds. Determination of the nutritional

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Objective 4; Optimal Supplementation of Practical Diets. Relative to terrestrial species,

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Objective 6; Identify Phenotypic Differences; To fully realize the potential of sustainable

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

APPROACHES AND RESEARCH PROCEDURES

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Sub-objective 1.B. Genetic mapping of the BG mutations.

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Objective 2: Ingredient Modification (Lead: Liu; Support: Barrows)

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Sub-objective 2.B. Improve nutritional and economic values of biofuel coproducts.

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Objective 3; Alternative ingredients and practical feed formulations. (Lead: Barrows;

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Sub-objective 3.B. Development and evaluation of alternative feed formulations.

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Objective 4: Refined mineral supplementation for plant-based diets (Lead;Barrows;

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Objective 5: Gene expression analyses (Lead; Overturf; Support; Barrows)

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Sub-objective 6.A. Identify variation in physiological performance of rainbow trout that is

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

Physical and Human Resources:

September 29, 2009 106 Barrows 5366-21310-003-00D PostPlan

John Michael Bonman_______________ 6/29/2009

John Michael Bonman_______________ 6/29/2009_