UTILIZATION OF SOYBEAN PRODUCTS AS FISH-MEAL PROTEIN REPLACEMENTS
IN YELLOW PERCH Perca flavescens FEEDS
BY
SCOTT C. SINDELAR
A thesis submitted in partial fulfillment of the requirements for the
Master of Science
Major in Wildlife and Fisheries Sciences
Specialization in Fisheries Sciences
South Dakota State University
2014
iii
ACKNOWLEDGEMENTS
I thank the following persons for making this study possible: Dr. Michael Brown
for his unparalleled mentoring and assistance throughout the study, Dr. David Willis for
admitting me into the program, Dr. William Gibbons for providing the soy ingredients,
Dr. Steven Chipps for serving on my committee, South Dakota Game, Fish, and Parks for
providing the fish, Michael Grey for helping with method development, Timothy Bruce
for assisting in analysis, Dustin Shultz for assisting in sampling, and Michael Barnes for
assisting with histology. I also thank Erinn Ipsen, Caleb Green, Hunter Brown, Cody
Treft, Tabor Martin, Janae Oein, and Jacob Fernholtz for their assistance. A special
thanks to my wife Alissa, son Timber, and daughter Melrose, for their support,
encouragement, and understanding throughout my difficult graduate study. Also, thanks
to my parents, Jim and Wanda for support and encouragement throughout my academic
career.
Support for this research was provided by the South Dakota Soybean Research
and Promotion Council, United Soybean Board, South Dakota Game, Fish and Parks,
South Dakota State University Department of Natural Resource Management, and South
Dakota Agricultural Experiment Station. Fish handling and sampling procedures were
conducted in compliance with the Animal Welfare Act (South Dakota State University
Animal Care and Use Committee protocol approvals #11-62A and 12-101A).
iv
TABLE OF CONTENTS
List of Abbreviations ........................................................................................................viii
List of Figures .................................................................................................................... xi
List of Tables .................................................................................................................... xii
Abstract ..............................................................................................................................xv
CHAPTER 1. ALTERNATIVE FEEDSTUFFS IN AQUACULTURE NUTRITION
RESEARCH
Introduction ..............................................................................................................1
Hypotheses and Research Objectives ......................................................................5
Soybean Products .....................................................................................................6
Antinutritional Factors ...........................................................................................10
Processing Methods ...............................................................................................15
Full-Fat Flours and Grits ..........................................................................16
Soy Oils ......................................................................................................16
Soybean White Flake..................................................................................18
Soybean Meal .............................................................................................18
Extruded Soy Protein .................................................................................19
Soy Protein Concentrate ............................................................................20
Soy Protein Isolate .....................................................................................22
Bioprocessed Soy Protein ..........................................................................22
Yellow Perch Culture .............................................................................................24
Recirculating Aquaculture System (RAS) .............................................................26
v
Water quality ..........................................................................................................28
Feed Formulation and Nutritional Requirements ..................................................33
Proteins and Amino Acids..........................................................................35
Lipids and Fatty Acids ...............................................................................36
Carbohydrates............................................................................................37
Minerals .....................................................................................................38
Vitamins .....................................................................................................39
Other Supplements .....................................................................................40
Pelleting .................................................................................................................41
Feeding...................................................................................................................42
Summary ................................................................................................................44
CHAPTER 2. BIOPROCESSED SOY DIGESTIBILITY IN JUVENILE YELLOW
PERCH
Introduction ............................................................................................................55
Methods and Materials ...........................................................................................57
Experimental Diets and Fish .....................................................................57
Culture System ...........................................................................................58
Feeding ......................................................................................................59
Sample Collection ......................................................................................59
Compositional Analysis .............................................................................60
Results ....................................................................................................................60
Discussion ..............................................................................................................62
Summary ................................................................................................................65
vi
CHAPTER 3. PERFORMANCE OF YELLOW PERCH FED BIOPROCESSED WHITE
FLAKE
Introduction ............................................................................................................71
Methods and Materials ...........................................................................................73
Experimental Design and Diet Formulation..............................................73
Compositional Analysis .............................................................................75
Culture System ...........................................................................................75
Feeding Trial .............................................................................................76
Physical Pellet Properties..........................................................................78
Statistical Analysis .....................................................................................79
Results ....................................................................................................................80
Soy Ingredient Composition.......................................................................80
Digestibility ................................................................................................81
Survival, Growth, and Health Performance ..............................................81
Physical Pellet Properties..........................................................................83
Discussion ..............................................................................................................84
Summary ................................................................................................................89
CHAPTER 4. PERFORMANCE OF YELLOW PERCH FED BIOPROCESSED
SOYBEAN MEAL
Introduction ............................................................................................................96
Methods and Materials ...........................................................................................97
Experimental Design and Diet formulation ...............................................97
Culture System ...........................................................................................98
vii
Feeding Trial .............................................................................................99
Statistical Analysis ...................................................................................101
Results ..................................................................................................................101
Discussion ............................................................................................................104
Summary ..............................................................................................................105
REFERENCES ................................................................................................................113
viii
LIST OF ABBREVIATIONS
ADC ................................................................................. Apparent digestibility coefficient
ADC-P.................................................................. Apparent digestion coefficient of protein
ADC-E .................................................................. Apparent digestion coefficient of energy
AOB ......................................................................................... Ammonia oxidizing bacteria
ANF..................................................................................................... Antinutritional factor
ANOVA ................................................................................................Analysis of variance
ANCOVA ......................................................................................... Analysis of covariance
BD ..................................................................................................................... Bulk density
BP....................................................................................................................Bioprocessing
CG ...................................................................................................... Compensatory growth
°C ................................................................................................................... Degree celsius
db............................................................................................................................ Dry basis
DDG .................................................................................................... Distillers dried grains
DDGS............................................................................ Distillers dried grains with solubles
DE ............................................................................................................. Digestible energy
df ............................................................................................................ Degrees of freedom
DHA ...................................................................................................Docosahexaenoic acid
dm ........................................................................................................................ Dry matter
DO ............................................................................................................. Dissolved oxygen
EAA ..................................................................................................... Essential amino acid
EPA .................................................................................................... Eicosapentaenoic acid
FCR ..................................................................................................... Feed conversion ratio
ix
FM ................................................................................................. Marine-derived fish meal
g.................................................................................................................................... Gram
GE .................................................................................................................... Gross energy
GMO .................................................................................... Genetically modified organism
HSI ....................................................................................................... Hepatosomatic index
K............................................................................................... Fulton-type condition factor
Kcal ......................................................................................................................Kilocalorie
Kg........................................................................................................................... Kilogram
L ..................................................................................................................................... Liter
Lpm ............................................................................................................ Liters per minute
Lys...............................................................................................................................Lysine
Met ...................................................................................................................... Methionine
MC ............................................................................................................. Moisture content
Min ............................................................................................................................. Minute
MJ ....................................................................................................................... Mega Joule
m .................................................................................................................................. Meter
µg .........................................................................................................................Microgram
mm ....................................................................................................................... Millimeter
NEAA ........................................................................................... Non-essential amino acid
NFE ............................................................................................................ Non-fiber extract
NOB .............................................................................................. Nitrite oxidizing bacteria
NSP ............................................................................................. Non-starch polysaccharide
n.................................................................................................................... Sample number
x
P ................................................................................................................. Probability value
PDI ..................................................................................................... Pellet durability index
PDII ............................................................................................Protein dispersability index
PER ................................................................................................... Protein efficiency ratio
RG ................................................................................................................ Relative growth
SBM ................................................................................................................ Soybean meal
SE ....................................................................................................................Standard error
SPC ..................................................................................................Soy protein concentrate
SPI ............................................................................................................Soy protein isolate
VFD................................................................................................Variable frequency drive
VFI ............................................................................................................ Visceral fat index
VSI .................................................................................................... Visceral somatic index
TAN ................................................................................................ Total ammonia nitrogen
TIA ................................................................................................ Trypsin inhibitor activity
TIU .....................................................................................................Trypsin inhibitor units
U.S. .................................................................................................................. United States
USD........................................................................................................ United States dollar
UV ........................................................................................................................ Ultraviolet
wb........................................................................................................................... Wet basis
WSI .................................................................................................... Water solubility index
xi
LIST OF FIGURES
Figure 1-1. Average commodity price of FM and SBM over the previous 10 years (World
Bank 2014) .............................................................................................................49
Figure 1-2. Total global fish production by primary production source (FAO 2012b) .....50
Figure 1-3. Total value of U.S. Atlantic Salmon imports for the previous 26 years (USDA
2014) ......................................................................................................................51
Figure 1-4. Total world production of FM and SBM over the previous 10 years (USDA
2014) ......................................................................................................................52
Figure 1-5. General soybean protein and oil processing flow diagram (modified from
Swick 1994; Brown and Hart 2010) ......................................................................53
Figure 1-6. Total production of Yellow Perch by method and source...............................54
Figure 4-1. Average relative growth (RG, %) response standardized to reference diet
([treatment average RG] – [reference average RG]). Dark bars indicate 40% FM
replacement, light bars indicate 70% FM replacement. Different overhead line
ranges indicate significant difference (P<0.05) among treatments. .....................112
xii
LIST OF TABLES
Table 1-1. Trypsin inhibitor activity (TIA) of soybeans and soy products .......................46
Table 1-2. Proximate composition of commercially processed soy products (g/100g dm
except where noted) ...............................................................................................47
Table 1-3. Various Microbes, process (solid state fermentation-SSF, and liquid state
fermentation-SLF), and feedstock used in fermentation of soy products. .............48
Table 2-1. Composition of the primary protein sources (g/100 g, dm unless noted)
incorporated into the experimental digestibility diets for Yellow Perch ...............67
Table 2-2. Digestibility feed formulations and estimated dietary composition of the
reference and test diets (g/100g, dm). Ranges are given for soy diets only ..........68
Table 2-3. Apparent digestibility coefficients (ADC) of protein (ADC-P) energy (ADCE) for test ingredients fed to Yellow Perch ............................................................69
Table 2-4. Apparent digestibility coefficients (ADC) of essential amino acids (EAA) and
non-essential amino acids (NEAA) for experimental bioprocessed soy products
fed to Yellow Perch ...............................................................................................70
Table 3-1. Feed formulations (g/100g dm) of the experimental Yellow Perch diets. .......90
Table 3-2. Composition of the primary protein sources (g/100 g, dm unless noted)
incorporated into the experimental diets. EAA=essential amino acids;
NEAA=non-essential amino acids; ANFs=antinutritional factors ........................91
Table 3-3. Analyzed diet compositions (g/100g dm unless noted) and digestibility results.
ADC=Apparent Digestibility Coefficient; GE=gross energy; DE=digestible
energy; PE=protein to energy ratio; PDE=protein to digestible energy ratio ........92
xiii
Table 3-4. Growth performance indices. Values given are treatment means±SE. Values
not significantly different (P>0.05) have the same letter within a given column. .93
Table 3-5. Necropsy variables for Yellow Perch. Values given are treatment means±SE.
Values not significantly different (P>0.05) have the same letter within a given
column GI=gastrointestinal distal inflammation score; HSI=hepatasomatic index;
VSI=viscerasomatic index; VFI=visceral fat index ...............................................94
Table 3-6. Physical properties of the feed extrudates. Values given are treatment
means±SE. Values not significantly different (P>0.05) have the same letter within
a given row.............................................................................................................96
Table 4-1. Primary ingredients, fish meal (FM) replacement levels, and coding of FM and
SBM treatments that are either Extruded (Ex), Bioprocessed (BP), or GMO
variety ..................................................................................................................107
Table 4-2. Proximate composition, trypsin inhibitor activity (TIA), and amino acid profile
of the main protein sources. Values are g/100 g dm unless noted.
ANF=antinutritional factor; EAA=essential amino acid; NEAA=non-essential
amino acid. ...........................................................................................................108
Table 4-3. Feed formulations and nutrient composition including only high level FM
replacement (70%) treatments. All values reported as g/100 g dm .....................109
Table 4-4. Feed formulations and nutrient composition including only low level FM
replacement (40%) treatments. All values reported as g/100 g dm .....................110
Table 4-5. Survival (S, %), mean relative growth (RG, %), specific growth rate (SGR),
Fulton-type condition factor (K), total tank consumption (TC, g), food conversion
ratio (FCR), and protein efficiency ratio (PER) for Yellow Perch fed experimental
xiv
diets. Values given are treatments means±SE. Significant differences (P<0.05)
from one-way analysis of variance (ANOVA) are indicated by different letters
within a given column ..........................................................................................111
xv
ABSTRACT
UTILIZATION OF SOYBEAN PRODUCTS AS FISH-MEAL PROTEIN
REPLACEMENTS IN YELLOW PERCH Perca flavescens FEEDS
SCOTT C. SINDELAR
2014
As a result of increasing demand, uncertain availability, and increasing cost for
fish meal (FM), fish nutritionists have been driven to find alternative sources of protein.
Particularly for carnivorous species such as Yellow Perch Perca flavescens (Mitchell),
alternative plant-based feedstuffs have shown decreased performance compared to FM.
Processed (mechanical, chemical, or microbiological) soybean products are of primary
interest as alternative feedstuffs due to their availability, domestic production, low price
and nutritional profile; however, use of processed soy has displayed varying degrees of
success. Novel soy ingredients created through bioprocessing technology potentially
provide more highly digestible nutrients with fewer anti-nutritional factors (ANFs) than
traditionally processed soy products. To examine the utility of certain bioprocessing
technologies, ingredient compositions were compared and incorporated into three Yellow
Perch feeding trials.
The first feeding trial was completed to measure apparent digestibility of protein
and energy of commercial and experimental bioprocessed soybean meal (BP-SBM), and
other common feed protein ingredients. Bioprocessing consistently upgraded the
bioavailability of protein in defatted soybean meal, while commercially bioprocessed
xvi
Hamlet HP 300 provided the highest digestibility of all soy products tested. Extrusion as
a pretreatment to bioprocessing had a negative effect on digestibility.
The second experiment was a 14-week feeding trial to evaluate growth
performance, digestibility, and organosomatic responses of Yellow Perch fed diets
containing commercial alcohol-washed soy protein concentrate (SPC), bioprocessed soy
white flake (BP-WF), or extrusion pretreated BP-WF as complete FM replacements.
Each soy protein source was replicated in diets with and without supplemental lysine +
methionine. BP diets consistently performed better than SPC diets. Extrusion
pretreatment led to improved consumption and growth performance over unextruded
products. Lysine and methionine supplementation further improved growth. SPC diets
provided the best feed conversion ratio (1.62) and highest apparent digestibility of protein
(91.8%), but examination of anatomical characteristics revealed significant deficiencies
in fish fed SPC diets.
In the final 16-week feeding trial we evaluated growth performance, feed intake,
and organosomatic responses of Yellow Perch fed diets containing various processed
SBM products. Treatment factors included soybean variety (GMO or non-GMO),
extrusion pretreatment, BP, and FM replacement levels (40 and 70%). An FM control and
14 soy-based diets were formulated on a dry matter basis to contain similar crude protein
(45%), lipid (9%), and gross energy (20.9 MJ/Kg). Five soy treatments displayed higher
relative growth (RG) than the FM control diet and nine were lower. A non-GMO, nonextruded, BP-SBM provided enhanced growth at both inclusion levels when compared to
FM. Both varieties of commercial SBM provided similar growth to the reference diet at
the low FM replacement level, but growth was depressed at the higher level.
1
CHAPTER 1. ALTERNATIVE FEEDSTUFFS IN AQUACULTURE NUTRITION
RESEARCH
Introduction
The future of aquaculture and aquafeed industry growth will depend greatly on
the use of alternative ingredients to displace fish meal (FM) and fish oil (FO) (Glencross
et al. 2007; FAO 2012a, 2014). FM has been a primary driver of high feed costs, which
can comprise over 50% of total production cost (FAO 2009). In June of 2014, the average
price of 64-65% protein FM ($2,001 per metric ton) was approximately 386% greater
than the price of soybean meal (SBM, $519 per metric ton) (Figure 1-1; World Bank
2014), and within the next decade, FM and FO price are forecasted to minimally increase
by 50% (FAO 2012a, 2014). Grain production is more stable and efficient than FM
production and will continue to cost much less than fixed supplies of FM products (FAO
2012a, 2014). Recent development of aquafeeds has been significant, which is apparent
from decreasing feed conversion ratios (FCR); 29.2 million tons were produced in 2008
with substantial increases forecasted (FAO 2012a).
World population growth has created an increased demand for seafood, a source
of high quality protein, which has been supported with increased aquaculture production
(Figure 1-2). The worldwide aquaculture industry has sustained an average growth of
8.8% per year for the last three decades, greater than any other food production sector
(FAO 2012a). For example, aquaculture production of Atlantic Salmon Salmo salar has
greatly surpassed production through wild capture fisheries, and has sustained a rapidly
growing import value for the previous 26 years (Figure 1-3). In 2013, the United States
imported $33.2 billion USD in seafood and other fisheries products (edible and non-
2
edible) while $23.0 billion was exported, creating a trade deficit of $10.2 billion (NOAA
2013). The U.S. contributes very little to world aquaculture volume compared to several
other countries around the world; especially China, which produced 60% of global
aquaculture volume in 2010 (FAO 2012a). Conversely, the U.S. imports at least 60% of
all fish products, including seafood, FM and FO, which were valued collectively at $17.5
billion in 2011 (FAO 2012a). By the year 2020, the world will require an estimated 23
million tons of seafood and other fish products if the current per-capita consumption
trend continues (FAO 2012a).
The most recent global production data (USDA 2014), shows that approximately
193 million metric tons more SBM was produced than FM, and over the past 10 years
SBM production increased by 126%, while FM production decreased by 35% (Figure 14). Although seafood production from wild-capture fisheries has remained relatively
constant (FAO 2014), production of both FM and fish oil from marine-derived sources
have shown reduced volumes in recent years and are forecasted to continue falling
(Jackson 2012). Additionally, aquaculture that utilizes FM as a primary protein source
actually consumes more volume of FM for feeding than volume in fish produced (i.e. fish
in:fish out; Naylor et al. 2000). Competition for FM and FO used in swine, poultry,
ruminants, and companion animal feeds also exacerbates inconsistency and uncertainty in
future availability. Additionally, the European Union’s stringent regulations including
banning on the use of animal-derived ingredients in feeds for ruminant animals destined
for human food (EU Regulations, 2009) has also compelled research on improving plant
ingredients for monogastrics. Understandably, the future development of aquafeeds and
3
aquaculture cannot sustainably utilize FM and fish oil as primary ingredients and
maintain cost-effective production.
In addition to sustainable and economic production, the ultimate goal of plantbased feed research is to produce highly bioavailable feeds that are low-polluting and
provide growth performance equivalent to FM-based feeds. Culture of carnivorous fishes
is particularly challenging given high quality nutrient requirements. Plant-based
aquafeeds have been successful in many omnivorous aquaculture species, such as carp
and tilapia (FAO 2009); however, carnivorous species such as Rainbow Trout
Oncorhyncus mykiss and Yellow Perch cannot utilize raw plant material effectively
(Gatlin et al. 2007; Yamamoto et al. 2010).
There has been considerable aquaculture research on a variety of processed plant
feedstuffs including soybeans (Kaushik et al. 1995), canola (Higgs et al. 1982), lupin
(Glencross et al. 2003), corn (Webster et al. 1991), and others (Aslaksen et al. 2007). The
high available protein content of soybeans and soy products make them one of the most
valuable alternative ingredients for carnivorous fish diets. Additionally, high production
and availability makes soybeans the most commonly used plant protein in aquaculture
feeds. However, the upper limit of inclusion for defatted SBM is between 20-30% in
carnivorous fish diets (Storebakken et al. 2000). Without further processing, SBM and
other plant products contain high levels of indigestible complex carbohydrates such as
fiber, oligosaccharides, and other non-starch polysaccharides (NSPs), which are not
efficiently utilized by carnivorous fish. Other antinutritional factors (ANFs) such as
trypsin inhibitors and phytate found in plant materials can also have negative impacts on
fish growth and health. For this reason, plant ingredient processing techniques are
4
essential to minimize carbohydrates and ANFs, meanwhile increasing usable nutrients
(Gatlin et al. 2007). Soy protein concentrate (SPC) or soy protein isolate (SPI) products
are better utilized by carnivorous fish (Storebakken et al. 2000). However, high price
hampers use of SPC and SPI as complete FM replacements. Local sourcing of ingredients
can also facilitate a more consistent supply and lower shipping costs. Biotechnology is
rapidly expanding in several industries, and has created great interest in the economic
production of enhanced soy and other feed products (Pandey et al. 2000; Chen et al.
2013).
In the U.S., soybeans are one of the largest and most important oilseed
commodities, with the Midwestern states producing the majority of soybeans and
soybean products (USDA 2007). Iowa, Illinois, Minnesota, Indiana, Ohio, Nebraska,
Missouri, South Dakota, North Dakota, and Arkansas continue to be the to be the top 10
producers, which collectively account for 82.8% of the total domestic soybean production
(USDA 2007). Because soybean products are considered to be the most promising
alternative to FM (Dersjant-Li 2002), there is considerable opportunity to develop new
aquafeed markets using plant-based alternative ingredients. Additionally, aquaculture
production is progressing in the Northern Great Plains, and the development of
sustainable, alternative feed production is well supported by the soybean industry
(USSEC 2008).
The purpose of this research is to assess composition, nutrient availability, and
fish performance responses to several soybean products produced through various
commercial and novel processing technologies. The ultimate goal is reducing dependence
on FM while stimulating the advancement of aquaculture for a relatively undeveloped
5
aquaculture species (Yellow Perch). This research also supports improving food
sustainability and security for a rapidly growing population. Increasing global food
demand requires more efficient food production technologies that improve the
sustainability of earth’s renewable natural resources.
Hypotheses and Research Objectives
Four main objectives were developed to examine the utility of different soy
processing technologies when used to replace FM in Yellow Perch feeds. Initially we
characterized composition with in vitro chemical analysis (Objective 1), progressing to in
vivo bioavailability (Objective 2), and ending with complementary growth, health, and
feed intake trials (Objectives 3 and 4). Based on a literature review of alternative
feedstuffs, bioprocessed soy has been shown to have beneficial properties for carnivorous
fish like Rainbow Trout (Yamamoto et al. 2010), Atlantic Salmon (Refstie et al. 2005),
Hybrid Striped Bass Morone chrysops x M. saxatilis (Rombenso et al. 2013) and
sensitive life stages of other domestic food animals (Hung 2008). Therefore, we
hypothesized that bioprocessing would significantly affect the performance and
efficiency of soy products in aquafeeds for Yellow Perch. Consequently, the primary
research objectives included:
1) Analysis of nutrient composition and antinutritional factors (ANF) of FM,
commercial SPC, and various forms of bioprocessed soybean meal (BP-SBM). The scope
of analysis included proximate composition, amino acids, minerals, and primary ANFs.
Commercial and bioprocessed ingredients were selected based on differences in primary
processing method.
6
2) Characterize primary nutrient bioavailability by utilizing in vivo apparent
digestibility determinations for nutrients in Yellow Perch feeds. Apparent digestibility
trials were completed for each main ingredient to determine potential inefficiencies,
processing effects, and also to provide recommendations for future feed formulations
based on digestible nutrients.
3) Evaluate Yellow Perch growth performance using practical feeds replacing FM
with commercial or bioprocessed soy products. Growth performance, health, and feed
intake were compared in two feeding trials. Supplements and inclusion rates were also
investigated using factorial designs.
Soybean Products
Soybeans Glycine max are legume oilseeds that have long been recognized as an
alternative or supplemental protein source to fish meal (FM). Soybean products (proteins,
oils, and lecithin) have shown promising results in several aquatic species, and some soy
products are routinely used in commercial feeds (Forster 2002). Soybean products are
generally preferred over other plant products, due to their high protein content, favorable
amino acid profile, high digestibility, low cost, consistency and domestic availability.
Additional to nutrition, the physical characteristics of soy provide functional benefits for
food and feed production (Thomas and Poel 2001; Dersjant-Li 2002).
Whole soybeans provide a substantial amount of protein (35-40% db) and lipid
(17-25%) (Wolf 1970). Whole beans are more valuable when separated into their
constituent carbohydrates, protein, and oil (Peisker 2001; Chen et al. 2013). Beans are
first crushed into flakes and the hulls are then removed which creates white flake (WF)
7
(Peisker 2001). Soybean oil is subsequently solvent extracted leaving soybean meal
(SBM), which is the most common soy product used in animal feeds (Drew et al. 2007).
Soy protein concentrates (SPC) and isolates (SPI) are further refinements of the
soybean’s valuable protein (Peisker 2001).
Soybeans often provide a more compositionally consistent product than FM;
however, soy products can vary in composition depending on growing conditions, genetic
strain and processing conditions (Swick 2007). These variations can influence price and
quality of soy products (Swick 2007). Like most plant ingredients soybeans are especially
well known to differ in nutritional characteristics depending on soil type, precipitation,
climate, and cultivar. In lupin products for example, the digestibility of protein and
energy differs substantially between cultivars (Glencross et al. 2004). Soybean strains
developed and grown in the U.S. generally provide a higher lysine to crude protein ratio
than other strains (Swick 2007).
Due to high levels of proteins and stachyose sugars, soybean plants are also
known for their ability to undergo periods of desiccation, and then continue to grow when
conditions are more favorable (Blackman et al. 1992). However, the components that
protect the plant (e.g., stachyose, trypsin inhibitors) are not utilized or are inhibitory in
carnivorous fishes and therefore must be removed through processing. Several
indigenous or genetically modified soybean strains have been found to contain varying
amounts of antinutritional factors and oligosaccharides (Parsons 2000), which may be
more suited for carnivorous fish feeds (Gatlin 2007).
Beneficial effects of soy processing on growth and digestibility have occurred in
herbivorous, omnivorous, and carnivorous fishes (Kim and Kaushik 1992; Gatlin 2002).
8
Various soy processing technologies affect ANFs differently, and their removal can
improve digestibility, palatability, and growth performance. One of the primary
processing technologies used to inhibit ANFs is thermal treatments. ANFs can be either
heat stable or heat labile, whereby the latter can easily be removed by commercial
toasting methods for SBM (Francis et al. 2001). Thermal treatments, however, can also
damage proteins making them unavailable for fish for growth (Glencross et al. 2007).
Lysine is one of the amino acids most susceptible to overheating, and the total amount of
lysine can diminish. The lysine to crude protein ratio or a reactive lysine assay are
common methods used to test for heat damaged soy products (Fontaine et al. 2007).
Other approaches to test the degree of soy processing include urease activity, protein
dispersibility index (PDII), and potassium hydroxide solubility (Batal 2000). The sulfur
amino acids and threonine have also been found to be sensitive to heat damage
(Dhurandhar and Chang 1990; Olli et al. 1994b). Heat stable ANFs include saponins,
NSPs, antigenic proteins, phytoestrogens, and phenolic compounds (Van der Poel 1989)
and must be removed using other processes such as alcohol washing, activated carbon
exposure, or chromatography. Heat damage has also been associated with color, where
lighter color indicates higher availability of lysine. For example, darker color of dried
distillers grains with solubles (DDGS) was found to have reduced amino acid digestibility
in swine and poultry (Fastinger and Latshaw 2006; Fastinger and Mahan 2006).
There are several other processes that can enhance the quality of protein. For
example, creating shorter-chain peptides or reducing allergenic protein fractions is
important. For this reason, PDII is commonly used as a measure of soy protein quality as
well as a measure of heat damage (Lusas and Riaz 1995; Thomas and Poel 2001). Higher
9
PDII values typically indicate higher solubility, higher enzyme activity, higher ANFs,
and lower protein efficiency in animals (Horan 1974; Lusas and Riaz 1995). However,
Barrows et al. (2007) found no significant relationship between PDII and growth of
Rainbow Trout.
The carbohydrate fraction of soy is made up of sucrose, oligosaccharides (αgalactosides), and mostly (20-30% of defatted SBM) non-starch polysaccharides (NSPs;
i.e. cellulose, hemicellulose, pectin, β-glucans, and gums) (Krogdahl et al. 2005; Gatlin et
al. 2007;Choct et al. 2010). Certain carbohydrates such as hemicelluloses, pectins, and
starches are also useful pellet-binding agents (Krogdahl et al. 2005). Other carbohydrate
fractions such as sucrose, are simple sugars which readily available to fish.
Oligosaccharides and most NSP’s are more complex and are indigestible by most fishes
(Refstie et al. 1998; Krogdahl et al. 2005; Choct et al. 2010) and other monogastric
animals (Zdunczyk et al. 2011).
The primary carbohydrate fraction in soy is NSP, which is associated with
reduced protein and lipid digestibility (Refstie et al. 1999) as well as a positive
correlation with increased water content of feces (Gatlin et al. 2007). NSPs have also
been linked to soy-induced enteritis in salmonids (Bureau et al. 1998; Yamamoto et al.
2008), but it appears that inflammation might be reversible if subsequently fed an FMbased diet (Baeverfjord and Krogdahl 1996). Hybrid Striped Bass Morone saxatilis x M.
chrysops (Gallagher 1994) and Rainbow Trout (de la Higuera et al. 1988) were also
found to have reduced feed intake when fed high levels of NSP.
Oligosaccharides occur in SBM primarily in the forms of sucrose (6-7%),
raffinose (1-2%), and stachyose (5-6%) (Francis et al. 2001). These complex
10
oligosaccharides can have a prebiotic effect by creating a fermentable substrate for
beneficial gastrointestinal microflora (Zdunczyk et al. 2011). However, the majority of
these sugars are generally considered antinutritional (Francis et al. 2001) and should be
kept at low dietary levels for all carnivorous fishes. Salmonids in particular are known to
exhibit a species-specific response to dietary oligosaccharides (Francis et al. 2001).
Beyond direct oligosaccharide inefficiencies, their removal has also been reported to
improve digestibility of the NSP portion in poultry diets (Slominski et al. 1994).
Antinutritional Factors
The use of soy products in carnivorous fish feeds is met with difficulty because
soybeans, like other plant products, contain several ANFs including protease inhibitors,
lectins, phytic acid, saponins, phytoestrogens, antivitamins, and antigenic compounds
(Francis et al. 2001). ANFs including NSPs and oligosaccharides are contained in various
levels in all plant products, and are created by the plant to protect itself from harmful
substances, environmental conditions, or predation from insects or herbivores (Osagie
and Eka 1998; Enneking and Wink 2000). Strategies for reducing ANFs or reducing the
impact from ANFs include processing (e.g., heating, fermentation), genetic manipulation
(e.g., artificial or natural selection), and diet supplementation (e.g., enyzmes) (Enneking
and Wink 2000).
One of the most inhibitory and important of the ANFs are protease inhibitors,
which directly disable the activation of digestive enzymes and reduce feed efficiency
(Francis et al. 2001). Trypsin inhibitors (Table 2-1) are well known protease inhibitors
that inhibit the activation of two protease enzymes: trypsin and chemotrypsin (Norton
11
1991). Trypsin is a powerful serine protease (digestive enzyme) produced in the pancreas,
which cleaves peptide bonds of proteins into smaller peptides or amino acids, which can
then be absorbed through the intestine and used for growth (Hedstrom 2002). Inhibition
of trypsin protease activation leads to reduced digestion, thus decreasing overall feed
efficiency. Soybeans contain two groups of trypsin inhibitors: the Kunitz trypsin inhibitor
and the Bowman-Birk protease inhibitor (Francis et al. 2001). The Bowman-Birk
inhibitor can block two protease molecules and is also more heat-stable than the Kunitz
inhibitor, which can block only one protease molecule (Norton 1991). The Kunitz
inhibitor is also more sensitive to acid treatments (Francis et al. 2001). Generally typical
thermal treatments for SBM or SPC reduce most of the trypsin inhibitor activity (TIA).
Extrusion cooking has also been shown to reduce TIA (Romarheim et al. 2006).
Sensitivity of TIA is particularly high in carnivorous fishes. Impairments in
Rainbow Trout digestion have been attributed to inhibition of both trypsin (Sandholm et
al. 1976) and chemotrypsin (Dabrowski et al. 1989). Fish have also been shown to
respond to protease inhibitor activity by secreting more trypsin and chemotrypsin, or
increasing the surface area of the intestine (Dabrowski et al. 1989; Olli et al. 1994a;
Francis et al. 2001). For most fish, trypsin inhibitor amounts below 5 mg/g do not seem
to inhibit digestibility significantly as enough extra proteases can be produced to mitigate
the inhibitory effect (Francis et al. 2001). However, soybean meal has been reported to
contain up to 15.0 mg/g of trypsin inhibitors (Gao et al. 2013).
Phytate is the major form of phosphorus storage in plants (Han and Wilfred
1988). Phytate (also known as phytic acid or inositol) is a heat-stable cyclic compound
that is found in almost all soy products unless enzymatically hydrolyzed (Liener 1994).
12
Soybeans and typical processed soy products regularly contain 1-2% (db) phytate (Liener
1994; Francis et al. 2001). This organic phosphorus is unavailable in monogastrics and
prevents phosphorus utilization as well as other di- and trivalent metals such as calcium,
magnesium, zinc, copper and iron (Liener 1994; Francis et al. 2001). Additionally,
phytate can form into phytate-protein complexes which reduce protein digestibility, as
well as inhibit nutrient absorption due to toxic damage in the pyloric ceca (Francis et al.
2001). Phytate also contributes to phosphorus pollution contained in effluent (Cain and
Garling 1995). Richardson et al. (1985) found dramatic changes is health, growth, and
mortality of Chinook Salmon Oncorhynchus tshawytscha due to dietary phytate. Those
researchers found that 25.8 g/kg dietary phytate increased mortality, depressed growth,
protein efficiency ratio, and thyroid function, as well as caused cataract formation and
increased vacuolization in the pyloric caeca (Richardson et al. 1985). Therefore, it
appears phytic acid is extremely detrimental to salmonids and possibly other carnivorous
fish at elevated levels. Usually phytate is found in SBM at lower levels than above, and
does not result in dramatic responses in most fishes (Francis et al. 2001).
Due to the concern about mineral deficiency and inefficiency when feeding soy
products containing phytate, the phytase enzyme is often included in dietary
formulations. Phytase can be produced through microbial fermentation methods and
subsequently purified (Han and Wilfred 1988). Hydrolyzing the phytate with phytase
during fermentation can significantly increase the availability of inositol as well as
inorganic phosphorus (Han and Wilfred 1988). Dietary exogenous phytase enzymes have
also been added into the diets of Rainbow Trout, which substantially improved
phosphorus availability of phosphorus in both SBM and FM-based diets (Riche and
13
Brown 1996). The maximum suggested amount of phytate in salmonid diets is about 5-6
g/kg (Francis et al. 2001).
Soybean lectins, or soybean agglutinin (SBA) are glycoproteins which are present
in soy protein products (Liener 1994) and soy oil (Klurfeld and Kritchevsky 1987), and
have been found to cause major digestive problems in rats; however, little information
exists on their effects on fish (Lajolo and Genovese 2002). It is known that the SBA
protein fraction is resistant to digestion in fish (Francis et al. 2001). One study found that
SBA has the ability to disrupt brush border surfaces of the intestine of Atlantic Salmon
(Hendriks et al. 1990). SBA are normally heat-labile, and denaturation requires moist
heating of at least 100°C for 10 min (Francis et al. 2001). The destruction of lectin also
typically parallels that of TIA, but there may be vast differences in lectin levels
depending on the soybean genetic strain (Liener 1994).
Saponins are heat stable steroid or triterpenoid glycosides including galactose,
arabinose, rhamnose, glucose, xylose, and glucuronic acid (Francis et al. 2001; Liener
1994). Both positive and negative effects from saponins have been alleged (Liener 1994).
Saponins exist in defatted toasted SBM up to 67 mg/kg (Fenwick et al. 1991). Saponins
have been linked to bitter taste (Liener 1994), significant intestinal damage, and when
dissolved in water can damage the respiratory epithelium of the gills (Bureau et al. 1998;
Francis et al. 2001). Other research has shown that saponins in solvent-extracted soybean
products did not produce significant negative effects on performance of Atlantic Salmon
(Krogdahl et al. 1995). Saponins have also been found to inhibit the chemotrypsin
protease, which resulted in reduced hydrolysis of total protein and allergenic proteins
(glycinin and β-conglycinin) (Shimoyamada et al. 1998). Although more research is
14
needed on species-specific responses to saponins, recommended levels for most species
are below 1 g/kg of diet (Francis et al. 2001).
Phenolic compounds such as tannins, phytoestrogens, and others (e.g., syringic
acid) are believed to play an important role in creating adverse organoleptic properties in
plant products, as well as affect normal bodily functions of animals (Liener 1994).
Tannins are generally lower in soybeans (0.45 g kg-1) than in other legumes (e.g., up to
20 g kg-1 in faba beans) and so little attention has been paid to this ANF in regard to
soybean products (Liener 1994). Phytoestrogens are also present in soybeans, usually in
the form of glycoside isoflavones (Francis et al. 2001). Their levels in soybean products
are typically low enough to cause little harm, but processing can concentrate some
isoflavones, in particular genistein (Liener 1994). These compounds can disrupt a wide
variety of body functions involving reproduction, as do other estrogens. Therefore,
careful management of phytoestrogens may be important for brood stock and early life
stage feeds. Additionally, phenolic compounds can be removed from soy products using
activated carbon, which results in improved odor and flavor (How and Morr 1982).
Antivitamins are also contained in raw soybean products, but are typically heat
labile and inactivated with a proper thermal treatments (Liener and Kakade 1980). Heat
labile antivitamins contained in untoasted soy products can inhibit availability of vitamin
B12, vitamin D, and potentially vitamin E (Liener 1994). However, there has been some
indication that lipoxygenase decreases the availability of vitamin A and carotene whether
or not a thermal treatment was done (Shaw et al. 1951).
In humans and other animals, allergenicity to unheated soybean products can also
be a problem, but most of the immunochemical reactivity in soybean products is
15
eliminated with heat treatment. Immunologically active allergenic compounds include
glycinin and β-conglycinin, which make up a large portion of soy protein (Liener 1994)
and have been shown to negatively affect rainbow trout growth performance (Rumsey
1993). These compounds have also been found to cause intestinal mucosal legions, villi
abnormalities, intestinal enteritis, compromised immune responses, and abnormal flow of
digesta through the intestine (Krogdahl et al. 2000).
Processing Methods
Processing technologies are necessary to transform soybeans into usable products
for a wide variety of applications including food, fuel, and other goods. Typical soybean
processing consists of several steps (Figure 1-1). Soy products are primarily sold as
ingredients for remanufacturing into other products or feeds with the exception of a few
products such as edamame or meat substitutes such as texturized soy protein for human
consumption. Most soybeans grown in the U.S. are processed for oil removal, which is
used in biodiesel production as well as food products such as vegetable oil for use in a
wide variety of processed foods.
Processing may alter the nutritional, functional, and antinutritional aspects of an
ingredient (Table 1-2). While one processing approach can provide a soy product with
adequate nutritional characteristics for one species, the same product does not always
provide a similar result in a different species. For this reason, the characterization of
ingredient processing technology is important to consider in relation to digestibility and
growth performance studies (Glencross et al. 2007). In addition, processing can also
affect the palatability of soy products. Soybean meal may impart a “beany” flavor, which
16
is caused by certain carbohydrates that can be removed by selective processing (Forster
2002).
Full-fat flours and grits
The first step in soybean processing is selecting, cleaning and sizing the beans to
remove any unwanted material. The hulls are then removed by aspiration after cracking
or crushing the bean. Hulls offer little value in diets for carnivorous fish, and are sold
loose or pelleted as low-value “filler” ingredients for ruminant feeds. Dehulling (NSP
reduction) has a large impact on improving digestibility in monogastrics and also
increases the nutritive value of plant products (Booth et al. 2001).
The product after dehulling is referred to as full-fat white flakes which can be
ground, extruded, micronized, or roasted to create full fat products, which contain about
35.2% crude protein (wb) (Swick 2007; NRC 2011). Full-fat products generally are
avoided in animal feeds due to their “beany” flavor created by lipases, primarily
lipoxygenase (Lusas and Riaz 1995). Many times, soy products can have refined soy oil
or lecithin added back to the products from which the crude oil was previously extracted
(Lusas and Riaz 1995).
Soy Oils
At this step in the process, the oil is extracted from soybeans by exposing full-fat
white flake to a fat extraction system using hexane (most common) or other solvents
(Muller and Schweiger 1973; Lusas and Riaz 1995; Brown and Hart 2010). Crude oil can
also be extracted by mechanically extracting, or “expelling”, which is not as efficient as
17
solvent extraction (Brown and Hart 2010). Expelled soy products typically contain higher
oil (4-9%) than solvent extracted products (<1%) (Kasper et al. 2007; Brown and Hart
2010).
Oil is a valuable component of the soybean, and is commonly used for biodiesel,
cooking, and other uses (Singh et al. 2008). Soy oil is regarded as a good source of
energy and n-6 fatty acids in fish feeds; however, soy oil lacks adequate long-chain (2022 carbon) n-3 fatty acids necessary for normal growth and health in species incapable of
desaturating and elongating shorter (18 carbon) n-3 fatty acids (Storebakken et al. 2000).
However, linoleic acid, phospholipids and natural antioxidants found in soy oil are
beneficial to fish (Storebakken et al. 2000; Swick 2007). Beneficial linoleic acid is
concentrated up to approximately 54% in crude soy lecithin (Hertrampf and PiedadPascual 2003). Soy lecithin is primarily composed of phospholipids, which are
indispensable nutrients for fishes. The primary soy oil phospholipids include
phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol (Hertrampf and
Piedad-Pascual 2003).
The crude soy oil can be further processed with a de-gumming process by adding
water to heated oil, which binds to lecithin and creates a gum that is then separated from
the soy oil (Hertrampf and Piedad-Pascual 2003). The gum or “sludge” can be purified by
various additional methods until either a standardized soy lecithin composition is
obtained or a pure, de-oiled lecithin is created (Hertrampf and Piedad-Pascual 2003).
18
Soybean White Flake
The majority of soy product produced comes in the intermediate form of defatted
white flakes (WF). These are created after exposing full-fat flakes to the hexane lipid
extraction process. WF can then be ground to make soy flour, toasted to make SBM, or
further processed to produce SPC or SPI. Defatted soy flour usually contains a minimum
of 50% protein on a wet basis (Lusas and Riaz 1995). WF can have extremely high PDII
values of 95, but can also be produced with PDII’s of 20, 70, or 90, depending on the
thermal exposure (Lusas and Riaz 1995). Therefore, adequate thermal treatments of WF
are recommended for use in carnivorous fish feeds to reduce PDII and ANFs.
Soybean Meal
Typically, defatted WF is flash desolventized using steam, followed by a toasting
process to drive off any residual solvents which are collected in a vacuum (Swick 2007).
This toasting process also inactivates certain heat-labile antinutritional factors (ANFs)
such as proteinase inhibitors (NRC 2011). The product after toasting is the industry
standard SBM (dehulled, solvent extracted, desolventized and toasted) and on average
contains about 48.5% (wb) protein (NRC 2011) and a low PDII of 10-50 (Lusas and Riaz
1995). SBM also contains approximately 5-8% (db) oligosaccharides (Zdunczyk et al.
2011).
Dehulled, defatted, toasted SBM provides adequate protein for most carnivorous
fish diets, and a favorable amino acid profile with methionine being the first limiting
amino acid (NRC 2011). However, cysteine is generally higher in SBM, which reduces
the influence of low methionine. These two amino acids are considered semi-essential or
19
conditionally essential, and their amounts are often added together and considered as total
sulfur amino acids (NRC 2011).
SBM is a popular component in herbivorous and omnivorous fish dies and is
sometimes included at high levels (Gatlin 2002; NRC 2011). Carnivorous fishes have a
low tolerance for SBM, and require further processing (van den Ingh et al. 1991; Olli et
al. 1994b; Baeverfjord and Krogdahl 1996; Kasper et al. 2007).
Extruded Soy Protein
Extrusion is often used to create texturized soy protein from soy flours or SPCs
(Lusas and Riaz 1995; USSEC 2008). This is accomplished by plasticizing the ingredient
at high temperature (120-180°C) and pressure within the extruder barrel (Lusas and Riaz
1995). When the product exits the die, immediate depressurization causes expansion,
creating a texturized structure similar to meat (Lusas and Riaz 1995). Typically, extruded
products contain a similar protein and lipid content as the starting material (Lusas and
Riaz 1995).
Extrusion of soy products has been shown to improve digestibility and growth
performance when included in carnivorous (Romarheim et al. 2006; Barrows et al. 2007)
or omnivorous (Allan and Booth 2004) fish diets. Extrusion cooking has been found to
improve carbohydrate digestibility through gelatinization of starch and fiber (Björck et al.
1984). The conditions of the extruder (e.g., temperature, moisture, screw speed, retention
time) can be manipulated to cause desirable physical and chemical changes within the
extrudate (Fallahi et al. 2014). This allows a greater susceptibility to enzymatic
20
hydrolysis (Björck et al. 1984; Karunanithy et al. 2012) either by gastrointestinal
enzymes, or through fermentation.
The extreme temperature and pressure during extrusion can also reduce soy ANFs
such as trypsin inhibitor activity (Barrows et al. 2007) and phytic acid (Allan and Booth
2004). Extrusion has also improved the digestibility of other legume seeds (Bangoula et
al. 1992; Burel et al. 2000b).
Soy Protein Concentrate
SPC can be produced from either WF or SBM (Muller and Schweiger 1973), but
is typically produced from WF (Lusas and Riaz 1995), and contains on average 63.6%
(wb) protein (NRC 2011). The United States Soybean Export Council (USSEC) describes
high-quality SPC as having a minimum of 65% (wb) protein (USSEC 2008). The primary
purpose of an SPC process is to reduce strong-flavor components and flatulence sugars
contained in WF and SBM; however, other soluble compounds and minerals are also
extracted (Lusas and Riaz 1995). This creates a bland product, which is preferred because
it readily inherits flavors of other ingredients when mixed into a feed blend. In the
traditional process, WF is desolventized and the material is passed through an ethanol
extractor, which removes some carbohydrates and ANFs, while concentrating protein and
fiber (Swick 2007). The material can then be optionally neutralized to pH 6.5-7 after the
acidic ethanol-water wash, dried, and milled (Lusas and Riaz 1995). The ethanol
extraction solution and processing time may also be modified to produce a “low antigen,
feed-grade” SPC (USSEC 2008). 21
SPC has provided promising results in carnivorous species like Atlantic Salmon,
likely due to additional heating and the removal of oligosaccharides and other alcoholsoluble components (Olli et al. 1994b). Additionally, SPC processing creates a more
concentrated and balanced amino acid profile, which makes SPC a more valuable feed
ingredient.
SPC generally contains fewer soluble carbohydrates, lectins, trypsin inhibitor
activity, glycinin, β-conglycinin, and saponins than regular SBM (USSEC 2008). Ingh et
al. (1996) completed an experiment where the alcohol-soluble extract (which contained
oligosaccharides, saponins, and other unidentified factors) was added to an Atlantic
Salmon diets containing either no SBM, 26% untoasted SBM, or 26% toasted SBM. The
authors found that SBM diets resulted in intestinal morphology changes, while the FM
control did not. In a study done by Bureau et al. (1998), Chinook Salmon suffered from
severe intestinal damage when fed alcohol-soluable extract, but it had minimal impact on
Rainbow Trout. Therefore, the alcohol washing process of SPC must remove heat stable
antinutritional components of soybeans, but there are obvious differences in speciesspecific responses to the ANFs. Commercial alcohol/water washing procedures are
known to remove the majority of the heat stable oligosaccharides (Choct et al. 2010), and
an enzymatic process is not required for removal (Slominski et al. 1994). However,
alcohol/water washing is not completely efficient at removing oligosaccharides from
SPC. Often, SPC contains about 3% (db) total oligosaccharides (Zdunczyk et al. 2011).
22
Soy Protein Isolate
Soy protein isolate (SPI) is the most purified protein produced in soy processing,
which contains about 80.7% (wb) protein (NRC 2011), and is created by dissolving and
precipitating proteins from defatted white flake in a pH 6.8-10 alkali solution (Lusas and
Riaz 1995). The precipitate centrifuged to remove fiber, acid precipitated (pH 4.5), and
centrifuged again to create a curd. The curd is then optionally neutralized to pH 6.5-7 and
spray-dried to create SPI (Lusas and Riaz 1995). SPI can also be processed further into
globulins, which are highly affected by pH and salinity (Lusas and Riaz 1995).
Sometimes prior to spray drying, the curd is treated with enzymes or chemicals (Lusas
and Riaz 1995). This SPI processed is commonly used as a whipping enhancer in aerated
confectionary food products such as marshmallows, while non-enzyme treated SPI can be
used in creamer or yogurt (Kolar et al. 1979; Lusas and Riaz 1995). As a feed ingredient, SPC is preferred over SPI because the increased price is not
substantiated by fish performance. Additionally, SPI has also been found to produce
similar digestibility compared with SPC in Rainbow Trout (Glencross et al. 2005). SPI
which was processed with reduced phytate and phenolic compounds was found to have
increased in vitro pepsin digestibility (Ritter et al. 1987). Only trace amounts of
oligosaccharides remain in SPI’s (Zdunczyk et al. 2011).
Bioprocessed Soy Protein
Recently there has been interest in utilizing the alcohol/water soluble portion in
the creation of an ethanol coproduct from fermentation (Karunanithy et al. 2012). The
development of novel bioprocessing methods that could eliminate ANFs further than the
23
typical alcohol-washing process, meanwhile producing a valuable fuel source, could
potentially yield a much more efficient, and economical SPC manufacturing process.
Removal of the costly alcohol washing step could also provide an opportunity to reduce
the overall cost of a complete feed.
The protein content (about 55-65% wb) of these products is typically less,
however, than ethanol-extracted SPC (Swick 2007). Bioprocessing technologies can
eliminate the need for the alcohol washing solvents, thus significantly reducing the cost
of processing.
A variety of microbes, conditions, and feedstocks have been tested in soy
bioprocessing (Table 1-3). Enzyme hydrolysis and/or fermentation can also be used to
produce SPC alternatively to alcohol washing, and these methods have been shown to not
only convert carbohydrates, but also hydrolyze phytate and allergenic proteins, making
the protein fraction more digestible (Swick 2007) and increasing mineral availability
(Liener 1994). Additionally, hydrolysis can reduce peptide length of the final product
(Swick 2007). Fermented soy ingredients have been shown to reduce various
physiological and morphological abnormalities in Rainbow Trout, normally caused by
feeding commercial soybean meal (Yamamoto et al. 2010).
Fermentation processes are often characterized as either submerged liquid
fermentation (SLF) or solid state fermentation (SSF) (Mienda et al. 2011; Subramaniyam
2012), although moisture content and solid loading rates lie on a continuum. The SSF
process minimizes the energy inputs required for drying which results in the most cost
effective and environmentally friendly process (Mienda et al. 2011). However, certain
microorganisms require high moisture content, and SLF has the potential to eliminate
24
water-soluble ANFs in the separation of mash and water. Both SLF and SSF processes
can reduce indigestible and antinutritional components in soy products. Gao et al. (2013)
used SSF to show a reduction in trypsin inhibitors from 15.0 mg/g in untreated SBM
down to 1.6 mg/g in fermented SBM. Song et al. (2008) used SLF for SBM to
demonstrate a significant reduction in the β-conglycinin subunits and subsequently a
greatly reduced immunoreactivity in humans. Gao et al. (2013) determined that SSF with
Aspergillus oryzae degraded trypsin inhibitors in SBM significantly better than with
Lactobacillus brevis. In other studies, SSF with Lactobacillus sp. lowered phytate,
reduced intestinal inflammation, and improved digestibility of starch and fat for wheat
flour, barley flour, or defatted soy WF when included in extruded Atlantic Salmon feeds
(Refstie et al. 2005; Skrede et al. 2002). Although bioprocessed products have been
successful, the industry is in its infancy and continued research in biotechnology is
necessary to reduce the influence on carbohydrates and other ANFs present in soybeans
and soy products.
Yellow Perch Culture
The increasing global demand for food is straining the earth’s capacity to sustain
natural food supplies, and animal protein production has recently focused on the
efficiencies in aquaculture production. Because of their aquatic environment, fish have
lower basal energy needs, and provide better feed conversion ratios than farmed
terrestrial animals (Kaushik and Seiliez 2010). Accordingly, aquaculture is the fastest
growing food production sector in the world (8.8% per annum); meanwhile, worldwide
capture fisheries are static or decreasing (FAO 2012a; FAO 2014). Aquaculture of
25
species such as Atlantic Salmon successfully produces far more human food (>99%) than
taken through capture fisheries (FAO 2014).
The research presented in this thesis is primarily focused on Yellow Perch Perca
flavescens, which is a small aquaculture species by volume but having high regional
recognition and demand in the Midwestern U.S. (Malison 2003). The market demand for
Yellow Perch cannot be sustained by wild capture fisheries (i.e. Great Lakes) or current
aquaculture production (Malison 2003). The commercial harvest of Yellow Perch has
diminished since the 1960’s and early 70’s (Figure 1-1), primarily due to the introduction
of aquatic nuisance species (e.g., zebra mussels), overharvest, and variable recruitment in
the Great Lakes (Knight et al. 1984). In the 1990s, abundance of Walleye Sander vitreus
and Yellow Perch was discovered to be significantly lower than the previous decade in
the Michigan waters of Lake Erie (Thomas and Haas 2000). Additionally, since the
outbreak of Viral Hemorrhagic Septicemia (VHS) in the Great Lakes, fish populations
have suffered and regulation of interstate trade has become much more stringent (Bowser
2009). This has further spurred an interest in developing Yellow Perch aquaculture
outside of states with VHS. In 2005, there were a total of 99 small commercial producers
raising Yellow Perch, with a value of about $692,000 USD (USDA-NASS 2006).
Yellow Perch are a high-value fish with flaky-white, low-oil flesh often exceeding
$12 per pound (retail) in the north-central region of the U.S. (Burden 2013). Yellow
Perch have also been found to be significantly better tasting than Walleye, with no
difference detected between farmed or wild-caught fish (Delwiche et al. 2006). Yellow
Perch have long been a popular food fish, but Midwestern retailers, restaurateurs, and
consumers often do not have the opportunity or availability to select Yellow Perch over
26
imported seafood (Malison 2003). Increasing demand for Yellow Perch has far outgrown
its current commercial fishing and aquaculture production capability, and so abundant
opportunity exists in the development of aquaculture production (Malison 2003). Hence,
there is great interest in improving culture methods for Yellow Perch from both
production and marketing perspectives (Burden 2013).
Many commercial Yellow Perch producers use extensive pond culture that suffer
from with high production variability due to natural food abundance and extant pond
conditions (Malison 2003). The use of supplemental aquafeeds can be used in extensive
pond rearing, but only a portion of the fish’s nutritional requirements would be supplied
by the introduced feed. Supplemental feeding in extensive aquaculture will not be
discussed here.
Genetic research on improved aquaculture performance of Yellow Perch is an
area of recent interest (Han-Ping et al. 2009) and investigators have made significant
advances in characterizing specific microsatellite loci for broodstock management (Li et
al. 2007). Furthermore, there are also distinct advantages to producing monosex female
Yellow Perch populations for food production (Malison and Garcia‐Abiado 1996). This
study will utilize wild, local Midwestern strains of mixed sex; however, continued
research is needed to further advance genetic pairing with plant-based feeds for increased
intensive production.
Recirculating Aquaculture System (RAS)
Growth of finfish aquaculture in the upper Midwest is reliant on increasing
efficiency not only in nutrition but in culture environments. Several problems have arisen
27
from high waste effluent and environmental contamination from aquaculture operations
(Boyd 2003). Commercial aquaculture commonly utilizes net pens and extensive pond
culture, which lead to aquatic pollution and natural habitat degradation (FAO 2012a). It is
well known that these environmental issues must be resolved for the industry to grow,
particularly for intensive culture operations in climate-restricted locations.
Advances in closed recirculating system (RAS) technology have proven
successful in producing economically sustainable amounts of fish with a reduced
environmental footprint (Bergheim et al. 2009), but RASs are frequently not
economically viable, so continued improvements are imperative (Badiola et al. 2012) to
enable broader species applications. Implementing RAS technology requires more costly
initial capital investments than extensive systems, with up to eight year payback (Badiola
et al. 2012). Most companies who have grown Yellow Perch in RASs have closed for
similar reasons (Malison 2003). However, recent engineering improvements have
allowed these systems to produce high densities of fish and reach almost 100% efficiency
in operating water usage (Timmons and Ebeling 2007).
Advantages of RASs also include increased biosecurity, smaller footprint,
sustainability, infinitely expandable, reduced effluent pollution, and ability to stringently
monitor and control water quality to maximize production and minimize risk (Timmons
and Ebeling 2007). Because of the operational complexity of these systems, technicians
must be highly trained (Badiola et al. 2012). A thorough understanding of water
chemistry, fish behavior and aquaculture engineering is needed to operate intensive
systems. The high level of water quality control, fish health maintenance, space
utilization, and solids removal efficiency of a RAS is growing in acceptance with rising
28
costs of land, water, and energy (Malison 2003). Even though intensive aquaculture
requires high startup cost and requires more frequent monitoring than extensive
aquaculture, intensification can also lead to more consistent, year-round production
(Badiola et al. 2012).
Water quality
Selecting a suitable water source and maintaining optimal water quality is critical
for healthy fish and a productive aquaculture operation (Piper 1986). There are several
guidelines published for water quality characteristics, and many (e.g., temperature) are
species specific for optimal production (Timmons and Ebeling 2007). Diligent water
quality monitoring is especially important in RAS (Summerfelt 2003; Timmons and
Summerfelt 2007).
The first step in maintaining RAS water quality is efficient solids removal.
Uneaten feed and fecal solids remaining in an intensive system contributes to many water
quality problems as well as directly affecting fish by interfering with oxygen transport
across gills, decreasing oxygen concentrations during decomposition, and harboring
pathogens (Cripps and Bergheim 2000; Timmons and Ebeling 2007). As a general rule,
for every kilogram of feed fed, fish can generate 0.3 to 0.4 kg total suspended solids
(TSS), or about 25% of feed fed (db) (Timmons and Ebeling 2007). The maximum
recommended level of TSS for freshwater fish is 10 mg TSS/L (Timmons and Ebeling
2007). Primary elimination of settleable and large suspended solids include bead
filtration, sand filtration, and screening systems (Terjesen et al. 2013). Although
settleable and some suspended solids can be removed through gravity clarification or
29
sedimentation processes, depending on their particle size and specific gravity, dissolved
solids are more difficult to remove (Timmons and Ebeling 2007; Terjesen et al. 2013).
Technologies such as ozonation, foam fractionation, and hydrocyclones can be used to
reduce dissolved solids (Summerfelt 2003; Timmons and Ebeling 2007; Terjesen et al.
2013). Ozonation (O3) works well in high-density intensive RASs to oxidize larger
materials as well as destroy organic molecules that cause water color issues (Summerfelt
2003).
Dissolved oxygen (DO) is the most important water quality parameter (Timmons
and Ebeling 2007). A DO concentration below about 2.0 mg/L was found to significantly
reduce growth rates of Yellow Perch, while DO above 3.5 mg/L did not affect growth
(Carlson et al. 1980). DO concentrations below about 0.84 mg/L is lethal to Yellow Perch
(Petit 1973). DO can be added by diffused air, pure oxygen injection, low head
oxygenators (LHO™), packed columns, U-tubes, spray towers, or ozonation with
subsequent ozone destruction via UV filter (Summerfelt et al. 2000). Air diffusers are not
recommended for high density systems, as the efficiency of oxygenation is very low (Colt
and Watten 1988). Daily monitoring of DO is especially important in intensive systems
containing high fish densities, where major fish kills can occur within minutes of an
oxygen system failure. One large drawback to maintaining DO is that the saturation point
of water decreases with increasing temperature. Increasing temperature also causes
increased metabolism, respiration rate, and thus an increased need for oxygen. Water also
contains much less oxygen than the ambient air (21% oxygen), and so fish are forced to
devote more energy towards respiration. DO saturation at the optimum temperature for
Yellow Perch is around 8.7 mg/L. Some species are more tolerant of low DO conditions.
30
Similar to Yellow Perch, Salmonids require a higher DO (6-8 mg/L) than catfishes
(Ictaluridae) or tilapia (Oreochromis spp.) (2-6 mg/L) (Timmons and Ebeling 2007).
Temperature is the second most important water quality parameter and can
directly affect several aspects of fish physiology (Tidwell et al. 1999) as well as influence
toxicity of other water quality parameters (Timmons and Ebeling 2007). Fish species are
generally grouped by thermal tolerance into three categories: Cold-water (~15°C), coolwater (15-20°C), and warm-water species (>20°C) (Timmons and Ebeling 2007).
Because fish are poikilotherms, their physiological processes are greatly affected by
ambient temperature (Stickney 1979). Therefore, the species optimum temperature for
efficient growth should be maintained in the intensive production system. Although
Yellow Perch are considered a cool-water fish, the optimum temperature for Yellow
Perch growth ranges from about 23 to 25.4°C (Fischer and Hall Jr. 1989; Tidwell et al.
1999; Brown and Smith 2004).
Un-ionized ammonia (NH3) is highly toxic to fishes and should be monitored on a
regular basis (Piper 1986). Even at low concentrations (LC50 0.08—2.2 mg/L) un-ionized
ammonia can be toxic, and should be maintained below 0.0125 mg/L for salmonids and
below 0.05 mg/L for other species (Timmons and Ebeling 2007). Unlike terrestrial
animals, aquatic animals excrete ammonia as the primary waste product of protein
catabolism (Wright 1995). It can also result from decomposition of uneaten feed (Piper
1986). The portion of total ammonia nitrogen (TAN) which is un-ionized increases with
increasing temperature and pH (Thurston et al. 1981).
A healthy biofilter containing chemoautotrophic nitrifying bacterial colonies (e.g.,
Nitrosomonas, Nitrobacter) is essential for reducing ammonia into a non-toxic form of
31
nitrogen. Starting a biofilter is sometimes difficult because different species of nitrifying
bacteria will colonize and proliferate at different times and rates; if not properly
stabilized before stocking fish, ammonia or nitrite levels may become a concern. There
are two main steps involved in the oxidation of ammonia. The first step involves
oxidation of ammonia to nitrite by ammonium oxidizing bacteria (AOB), which is
typically represented by Nitrosomonas species. The nitrite (NO2-) produced by AOB
during this step is also very toxic to fish and must be monitored on a regular basis. The
AOB are the first species to proliferate in the biofilter with high levels of ammonia and
nitrite spikes occur several days later. The second step involves nitrite oxidizing bacteria
(NOB), which are represented usually by species of the Nitrobacter genus. This step
produces the relatively non-toxic nitrate (NO3-) which spikes shortly following nitrite
reduction (Timmons and Ebeling 2007). One of the benefits of ozone treatment is the
ability to oxidize the toxic nitrite to the non-toxic nitrate (Summerfelt 2003).
The pH is also important in that extreme levels can be stressful or lethal, but pH
also has an effect on toxicity of other water quality characteristics such as ammonia
(Timmons and Ebeling 2007). Yellow Perch have been considered to be relatively
tolerant of acidic conditions compared to other species like juvenile Rock Bass
Ambloplites rupestris, Black Crappie Pomoxis nigromaculatus, and Largemouth Bass
Micropterus salmoides (McCormick et al. 1989). Juvenile Yellow Perch have been found
to endure soft water of pH 5.0, but at pH 4.0 the perch do not survive (McCormick et al.
1989). Various life stages of Yellow Perch have been found to respond differently to pH
(Fischer and Hall Jr. 1989). Additionally, pH has been found to affect the taste
preferences in fish (Kasumyan and Doving 2003).
32
Alkalinity and hardness also share important interactions with pH. Alkalinity is a
quantitative measure of the waters ability to neutralize an acid. Carbonates and
bicarbonates can be added to water in several forms (Timmons and Ebeling 2007) to
increase alkalinity. Alkalinity is closely related to hardness (mineral content), which at
low levels can result in acute toxicity of copper (Taylor et al. 2003). Yellow Perch have
been found to be more tolerant of copper binding to gills than Rainbow Trout (Taylor et
al. 2003).
Carbon dioxide (CO2) can also be a problem in RAS culture, and is directly
associated with pH and the carbonate system (e.g., carbonic acid, bicarbonate ions,
carbonate ions) (Timmons and Ebeling 2007). Yellow Perch have been found to be more
sensitive to CO2 than Brown Bullhead Ameiurus nebulosus, but similar in sensitivity to
Brook Trout Salvelinus frontinalis and White Sucker Catostomus commersonnii (Black et
al. 1954). Carbon dioxide is created through respiration as well as solids decomposition
and should be maintained at low levels (<20 mg/L) (Timmons and Ebeling 2007). High
levels of CO2 can cause physiological problems including respiratory acidosis, but there
has been some research in using high levels of CO2 as an anesthetic (Timmons and
Ebeling 2007). CO2 removal in RASs includes both gas transfer and chemical processes
in addition to simply increasing water replacement rates (Summerfelt et al. 2007). Tank
aeration and degassing towers can off-gas to the atmosphere, while pH control will
equilibrate the carbonate acid-base system to help reduce CO2 concentrations
(Summerfelt et al. 2007).
Other water quality parameters such as salinity, chlorine, phosphorus, and
conductivity can present problems at certain levels. Water quality measurements also
33
depend on the specific situation (e.g., testing for chlorine while using a dechlorinated
municipal water supply). However, the above water quality parameters are important to
control in every type of system and they will often be the first to deteriorate when there is
a problem. Often when a water quality problem is detected, fish are already stressed or
dying. It is for this reason why frequent water quality measurements and monitoring
systems are of utmost importance to high density RAS aquaculture.
Feed Formulation and Nutritional Requirements
Commercial fish feeds are often least-cost formulated based on availability and
pricing constraints of certain ingredients and not necessarily optimal performance. The
feeds not only require complete nutrition and palatability but also functionality due to
water immersion. Ingredients may be included in diets to act as binders for pellet stability
and durability purposes, while others may be included for enhanced immunity and
improved organoleptic properties (Glencross et al. 2007).
Feeds which rely on not only FM and oil, but any single ingredient pose a higher
risk associated with fluctuating prices, quality, or availability. Feed costs can represent
40-60% of operational costs of aquaculture production (Brown et al. 1996). It is
suggested that a combination of plant-based ingredients would be required to fully
replace FM, including supplemental amino acids, palatability enhancers, and pellet
property augmentations (Gatlin et al. 2007).
Formulation software is available from several commercial providers and also can
be produced on spreadsheets by nutritionists. Feed formulation software is often based on
linear, least-cost software which may provide an optimal recipe based on user-defined
34
limitations of cost, availability, digestibility, and nutritional requirements. Some software
products have the capability of implementing stochastic variation based on changing
commodity growing conditions and also seasonal variation in ingredient composition
(Pesti and Seila 1999). Using stochastic software is often more accurate at providing a
guaranteed nutritional composition (Pesti and Seila 1999). Regardless of software,
ingredient quality and variability must be considered in any formulation.
The nutritional requirements of fish vary greatly between carnivorous species
which need high amounts of protein compared to omnivorous or herbivorous species
which grow favorably on feeds made similar to poultry or swine feeds (Storebakken et al.
2000). Formulations in the present research were based on a compilation of information
from requirements suggested by Hart et al. (2010), Twibell and Brown (2000), Dr. M.
Brown (personal communication), and unknown nutrient requirements were based on
Rainbow Trout requirements (NRC 2011). Diet formulations included supplements due
to an incomplete nutritional profile of plant ingredients (including soybeans), compared
with animal ingredients (NRC 2011). The nutritional components, antinutritional
components and known interactions of ingredients must also be characterized and
combined with other ingredients to provide all essential proteins, lipids, carbohydrates,
vitamins, and minerals.
Palatability and attractability are the first determinants of whether a feed
ingredient is “good” or “bad,” and are considered some of the most important
components of a feed, although there is sometimes no apparent relationship between the
two determinants (Kasumyan and Doving 2003). Regardless of the nutritional quality, if
fish do not consume a feed because of poor palatability, the nutritional quality cannot
35
support growth and results in wasted feed and unwanted water contamination. There have
been several palatability enhancing supplements identified in the literature, however
these enhancements are highly species-specific (Kasumyan and Doving 2003; Yackey
1998). Ingredient processing can also impart certain flavors on soy products as well as
the final pelleted feeds (Refstie et al. 1998).
Proteins and Amino Acids
Dietary protein is one of the most important components of a complete feed
because it is used for energy, growth and maintenance (Kaushik and Médale 1994). FM
has been the traditional protein source in carnivorous aquafeeds because it has
outperformed plant products in feed uptake and growth performance. Some commercial
feed formulations for carnivorous fish include FM in excess of 50% (Glencross et al.
2007). However, due to the rising price of FM along with fluctuating availability and
uncertain quality, producers and feed manufacturers who rely on FM are faced with a
considerable risk. Therefore it has been suggested that future FM-based feeds will be
used only for expensive starter, finisher and broodstock feeds (Tacon and Metian 2008)
Plant-derived protein products can be added as a protein substitute or supplement
to FM, only as long as the final amino acid profile meets or exceeds species
requirements. In a review of soy proteins in aquafeeds, Storebakken et al. (2000)
mentioned several species of carnivorous fish for which 75 to 100% of the FM protein
was successfully replaced with SPC. Protein concentrates generally work well to supply
the bulk of essential amino acids, but supplementation with lysine and methionine is
often necessary to meet all requirements (Storebakken et al. 2000). Multiple plant meals
36
is one way to provide all the essential amino acids (Gatlin et al. 2007). Corn gluten,
wheat gluten, wheat midds, and others are commonly used in aquafeeds to provide
different and complementary amino acid profiles as well as for improving pellet
functionality or taste (Gatlin et al. 2007).
There have been mixed results on protein requirements of Yellow Perch, and
evidence seems recommend higher levels of protein (at least 36% db) than previously
thought (Brown et al. 1996, Ramseyer and Garling 1998). Other preliminary studies
indicate Yellow Perch may require up to 45% protein for maximum growth (Dr. M.
Brown, Personal Communication).
Lipids and Fatty Acids
Most plant oils including soybean oil lack adequate amounts of essential long
chain n-3 fatty acids Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA)
which are found in FM and fish oil. EPA and DHA are known to be important in the
nervous, circulatory and immune systems in humans (Simopoulous 2002) and they are
also important for Yellow Perch (Mjoun et al. 2012). Soybean oil contains high n-6 fatty
acids and cannot fully replace fish oil to maintain health and growth of Yellow Perch
(Mjoun et al. 2012). Additionally, replacing fish oil with soybean oil can also change the
organoleptic properties of the fish flesh (Storebakken et al. 2000). In feeding trials with
Red Drum Sciaenops ocellatus, Tucker et al. (1997) found no difference in growth when
low levels of soybean oil were used to replace menhaden fish meal. Growth was
restricted when those fish were fed higher levels of soybean oil, which may have been
caused by the decrease in EPA and DHA (Tucker et al. 1997).
37
Phospholipids, which are the major constituent of soy lecithin, are vital to cell
structure and function in fish (Hertrampf and Piedad-Pascual 2003). However, there is
some evidence that smaller, fast growing fish have a higher requirement than larger fish
(Poston 1990a; Poston 1990b). Choline, inositol, and phosphorus are all essential
nutrients which are present in soy lecithin in the form of phospholipids. Inadequate or
absent dietary phosphatidylcholine can lead to accumulation of fat in the liver due to
interference with lipid metabolism, fatty acid transport, and fat-soluble vitamin
absorption (Hertrampf and Piedad-Pascual 2003). Additionally, soy lecithin has been
found to act as an antioxidant as well as an attractant (Hertrampf and Piedad-Pascual
2003). There has been success replacing soy lecithin with artificial choline chloride
(Hertrampf and Piedad-Pascual 2003); however, Poston (1990a) found improved growth
in Atlantic Salmon when fed soy lecithin over artificial choline chloride. The dietary
requirements of these phospholipids depend on the age of the fish, total fat content of the
feed, and cold water species require more than warm-water species (Hertrampf and
Piedad-Pascual 2003).
Carbohydrates
It is well known that plant products contain more complex indigestible
carbohydrates than FM and are normally not as easily digested by carnivorous fish
(Glencross et al. 2007). This variability is attributed to structural and functional
differences within the intestine and related organs of herbivorous or omnivorous fish.
While sucrose is an oligosaccharide which is readily available to carnivorous fish,
the other complex oligosaccharides (i.e. raffinose and stachyose) are not digestible due to
38
the lack of α-galactosidase enzyme production in fish (Gatlin et al. 2007). Before nutrient
absorption can occur, a polysaccharide must be broken down to a monosaccharide by
various enzymes produced by microbes within the intestine. Sometimes, the limiting
enzyme may be available in the intestine, but intestinal conditions (e.g., pH, temperature)
are not conducive to carbohydrate digestion.
It has also been noted that fish may be able to adapt in different ways to various
carbohydrate levels (Krogdahl et al. 2004; Krogdahl et al. 2005). Genetic selection
programs may also provide strains more suitable for maximizing production when fed
alternative plant-based protein sources (Hardy 1998), which tend to have higher
carbohydrate levels.
Minerals
Mineral requirements of Yellow Perch are essentially unknown, so salmonid
requirements are typically substituted. There are, however, several suggested
requirements for various cold, cool, and warm-water fish species summarized in NRC
(2011). Quantifying mineral requirements of any species is difficult due to the incredible
amount of interaction between various minerals. Quantifying requirements of a fish
species presents an especially difficult task, due to the fish’s ability to absorb minerals
directly from the water, feed leaching, and significant variability in bioavailability (NRC
2011). FM appears to provide a better bioavailable source of minerals than plant-based
products (NRC 2011). Therefore, it is well known that supplementing plant-based diets is
critical (NRC 2011). Additionally, ingredient processing and the addition of exogenous
enzymes such as phytase can increase the bioavailability of minerals (Riche and Brown
39
1996; Storebakken et al. 1998b). Some of the major signs of mineral deficiency in fish
include impaired growth, skeletal deformities, anemia, cataracts, and impaired nutrient
utilization (NRC 2011).
Vitamins
Vitamin content and bioavailability varies widely between different dietary
ingredients and are influenced by processing methods of both feed ingredients and final
feeds (Barrows et al. 2008; NRC 2011). Vitamins can be classified as either water soluble
or fat soluble. Water soluble vitamins include the B complex vitamins, ascorbic acid,
choline, and inositol (NRC 2011). Fat soluble vitamins include vitamins A, D, E, and K
are stored by the body, unlike water soluble vitamins, which require a constant dietary
supply (NRC 2011).
Very little information exists on vitamin requirements for Yellow Perch, but
relatively complete requirements have been determined for Chinook Salmon, Rainbow
Trout, and several warm-water species (Barrows et al. 2008; NRC 2011). However, due
to the lack of adequate vitamin bioavailability data, formulations are generally overcompensated with additional vitamin mixtures assuming minimal bioavailability from
primary ingredients (Gatlin et al. 2007; NRC 2011). Plant products that are vigorously
processed such as through extrusion have been found to require a different supplemental
vitamin profile compared with previous formulations containing fish and animal
ingredient-based diets (Barrows et al. 2008). For example, ascorbic acid (vitamin C) is
often inactivated by heat produced in the feed making process, so additional
40
phosphorylated (heat stable) ascorbic acid must be supplemented. Ascorbic acid, choline,
and inositol are required in larger quantities than other vitamins (NRC 2011).
Barrows et al. (2008) observed that Rainbow Trout fed a plant-based diet with an
inadequate vitamin profile, exhibited reduced survival, feed intake, protein digestibility,
energy digestibility, hematocrit, and Hepatosomatic Index (HSI). Other signs of vitamin
deficiency include decreased lipid utilization, abnormal swimming, emaciation, cataracts,
skeletal deformities, and liver abnormalities (NRC 2011).
Other Supplements
A variety of feed additives, can be included in a diet to enhance the physical or
chemical properties of a feed. These include antimicrobial agents, antioxidants, binders,
pigmentation, enzymes, organic acids, palatability enhancers, immunostimulants,
prebiotics, and probiotics (NRC 2011). Commercial formulations commonly include a
variety of feed additives for specific purposes (NRC 2011).
Enzymes are now commonly used in complete feeds. The addition of phytase can
improve the availability of phosphorus as well as minerals that are normally bound in
SBM and other plant products by phytic acid (Gatlin et al. 2007). Maximizing utilization
of intact phosphorous by releasing the bound phosphorus from phytic acid reduces
phosphorus pollution (Storebakken et al. 1998b). Unfortunately, commercial phytase is
heat labile and also less effective at low water temperatures, which creates problems
when included in extruded feeds fed to cold-water species (Storebakken et al. 2000).
Anti-microbial chemicals are commonly added to feeds as either a therapeutic or
to extend shelf-life (NRC 2011). Depending on culture system and logistics, oral delivery
41
of antibiotics is more feasible than immersion treatment in a large system. A variety of
compounds can be added to increase product shelf-life, including benzoic acid, propionic
acid, sorbic acid, and other salts (NRC 2011).
Pigments such as astaxanthin have been supplemented to salmonid diets, to mimic
natural coloration of the fillet (NRC 2011). This is because most natural pigments in fish
and shrimp flesh is the result of natural dietary items (Barrows et al. 2007). Plant-based
diets are particularly lacking of the pigments necessary to create a natural salmon fillet
color (NRC 2011).
It is also important to consider functionality when selecting ingredients. The
functional color, size, moisture, shape or texture of pellets may also have an effect on
ingestion. Both nutritional functionality and physical strength of the pellets are important,
so several functional agents can be added. For example, wheat gluten’s viscoelasticity
properties are related to the quality of bread (Shewry et al. 2002). It is also important to
predict ingredient functionality prior to processing, especially when using cooking
extrusion, which can impart changes in chemical structure (Barrows et al. 2007).
Pelleting
Extrusion cooking technology provides feed manufacturers the capability to
produce floating, slow sinking, or sinking feeds which are more stable in water than other
types of feed production. Major aquafeed manufacturers often use extrusion for larger
pellets (>1 mm) and spheronizers (marumerizers) or sieved crumbles for smaller pellet
sizes. However, extrusion of smaller sizes (~200 µm) is also possible. Steam-pelleting
and screw-pressing pellets can also be used to create aquafeeds, but are typically less
42
durable and water stable than extruded pellets. Extruding the final formulation into a
complete pellet improves characteristics such as pellet stability and durability, due to
carbohydrate gelatinization (Fallahi et al. 2014).
Pelleting temperature is critical in both extruded and non-extruded aquafeed
production. Extrusion usually offers a much higher degree of temperature control than
screw-pressing due to steam injection, jacketed barrel segments, and temperature
monitoring. Heating feeds via extrusion has also been shown to reduce trypsin inhibitors
(Romarheim et al. 2006) and increase the digestibility of plant-based feeds in several fish
species (Burel et al. 2000a; Stone et al. 2003; Stone et al. 2005). However, adverse
processing conditions from excessive heat are known to cause decreased quality in feed
constituents (Öste and Sjödin 1984). The amount and type of protein, carbohydrate, lipid
and moisture all have an effect on reactions experienced in cooking extrusion (Öste and
Sjödin 1984). Hence, caution must be used in extrusion processing because overheating
can cause a Maillard reaction wherein amino acids react with sugars and interfere with
amino acid digestion of the feed (Öste and Sjödin 1984). Additionally, overheating
soybean products will result in reduced lysine bioavailability (Swick 2007). Because
under-heating will produce less stable pellets, an optimum, stable extrusion temperature
is necessary.
Feeding
Feeding a prepared feed is generally easier and more consistent than feeding live
feeds. However, feeds and feeding methods change from one life stage to the next. Feed
43
can be distributed by hand, belt, vibratory, pneumatic, or demand systems. Both feeding
level and feeding frequency can be optimized for a particular species, life stage, and feed.
Feeding frequency of Yellow Perch can be several times per day, and is typically
more frequent during early life stages (≥5 feedings per day), and can reduce to 1-4
feedings per day at later life-stages. There is some interest in inducing compensatory
growth (CG) of Yellow Perch (Schaeffer et al. 2012) as well as other species (Bavcevic et
al. 2010), to improve feed conversion ratios by manipulating feed-fast cycles. CG is a
natural compensatory response experienced by wild fish under harsh conditions or food
shortages (Jobling 1994). CG is a potential opportunity to increase the economic viability
of aquaculture production, however, more research is needed on this topic.
Feed acclimation is important when feed training larvae transitioning from
endogenous to exogenous feeding, switching juveniles from a live diet to a prepared diet,
and acclimating fish from an FM-based diet to a plant-based diet. Switching from FMbased diets to SPC-based diets often leads to temporary decreased feed consumption and
growth in salmon (Storebakken et al. 1998b). Yellow Perch often show a similar reaction
when using soy-based feeds with inadequate palatability (Malison 2003). Providing
mixtures of feeds during feed transitioning is often used in commercial aquaculture to
minimize negative reactions.
Accurate feed intake is also critical for the assessment of palatability by
discriminating the effects of voluntary feeding and nutrient utilization (Glencross et al.
2007). Feed intake estimates can be measured in several ways including specialized
collection structures, collecting and measuring, or pellet counting (Helland et al. 1996).
44
Feeding levels used in this research were based primarily on consumption estimates of
juvenile Yellow Perch through the pellet counting method.
Summary
The success of soy ingredients in human food and animal feed is attributed to
processing technologies. While a single processing method will not be adequate for a
complete aquafeed, soy processing methods optimized to meet the requirements of
carnivorous fish will definitely help in overcoming the issues associated with alternative
proteins for aquafeeds.
There are still considerable research needs to characterize species-specific fish
responses to dietary soy ANFs and improvements in processing. Bioprocessing is one of
the fastest growing and most promising methods of creating lower-cost processing
technologies that can create more cost-efficient soy products for use in carnivorous fish
diets.
Little is known about the nutritional requirements of Yellow Perch and there are
very few domestic producers in comparison to more commonly culture species of
Rainbow Trout, Atlantic Salmon, Channel Catfish, Hybrid Striped Bass, and Tilapia.
Greater knowledge of Yellow Perch dietary needs and culture methods must be
developed to facilitate production. Ultimately, cost-effective, commercial feeds designed
for Yellow Perch life stages that provide high conversion, good growth, and minimal
solids would be ideal for RAS production. Fish fed nutritionally complete diets exhibit
higher growth rates and produce less waste than fish fed natural forage or nutritionally
incomplete artificial diets (NRC 2011). Meeting the energetic needs of a Yellow Perch,
45
while providing optimal protein for somatic growth enhances protein efficiency and
reduces diet cost. In addition to the nutritional aspects of Yellow Perch culture, research
on genetics, optimum water quality requirements, disease prevention, and intensive RAS
technology are also critical for efficient and economical production.
46
Table 1-1. Trypsin inhibitor activity (TIA) of soybeans and soy products.
Material
Defatted white flake
TIA (mg/g wb)
28-32
Source
Rackis et al. 1985
Soybean Meal
2-15.0
Rackis et al. 1985; Synder and
Kwon 1987; Gao et al. 2013
Soy Protein
Concentrate
5.4-7.3
Peace et al. 1992
Soy Protein Isolate
Bioprocessed SBM
1.2-30
1.6-6.4 (db)
Rackis et al. 1985; Peace et al. 1992;
Gao et al. 2013
47
Table 1-2. Proximate composition of commercially processed soy products (g/100g dm except
where noted).
Moisture
Material Protein (% wb)
Full-fat soy
42
≤10
flour
Fat
Fiber
Ash
NFE
20-21
5.6
4.7
27-28
Source
Lusas and Riaz (1995);
Forster (2002)
Defatted
White flake
56-59
6-8
0.5-1.1
2.7-3.8 5.4-6.5
32-34
Lusas and Riaz (1995)
Soybean
Meal
50-59
6-11
0.5-1.4
2.7-6.9 5.4-6.9
32-34
Lusas and Riaz (1995);
Forster (2002); NRC
(2011)
Soy Protein
Concentrate
65-84
4-8
0.5-1.0
0.1-5.0 3.8-6.5
20-22
Lusas and Riaz (1995);
Forster (2002); NRC
(2011)
Soy Protein
Isolate
90-92
4-6
0.5-1.0
0.1-0.2 4.0-5.0
3-4
Lusas and Riaz (1995)
48
Table 1-3. Various Microbes, process (solid state fermentation-SSF; liquid state
fermentation-SLF), and feedstock used in fermentation of soy products.
Microbe
Aspergillus ficcum
Process
SSF
Feedstock
SBM
Aspergillus oryzae
SSF
SBM
50% MC
Aspergillus usami
Aspergillus oryzae+
40-60% MC
Lactobacillus casei
Bacillus subtilis
~50% MC
Aspergillus+Bacillus spp.
Bacillus spp. compound
Bifidobacterium lactis
Lactobacillus brevis
Lactobacillus brevis
Lactobacillus plantarum
Rhizopus oligosporus
Saccharomyces cereviseae
Proprietary
SSF
SLF
SSF
SSF
SLF
SSF
SLF
SBM
Source
Han and Wilfred 1988
Hong 2004; Gao et al.
2013
Matsui et al. 1996
SBM
Chen et al. 2010
Texturized
soy protein
PepSoyGen
SBM
SBM
SBM
WF
SBM
SBM
SBM
Kim et al. 2010
Barnes et al. 2014
Yamamoto et al. 2010
Song et al. 2008
Gao et al. 2013
Refstie et al. 2005
Song et al. 2008
Buckle 1985
Song et al. 2008
Jul‐04
Dec‐04
May‐05
Oct‐05
Mar‐06
Aug‐06
Jan‐07
Jun‐07
Nov‐07
Apr‐08
Sep‐08
Feb‐09
Jul‐09
Dec‐09
May‐10
Oct‐10
Mar‐11
Aug‐11
Jan‐12
Jun‐12
Nov‐12
Apr‐13
Sep‐13
Feb‐14
Average Price (US Dollars per Metric Ton)
49
2500
Fish meal
Bank 2014).
Soybean Meal
2000
1500
1000
500
0
Date
Figure 1-1. Average commodity price of FM and SBM over the previous 10 years (World
50
Aquaculture
200
180
160
140
120
100
80
60
40
20
0
1950
1953
1956
1959
1962
1965
1968
1971
1974
1977
1980
1983
1986
1989
1992
1995
1998
2001
2004
2007
2010
Total Production (Million Metric Tons)
Capture Fisheries
Year
Figure 1-2. Total global fish production by primary production source (FAO 2012b).
Total Value of Atlantic Salmon
imports (1 million U.S. dollars)
51
2500
2000
1500
1000
500
0
Figure 1-3. Total value of Atlantic Salmon imports to the U.S. for the previous 26 years
(USDA 2014).
52
SBM
7000
190000
6500
170000
6000
150000
5500
130000
5000
110000
4500
90000
4000
70000
3500
50000
3000
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
210000
Total FM Production (1000 metric tons)
Total SBM Production (1000 metric tons)
FM
Year
Figure 1-4. Total world production of FM and SBM over the previous 10 years (USDA
2014).
53
Figure 1-5. General soybean protein and oil processing flow diagram (modified
from Swick 1994; Brown and Hart 2010).
54
1000
Capture Fisheries (USA)
900
14000
Capture Fisheries (Canada)
12000
700
Aquaculture
10000
800
600
8000
500
6000
400
300
4000
200
2000
100
2010
2006
2002
1998
1994
1990
1986
1982
1978
1974
1970
1966
1962
1958
1954
0
1950
0
Year
Figure 1-6. Total production of Yellow Perch by method and source. (FAO 2012b).
Aquaculture Production (Metric Tons)
Capture Fisheries Production (Metric Tons)
16000
55
CHAPTER 2. BIOPROCESSED SOY DIGESTIBILITY IN JUVENILE YELLOW
PERCH
Introduction
Considerable research has been recently done to determine sustainable, plantbased alternatives that can support growth equivalent to marine-derived protein (i.e., fish
meal; FM) (Gatlin et al. 2007; NRC 2011). Plant-based aquafeeds have been successful
in omnivorous and herbivorous fishes (Gatlin 2002), but carnivorous fishes do not readily
accept or digest plant-based feeds (Gatlin et al. 2007). Soybean Glycine max products
have been of particular interest as an FM replacer due to the dense nutritional content;
however, indigestible, deficient, and antinutritional (ANF) components limit the
bioavailability of nutrients in soy and other plant products (Tacon and Jackson 1985;
Francis et al. 2001; Sinha et al. 2011).
Bioprocessing (BP) technology has been shown to enhance the nutritional quality
of soybean meal (SBM) (Hong et al. 2004). Although fermentation of soy products is a
common processing method for human food production and some animal feedstuffs,
alternative BP methods have been developed with the goal of improving the palatability
and bioavailability of soy nutrients in carnivorous fish feeds. Researchers have related
improved composition and fish performance to BP (Rombenso et al. 2013; Trushenski et
al. 2014). BP-soy contains higher protein, reduced antinutritional factors (ANF) (Han and
Wilfred 1988; Chen et al. 2013; Gao et al. 2013), shorter peptides (Hong et al. 2004), and
in some cases, improved digestibility (Skrede et al. 2002).
Digestibility is the measurement of nutrient absorption from a feed or a feed
ingredient and can be expressed as true or apparent digestibility. Nutrient digestibility
56
information can be very useful in the comparative evaluation of feedstuff processing
technologies, however it is also difficult to measure (Austreng 1978b). Applied fish
nutrition research is generally focused on apparent digestibility due to the limitations of
collecting feces and quantifying nitrogenous waste excreted from gills. Usually,
digestibility measurements are reported as apparent digestibility coefficients (ADC) (e.g.,
protein [ADC-P] or energy [ADC-E]), but the estimates can be considered biased due to
many factors interfering with digestion (Alarcón et al. 1997; Glencross et al. 2007).
Nutrient utilization interference is complex and difficult to analyze, even when changing
one ingredient (Alarcón et al. 1997; Glencross et al. 2007). Nutrient absorption rates are
constantly changing, especially within a variable feeding strategy or with temperature
(Kitchell et al. 1977; Post 1990). ANFs, amount ingested (due to appetite or palatability),
and amount of stomach distention can all have an effect on the digestive enzymes
produced, digesta mobility, and nutrient absorption (Garber 1983; Alarcón et al. 1997;
Glencross et al. 2007).
Given the need to develop improved soy products as FM replacers and understand
fish responses to soy ingredients, the aim of this set of experiments was to evaluate the
effect of soy processing on protein and energy digestibility in Yellow Perch Perca
flavescens. This study was done to provide data for several forms of BP-soy to assist
optimization of nutrient upgrading approaches.
57
Methods and Materials
Experimental Design and Diet Formulation
A completely randomized design was applied to soybean white flake (WF) and
soybean meal (SBM) of two genetic types (GMO or non-GMO) bioprocessed with and
without extrusion pretreatment. BP and extrusion parameters were held constant between
products. Soy products were extruded with a Plasti-Corder PL2000 single-screw
laboratory extruder (C.W. Brabender, South Hackensack, NJ) using conditions optimized
for oligosaccharide hydrolysis (Karunanithy et al. 2012; Karuppuchamy 2011).
Experimental BP-soy was obtained from Prairie AquaTech (Brookings, SD). In addition,
commercially available enzymatically treated soy (Hamlet HP 300), fermented soy
(PepSoyGen), FM, wheat flour, and wheat gluten were also tested. Each treatment was
delivered to tanks containing 100 to 125 juvenile Yellow Perch of about 30-70 g each.
Reference feed blends were slightly modified from a formulation used for
Rainbow Trout Oncorynchus mykiss and Hybrid Striped Bass Morone chrysops× M.
saxatilis ADC measurements (Barrows et al. 2012). Test diets were blended using 70% of
the bulk reference diet (db) and 30% of the test ingredient (db); a chromic oxide tracer
(0.5%, db) was kept constant in diets.
Large particle ingredients were ground through a 1.27 mm screen using a
Fitzpatrick comminutor (Elmhurst, IL) prior to dry blending. Dry ingredients were then
blended in a Hobart HL200 mixer (Troy, OH) where oil and water are added and blended
until homogenous for approximately 5 min. Dough was then screw-pressed using a
Hobart 4146 grinder equipped with variable speed feed screw and cutting head with a
3.5-mm die plate. Extrudates were then dried for ~12 min in a Despatch UDAF electric
58
conveyor drier (Minneapolis, MN) with maximum temperature set at 120°C. Feeds were
then cooled with forced air prior to packaging, and stored at -20°C pending use.
All calculations of digestibility were based on the dry matter composition of
nutrients in both feeds and feces. Apparent digestibility coefficients were calculated using
the formulas described by Kleiber (1961) and Forster (1999):
100
100 %
%
∗
∗%
∗%
∗
where a = nutrient contribution of the reference diet to the test diet = (level of nutrient in
reference diet) * (100-i); b = nutrient contribution of test ingredient to nutrient content of
test diet = (level of nutrient in test ingredient) * i; i = level of test ingredient in combined
diet (%); and (a + b) = level of nutrient in combined diet (%). Statistical analysis was not
used to compare ADC values due to a lack of sample replications.
Culture System
The 5,678 L recirculating aquaculture system (RAS) consisted of six, 719 L
circular tanks, radial flow settler, fluidized biofilter, bead filter, UV filter, heat pump, and
a secondary sump equipped with water level control and electronic water quality
monitoring probes. Temperature was maintained at 20-23°C, and flow rates were
maintained at 1.5 tank water turnovers per hour (18.0 Lpm). Tanks and bioreactors were
oxygenated with forced air diffusers. Replacement water was sourced from a municipal
water supply and dechlorinated using activated carbon filters and stored in an indoor
15,200 L head tank.
59
Feeding
Prior to feeding treatment diets, Yellow Perch fingerlings were fed a complete
FM reference diet and were fed to apparent satiation twice daily. Satiation was
determined by hand-feeding each tank of fish while monitoring feeding activity. When
feeding activity slowed and a significant amount of pellets were visible on the tank
bottom, apparent satiation was assumed. The same feeding strategy was maintained after
treatment diet feeding began; diets were then fed for at least 10 days prior to stripping
fecal material.
Sample Collection
Fecal samples were collected by distal gastrointestinal stripping (Austreng 1978a;
Gaylord et al. 2008). Prior to collecting, anesthetic (80 ppm buffered tricaine
methanesulfonate, MS 222) and recovery baths (6 ppt NaCL, 250 ppm KCL, 163 KPO4,
118 ppm MgSO4) were prepared using water from the RAS. Approximately 20 fish were
sampled in batches to minimize handling and exposure time in the anesthetic bath. After
equilibrium was lost, fish were individually removed and prepared by holding upside
down and gently towel drying the ventral area. A consistent amount of external
abdominal pressure was applied in a peristaltic motion to only the distal portion of the
intestine as described by Austreng (1978b). In an effort to reduce dilution of sample from
water splashing or dripping into the sample, fecal matter was expressed onto a latex
gloved finger, then immediately transferred with tweezers to a pre-weighed aluminum
tin. Fecal samples were pooled by tank. Fish batches were placed in an aerated recovery
60
baths for approximately 10 minutes prior to returning to their original RAS tank. Sample
moisture loss was mitigated by covering the aluminum tin between subsets.
After all fish from a tank were sampled, sample tins were immediately weighed
and frozen at -20°C, and then freeze-dried for analysis. Freeze-dried feces were then
weighed to the nearest 0.1 mg to determine moisture loss from freeze drying.
Compositional Analysis
Ingredients, diets, and freeze-dried fecal samples were ground to a fine powder
using a mortar and pestle prior to analysis. Dry matter was analyzed according to NFTA
method I (Undersander et al. 1993). Analysis of crude protein (%N x 6.25, AOAC 2006,
method 990.03), amino acids (AOAC 2006, method 982.30 E [a,b,c]) and chromic oxide
(acid digestion and spectrophotometry) were completed by a certified laboratory. Gross
energy was analyzed with a Parr model 6200 Isoperibol calorimeter (Parr Instrument
Company, Moline, Illinois) equipped with a semi-micro bomb to accommodate small
samples (~0.25 g).
Results
Nutrient analysis of primary protein sources revealed notable differences in
proximate (Table 2-1) and amino acid composition. Average crude protein of
experimental BP-soy products (61.8 g/100g dm) was 4.7% higher than unconverted nonGMO SBM (57.1 g/100g dm). Crude fiber was also reduced from 6.2 g/100g dm in BPsoy products to 3.4 g/100g dm in unconverted SBM. Non-fiber extract (NFE) was lower
in extruded BP-soy (23.5 g/100g dm) than all other soy products including unextruded
61
BP-soy (27 g/100g dm). Extruded BP-soy products also contained higher ash (9.7 g/100g
dm) than all other soy products. Hamlet HP-300 contained a similar protein level (59.3
g/100g dm) to the experimental BP-soy products (61.2 g/100g dm), while crude protein
in PepSoyGen (54.9 g/100g dm) was lower.
Feed formulations were identical and resulting estimated compositions were
similar among soy diets (Table 2-2). Dietary crude protein was considerably higher in test
diets containing Empyreal (54.2 g/100g dm) and wheat gluten (52.7 g/100g dm), but
considerably lower for wheat flour (34.2 g/100g dm). Crude protein in the FM diet (49.6
g/100g dm) was comparable to the experimental soy diets (range 45.9 to 49.2 g/100g
dm).
Apparent protein and energy digestibility for the feedstuffs are given in Table 2-3.
The highest apparent digestibility of protein (95.3) of any ingredient measured was
observed for Hamlet HP 300, followed by bioprocessed, unextruded SBM (GMO
SBM#2, 94.4). Soy bioprocessing generally provided higher protein digestibility than
commercial soybean meal. Protein in commercial bioprocessed products was generally
more digestible than experimental bioprocessed products. However, the commercial
products provided lower energy digestibility than any other ingredients. Hamlet HP-300
delivered the lowest energy digestibility of soy products (50.7), but wheat flour was the
lowest of all ingredients (44.9). The highest energy digestibility was observed for FM
(97.4), which was considerably higher than found for all other products (range 44.9 87.0).
Two bioprocessed, unextruded GMO SBM products (GMO SBM#2 and #4)
produced high ADC-P values (94.4 and 93.5, respectively) and relatively high ADC-E
62
(87.0 and 67.7, respectively). GMO SBM was the best performing feedstock over nonGMO SBM. ADC-P was also high for wheat gluten (94.1).
Apparent amino acid digestibility was on average 4.9% higher for unextruded
products than extruded products (Table 2-4). ADCs of the essential amino acids lysine,
methionine, arginine, leucine, and valine were improved from extrusion of WF, but
negatively impacted from extrusion of SBM. ADCs for lysine (89.2), methionine (91.3),
cysteine (60.7), and total amino acids (90.8) were highest for unextruded, non-GMO, BPSBM. Digestibility of taurine fluctuated drastically between diets, and showed an average
ADC reduction of 22.2% in extruded products. Non-essential amino acids hydroxylysine
and hydroxyproline also exhibited a large reduction in extruded products (16.2 and 15.8%
lower ADCs, respectively).
On average, unextruded BP-soy products performed favorably to extruded
products. Extrusion caused a reduction in protein and energy digestibility for both NonGMO SBM and GMO SBM; however, digestibility of GMO WF was improved from
extrusion. Over-processing of SBM could be the cause of decreased improvement. Unlike
SBM, WF is not toasted and thus benefited to a greater extent from extrusion
pretreatment.
Discussion
ADCs of protein and energy of FM, SBM, Empyreal, wheat gluten, and wheat
flour found in this study were similar to values obtained for Rainbow Trout and Hybrid
Striped Bass (Barrows et al. 2012). The special select FM and unconverted SBM in this
63
study also performed similar to the high-quality FM and SBM used in another study with
Rainbow Trout (Aksnes and Opstvedt 1998).
Energy digestibility of soybean meal was higher than wheat flour and corn gluten,
but was similar to wheat gluten, and much lower than FM. Allan and Booth (2004) also
found soybeans to provide higher energy digestibility than other plant-meals in Silver
Perch Bidyanus bidyanus (Mitchell). Although firm relationships could not be discerned
here, lipid constituents could greatly affect energy digestibility, and may be the cause for
lower energy digestibility in plant meals than in FM.
Results indicate extrusion of WF improved digestibility of protein and amino
acids, but extrusion of SBM degraded digestibility. Extrusion reduced NFE and
consequently non-starch polysaccharides, which are negatively correlated with protein
and lipid digestibility (Refstie et al. 1999). The reduction of NFE along with improved
digestibility of WF would be in agreement with other research that has found extrusion
processing of raw ingredients to improve nutrient digestibility in many species (Barrows
and Hardy 2001; Romarheim et al. 2006; Barrows et al. 2007), especially those
ingredients with high levels of starch or heat-labile antinutritional factors (Glencross et
al. 2011). Other researchers have found extrusion improved digestibility of dry matter,
organic matter, and energy in soybean meal by about 3 to 14% (Allan and Booth 2004).
Also, extrusion has been shown to reduce soy ANFs such as trypsin inhibitor activity
(Barrows et al. 2007) and phytic acid (Allan and Booth 2004), but excessive heat damage
is known to make proteins of several plant meals unavailable for fish growth (Glencross
et al. 2007). The extruded SBM used in this study may have been exposed to heat damage
due to the subsequent thermal treatments (toasting/desolventizing and extrusion cooking).
64
These products showed reduced protein and amino acid digestibility, which supports the
probability of excessive heat damage. Since extrusion parameters were not optimized for
digestibility of these individual ingredients, ADCs found for Yellow Perch in this study
may be conservative values.
In addition to ingredient processing, experimental conditions and diet formulation
have been suggested to cause variability in results (Bailey and Alanara 2006). These
conditions were maintained consistent for test diets in this study. Although relative
differences in digestion may be experimentally compared, several factors have been
found to affect nutrient absorption rates including sampling method, developmental stage
of fish, feeding regime, amount ingested, stomach distention, evacuation rates, and
antinutritional factors (Garber 1983; Henken et al. 1985; de la Higuera et al. 1988;
Andersen 1998; Francis et al. 2001; Jobling et al. 2007).
The feeding strategy of apparent satiation was adopted in this study as a means to
reduce variability and increase repeatability in performance results (Petrell and Ang
2001). In addition, a feeding rate of twice per day was used, which was found to be the
optimal feeding schedule for Silver Perch (Rowland et al. 2005), and was generally easily
implemented with hand-feeding. Feeding to apparent satiation, however, has also been
found to decrease digestibility due to larger meal size and subsequently faster gastric
evacuation rates (Henken et al. 1985). This may be one source of bias in comparing the
results of this study with others (Aksnes and Opstvedt 1998; Barrows et al. 2012).
Fecal stripping generally provides a representative fecal sample as long as care is
taken to squeeze only the distal third of the intestine with a consistent amount of pressure
(Austreng 1978b). The stripping method has been found to underestimate digestibility,
65
most likely because of the incomplete digestion in the hindgut (Austreng 1978b;
Vandenberg and De La Noüe 2001). However, other collection methods which involve
collecting feces after it is evacuated often causes overestimation due to leaching (Rawles
et al. 2010). Stripping limitations include the possibility of including undigested matter,
and also contaminating feces with intestinal epithelium and mucus, which can all lead to
underestimation of digestibility (Storebakken et al. 1998a). In order to more accurately
define ADC values using the stripping method, the amount of nutrient absorption in the
hindgut should be studied.
Chromic oxide is widely used as a tracer in digestibility studies, and is thought to
be predominantly inert. However, the inclusion of chromic oxide at higher levels (≥1.5%)
has been shown to affect digestibility and reduce growth and carbohydrate digestibility in
tilapia (Shiau and Liang 1995). Considerable research has been done on the influence of
chromic oxide, but the effects are relatively minor at low inclusion levels and probably
do not change digestibility results (Ng et al. 2004). We assumed our inclusion rate of
0.5% had a negligible effect on digestibility.
Summary
Very little information exists on feedstuff digestibility in Yellow Perch, a species
suited for intensive production growth. Therefore, characterization of plant-based
alternative ingredients to FM are critical. Feed formulation on a digestible basis can allow
for more economical production, with less waste and faster production.
Bioprocessing consistently improved digestibility over unconverted material.
Unextruded BP-soy provided a slight advantage in protein digestibility, including FM.
66
However, there was considerable variation in ADC-E values detected between BP
conditions.
With the growing cost and decreased availability of FM, sustainable plant-based
sources are an essential alternative for use in aquaculture feeds. This study has indicated
that commercial and novel soy bioprocessing technologies can improve bioavailability of
nutrients in Yellow Perch. Further research in optimizing processing technology and
carnivorous fish responses will help create more consistent production and better feed
ingredients, able to be utilized to a greater extent than conventional plant-based products.
67
Table 2-1. Composition of the primary protein sources (g/100 g, dm unless noted)
incorporated into the experimental digestibility diets for Yellow Perch.
Constituent
Ingredient
Fish Meal
Special Select
Moisture
Protein
(wb)
Lipid
Crude
fiber
Ash
Energy
(MJ/kg)
NFE
67.2
7.6
5.2
0.2
25.3
19.2
2.1
Soybean Meal
Non-GMO SBM
57.1
8.3
1.1
6.2
7.2
19.7
28.5
Commercial BP-soy
PepSoyGen
Hamlet HP-300
54.9
59.3
6.5
9.5
1.2
1.7
2.6
5.1
7.2
7.7
20.1
20.1
34.1
26.2
Unextruded BP-soy
GMO WF
GMO SBM #1
GMO SBM #2
GMO SBM #3
GMO SBM #4
Non-GMO SBM
55.9
61.5
61.3
61.6
61.1
65.8
4.1
3.5
4.7
6.0
3.8
5.9
0.9
0.8
0.4
0.4
0.5
0.8
3.6
3.9
4.4
2.4
3.4
1.9
11.8
9.5
5.6
5.0
4.2
11.1
19.2
19.7
19.0
19.4
19.5
20.2
27.8
24.3
28.3
30.6
30.8
20.3
Extruded BP-soy
GMO WF
GMO SBM
Non-GMO SBM
62.3
62.7
62.3
3.6
3.1
6.3
0.6
1.0
0.8
2.6
2.0
6.3
9.1
11.0
9.0
19.9
19.6
20.1
25.5
23.4
21.7
Other feed ingredients
Empyreal
Wheat Flour
Wheat Gluten
83.3
15.7
77.5
8.5
10.7
7.1
4.9
1.8
0.0
1.1
2.2
0.2
1.4
2.0
0.7
25.4
18.7
23.4
5.8
78.4
21.7
68
Table 2-2. Digestibility feed formulations and estimated dietary
composition of the reference and test diets (g/100g, dm). Ranges are
given for soy diets only.
Ingredient
Fish meala
Test Ingredient
Whole wheat flourb
Vitamin premixc
Mineral premixd
Stay-Ce
Fish Oilf
Chromic Oxideg
Reference
Diet
55.0
0.0
33.2
1.0
1.0
0.3
9.0
0.5
Test
Diets
38.5
29.9
23.2
0.7
0.7
0.2
6.3
0.5
Estimated Composition
Crude Protein
42.2
45.9-49.2
Crude Fat
12.5
8.8-9.2
Crude Fiber
0.8
1.1-2.4
Ash
16.9
13.1-15.3
NFE
24.1
22.9-27.0
Gross Energy (GE, MJ/kg)
19.2
19.1-19.5
Protein Energy Ratio (g/MJ)
22.0
23.8-25.5
a
b
Special Select, Omega Protein, Houston, TX; Bob’s Red Mill
Natural Foods, Milwaukie, OR; cARS 702 vitamin premix, Nelson
and Sons, Murray, UT; dARS 640 trace mineral premix, Nelson and
Sons, Murray, UT; eArgent Laboratories, Redmond, WA; fVirginia
Prime Gold, Omega Protein, Houston, TX; gFisher Scientific,
Pittsburg, PA.
69
Table 2-3. Apparent digestibility coefficients (ADC) of protein
(ADC-P) energy (ADC-E) for test ingredients fed to Yellow Perch
Ingredient
ADC-P
ADC-E
Fish Meal
Special Select
88.8
97.4
Soybean Meal
Non-GMO SBM
85.2
57.8
Commercial BP-soy
Pep Soy Gen
Hamlet HP 300
84.7
95.3
57.9
50.7
Unextruded BP-soy
GMO WF
Non-GMO SBM
GMO SBM #1
GMO SBM #2
GMO SBM #3
GMO SBM #4
78.4
89.6
86.8
94.4
90.4
93.5
54.7
76.6
71.1
87.0
70.9
67.7
Extruded BP-soy
GMO WF
Non-GMO SBM
GMO SBM
79.9
76.5
85.2
67.9
66.5
64.4
83.7
70.2
94.1
57.4
44.9
74.5
Other feed ingredients
Empyreal
Wheat Flour
Wheat Gluten
70
Table 2-4. Apparent digestibility coefficients (ADC) of essential amino acids
(EAA) and non-essential amino acids (NEAA) for experimental bioprocessed soy
products fed to Yellow Perch.
Non-Extruded
FM
Ref
Diet
GMO
WF
Extruded
GMO
SBM
NonGMO
SBM
GMO GMO
WF SBM
NonGMO
SBM
EAA
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Valine
NEAA
Alanine
Aspartic Acid
Cysteine
Glutamic Acid
Glycine
Hydroxylysine
Hydroxyproline
Ornithine
Taurine
Proline
Serine
Tyrosine
86.4
84.8
88.6
90.1
89.5
86.6
88.2
84.7
87.0
89.7
84.1
84.0
83.9
80.6
80.6
86.9
72.3
83.5
97.9
91.2
90.2
89.5
86.8
86.9
93.9
80.4
90.4
97.3
92.9
92.6
91.6
89.2
91.3
93.1
82.6
94.2
92.4
84.0
84.0
84.6
82.0
83.5
86.1
72.1
84.3
94.5
89.1
89.0
88.6
84.2
87.3
90.6
79.4
89.6
89.5
79.5
81.5
82.7
77.2
81.8
84.4
70.1
80.8
82.5
81.3
56.9
88.4
68.8
45.3
45.4
81.8
69.1
79.1
83.0
88.4
82.0
78.3
44.5
86.4
79.2
25.4
39.9
109.9
7.4
84.5
76.5
85.5
91.1
83.5
46.2
91.7
91.1
64.3
59.1
83.8
35.8
90.7
84.8
92.1
92.1
85.7
60.7
92.8
91.4
45.0
40.9
93.8
25.5
92.0
87.5
91.8
83.5
75.6
45.0
86.3
79.4
18.3
23.3
89.0
-28.2
83.6
80.1
85.2
87.1
79.2
54.8
89.5
82.7
27.5
30.4
95.6
29.6
87.1
84.1
88.6
81.9
68.4
34.5
84.5
75.0
40.2
38.9
82.8
0.8
80.4
77.8
84.0
Total AA
83.6
82.1
89.2
90.8
82.3
86.3
78.9
71
CHAPTER 3. PERFORMANCE OF YELLOW PERCH FED BIOPROCESSED SOY
WHITE FLAKE
Introduction
Expansion of Yellow Perch Perca flavescens intensive aquaculture is highly
dependent upon creating more efficient and cost effective feeds. Commercial feeds
commonly fed to Yellow Perch contain high levels of fish meal (FM) as the primary
protein and essential amino acid source (Kasper et al. 2007); however FM is becoming
more expensive due to decreasing availability and increasing market competition (Tacon
and Metian 2008). The current world supply of FM is relatively stable and arguably
sustainable, but demand will exceed supply if aquaculture and aquafeed continue to grow
using FM-based formulations (Gatlin et al. 2007). Therefore, FM must be replaced by
alternative feedstuffs for the sustainability of FM stocks as well as for aquaculture and
aquafeed industry growth. Additionally, most Yellow Perch feeds are formulated based
on the requirements of Rainbow Trout Oncorhyncus mykiss (Brown et al. 1996), which
contain higher levels of FM than feeds for herbivorous or omnivorous species (Kasper et
al. 2007).
Defatted soybean Glycine max meal (SBM) is one of the most commonly used
alternatives to FM, due to a well-balanced amino acid profile, moderately high protein
content, consistent quality, relatively low cost, and high domestic availability (Sales
2009). Compared to FM, SBM has lower methionine, which is considered to be the first
limiting amino acid for fish (Storebakken et al. 2000), while lysine and threonine are also
known to be limiting in SBM (Gatlin et al. 2007). SBM and other processed (mechanical,
chemical, and/or microbiological) soy products have been used to replace FM in
72
aquaculture feeds for several species with varying degrees of success (Kaushik et al.
1995; Refstie et al. 1997; Brown 2008). Experiments which utilized carnivorous species
have revealed limited inclusion levels of dietary SBM (Olli et al. 1994b; Baeverfjord and
Krogdahl 1996;). For example, Olli et al. (1994b) found that Atlantic Salmon Salmo
salar, L., fed either full-fat or defatted SBM impaired growth performance with
increasing inclusion levels, while a solvent-extracted soy protein concentrate (SPC) was
comparable to FM up to 56% of the total protein (37.6% of diet).
SPC and soy protein isolate (SPI) with reduced antinutritional factors (ANFs such
as trypsin inhibitors and phytic acid) and bioavailable protein have been successful, but
high processing costs hamper their use as complete FM replacements (Gatlin et al. 2007).
Soy bioprocessing (BP) technologies are being optimized to increase protein content,
reduce indigestible components (e.g., oligosaccharides), reduce ANF’s, or improve
digestibility (Refstie et al. 2005).
Cooking extrusion as a BP pretreatment has been found to increase the hydrolysis
of oligosaccharides into simpler, fermentable sugars for improved bioprocessing
(Karunanithy et al. 2012; Karuppuchamy 2011). Extrusion causes a change in
carbohydrate structure and peptide length and is accomplished under extreme pressure,
sheer, and high temperature (Barrows and Hardy 2001). Final feeds used in this study
were produced via warm extrusion (<50°C), but extrusion cooking is also the primary
method of producing commercial aquaculture feeds, and can improve both nutritional and
physical qualities of a feed.
Most fish nutrition studies do not report physical pellet characteristics, while both
nutritional and functional aspects of the final feeds are important. Aquaculture feeds
73
should have good pellet durability, stability, and other physical traits so that feed waste
(fines, leaching, spoilage, etc.) is minimized. Plant-based ingredients such as glutens,
starches, or other ingredients are commonly added to conventional diets to improve pellet
functionality.
The primary objective of this study was to evaluate Yellow Perch growth
performance, feed efficiency, general health, and gastrointestinal responses to
conventional and BP-soy products fed as main protein sources. We also assessed the
nutritional composition, ANF’s, and functionality of the primary protein sources. Lastly,
we examined the effects of soy processing technologies on physical pellet characteristics.
Methods and Materials
Experimental Design and Diet Formulation
A factorial design with an FM reference was used to test the effects of complete
FM replacement with one of three soy protein sources with and without amino acid
supplementation. Soy protein sources included one commercial SPC, and two BP-soy
white flake (BP-WF) products obtained from Prairie AquaTech (Brookings, SD).
One lot of white flake was originally obtained from South Dakota Soybean
Processors (Volga, SD), from which half was immediately subject to BP and the
remaining half was extruded prior to BP. White flake was extruded with a Plasti-Corder
PL2000 single-screw laboratory extruder (C.W. Brabender, South Hackensack, NJ) using
conditions optimized for oligosaccharide hydrolysis (Karunanithy et al. 2012;
Karuppuchamy 2011).
74
Seven dietary treatments were used with four replications. Diets were coded as
follows: FM control (Ref), commercial SPC, SPC with Lys + Met (SPCaa), unextruded
BP (BPWF), BPWF with Lys + Met (BPWFaa), extruded BP WF (ExBPWF), and
ExBPWF with Lys + Met (ExBPWFaa).
Main protein sources were incorporated into diets specifically formulated for
Yellow Perch (Table 3-1) based on requirements suggested by Twibell and Brown
(2000); Hart et al. (2010); and Dr. M. Brown (personal communication); unknown
requirements were based on Rainbow Trout requirements (NRC 2011). Lipid
composition of the feeds in this study incorporated flaxseed oil as a partial replacement
for fish oil, but effects were not specifically examined.
Diets were formulated to balance mass of the primary soy protein sources as well
as other ingredients, and also to be similar in composition to the reference diet.
Formulations targeted a composition of 42% crude protein, 10% crude lipid, and protein
to energy (PE) ratio of 30 MJ/g protein by modifying wheat flour, wheat gluten, celufil,
fish oil, and flax oil. Other supplements, except lysine and methionine, were included
equally in all diets based on the most limiting diet.
Large particle ingredients were ground with a Fitzpatrick comminutor (Elmhurst,
IL) with 1.27 mm screen prior to dry blending. Dry ingredients were blended for 20 min
using a V-10 mixer containing an intensifier bar (Vanguard Pharmaceutical Machinery,
Inc., Spring, TX). Dry blended feedstuffs were then transferred to a Hobart HL200 mixer
(Troy, OH) where oils and moisture content (30% wb) were added and blended for ~5
min. Feeds were then screw-pressed using a Hobart 4146 grinder with a 2.5 mm die and
dried to ~10% MC under cool, forced-air conditions. Following drying, feeds were milled
75
into pellets using a food processor, sieved to achieve consistent pellet size, and placed in
frozen storage at -20o C, pending use for feeding or analysis.
Compositional Analysis
Chemical analyses of primary protein sources and feeds were completed by
certified private laboratories. Analyses were completed for crude protein (%N x 6.25,
AOAC 2006, method 990.03), crude fat (AOAC 2006, method 921.39), crude fiber
(AOAC 2006, method 978.10), moisture content (MC; AOAC 2006, method 934.01), ash
(AOAC, method 942.05) and amino acids (AOAC 2006, method 982.30 E (a, b, c)).
ANF’s were also analyzed by certified private labs and included oligosaccharides (Bhatti
et al. 1970; Churms et al. 1982), phytic acid (AOAC 2006 method 986.11), and trypsin
inhibitor activity (AACC 2006, method 22-40).
Culture System
The feeding trial was done in a 3,370 L RAS composed of 30, 110 L circular
tanks, radial flow settler, bead filter, secondary sump with water level control and
electronic water quality monitoring devices, moving bed bioreactor, UV filter, and
heating/chilling unit. Individual tanks were also equipped with a solids settling column
attached to the drain line for heavy solids removal and tank water level control. Tanks
and bioreactor were each supplied with air diffusers fed by a regenerative blower. The
RAS was supplied with municipal water, dechlorinated with adsorptive carbon and stored
in a 15,200 L head tank. Temperature was maintained at 22°C (range 19-23°C). Flow rate
was monitored daily using calibrated manometers installed on each tank supply.
76
Dissolved oxygen, pH, ammonia, nitrite, and alkalinity were also monitored and
maintained within acceptable culture levels.
Feeding Trial
A total of 588 juvenile Yellow Perch were randomly stocked into 28 tanks. The
fish were previously held on a commercial trout diet (BioVita Fry; Bio-Oregon,
Warrenton, OR). After a two-day period of system acclimation, tanks were randomly
assigned a treatment diet, and fish were fed a graded mixture of the commercial diet and
the assigned treatment for a week and then fed 100% of the treatment diets. To monitor
growth performance, tank biomass was sampled (mean±SE, 4.13±0.64g) after feed
acclimation and subsequently every other week until trial end. Individual lengths (mm)
and weights (+0.01g) were measured on four randomly sampled fish from each treatment
during each sampling period. Tank density was initially 789±79 g/m3 with 21 fish per
tank providing a tank biomass of 86.78±2.94g. Sampled individuals were also briefly
examined for health issues (e.g., fin erosion, gill color, skin color).
Fish were fed to apparent satiation twice daily (0800 and 1600 hr) and feeding
rates were modified to slightly overfeed based on feed consumption assessments at least
twice per week. Consumption (%) was estimated by counting uneaten pellets 30 min after
feeding. This method assumes fish consume feed independent of pellet size (Helland et
al. 1996), although the feeds made in this study were sieved and composed of similar size
pellets. On a weekly basis, tank consumption averages were used to estimate actual
consumption (g). Palatability was inferred from the amount of feed consumed or rejected,
especially early in the trial.
77
Upon completion of the trial, five fish per tank were euthanized with 300 ppm
buffered MS-222 and examined for ending performance measures including specific
growth rate (SGR), relative growth, Fulton condition factor (K), feed consumption,
survival, feed conversion ratio (FCR), protein efficiency ratio (PER), fillet weight ratio,
fillet composition, hepatosomatic index (HSI), liver color, viscerosomatic index (VSI),
apparent protein and energy digestibility, and energy conversion efficiency.
Fulton-type condition factor (K) was calculated as:
100,000.
Δmass wet, g
initialmass wet, g
100%
Relative growth (RG) was calculated as:
Specific growth rate (SGR) calculated as:
ln finalwt g
ln startwt g
100
Estimated feed intake values were used in the conversion calculations:
Feed conversion ratio (FCR) was calculated as:
,
,
Protein efficiency ratio (PER) was calculated as:
growth wet, g
massofproteinconsumed dry, g
Organosomatic parameters were calculated by the following equations:
Hepatosomatic Index (HSI) was calculated as: HSI
Viscerosomatic Index (VSI) was calculated as: VSI
Visceral Fat Index (VFI) was calculated as: VFI
100, and
100.
100,
78
Intestinal sections were also analyzed for dietary induced inflammation (enteritis)
with histological examination. Small sections (~2mm) of proximal and distal intestines
were sampled from all euthanized fish. Sections were fixed in 10% buffered formalin for
48 hr and subsequently stored in 70% ethanol until being sent to the South Dakota
Animal Disease Research and Diagnostic Laboratory (ADRDL) for sectioning and
mounting. Intestine sections were qualitatively scored using the protocol described by
Colburn et al. (2012).
After the end of the growth trial, the remaining fish in the system were offered the
digestibility diets with 1.0% chromic oxide Cr2O3 (Austreng 1978b) for 10 days. Fecal
material was collected via gastrointestinal stripping under anesthesia (80 ppm MS-222).
Diets and dried feces were then analyzed for gross energy (Parr 6200 bomb calorimeter)
and chromic oxide and crude protein (%N x 6.25) analysis. The apparent digestibility
coefficient (ADC) for protein (ADC-P) and energy (ADC-E) in the test diets used the
following calculation (Kleiber 1961).
100
100 %
%
%
%
Physical Pellet Properties
Samples of each feeding trial diet were analyzed in triplicate for water activity
(aw-), bulk density (BD), pellet durability index (PDI), water solubility index (WSI) and
color (Hunter L,a,b-); compressive strength (CS), and diameter (mm) were determined
for 10 replications. Water activity of 2 g pellet samples was measured with a Lab Touch
aw analyzer (Novasina, Lachen SZ, Switzerland). Three pellet color variables were
79
measured with a spectrophotocolorimeter (LabScan XE, HunterLab, Reston, VA) as
Hunter L (brightness/darkness), Hunter a (redness/greenness), and Hunter b
(yellowness/blueness); the illumination/observer was D65/10°. BD was estimated by
weighing 100mL of pellets and dividing the mass by 0.0001m3 and then converting to
kg/m3. PDI was determined according to ASAE standard method S269.4. PDI was
calculated as: PDI (%) = (Ma/Mb) x 100, where Ma was the mass (g) after tumbling and
Mb was the mass (g) before tumbling. WSI was determined by the static method, and was
calculated as loss of weight from leaching/dry weight of initial sample (Obaldo et al.
2002). Pellet diameter was measured using a digital Starrett caliper (Arthol, MA). CS was
calculated as peak fracture force of the stress-strain curve from a perpendicular axial
direction. Pellets were tested for CS using a TA.XT Plus Texture Analyzer (Scarsdale,
NY).
Statistical Analysis
Performance measures were analyzed for treatment effects with single-factor
analysis of variance ANOVA (a priori α = 0.05). Two-way analysis of covariance
ANCOVA was used to test the influence of amino acid supplementation in soy diets.
Tukey’s HSD test was used for all post-hoc comparisons. Where needed, data were
transformed to the confines of a normal distribution and equal variance prior to analysis.
80
Results
Soy Ingredient Composition
Analysis of primary protein sources indicated substantial differences in proximate
composition, amino acids, and ANFs (Table 3-2). Crude protein was highest in SPC
followed by FM, BPWF, and extruded BPWF. Crude lipid was highest in FM followed
by BPWF, extruded BPWF, and not detected in SPC. Crude fiber was significantly higher
in SPC than the other ingredients. Contrary to expectation, fiber was also higher in
extruded BPWF than unextruded BPWF.
Corresponding to higher protein, SPC also had a greater concentration of essential
amino acids (EAA) compared to BP-soy. FM had a greater concentration of methionine,
and slightly higher lysine than SPC. All other EAAs were higher in SPC. FM provided
more alanine, glycine, hydroxyproline, and taurine than any soy product. Extruded
BPWF provided a greater total of amino acids than unextruded BPWF, with a greater
concentration of Arginine and a lower concentration of hydroxylysine.
Raffinose was not detectible in any soy products. Stachyose was 0.24% (dm) in
SPC while bioprocessing completely eliminated stachyose. Phytic acid was reduced from
0.39% in unextruded BPWF to 0.18% (dm) in extruded BPWF, which was similar to
levels in SPC (0.23% dm). Trypsin inhibitor activity was substantially reduced from
16,750 TIU/g in unextruded BPWF to 6,538 TIU/g in extruded BPWF. Trypsin inhibitor
activity was also high in the commercial SPC (13,630 TIU).
Diet analyses revealed similar but not isonitrogenous diets (Table 3-3). Analyses
showed that crude protein of unsupplemented diets was highest for FM (44.9%) followed
by SPC (43.2%), BPWF (36.8%), and lowest for ExBPWF (37.5%). Differences in other
81
constituents except for ash were negligible between diets. Crude lipid was approximately
10% for all diets. Amino acid analysis of feeds did not reveal deficiencies among any of
the trial diets in comparison to estimated Yellow Perch requirements.
Digestibility
The SPC diet provided the highest ADC-P (91.8) and the FM diet provided the
highest ADC-E (81.9) (Table 3-3). Lower consumption or possibly slower gastric
evacuation rates may have improved ADC-P of the SPC by increasing contact time with
digestive enzymes. BPWF showed similar ADC-P to FM, while ExBPWF produced the
lowest ADC-P (84.5) and also lowest ADC-E (63.6). SPC diets also had a moderate
protein to digestible energy ratio (PDE). The highest PDEs were observed for FM (29.2)
ExBPWF (28.6) and ExBPWFaa (28.6), corresponding with the order of relative growth.
Survival, Growth, and Health Performance
Survival was significantly different among treatments (P<0.01). Fish fed the FM
control had the highest survival (98%) but was not significantly different from ExBPWF
(95%), ExBPWFaa (92%), or BPWFaa (85%). All other diets provided lower survival but
were not significantly different.
Initial tank weights were not significantly different (P=0.76) among tanks or
treatments. All endpoint growth performance parameters were significantly different
among treatments (P<0.05) (Table 3-4). The FM diet produced significantly higher RG
(401.8%) than SPC, SPCaa, and BPWF (205, 294, and 275%, respectively), but more
comparable results to BPWFaa, ExBPWF, and ExBPWFaa (305, 310, and 338%,
respectively). The commercial SPC provided significantly lower growth than both FM
82
and ExBPWF; but not significantly different from BPWF. RG of supplemented diets was
greater than unsupplemented diets. SGR followed a similar trend with FM (0.71)
outperforming SPC diets, but not significantly different from ExBPWFaa (0.65). The
SPC diet (0.49) had a significantly lower SGR than all other treatments.
Fulton’s condition factor (K) differed among between treatments (P<0.01), and
was highest for fish fed ExBPWF (1.22), which was significantly higher than SPC and
BPWF (1.05 and 1.10, respectively). Diets with low K values were correlated with low
consumption and growth, which may indicate lower palatability.
Total dry matter consumption was significantly different (P<0.01) and was
highest for FM (658 g), followed by ExBPWF (525 g), and ExBPWFaa (491 g), and then
lowest for BPWF (335 g), SPCaa (305 g), and SPC (198 g). Feed conversion ratio (FCR)
was significantly different between diets (P=0.04). SPCaa fed fish provided the best FCR
(1.62), but was not significantly different from SPC (1.74) and BPWF (1.81). Both
supplemented BP diets BPWFaa (1.96) and ExBPWFaa (1.95) produced the highest
FCRs and were only significantly different from SPCaa. Protein efficiency ratio (PER)
was significantly different between treatments (P<0.01). PER was highest in BPWF
(1.50) followed by SPCaa (1.49), and these were significantly different from the FM
control (1.16).
There were significant differences in fish anatomical measurements resulting
from of the experimental diets (Table 3-5). The BP diets produced fish which on average
had a higher VFI than the FM control or commercial SPC diets. Significant differences
existed in VFI (P<0.01). The ExBPWF treatment had the highest VFI (3.4) and SPC had
the lowest (1.7), which was significantly lower than all BP diets. The HSI was not
83
significant between diets. However, qualitative examination of liver color did vary
among treatments wherein livers of SPC fish tended to be pale in comparison to the
control fish or fish fed bioprocessed soy. Color was most variable in the FM control.
No significant differences existed in fillet yield, HSI, VSI, and Hunter color
analysis of the fillets. Additionally, there were no statistically significant differences in
the gastrointestinal histology scores but there was a trend. The ExBPWF treatment
provided the highest average score (13.3) for distal sections, and ExBPWFaa provided
the lowest score (9.8). FM scored a moderate 11.9, which was considered to be a normal
baseline.
Physical Pellet Properties
Pelleted feeds exhibited significant differences among treatments (Table 3-6).
Water activity (aw) ranged from 0.58 for FM to 0.74 for ExBPWFaa. Water stability
index (WSI) was moderate in all diets (range 9.65 to 14.43). Pellets did not visibly
dissolve until at least 30 min after feeding, which allowed sufficient time for feeding. The
highest WSI was for SPC (11.9), which differed significantly (P<0.01) only from
ExBPWFaa (6.7). Due to the nature of screw-pressed pellets, WSI was expected to be
low to moderate.
PDI was very high in all diets (range 98.05 to 99.48%). However, there was a
significant difference between BPWFaa, ExBPWF, and ExBPWFaa diets compared to
the others. CS varied significantly from 24.36 to 67.03 g force and the FM diet exhibited
the lowest CS (24.36). BD ranged from 634.87 to 695.9 kg/m3. FM had a lower BD as
well as a visually greater pellet heterogeneity.
84
Hunter color parameters (L, a, b) revealed an association with growth
performance. Hunter a (redness) was highest in FM (5.15) and lowest in SPC (2.80).
Hunter b (yellowness) was highest in BPWF (22.81) and FM (22.76) and lowest in SPC
(18.07). Hunter L (brightness) was highest in SPCaa (68.48) and lowest in FM (47.91).
FM provided the largest diameter pellets which was on average 200 µm larger
than the 2.5 mm die. Pellet expansion due to pressure changes when exiting die were
expected to be minimal due to low dietary starch as well as low processing temperature
and pressure of screw-pressed pellets.
Discussion
The soybean products used in this study demonstrated fish dietary responses to a
range of processes, which resulted in differing performance. It is evident that commercial
SPC processing results in higher protein than the BP methods used in this study.
However, there appears to be other advantages of BP including reduction of
antinutritional factors, which is essential for improved growth for Yellow Perch.
Regardless of extrusion pretreatment, BP eliminated oligosaccharides from 0.8%
raffinose and 4.8% stachyose in commercial WF (Karunanithy et al. 2012). This result
was supported by the observation of Dust et al. (2004) who found extrusion had minimal
effect on soy flour when compared to other ingredients having higher starch and fiber
contents. These results indicate that oligosaccharide removal occurs from either
commercial or biological processing, but a small amount (0.24%) of stachyose remains in
SPC.
Trypsin inhibitors were reduced primarily due to extrusion. Unextruded BPWF
and SPC ingredients contained high levels of trypsin inhibitors. Trypsin inhibitors are one
85
of the largest and most diverse groups of ANFs (Rawlings et al. 2004) and their effects
could be a factor in diet consumption and utilization. Soybeans contain natural trypsin
inhibitors which are effective for insect resistance; contributing to the disruption of
digestive enzyme activation. The elimination of natural soybean trypsin inhibitors from
thermal treatments and extrusion has been recognized as an essential component of
soybean based aquafeeds (Francis et al. 2001). These processes can reduce trypsin
inhibitors to safe levels (<5 mg/g or 9500 TIU/g assuming 1µg of trypsin inhibitor has a
TIU of 1.9) for most fishes (Viola et al. 1983; Olli et al. 1994b; Anderson and Wolf 1995;
Francis et al. 2001).
ExBPWF contained lower protein than the BPWF, which could either be an effect
of extrusion, BP, starting material, or their combination. This is similar to the results of
Allan and Booth (2004) in which soy extrusion resulted in slightly less protein. Extrusion
has been reported to reduce certain protein fractions in wheat flour (Rebello and Schaich
1999). This may also indicate a higher amount of non-protein nitrogen (NPN) remaining
in the unextruded BPWF, however this is not confirmed. NPN is used in ruminant
nutrition to supply gastrointestinal flora with additional nitrogen which is converted into
cellular protein (Loosli and McDonald 1968). NPN remaining in bioprocessed material
may indicate a limiting agent for monogastric organisms.
Growth performance overall was best for the FM control, but the ExBPWFaa
treatment was highly comparable and more favorable than SPC diets, despite having
lower protein. The considerable variation in protein between reference and BP diets may
have been an important factor in the observed performance. We speculate that if diets had
been isonitrogenous, there might be a potential for higher RG of fish fed the lower
86
protein diets. For example, adjusting observed RG values by linearly standardizing to the
protein content of the control diet would provide an adjusted mean RG of 405% for
ExBPWFaa, which would be similar to the control diet (401%). By standardizing RG to
protein content, all diets were not significantly different except for the SPC diet (213%).
Daily changes in feed intake were observed throughout the trial, independent of
dietary treatment. Although this may suggest an environmental fluctuation or disturbance,
treatments were randomly distributed and each tank was maintained from a collective
water source. Direct comparisons of feed intake, or consumption, can be indicative of
palatability (Helland et al. 1996). In addition to a complete nutritional profile, providing
feeds with high palatability with high feed intake contributes to increased growth and
production. Lack of palatability and reduced consumption can lead to malnutrition,
emaciation and death, which was thought to be the cause of significantly lower survival
for diet 2 (82.3%), the commercial SPC diet. Lower consumption of the commercial SPC
diets may also have increased apparent digestibility as a result of slower feed evacuation
rates with increased contact with digestive enzymes (Garber 1983).
PDE values showed a greater correlation with RG than did PE. Variable PDE with
negligible differences in dietary lipid content between diets may be an indication that
protein exhibited a greater nutritional effect than lipid. The diets used in this study
consisted of fish oil and flaxseed oil which are fairly well balanced sources of essential
fatty acids. Other studies that have found a correlation with lipid content and reduced
growth and feed efficiency (Francis et al. 2007; Montero et al. 2008). This is because
most plant material lacks important long chain omega-3 fatty acids such as
Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) which are found in fish
87
oil. While fatty acid composition was not specifically examined in this study, we noticed
that fish fed the SPC diet had generally paler livers than other treatments. A pale liver
color has been found in other species that have been fed diets with essential fatty acid
deficiencies (Takeuchi and Watanabe 1982; Watanabe et al., 1989; Ruyter et al., 2006).
In addition, excess fat in the visceral cavity is sometimes considered an indication of poor
health (Craig et al. 1999; Mathis et al. 2003) and is not desired in cultured food fish.
Excess lipids can affect the visual sense and odor of the final product (Grigorakis 2007)
and decrease the carcass yield (Mathis et al. 2003).
No enteritis was observed and all intestines appeared relatively normal; however,
distal intestine sections provided more variance in inflammation scores than did proximal
sections, as observed in other research (UrÁN et al. 2008). Other researchers have linked
compounds in soy products to enteritis in salmonids (Baeverfjord and Krogdahl 1996;
Krogdahl et al. 2000; UrÁN et al. 2008).
Significant differences in MC were likely due to textural inequalities resulting in
uneven moisture loss during pellet drying. The pellets used in this experiment were dried
at room temperature on a forced air drying rack, so drying at or below ambient humidity
was limiting. MC may have had an effect on pellet performance parameters such as PDI,
BD, CS, and color. Higher moisture contents may lead to better acceptance due to a
“softer” texture with lower CS.
Water activity was high in all diets. Higher water activity may lead to shorter
storage life due to microbiological growth. aw values over 0.50 tend to allow diverse
microbial growth at room temperatures. Feeds were stored in a freezer at -20° C to
preserve the microbiological and chemical stability.
88
Low CS of the FM diet can be partially attributed to increased heterogeneity of
pellet constituents and also that FM and other animal proteins are cooked during
processing, which generally decreases their functionality. The softer texture of the FM
diet may be associated with higher consumption. A softer texture is also preferred by
other species such as Gilthead Sea Bream Sparus aurata L. (Andrew et al. 2004). CS was
considerably higher for all soy diets compared with the FM control, and there was no
consistent soy processing effects on CS.
Our analysis was unable to find any differences associated with PDIs and diet
compositions. The differences could be the result of inconsistencies with the pelleting
process (e.g., feed rate, temperature) or possibly a fraction of carbohydrate or the addition
of carbomethylcellulose (CMC), which was added as a binder to increase the PDI and aw
of screw pressed feeds (Ruscoe et al. 2005). A high PDI for transport and handling
coupled with a low CS for fish acceptability and palatability would be ideal for
commercial aquafeed and aquaculture producers.
The FM reference feed in this study was redder, yellower, and darker than soy
diets. The commercial SPC diet was brighter (+L), less yellow (-a), and less red (-b) than
other soy products tested. Contradictory to these results, others have found lighter colored
feeds to contain higher concentrations and availability of lysine than darker colored feeds
(Cromwell et al. 1993; Fastinger et al. 2006). SPC and FM diets contained similar
amounts of lysine, but BP-soy contained lower lysine. Both SPC and ExBPWF showed
an increased L value with lysine supplementation, but BPWF did not. Our results
generally agreed with studies that have found that more yellow equates to better nutrient
quality (Goihl 1993; Ergul et al. 2003).
89
Summary
This nutritional and functional study provides useful information on the value of
biological and mechanical soy processing technologies for production of main protein
sources in formulated Yellow Perch feeds. We noticed a consistent increase trend in
growth with amino acid supplementation within each soy protein source; however, when
compared between protein sources, SPC provided more lysine, but also the most
restricted growth and health. This indicates that an alternative nutrient limitation or lower
palatability affected growth performance of the commercial SPC diets.
Our results indicate overall the FM control diet to be a more balanced and
bioavailable nutritional composition than the experimental bioprocessed soy proteins.
Yellow Perch aquafeed containing soy proteins as a main ingredient is at the early stages
of development, and there is a definite potential for these ingredients to be utilized for
commercial aquaculture performance, economic sustainability and ultimate success.
These results have developed a baseline for designing the next round of
experimental diets. The information does inquire the need to develop higher quality BPsoy which would support growth performance equivalent to or superior than diets
containing FM, hence reducing cost of commercial food fish production. The
improvement of soy macronutrient composition, digestibility, and palatability through
extrusion pretreatments and BP optimization depends on information like that presented
in this research.
90
Table 3-1. Feed formulations (g/100g dm) of the experimental Yellow Perch diets.
Diet #
Ingredient
FM
SPC
SPCaa BPWF BPWFaa ExBPWF ExBPWFaa
Menhaden FM a
50.0
0.0
0.0
0.0
0.0
0.0
0.0
Commercial SPC
0.0
45.0
45.0
0.0
0.0
0.0
0.0
HQSPC Trial 5
0.0
0.0
0.0
45.0
45.0
0.0
0.0
HQSPC Trial 6
0.0
0.0
0.0
0.0
0.0
45.0
45.0
Yellow corn gluten b
5.0
5.0
5.0
5.0
5.0
5.0
5.0
Wheat flour c
18.0
21.0
21.0
21.0
21.0
21.0
21.0
6.0
5.0
5.0
5.0
5.0
5.0
5.0
Wheat gluten b
CMC d
5.0
5.0
5.0
5.0
5.0
5.0
5.0
d
Celufil
3.7
3.6
3.6
3.5
3.0
3.5
3.0
Menhaden oil e
4.59
8.19
8.19
8.37
8.37
8.37
8.37
Flax oil f
0.51
0.91
0.91
0.93
0.93
0.93
0.93
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Vitamin premix g
Mineral premix h
0.1
0.1
0.1
0.1
0.1
0.1
0.1
i
Vitamin C
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.2
0.2
0.2
0.2
0.2
0.2
0.2
Choline j
k
Phytase
0.037 0.037
0.037
0.037
0.037
0.037
0.037
Brewer’s yeast l
2.0
2.0
2.0
2.0
2.0
2.0
2.0
j
L-Lysine
0.0
0.0
0.3
0.0
0.3
0.0
0.3
L-Betaine j
0.5
0.5
0.5
0.5
0.5
0.5
0.5
j
0.0
0.0
0.2
0.0
0.2
0.0
0.2
L-Methionine
Sodium chloride m
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Potassium chloride m
0.8
0.8
0.8
0.8
0.8
0.8
0.8
m
Magnesium oxide
0.05
0.05
0.05
0.05
0.05
0.05
0.05
Calcium phosphate m
2.0
1.0
1.0
1.0
1.0
1.0
1.0
a
b
Special Select, Omega Protein, Houston, TX; Consumers Supply Distributing, Sioux City, IA; c
Bob’s Red Mill Natural Foods, Milwaukie, OR; d USB Corporation, Cleveland, OH; e Virginia
Prime Gold, Omega Protein, Houston, TX; f Thomas Laboratories, Tolleson, AZ; g ARS 702
premix, Nelson and Sons, Murray, UT; h ARS 640 trace mineral premix, Nelson and Sons, Murray,
UT; i U.S. Nutrition, Bohemia, NY; j Pure Bulk, Roseburg, OR; k DSM Nutritional Products,
Parsippany, NJ; l Diamond V Mills, Cedar Rapids, IA; m Fisher Scientific, Pittsburg, PA.
91
Table 3-2. Composition of the primary protein sources (g/100g, dm unless noted)
incorporated into the experimental diets. EAA=essential amino acids; NEAA=nonessential amino acids; ANFs=antinutritional factors.
FM
Commercial
Unextruded
Extruded
Constituent
(reference)
SPC
BPWF
BPWF
Protein
66.77
72.18
61.61
56.86
Moisture (wb)
7.62
9.73
5.14
7.89
Lipid
5.21
0.00
1.70
1.26
Crude fiber
0.18
10.08
0.81
4.86
Ash
25.33
7.10
8.82
5.21
EAA
Arginine
3.69
5.30
2.44
3.65
Histidine
1.26
1.84
1.41
1.40
Isoleucine
2.73
3.30
2.89
2.92
Leucine
4.47
5.61
4.64
4.87
Lysine
4.58
4.56
3.47
3.41
Methionine
1.72
1.00
0.83
0.90
Phenylalanine
2.51
3.62
2.89
3.08
Threonine
2.32
2.80
2.36
2.31
Tryptophan
0.58
1.00
0.79
0.82
Valine
3.10
3.51
3.13
3.10
NEAA
Alanine
3.97
3.03
2.71
2.66
Aspartic acid
5.47
8.08
6.72
6.45
Cystine
0.48
0.97
0.87
0.88
Glutamic acid
7.73
12.51
8.70
8.85
Glycine
4.81
2.96
2.67
2.51
Hydroxylysine
0.27
0.04
0.81
0.10
Hydroxyproline
1.19
0.08
0.10
0.07
Lanthionine
0.00
0.02
0.00
0.00
Ornithine
0.14
0.04
0.14
0.04
Proline
3.31
3.65
3.17
2.92
Serine
1.85
3.07
2.28
2.73
Taurine
0.42
0.08
0.09
0.10
Tyrosine
2.01
2.57
1.98
2.25
Total AA
58.61
69.64
55.09
56.02
ANFs
Raffinose
-0.00
0.00
0.00
Stachyose
-0.24
0.00
0.00
Phytic acid
-0.23
0.39
0.18
Trypsin inhibitor
(TIU/g)
-13,630
16,750
6,538
92
Table 3-3. Analyzed diet compositions (g/100g dm unless noted) and digestibility results. ADC=
Apparent Digestibility Coefficient; GE=gross energy; DE=digestible energy; PE=protein to energy
ratio; PDE=protein to digestible energy ratio; EAA=essential amino acid; NEAA=non-essential
amino acid.
Diet
Constituent
Protein
Moisture (wb)
Lipid
Crude fiber
Ash
ADC-P
ADC-E
GE (MJ GE/kg)
DE (MJ DE/kg)
PE (g/MJ GE)
PDE (g/MJ DE)
EAA
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Valine
NEAA
Alanine
Aspartic acid
Cysetine
Glutamic acid
Glycine
Hydroxylysine
Hydroxyproline
Proline
Serine
Taurine
Tyrosine
FM
44.93
7.90
10.60
3.44
16.80
87.5
81.9
18.77
15.37
23.94
29.22
SPC
43.23
10.10
9.09
3.84
6.90
91.8
76.1
21.05
16.02
20.54
26.99
SPCaa
43.63
10.90
9.57
4.62
6.84
NA
NA
21.05
16.02
20.72
27.23
BPWF
36.76
13.90
10.10
4.01
8.65
87.1
68.7
20.66
14.20
17.79
25.89
BPWFaa
37.13
10.00
10.19
4.04
8.90
NA
NA
20.70
14.22
17.94
26.11
ExBPWF
37.50
9.68
10.20
3.80
8.77
84.5
63.6
20.62
13.12
18.19
28.58
ExBPWFaa
37.71
11.80
9.69
2.87
8.71
NA
NA
20.72
13.19
18.20
28.59
2.30
0.91
1.76
3.36
2.56
1.04
1.89
1.60
2.06
2.78
1.07
1.96
3.62
2.30
0.58
2.20
1.49
2.22
2.75
1.05
1.91
3.57
2.46
0.74
2.21
1.50
2.09
2.06
0.87
1.74
3.28
1.80
0.55
1.96
1.37
1.96
2.01
0.87
1.78
3.22
1.99
0.70
1.96
1.29
1.94
2.07
0.88
1.82
3.29
1.85
0.56
1.99
1.36
2.05
2.05
0.87
1.85
3.34
2.05
0.74
2.02
1.36
2.04
2.62
3.36
0.46
7.56
2.89
0.14
0.56
2.84
1.51
0.25
1.37
1.95
4.12
0.63
9.02
1.77
0.02
0.03
2.87
1.64
0.03
1.50
1.89
4.05
0.63
8.08
1.74
0.00
0.00
2.70
1.72
0.00
1.46
1.77
3.40
0.58
7.15
1.56
0.05
0.03
2.49
1.60
0.03
1.34
1.70
3.31
0.57
6.56
1.53
0.00
0.00
2.29
1.46
0.09
1.29
1.78
3.43
0.58
7.10
1.57
0.03
0.04
2.46
1.51
0.03
1.34
1.80
3.44
0.58
6.85
1.58
0.00
0.00
2.35
1.64
0.09
1.32
93
Table 3-4. Growth performance indices. Values given are treatment means+SE. Values not
significantly different (P>0.05) have the same letter within a column.
Performance Characteristic
Diet
RG
SGR
S
TC
FCR
PER
K
FM
401.8±20.2a
SPC
205.1±9.4b
SPCaa
293.8±17.8cd
BPWF
274.6±22.7d
BPWFaa 304.7±25.9cd
ExBPWF 309.7±7.8cd
ExBPWFaa338.0±23.7c
0.71±0.02a
0.49±0.01b
0.60±0.02c
0.58±0.03cd
0.61±0.03cd
0.62±0.01cd
0.65±0.02ad
97.6±1.4a
71.4±4.3b
77.4±1.2bc
79.8±3.0bc
84.5±4.5c
95.2±0.0a
91.7±3.6ac
658.4±49.9a
198.4±13.1b
305.1±13.0bc
335.2±40.6c
415.7±64.9cd
525.0±50.9d
491.3±40.9d
1.92±0.05a
1.74±0.34ab
1.62±0.10b
1.81±0.07ab
1.96±0.10a
1.92±0.09a
1.95±0.05a
1.16±0.03a
1.33±0.03b
1.49±0.04b
1.50±0.07b
1.40±0.07b
1.40±0.07b
1.37±0.03b
1.21±0.02a
1.05±0.04b
1.16±0.04ac
1.10±0.02bc
1.21±0.02a
1.22±0.02a
1.20±0.02a
Relative growth (RG, %), specific growth rate (SGR), survival (S, %) total consumption (TC,
dmb, g), food conversion ratio (FCR), protein efficiency ratio (PER), and Fulton-type condition
factor (K)
94
Table 3-5. Necropsy variables for Yellow Perch. Values given are treatment
means+SE. Values not significantly different (P>0.05) have the same letter within a
given column. GI=gastrointestinal distal inflammation score; HSI=hepatasomatic
index; VSI=viscerasomatic index; VFI=visceral fat index.
Diet
FM
SPC
SPCaa
BPWF
BPWFaa
ExBPWF
ExBPWFaa
Fillet/Wt.
34 ± 1a
32 ± 1a
36 ± 2a
33 ± 1a
34 ± 1a
32 ± 2a
35 ± 1a
Anatomical Characteristic
GI Score
HSI
VSI
11.9 ± 0.9a
11.0 ± 0.8a
11.0 ± 1.1a
11.8 ± 1.4a
11.6 ± 1.2a
13.3 ± 1.3a
9.8 ± 0.5a
2.0 ± 0.1a
1.6 ± 0.1b
1.9 ± 0.1ab
2.0 ± 0.1a
1.9 ± 0.1ab
1.9 ± 0.1ab
1.9 ± 0.1ab
5.1 ± 0.3a
4.9 ± 0.3a
5.9 ± 0.4a
5.4 ± 0.3a
5.5 ± 0.3a
5.3 ± 0.3a
5.9 ± 0.4a
VFI
2.2 ± 0.2ab
1.7 ± 0.2a
2.4 ± 0.2abc
3.0 ± 0.3bcd
3.1 ± 0.3cd
3.4 ± 0.3d
3.1 ± 0.3cd
95
Table 3-6. Physical properties of the feed extrudates. Values given are treatment means±SE.
Values not significantly different (P>0.05) have the same letter within a given row.
Diet
Properties
FM
SPC
SPCaa
BPWF
BPWFaa ExBPWF ExBPWFaa
aw (-)
BD (kg/m3)
CS (g)
PDI (%)
WSI (%)
L (-)
a (-)
b (-)
Dia (mm)
0.58±0.02a
634.8±3.0a
24.4±0.7a
98.1±0.4a
10.2±0.0ab
47.9±0.2a
5.2±0.0a
22.8±0.1a
2.2±0.0a
0.67±0.00b
648.6±3.2b
56.1±1.8b
98.1±0.8a
11.9±0.0a
58.5±0.1b
2.8±0.0b
18.1±0.1b
2.1±0.1a
0.69±0.00c
659.4±2.4bc
42.9±2.3c
98.1±0.7a
9.1±0.0ab
68.5±0.5c
3.2±0.1c
20.2±0.1c
2.1±0.0a
0.68±0.00bc
674.5±0.5d
44.9±3.8bc
98.3±0.3a
8.9±0.0ab
59.9±0.3bd
4.3±0.0d
22.8±0.1a
2.0±0.0a
0.68±0.00bc
688.9±0.6e
54.8±2.3b
99.3±0.3b
9.1±0.0ab
53.7±0.2e
4.4±0.0d
21.4±0.1d
2.0±0.0a
0.68±0.00bc
669.3±2.8cd
67.0±2.2b
99.5±0.2b
8.2±0.0ab
60.6±0.3d
2.9±0.0b
20.3±0.0ce
2.0±0.0a
0.74±0.01d
695.9±2.2e
60.1±3.9b
99.5±0.3b
6.7±0.0b
63.8±0.4f
2.9±0.0b
20.7±0.1e
2.0±0.0a
MC (% db) = moisture content; aw (-) = water activity; BD (kg/m3) = bulk density; CS (g) =
compressive strength; PDI (%) = pellet durability index; WSI (%) = water solubility index in
still water; L (-) = Hunter brightness; a(-) = Hunter yellowness; b(-) = Hunter redness; Dia (mm)
= diameter.
96
CHAPTER 4. PERFORMANCE OF YELLOW PERCH FED BIOPROCESSED
SOYBEAN MEAL
Introduction
The increasing cost, demand, and fixed availability of FM has stimulated an
increased use of plant-based aquafeed (Gatlin et al. 2007; Glencross et al. 2007).
Soybeans Glycine max are generally considered to be the most favorable plant material
for use in carnivorous fish feeds, due to their high yields and nutritional quality (Gatlin et
al. 2007). However, there are still apparent and unidentified biologically active
compounds in soy products that inhibit the complete replacement of FM in carnivorous
fish feeds (Gatlin et al. 2007; Barnes et al. 2014). A balance between feed cost and
improved nutritional factors much be reached for Yellow Perch Perca flavescens
producers to become more profitable (Malison 2003).
Soy proteins are known to vary in composition based on processing method, plant
growing conditions, and genetic variety (Swick 2007). Soybeans which have been
processed into soy protein concentrate (SPC) have provided favorable growth
performance in carnivorous fish culture but are currently too costly for broad application
(Olli et al. 1994b; Ingh et al. 1996). Typical soybean meal (SBM), however, is not as well
tolerated by carnivorous fish (van den Ingh et al. 1991; Olli et al. 1994b; Baeverfjord and
Krogdahl 1996; Kasper et al. 2007). Bioprocessing (BP) of soy white flake (WF) and
SBM have been shown to provide favorable growth along with a reduction in
physiological and morphological problems normally caused by WF or SBM (Skrede et al.
2002; Yamamoto et al. 2010).
The present research explored the effects of additional processing to commercial
SBM using three primary soy processing factors (extrusion pretreatment, BP, typical
97
SBM) and two secondary factors (soybean variety and FM replacement level) in
experimental diets for juvenile Yellow Perch. The objective was to determine growth or
health benefits from these processes, and illustrate the effect of increasing dietary
inclusion levels.
Methods and Materials
Experimental Design and Diet Formulation
A 16-week Yellow Perch feeding trial was conducted using a factorial design with
four sampling points to assess growth performance and organosomatic responses of soy
products. Fourteen soy-based diets and an FM control were designed to examine
differences in SBM variety (GMO vs. Non-GMO) and BP with or without extrusion
pretreatment. Diet descriptions and codes are provided in Table 4-1. Soy products were
extruded with a Plasti-Corder PL2000 single-screw laboratory extruder (C.W. Brabender,
South Hackensack, NJ) using conditions optimized for oligosaccharide hydrolysis
(Karuppuchamy 2011; Karunanithy et al. 2012). BP parameters were held constant with
the exception of GMO-BP-SBM (2), which used a modified process but duplicated
feedstock to GMO-BP-SBM (1). Bioprocessed products were obtained from Prairie
AquaTech (Brookings, SD). The primary soy ingredients were included in the diet at two
levels of FM replacement (40% or 70%).
Chemical analyses of primary protein sources were completed by certified
laboratories (Table 4-2). Analyses were completed for crude protein (%N x 6.25, AOAC
2006, method 990.03), crude fat (AOAC 2006, method 921.39), crude fiber (AOAC
2006, method 978.10), moisture content (AOAC 2006, method 934.01), ash (AOAC,
method 942.05) and amino acids (AOAC 2006, method 982.30 E (a, b, c)). Trypsin
98
inhibitor activity (AACC 2006, method 22-40) was measured using the same certified
laboratory.
Main protein sources were incorporated into diets formulated for Yellow Perch
based on requirements suggested by Twibell and Brown (2000); Hart et al. (2010); and
Dr. M. Brown (personal communication); unknown requirements were based on Rainbow
Trout Oncorynchus mykiss requirements (NRC 2011). Diets were formulated on a dry
matter basis with a targeted composition of 45% crude protein, 9% crude lipid, and
protein to energy (PE) ratio of 23.2 g protein/MJ by modifying celufil and soybean oil.
High (70%) FM replacement rate formulations (Table 4-3) contained a higher amount of
soybean oil than low (40%) replacement diets (Table 4-4) in order to maintain isocaloric
diets. Feed supplements were applied in equal amounts among all diets.
Large particle ingredients were ground with a Fitzpatrick comminutor (Elmhurst,
IL) prior to dry blending. Dry ingredients were then blended in a Hobart HL200 mixer
(Troy, OH) where oil and water are added and blended until homogenous for
approximately 10 min. Dough was screw-pressed using a Hobart 4146 grinder equipped
with variable speed feed screw, 2.5 mm die, and cutting head. Extrudates were then dried
in a Despatch UDAF electric conveyor drier (Minneapolis, MN) with maximum
temperature set at 120°C. Feeds were then allowed to cool prior to packaging, and stored
at -20°C pending use.
Culture System
The 6,738 L recirculating aquaculture system (RAS) consisted of 60, 110 L
circular tanks equipped with Cornell-style dual-drains, clarifier sump, bead filter,
secondary sump with water level control and electronic water quality monitoring devices,
99
moving bed bioreactor, UV filter, and heater/chiller. Tanks and bioreactor were each
individually oxygenated with air diffusers fed by a regenerative blower. The RAS was
supplied with municipal water, dechlorinated with adsorptive carbon and stored in a
15,200 L head tank. Temperature was maintained at 22°C (range 19-23). Flow rate was
monitored daily (3-4 L/min) using calibrated manometers installed on each tank supply.
Temperature, dissolved oxygen and pH were measured daily, while ammonia, nitrite,
alkalinity, and chlorine were measured weekly and maintained within acceptable levels
for cool-water fish culture (Timmons and Ebeling 2007; NRC 2011).
Feeding Trial
Juvenile yellow perch of mean weight 6.81g (0.10 SE) were randomly stocked
(n=1,200) at a density of 20 fish per tank. Each of the 15 treatments were randomly
assigned to four experimental units. After stocking, fish were acclimated to experimental
feeds by incrementally increasing test diets mixed with the control for three days.
Following the feed acclimation period, fish were fed slightly above apparent satiation as
determined by the pellet counting method (Helland et al. 1996). Fish were fed to apparent
satiation twice daily (0800 and 1600 hr) and feeding rates were modified to slightly
overfeed based on feed consumption assessments at least twice per week. Consumption
was estimated by counting the uneaten pellets 30 to 45 min after feeding. This method
assumes fish do not eat after the pellet count and that pellet size variation has no effect on
intake (Helland et al. 1996). Pellets were sieved to minimize pellet size variability.
Estimated feed intake values were used for the indirect inference of palatability, and in
the conversion calculations as follows:
100
Feed conversion ratio (FCR) was calculated as:
,
Protein efficiency ratio (PER) was calculated as:
,
,
,
Tank biomass was sampled a total of 5 times (stocking, wk 4, wk 8, wk 12, wk
16) in order to monitor changes in growth throughout the trial. Mortalities were
accounted for in growth metrics by using average weight per fish from dividing the tank
biomass by the number of individuals present. Average final weight per fish was then
used to calculate growth performance metrics. Upon completion of the trial, three fish per
tank were euthanized (300 ppm buffered MS-222) and examined for ending
organosomatic indices.
Relative growth (RG) was calculated as:
Δmass wet, g
initialmass wet, g
100%
Specific growth rate (SGR) calculated as:
ln finalwt g
ln startwt g
100
Fulton-type condition factor (K) was calculated as:
100,000.
Hepatosomatic Index (HSI) was calculated as: HSI
Viscerosomatic Index (VSI) was calculated as: VSI
Visceral Fat Index (VFI) was calculated as: VFI
x 100,
x 100, and
x 100.
101
Statistical Analysis
Treatment data were examined for normality and homogeneity of variance (SAS
Institute Inc. 2011); transformations were completed as necessary. Results were analyzed
first against the reference diet using one-way Analysis of Variance (ANOVA).
Subsequently, the reference diet was removed and data were analyzed for effects of soy
ingredients and FM replacement level. Here, two-way Analysis of Covariance
(ANCOVA) was used with soy ingredient as a main factor and FM replacement level as a
covariate. If either analysis was significant (a priori α=0.05), Duncan’s multiple range
post hoc test was implemented. Survival data were analyzed with the chi-squared statistic
from a non-parametric Kruskal-Wallis test.
Results
There was apparent differences in proximate composition, amino acids, and
trypsin inhibitors among SBM and BP-SBM products (Table 4-2). Average crude protein
of BP-SBM products was improved by an average of 9.8% over GMO-SBM and 5.6%
over non-GMO SBM. The GMO SBM had less protein, fiber, and ash, but higher gross
energy and lipid than the non-GMO variety. Fiber was reduced in BP-SBM, but not
others. Ash was higher in all BP-SBM products than SBM. Leucine, Threonine, aspartic
acid, and glycine were higher in BP-SBM than both varieties of un-converted SBM.
Trypsin inhibitors were substantially reduced in all BP-SBM products regardless of
extrusion pretreatment. Typical SBM products averaged 6,298 TIU/g (dm) and
bioprocessed products averaged 379 TIU/g (dm).
102
Water quality in the RAS was maintained within reasonable limits throughout the
trial duration (temperature 19.2-23.0°C, D.O. 6-9 mg/L, pH 6.6-8.6, unionized ammonia
<0.1 mg/L, free chlorine <0.06 mg/L). Sodium bicarbonate and sodium chloride were
added when necessary to maintain a consistent water chemistry (pH, chloride
concentration, and alkalinity) throughout the trial.
Soy consumption varied significantly among treatments (P=0.03) and revealed
that differences were a result of BP (P=0.03) to a greater effect than FM replacement
level (P=0.29) (Table 4-5). The fish accepted most diets readily, with the exception of the
low pH GMO-BP-SBM (2) diets 12 and 13 (pH 5.05 and 4.45, respectively) which were
not accepted readily by the fish, indicating a problem with palatability. All other diets
ranged from pH 5.88 (diet 3) to 6.24 (diet 14) and did not show a correlation with
consumption or growth performance parameters. Ex-GMO-BP-SBM at a low inclusion
level (diet 8) provided the highest consumption (623g, dm) and resulted in moderate
growth and efficiency parameters.
Survival was not significantly affected by main ingredient (P=0.92; chisquared=7.3; df=14) or FM replacement level (P=0.36; chi-squared=0.8; df=1). Survival
was high and the few mortalities observed were attributed to stress from handling during
sampling events. The lowest survival was observed for two BP soy products Ex-GMOBP-SBM and GMO-BP-SBM (2) which also presented reduced relative growth rates and
increased FCR.
Relative growth (RG) was improved over an FM-based reference diet when 40%
of the FM was replaced with most BP-SBM products. The ANCOVA for RG was
significant (P=0.02) with covariate FM replacement level also significant (P<0.01). Five
soy treatments displayed higher RG than the FM reference diet and nine were lower
103
(Figure 4-1). Dietary soy product and inclusion rate effects created a range of RG from
62.1% greater than FM to 124.0% lower than FM. The BP-SBM protein source improved
RG over the reference diet at both inclusion levels. At a low FM replacement level, three
processed soy products (BP-SBM, Ex-BP-SBM, and GMO-BP-SBM (1)) and one
conventional soy product (GMO-SBM) improved RG over the reference diet. Both
conventional soybean meals provided similar RG to the reference diet at low FM
replacement levels. The slowest RG was observed when 70% of FM was replaced with
Ex-GMO-BP-SBM, GMO-SBM, or GMO-BP-SBM (2). Specific growth rate (SGR)
analysis revealed a pattern similar to RG and provided a significant ANCOVA (P=0.02)
with covariate FM replacement rate (P<0.01).
Fulton’s condition factor (K) provided insignificant (P=0.08) influence from
primary protein source. However, when low inclusion soy and FM diets were removed
from the data, significant differences existed (P=0.02). Two bioprocessed soy products
GMO-BP-SBM (1) and Ex-BP-SBM produced significantly higher (P<0.05) condition
factor than GMO-SBM, conventional soybean meal. Feed conversion ratio (FCR) was
significantly affected by treatment (P=0.01). Most of the differences were attributed to
FM replacement level (P<0.01) over soy treatment (P=0.25). FCR was lowest for diets 6
(0.97) and 1 (0.99) and highest for diets 7 (1.26) and 13 (1.26). Protein efficiency ratio
(PER) was also significant (P=0.02) and produced identical results to FCR as is expected
for highly isonitrogenous diets. Opposite of FCR, PER signifies a unit of growth for each
unit of protein, so higher values indicate greater protein efficiency.
Hepatasomatic index (HSI) was significantly different (P<0.01) among diets. HSI
of both unconverted SBM (1.59) and GMO-SBM (1.63) and FM (1.74) showed lower
HSI than BP products (average 2.15). Diet 2 provided a significantly higher HSI (3.37)
104
than all other diets, indicating possible problems with liver function and lipid storage.
Viscerasomatic index (VSI) was also significant (P<0.01) with high VSI values when fed
high FM replacement levels of BP-SBM and GMO-BP-SBM (2). Lower inclusion of the
duplicate products responded similar to FM. The FM ref diet provided the lowest fillet
yield (33.4%), and soy products maintained an average of approximately 40% with the
exception of diet 3 which reached 57.5%.
Discussion
Bioprocessing had a substantial effect on nutritional composition, which is
consistent with other compositional results of BP-soy (Osagie and Eka 1998; Refstie et
al. 2005) where protein was increased and ANFs were removed. Antinutritional trypsin
inhibitors were also reduced substantially from previously measured BP-WF (16,750
TIU/g), SPC (13,630 TIU/g) to extruded BP-WF (6,538 TIU/g), SBM (5647-6950
TIU/g), and much lower levels in the present experimental soy. All BP products were
also exposed to the same degree of forced-air drying (<100°C) subsequent to BP. The
lowest TIU value was obtained in the low pH GMO-BP-SBM (2) (131 TIU/g), indicating
some susceptibility of trypsin inhibitors to the specific BP conditions.
Severely reduced growth was found with high inclusions of traditional GMOSBM and the low pH GMO-BP-SBM (2) indicated a critical buffering or electrolyte
misbalance that was interfering with the final palatability or digestibility. Organic
acidifiers have been recommended as feed supplements (Luckstadt 2008), but the acidity
of GMO-BP-SBM (2) may have decreased palatability resulting in lower growth of diets
12 and 13.
105
Accurate measures of palatability and consumption are difficult to obtain, and
common errors in bioenergetics modeling has been attributed to inaccurate consumption
estimates (Bajer et al. 2003). We used pellet counting as the consumption method of
choice, due to the ease of implementation, and the consistency of pellets (Helland et al.
1996). This method, however, assumes that feeding is not selective on certain pellet sizes,
and that no pellets went down the drain (Helland et al. 1996). Drain restrictors were
placed on drains, which blocked uneaten pellets from entering the waste streams.
Although extruded soy products can offer improved digestibility (Opstvedt et al.
2003), extrusion in this study was primary accomplished to improve the degree of
hydrolysis during bioprocessing. Research has found extruded soy to have a greater
susceptibility to enzymatic hydrolysis than unextruded soy (Björck et al. 1984). In
addition, extrusion can reduce ANFs such as trypsin inhibitor activity (Barrows et al.
2007) and phytic acid (Allan and Booth 2004) prior to bioprocessing.
Generally, the FM reference tended to produce intermediate growth performance
compared with soy treatments which provided either improved or depressed performance
in each metric, depending on product and inclusion rate. Lesser inclusion rates (Barnes et
al. (2014) also found supporting evidence that 35% fermented soybean meal provided
equivalent growth FM in Rainbow Trout Oncorhynchus mykiss diets.
Summary
In conclusion, implications of this research could provide a better understanding
of Yellow Perch nutritional requirements and soybean bioprocessing technology. This
research has shown that soy processing method has a significant effect on growth
performance of Yellow Perch, and extrusion pretreatment had a lesser effect. Growth
106
performance was also sensitive to specific BP parameters, resulting in both commendable
and defective soy products. As the present study suggests, the future of alternative
aquafeeds and the advancement of Yellow Perch aquaculture depend on continued
research into replacing FM through developing novel feed and feed ingredient processing
technologies.
107
Table 4-1. Primary ingredients, fish meal (FM) replacement levels, and coding of
FM and SBM treatments that are either Extruded (Ex), Bioprocessed (BP), GMO
variety.
Diet
Label
1
2
3
4
5
6
7
FM
Replace
Level
40%
70%
40%
70%
40%
70%
Main Ingredient
Reference
Ex-BP-SBM
Ex-BP-SBM
GMO-BP-SBM (1)
GMO-BP-SBM (1)
GMO-SBM
GMO-SBM
Diet
Label
8
9
10
11
12
13
14
15
FM
Replace
Level
40%
70%
40%
70%
40%
70%
40%
70%
Main Ingredient
Ex-GMO-BP-SBM
Ex-GMO-BP-SBM
BP-SBM
BP-SBM
GMO-BP-SBM (2)
GMO-BP-SBM (2)
SBM
SBM
108
Table 4-2. Proximate composition, trypsin inhibitor activity (TIA), and amino acid
profile of the main protein sources. Values are g/100 g dm unless noted.
ANF=antinutritional factor; EAA=essential amino acid; NEAA=non-essential amino
acid.
Soy Product
Constituent
Protein
Moisture (wb)
Lipid
Crude fiber
Ash
Energy (MJ/kg)
EAA
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Taurine
Threonine
Tryptophan
Valine
NEAA
Alanine
Aspartic acid
Cysteine
Glutamic acid
Glycine
Hydroxylysine
Hydroxyproline
Lanthionine
Ornithine
Taurine
Proline
Serine
Tyrosine
Total AA
ANF
TIA (TIU/g)
SBM
57.06
9.02
1.05
6.16
7.24
19.7
GMOSBM
52.94
12.37
1.13
2.93
6.38
21.3
BP- Ex-BP- GMO-BP- GMO-BP- Ex-GMOSBM SBM SBM (1) SBM (2) BP-SBM
65.83 62.25
61.53
61.19
62.71
5.86
6.31
3.45
4.74
3.11
0.83
0.79
0.79
0.39
1.02
1.91
6.25
3.93
4.66
1.96
11.11
9.00
9.47
5.72
10.95
20.2
20.1
19.7
19.0
19.6
4.02
1.33
2.38
3.94
3.26
0.70
2.58
0.09
1.86
0.64
2.53
3.81
1.36
2.45
4.01
3.34
0.71
2.63
0.03
1.91
0.83
2.74
4.06
1.57
3.55
5.72
3.88
1.03
3.65
0.03
2.72
1.00
3.83
3.45
1.38
3.13
5.10
3.22
0.94
3.26
0.03
2.55
0.87
3.43
3.93
1.50
3.14
5.10
3.61
0.95
3.16
0.02
2.50
0.93
3.38
4.00
1.51
3.01
4.92
3.65
0.92
3.13
0.05
2.39
0.88
3.13
3.94
1.54
3.22
5.16
3.62
0.93
3.32
0.02
2.48
0.95
3.45
2.12
5.89
0.73
9.01
2.16
0.02
0.13
0.02
0.04
0.09
2.76
2.12
1.81
50.14
2.24
5.85
0.70
8.88
2.18
0.03
0.08
0.00
0.06
0.03
2.60
2.02
1.78
50.24
3.22
7.45
0.96
9.44
2.95
0.05
0.06
0.00
0.12
0.03
3.34
2.93
2.50
64.06
2.95
6.78
0.91
8.74
2.82
0.13
0.12
0.00
0.11
0.03
3.22
2.76
2.32
58.22
2.82
6.85
0.90
9.28
2.78
0.02
0.07
0.00
0.05
0.02
3.26
2.74
2.25
59.24
2.70
6.82
0.94
9.59
2.63
0.15
0.06
0.00
0.05
0.05
3.03
2.61
2.23
58.4
2.85
6.95
0.91
9.38
2.81
0.04
0.07
0.00
0.10
0.02
3.18
2.69
2.36
59.97
5647
6950
853
353
269
131
291
109
Table 4-3. Feed formulations and nutrient composition including only high level FM
replacement (70%) treatments. All values reported as g/100 g dm.
Diet
Diet Composition
Crude Protein
Crude Lipid
Crude Fiber
Ash
NFE
GE (MJ/kg)
PE (g CP/MJ)
pH
Ingredient
Fishmeal
Ex-BP-SBM
GMO-BP-SBM (1)
GMO-SBM
Ex-GMO-BP-SBM
BP-SBM
GMO-BP-SBM (2)
SBM
Empyreal 75
Wheat flour
Wheat gluten
CMC
Celufil
Vitamin premix
Mineral premix
Stay-C
L-Lysine
Histidine
Taurine
Sodium chloride
Potassium chloride
Magnesium oxide
Calcium phosphate
Calcium propionate
Menhaden Oil
Soybean Oil
Totals
1
45
9
12.7
17.1
15
19.6
23.0
6.01
3
45
9
11.4
17.6
15.7
20.6
21.8
5.88
5
45
9
10.4
17.7
16.7
20.8
21.6
5.93
7
45
9
5.6
19
20.1
21
21.4
6.22
9
45
9
10.3
17.5
16.8
21.5
20.9
5.94
11
45
9
11.6
17.2
16
20.9
21.5
6.15
13
45
9
10.2
17.7
16.8
21.5
20.9
4.45
15
45
9
8.8
12.2
23.7
21.6
20.8
5.91
41.7
12.51 12.51 12.51 12.51 12.51 12.51 12.51
0
31.3
0
0
0
0
0
0
0
0
31.67
0
0
0
0
0
0
0
0
36.81
0
0
0
0
0
0
0
0
31.08
0
0
0
0
0
0
0
0
29.6
0
0
0
0
0
0
0
0
31.89
0
0
0
0
0
0
0
0
34.39
8.79
8.79
8.79
8.79
8.79
8.79
8.79
8.79
15
15
15
15
15
15
15
15
8
8
8
8
8
8
8
8
3
3
3
3
3
3
3
3
10.12
6.74
6.37
1.4
7.03
8.44
6.04
3.76
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.04
0.04
0.04
0.04
0.04
0.04
0.04
0.04
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.27
1.27
1.27
1.27
1.27
1.27
1.27
1.27
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
0.0025 0.0025 0.0025 0.0025 0.0025 0.0025 0.0025 0.0025
6.13
6.13
6.13
6.13
6.13
6.13
6.13
6.13
0
1.27
1.27
1.1
1.2
1.27
1.38
1.16
100
100
100
100
100
100
100
100
110
Table 4-4. Feed formulations and nutrient composition including only low level FM
replacement (40%) treatments. All values reported as g/100 g dm.
Diet Composition
2
Crude Protein
45
Crude Lipid
9
Crude Fiber
12.0
Ash
18.5
NFE
15.4
GE (MJ/kg)
20.5
PE (g CP/MJ)
22.0
pH
5.91
Ingredient
Fishmeal
25.02
Ex-BP-SBM
17.89
GMO-BP-SBM (1)
0
GMO-SBM
0
Ex-GMO-BP-SBM
0
BP-SBM
0
GMO-BP-SBM (2)
0
SBM
0
Empyreal 75
8.79
Wheat flour
15
Wheat gluten
8
CMC
3
Celufil
8.1875
Vitamin premix
1
Mineral premix
1
Stay-C
0.1
L-Lysine
0.8
Histidine
0.04
Taurine
0.5
Sodium chloride
1.23
Potassium chloride 1.27
Magnesium oxide
0.02
Calcium phosphate
1.3
Calcium propionate 0.0025
Menhaden Oil
6.13
Soybean Oil
0.72
Totals
100
4
45
9
11.4
18.5
16.0
21
21.4
5.9
6
45
9
8.6
19.3
17.9
20.9
21.5
6.09
Diet
8
45
9
11.3
18.4
16.1
21.2
21.2
5.9
25.02
0
18.1
0
0
0
0
0
8.79
15
8
3
7.9775
1
1
0.1
0.8
0.04
0.5
1.23
1.27
0.02
1.3
0.0025
6.13
0.72
100
25.02
0
0
21.03
0
0
0
0
8.79
15
8
3
5.1375
1
1
0.1
0.8
0.04
0.5
1.23
1.27
0.02
1.3
0.0025
6.13
0.63
100
25.02
0
0
0
17.76
0
0
0
8.79
15
8
3
8.3575
1
1
0.1
0.8
0.04
0.5
1.23
1.27
0.02
1.3
0.0025
6.13
0.68
100
10
45
9
12.0
18.2
15.6
20.9
21.5
6.19
12
45
9
11.3
18.6
16.0
20.8
21.6
5.05
14
45
9
10.5
15.4
20.0
21.1
21.3
6.24
25.02
0
0
0
0
16.92
0
0
8.79
15
8
3
9.1475
1
1
0.1
0.8
0.04
0.5
1.23
1.27
0.02
1.3
0.0025
6.13
0.73
100
25.02
0
0
0
0
0
18.22
0
8.79
15
8
3
7.7875
1
1
0.1
0.8
0.04
0.5
1.23
1.27
0.02
1.3
0.0025
6.13
0.79
100
25.02
0
0
0
0
0
0
19.65
8.79
15
8
3
6.4875
1
1
0.1
0.8
0.04
0.5
1.23
1.27
0.02
1.3
0.0025
6.13
0.66
100
111
Table 4-5. Survival (S, %), mean relative growth (RG, %), specific growth rate (SGR),
Fulton-type condition factor (K), total tank consumption (TC, g), food conversion ratio
(FCR), and protein efficiency ratio (PER) for Yellow Perch fed experimental diets. Values
given are treatments means±SE. Significant differences (P<0.05) from one-way analysis
of variance (ANOVA) are indicated by different letters within a given column.
Diet
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
S
97.5±2.5
96.3±2.4
96.3±3.4
96.7±2.9
96.3±1.3
97.5±2.5
95.0±5.0
96.3±2.4
91.3±4.3
96.3±2.4
97.5±2.5
91.3±5.2
92.5±3.2
98.8±1.3
96.3±2.4
Performance Characteristic
SGR
K
TC
RG
abc
433±20
462±18ab
427±24abc
452±33ab
394±20abcd
444±31abc
342±32cd
403±37abcd
368±33bcd
495±20a
437±21abc
416±37abc
309±47d
430±17abc
375±41bcd
abc
1.49±0.03
1.54±0.03ab
1.47±0.03abc
1.52±0.05ab
1.42±0.04abcd
1.51±0.05abc
1.32±0.07cd
1.44±0.07abcd
1.37±0.06bcd
1.59±0.03a
1.48±0.04abc
1.44±0.08abc
1.24±0.10d
1.49±0.03abc
1.38±0.08bcd
1.25±0.05
1.26±0.05
1.25±0.05
1.22±0.03
1.29±0.07
1.21±0.04
1.15±0.03
1.20±0.04
1.20±0.04
1.19±0.05
1.18±0.05
1.19±0.06
1.21±0.03
1.21±0.05
1.21±0.03
abcd
561±16
608±13ab
596±2abc
590±28abc
575±29abcd
572±22abcd
613±29ab
623±14a
605±44ab
598±25abc
558±11abcd
529±25cd
509±14d
553±5abcd
539±19bcd
FCR
PER
ab
0.99±0.03
1.01±0.04ab
1.06±0.04ab
1.05±0.05ab
1.11±0.05abc
0.97±0.03a
1.26±0.06c
1.02±0.05ab
1.15±0.05bc
1.01±0.06ab
1.04±0.04ab
1.05±0.05ab
1.26±0.10c
1.03±0.03ab
1.12±0.06abc
2.33±0.07ab
2.32±0.10ab
2.20±0.08abcd
2.24±0.11ab
2.13±0.10abcd
2.40±0.07a
1.91±0.09cd
2.27±0.11ab
2.06±0.08abcd
2.30±0.12ab
2.28±0.08ab
2.23±0.12abc
1.90±0.16d
2.26±0.06ab
2.04±0.11bcd
112
Standardized Relative Growth (%)
100.0
50.0
62.1
28.3 19.1
11.1
FM
3.7 Ref.
0.0
-2.9
-50.0
-6.2
-17.4
-29.9 -39.4
-58.0
-100.0
-64.8
-91.8
-150.0
-124.0
-200.0
10 2 4 6 11 1 14 3 12 8 5 15 9 7 13 Diet
Figure 4-1. Average relative growth (RG, %) response standardized to reference
diet ([treatment average RG] – [reference average RG]). Dark bars indicate 40%
FM replacement, light bars indicate 70% FM replacement. Different overhead line
ranges indicate significant difference (P<0.05) among treatments.
113
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