Microbial conversion of plant-based polyunsaturated fatty ...
Improvement of Yellow Perch Larvae Culture via Live Food Enrichment with Polyunsaturated Fatty
Transcript of Improvement of Yellow Perch Larvae Culture via Live Food Enrichment with Polyunsaturated Fatty
Improvement of Yellow Perch Larvae Culture via Live Food Enrichment with
Polyunsaturated Fatty Acids
THESIS
Presented in Partial Fulfillment of the Requirements for the Degree Master of
Science in the Graduate School of The Ohio State University
By
John David Grayson
Graduate Program in Environment and Natural Resources
The Ohio State University
2014
Master's Examination Committee:
Dr. Konrad Dabrowski, Advisor
Dr. Suzanne Gray
Dr. Robert Gates
Dr. Ana Hill
Copyrighted by
John David Grayson
2014
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Abstract
Limited experience with live food regimes and fragmentary knowledge of
nutritional requirements have been inhibitors for the indoor-intensive production of
Yellow Perch Perca flavescens larvae. Live food enrichment with polyunsaturated
fatty acids (PUFA) is a proven means of increasing the growth and survival of larval
fish, but no studies to date have tested this method on Yellow Perch. This thesis
consists of two live food enrichment experiments carried out in May/June of 2013
and 2014, as well as lipid analysis of live food and fish samples from both years.
The 2013 study examined the effect of live food enrichment with docosahexaenoic
acid (DHA; C22:6[n-3]) and arachidonic acid (ARA; C20:4[n-6]) on the growth,
survival, and swim bladder inflation of larval Yellow Perch. The 2014 experiment
was similar in design, but compared PUFA enrichments in ethyl ester (EE) and
triglyceride (TG) forms. Both experiments were conducted in two phases. The first
phase was carried out in a recirculating system with nine 50 L conical tanks,
initially stocked at 50-70 larvae/L. Live rotifers Brachionus plicatilis were
provided to larvae for the first two days of exogenous feeding, before transitioning
to Artemia franciscana nauplii for the remaining eight days of this phase. The
second phase was carried out in nine 60 L cylindrical flow-through tanks, initially
stocked with 10 larvae/L. During this phase, fish were fed Artemia nauplii for 3
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days, then gradually transitioned to a formulated starter diet (Otohime A®) over a
7 day period. At the end of the first phase the ARA and DHA enriched groups had
significantly (p≤0.05) improved swim bladder inflation rates when compared to the
control group. For the second phase, enriched groups had significantly larger mean
weights and growth rates than the control. The EE-TG experiment was similar in
design to the DHA-ARA experiment, except that the second phase was concluded
after seven days of feeding. At the end of the first phase, the EE group had a
significantly improved average weight and growth rate than the TG group. No
significant trends were seen in the second phase. Following enrichment
experiments, the fatty acid composition of live feeds and experimental fish were
analyzed using the gas chromatography method. Fatty acid composition of
zooplankton was heavily influenced by enrichments, and composition of
larvae/juveniles generally reflects that of their live prey. Arachidonic acid was
assimilated poorly in ARA enriched Artemia, but DHA was found in abundance.
Also, rotifers tended to assimilate PUFA better in EE form, while Artemia achieved
higher PUFA contents with TG enrichments. The data support that PUFA
enrichment of live food can be utilized to increase the success of Yellow Perch
culture by increasing growth and swim bladder inflation rates during the critical
period of larval development.
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Acknowledgments
I would like to first and foremost thank my advisor, Dr. Konrad Dabrowski, for
his patient instruction, seemingly endless knowledge, and long hours of help. I
would also like to thank the other members of my examination committee and the
helpful staff within at the School of Environment and Natural Resources. I would
like to thank Dr. Karolina Kwasek and Dr. Michal Wojno for their guidance with
culturing larvae and Dr. Malgorzata Korzeniowska for assisting me greatly with
lipid analysis. Lastly, I would like to thank Tim Parker, Nevine Shabana, Abigail
King, Mackenzie Miller, Megan Kemski, and Mohammed Alam for their
assistance and support.
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Vita
June 2008 ............................Keystone High School, LaGrange, Ohio
June 2012 ............................B.S. Environment and Natural Resources, The Ohio
State University
Sep. 2012 to April 2014 .... Graduate Administrative Assistant, SENR, The Ohio
State University
Aug. 2014 to present ..........Graduate Teaching Assistant, SENR, The Ohio State
University
Field of Study
Major Field: Environment and Natural Resources
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Table of Contents
Abstract ................................................................................................................... ii
Acknowledgments.................................................................................................. iv
Vita .......................................................................................................................... v
Field of Study .......................................................................................................... v
Table of Contents ................................................................................................... vi
Chapter 1: Comparison of DHA-based and ARA-based Enrichments ................... 1
Introduction ......................................................................................................... 1
Methods ............................................................................................................... 4
Facilities and Fish ............................................................................................ 4
Enrichment Procedure ..................................................................................... 8
Statistical Analysis ........................................................................................ 10
Results ............................................................................................................... 11
Discussion ......................................................................................................... 11
Culture System .............................................................................................. 11
Fish Performance ........................................................................................... 13
Chapter 2: Comparison of Enrichments in TG and EE forms .............................. 17
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Introduction ....................................................................................................... 17
Methods ............................................................................................................. 18
Facilities and Fish .......................................................................................... 18
Enrichment Procedure. .................................................................................. 21
Sample Collection and Measurement. ........................................................... 22
Statistical Analysis. ....................................................................................... 22
Results ............................................................................................................... 24
Discussion ......................................................................................................... 24
Fish Performance ........................................................................................... 24
Chapter 3: Lipid Analysis of Yellow Perch and Enriched Live Feeds ................. 29
Introduction ....................................................................................................... 29
Methods ............................................................................................................. 30
Lipid Separation and Analysis ....................................................................... 30
Statistical Analysis ........................................................................................ 31
Results ............................................................................................................... 32
DHA-ARA Enrichment Experiment ............................................................. 32
EE-TG Enrichment Experiment .................................................................... 33
Discussion ......................................................................................................... 38
DHA-ARA Experiment ................................................................................. 38
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EE-TG Experiment ........................................................................................ 44
Culture Success.............................................................................................. 46
Future Research ............................................................................................. 48
Literature Cited (AFS) .......................................................................................... 51
Appendix A: Weight Corrections for DHA-ARA Enrichment Experiment ......... 57
Appendix B: T Tests and ANOVA Tables ........................................................... 59
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List of Tables
Table 1. Mean (±SD) water chemistry parameters within culture tanks during the
first 10 days of feeding (Phase I) in the DHA-ARA enrichment experiment. ........ 6
Table 2. Mean (±SD) weight (mg) of Yellow Perch juveniles at the end of Phase I
(10 days feeding) and Phase II (24 days feeding) with unenriched (Control), DHA
enriched, and ARA enriched live feeds. Larvae/juveniles are further stratified into
those with inflated swim bladders (SB+) and those without inflated swim bladders
(SB-). Thirty inflated and 30 non-inflated fish were measured per tank for both
phases. The mean weight was calculated as the average of three tanks per
treatment. Superscript a,b, and c indicate statistical differences between dietary
treatment groups (P≤0.05), while superscript x and y indicate statistical
differences between fish with inflated and uninflated swim bladders (P≤0.05). .. 13
Table 3. Mean (±SD) water chemistry parameters within culture tanks during the
first 10 days of feeding in EE-TG enrichment experiment (Phase I). .................. 20
Table 4. Mean (±SD) weight (mg) of Yellow Perch juveniles at the end of Phase I
(10 days feeding) and Phase II (17 days feeding) with oleic acid enriched
(Control), EE enriched, and TG enriched live feeds. Juveniles are further stratified
into those with inflated swim bladders (SB+) and those without inflated swim
bladders (SB-). Thirty inflated and 30 non-inflated fish were measured per tank
for both phases. The mean weight was calculated as the average of three tanks per
treatment. Superscript a,b, and c indicate statistical differences between dietary
treatment groups (P≤0.05), while superscript x and y indicate statistical
differences between fish with inflated and uninflated swim bladders (P≤0.05). .. 25
Table 5. Lipid and fatty acid composition of neutral lipid (NL) and polar lipid
(PL) fractions of rotifers and Artemia enriched for four hours with DHA-based
and ARA-based lipid emulsions (mean±SD, n=2). Marked letters represent
significant differences within the zooplankton type (P≤0.05). ............................. 35
Table 6. Lipid and fatty acid composition of neutral lipid (NL) and polar lipid
(PL) fractions of whole Yellow Perch after 10 days (Phase I) and 24 days (Phase
II) of feeding with DHA and ARA enriched zooplankton (mean±SD, n=3).
Marked letters represent significant differences within the experimental phase
(P≤0.05). ............................................................................................................... 37
x
Table 7. Lipid and fatty acid composition of neutral lipid (NL) and polar lipid
(PL) fractions of rotifers and Artemia enriched for ≥13 hours with PUFA
emulsions in EE and TG form (mean±SD, n=3). Marked letters represent
significant differences within the zooplankton type (P≤0.05). ............................. 41
Table 8. Lipid and fatty acid composition of neutral lipid (NL) and polar lipid
(PL) fractions of whole Yellow Perch after 10 days (Phase I) and 17 days (Phase
II) of feeding with EE and TG enriched zooplankton (mean±SD, n=3). Marked
letters represent significant differences within the phase (P≤0.05). ..................... 43
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List of Figures
Figure 1. Experimental recirculating larviculture system used for Phase I Yellow
Perch rearing. System includes nine 50 L cylindrical culture tanks (A), a 100 L
reservoir tank (B), a 1800 gph submersible pump (C), sprayer heads for culture
tank inflow (D), 50 µm exchangeable mesh outlets (E), inflow of tap water, salt
stock solution, and algae stock solution (F), and 240 watt heater (G). ................... 7
Figure 2. Experimental tanks used for Phase II Yellow Perch culture. The
production system consists of nine 60 L cylindrical tanks (A), inflow of
dechlorinated city water (B), 100 µm mesh outlets (C), air stones (D), and
automated belt feeders (E). ..................................................................................... 7
Figure 3. Feeding regime of larval/juvenile Yellow Perch during both phases of
the DHA-ARA enrichment experiment. Rotifers were provided at a density of
10/mL, while Artemia was provided at a density of 4 nauplii/mL, and Otohime A
diet was fed up to 10% tank bio .............................................................................. 9
Figure 4. Mean (±SD) daily growth rate, survival and percent swim bladder
inflation of Yellow Perch after 10 days of feeding (Phase I) and 24 days of
feeding (Phase II) with unenriched (Control), DHA enriched and ARA enriched
rotifers and Artemia (n=3 tanks/trea ..................................................................... 14
Figure 5. Feeding regime of larval/juvenile Yellow Perch during both phases of
the EE-TG enrichment experiment. Rotifers were provided at a density of 10/mL,
while Artemia were provided at a density of 4 nauplii/mL, and Otohime A diet
was fed up to 10% tank biom ................................................................................ 21
Figure 6. Enrichment station, including 50 mL tubes of enrichment solution (A)
and 5 L McDonald jars (B) where rotifers and Artemia were placed in lipid
emulsion for ≥12 h prior to entry into larvae culture tanks (EE-TG enrichment
experiment). .......................................................................................................... 24
Figure 7. Mean (±SD) daily growth rate, survival and percent swim bladder
inflation of Yellow Perch after 10 days of feeding (Phase I) and 17 days of
feeding (Phase II) with OE enriched (Control), EE enriched and TG enriched
rotifers and Artemia nauplii (n=3 tanks/treatment). Different letters indicate
statistical differences between treatment groups (P≤0.05). .................................. 26
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Figure 8. Relationship between the measured weight of fish samples when
weighed immediately after removal from 70% ethyl alcohol (wet) and after
5.5±0.3 minutes of air exposure (“semi-dry”). ..................................................... 58
1
Chapter 1: Comparison of DHA-based and ARA-based Enrichments
Introduction
Yellow Perch Perca flavescens is an attractive aquaculture species in the Great
Lakes region with the potential market of over 50 million pounds annually (Malison
2003). While the primary method of producing Yellow Perch juveniles in the
Midwest is with fertilized ponds, indoor-intensive tank culture offers a promising
alternative (Garling et al. 2007). This method of producing larvae/juvenile Yellow
Perch has lower demand for space and water resources, while providing more
environmental stability and supporting faster growth (Timmons and Losordo 1994).
Yellow Perch have several attributes that make the species an excellent candidate
for indoor-intensive culture, including a high tolerance for crowding, handling and
marginal water quality. They are relatively easy to train to accept commercial diets
and show very little aggressive or cannibalistic behavior. While these attributes
seem promising, several significant limitations during the first few weeks of life
have prevented widespread adoption of indoor-intensive culture methods for
Yellow Perch. Culturist inexperience with feeding live foods, lack of formulated
starter-diets, limited knowledge of nutritional requirements, and the inability to
inflate the swim bladder are associated with high mortality of Yellow Perch larvae
in indoor-intensive culture systems (Garling et al. 2007; Malison 2003).
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One practice that could potentially increase the survival and growth of indoor-
intensive Yellow Perch larvae culture is enrichment of live feeds. This is a
commonly used practice for improving the nutritional quality and acceptance of
larval diets in both freshwater (Akbary et al. 2011; Lund et al. 2012) and marine
(Boglino et al. 2012; Gapasin and Duray 2001; McKenzie et al. 2008) fish. Many
of the zooplankton used as live feeds for larval fish, such as rotifers Brachionus
plicatilis and brine shrimp nauplii Artemia franciscana, are deficient in key
nutrients needed for fish growth (Copeman et al. 2002; Navarro et al. 1992; Ritar
et al. 2004). Long-chain polyunsaturated fatty acids (PUFA) are one of the
commonly studied nutrients in larval fish. These fatty acids are important for early
physiological and neurological development, and dietary deficiencies are
associated with increased stress response, impaired brain development, abnormal
swimming behavior, and mortality (Lund et al. 2012). DHA, ARA, and
eicosapentaenoic acid (EPA; C20:5[n-3]) have been identified to be especially
important for early development, as they are functionally important and de novo
synthesis is often limited (Copeman et al. 2002; Martins et al. 2013; Matsunari et
al. 2013). While dietary availability of all three fatty acids benefit most fish species,
evidence suggests that DHA and ARA are more important than EPA (Rainuzzo et
al. 1994). DHA is one of the largest unsaturated fatty acids found in abundance in
aquatic animals, and deficiencies have been associated with non-inflation of the
swim bladder (Tandler et al. 1995), reduced stress tolerance (Lund et al. 2012),
impaired vision (Bell et al. 1995), malpigmentation, and reduced growth and
survival (Copeman et al. 2002). DHA and EPA have competitive interactions
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regarding enzymes responsible for esterification, thus nutritional requirements are
often expressed in DHA/EPA ratio (Sargent et al. 1995). Survival and normal
pigmentation of Yellowtail Flounder Limanda ferruginea larvae are maximized at
a DHA/EPA ratio of 5.6:1 (Copeman et al. 2002), while growth and stress tolerance
of Pikeperch Sander lucioperca were maximized between 3.7:1-6.9:1 DHA/EPA
ratio (Lund et al. 2012). ARA is not as abundant as DHA and EPA in fish bodies
(Copeman et al. 2002), but it is also strongly associated with larval stress response
(Martins et al. 2013), hormone production (Sargent et al. 1995), and morphological
development (Boglino et al. 2012; Boglino et al. 2013). Correlations between
relative PUFA levels and the production of eicosanoid hormones has also been
investigated by aquaculture researchers. ARA has a competitive interaction with
other n-6 PUFA that inhibits eicosanoid activity when n-6 PUFA are in relatively
high concentrations (Sargent et al. 1995). Ratios of n-3:n-6 PUFA are thus
important considerations in larval diets. Bell et al. (1991) found that stress-induced
mortality of Atlantic Salmon Salmo salar was minimized when fish were fed a diet
with a n-3:n-6 of 10.
While PUFAs contribute approximately 46.0±0.8% of the total lipid content of wild
Yellow Perch juveniles (Czesny et al. 2011), live zooplankton have significantly
lower levels. Polyunsaturated fatty acids account for only 35.0% of the total lipids
in unenriched Artemia nauplii (Ritar et al. 2004), and only 19.8±0.5% of total lipids
in unenriched rotifers (Copeman et al. 2002). Artemia nauplii are of particular
concern, due to their low DHA and ARA concentrations (0.1% and 0.6%
respectively; Ritar et al. 2004). In contrast, Yellow Perch larvae and juveniles
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grown in pond culture have 5-10% DHA and 3-5% ARA fatty acid content
(Dabrowski et al. 1993).
While the digestive track of Yellow Perch is not fully developed until a size of
approximately 100 mg, development of a distinct stomach occurs in the 10-20 mg
size range (Dabrowski et al. 1993). The first 24 days of exogenous feeding represent
the most significant period of morphological development in the life cycle of
Yellow Perch, and deficiencies in essential fatty acids during this period can result
in irreparable, and often fatal, deformities (Dabrowski et al. 1993). Emulsion of live
feeds in PUFA-rich solutions is one method of improving the nutritional content of
larval feed, and thus increasing the success of intensive larviculture.
The first enrichment experiment carried out as part of this thesis compared DHA
and ARA based enrichments, and was the first investigation on the enrichment of
live feeds with PUFA as a means to increase the production of Yellow Perch
larvae/juveniles in indoor-intensive condition. Growth, survival, and swim bladder
inflation rate of Yellow Perch larvae were compared after 10 and 24 days of feeding
with unenriched, ARA enriched, and DHA enriched rotifers and Artemia nauplii.
Methods
Facilities and Fish
Yellow Perch larvae used in this experiment were spawned from 5-6 year old OSU
aquaculture facility broodstock. Eggs were stripped from five females and fertilized
by two males, then set to incubate on wire cages within large tanks. Limited
broodstock diversity reduced the influence of genetics on production outcomes.
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Embryos were force-hatched using strong agitation (syphoning through 0.8 cm
tube) after 1,440 degree-hours of incubation, then transferred to the Phase I
experimental system in the OSU Aquaculture greenhouse facility.
For Phase I, 50 L conical tanks were initially stocked with 2,806 larvae/tank (Figure
1). Larvae were initially held in a 19 L bucket that was placed under strong aeration
and repeatedly sampled for larval density. The density within the bucket was
multiplied by the volume of water distributed to each tank in order to estimate initial
stocking density of larvae in Phase I tanks. Phase I began with the first feeding of
larvae at 5 days post-hatch (dph) and continued throughout the first 10 days of
exogenous feeding. During the first phase, fish were reared in a specially designed
recirculating system consisting of nine 50 L cylindrical tanks and a 100 L reservoir
(3 tanks per dietary treatment; Figure 1). The system was equipped with a constant
inflow of evaporated sea salt (100 ppt Instant Ocean®) and Nannochloropsis algae
paste (Nanno 3600 Instant Algae®). Each tank was set up with a sprinkler head inlet
providing 300 mL/min inflow. Individual tanks also had removable screen outlets
that were exchanged daily for cleaning. The size of the outlet mesh was changed
sequentially depending on food type (50 µm for rotifers and 100 µm for Artemia
nauplii). Temperature and water quality were monitored daily to ensure acceptable
conditions were maintained (Table 1).
After 10 days of feeding in the greenhouse facility, 500 larvae were randomly
sampled from each
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Table 1. Mean (±SD) water chemistry parameters within culture tanks during the
first 10 days of feeding (Phase I) in the DHA-ARA enrichment experiment.
Temperature (˚C) Dissolved oxygen (%) pH NH4 (mg/L) Turbidity (NTU) Salinity (‰)
23.1±1.0 95.8±4.7 7.5±0.1 1.6±0.4 11.7±4.5 3.1±0.4
tank and moved to the indoor laboratory facility for the second phase of the
experiment. Fish from
Phase I tanks were moved to Phase II tanks of the same tank number and
corresponding dietary treatment. Phase II lasted from the 11th-24th day of
exogenous feeding. During this phase, fish were reared in nine 60-L cylindrical
tanks with constant inflow of dechlorinated tap water (Figure 2). Tanks were
outfitted with 100 µm mesh outlets and no additional salt or algae inputs were used.
Temperature remained at 19.6±0.4 ˚C. In both phases, tanks were cleaned daily to
remove solid waste and dead individuals. The number of dead perch per tank were
counted and removed at this time.
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Figure 2. Experimental tanks used for Phase II Yellow Perch culture. The
production system consists of nine 60 L cylindrical tanks (A), inflow of
treated city water (B), 100 µm mesh outlets (C), air stones (D), and
automated belt feeders (E).
Figure 1. Experimental recirculating larviculture system used for Phase I Yellow
Perch rearing. System includes nine 50 L cylindrical culture tanks (A), a 100 L
reservoir tank (B), a 1800 gph submersible pump (C), sprayer heads for culture tank
inflow (D), 50 µm exchangeable mesh outlets (E), inflow of tap water, salt stock
solution, and algae stock solution (F), and 240 watt heater (G).
Figure 2. Experimental tanks used for Phase II Yellow Perch culture. The
production system consists of nine 60 L cylindrical tanks (A), inflow of
dechlorinated city water (B), 100 µm mesh outlets (C), air stones (D), and
automated belt feeders (E).
G
F
A
A
D
D
C
B
E E
C
D
A
E
A
B
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Feeding Regimes
The rotifers used in this experiment came from a continuous culture maintained at
the OSU aquaculture facility, and were fed on a diet of yeast and Nannochloropsis
algae paste prior to enrichment. Artemia nauplii were hatched from cysts 6-15 hours
prior to enrichment. Argentemia Platinum® and Argentemia Silver® Artemia cysts
(Argent Chemical Laboratories) were used for the first and second phase,
respectively. At the onset of Phase I, larvae were provided with rotifers up to a
density of 10/mL. After two days of feeding with rotifers, fish were transitioned to
Artemia nauplii at a density of 4 nauplii/mL (Figure 3). The density of live food
was monitored regularly and additional food was added 3-4 times a day. During the
second phase, fish were initially provided with Artemia nauplii, then gradually
transitioned to Otohime A® (Marubeni Nisshin Feed Co, Tokio, Japan) formulated
starter diet (Figure 3). Fish were manually fed 4-6 times throughout the day during
this phase, until automated belt feeders were introduced in the last five days of the
experiment (Figure 2). In both phases, three clustered tanks (non-random) were
assigned to each dietary treatment, and dietary enrichments were the only
differences between treatments.
Enrichment Procedure
Rotifers and Artemia nauplii used as live feeds in the experiment were enriched
using the same procedure. Initially, 1 mL of DHASCO® (Martek Biosciences,
Columbia, MD) or ARASCO® (Martek Biosciences, Columbia, MD) concentrate
and 0.4 mL chicken egg yolk were homogenized in 50 mL distilled water for 2 min.
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This solution was then added to a 5 L McDonald jar containing 4,500,000 rotifers
or 2,250,000 Artemia nauplii. Live feed was maintained in the enrichment solution
for four hours before separation and feeding. After separation, portions of 500,000
rotifers or 200,000 Artemia nauplii were placed to petri dishes and refrigerated at
6˚C until feeding to culture tanks. The control diets were not placed to enrichment
jars and were drawn from their original culture (continuous culture for rotifers and
5 L hatching jar for Artemia) as needed.
Sample Collection and Measurement
At the end of the first phase approximately 100 fish per tank were sampled for
analysis. At the end of the second phase the entire experimental population was
sampled for analysis. Samples were initially preserved in a 10% neutral formalin
solution, and transferred to a 70% ethyl alcohol solution after 24 hours. After
collection in both phases, fish from each tank were sorted into inflated (SB+) and
uninflated (SB-) groups. Thirty fish from each group were randomly selected for
weight and length measurement. Corrections for evaporative drying during the
Figure 3. Feeding regime of larval/juvenile Yellow Perch during both phases of the
DHA-ARA enrichment experiment. Rotifers were provided at a density of 10/mL,
Phase I
Day
1
Day
3
Day
5
Day
10
- Rotifers
- Artemia
- Otohime A
Phase II
Day
11
Day
14
Day
20
Day
24
10
while Artemia was provided at a density of 4 nauplii/mL, and Otohime A diet was
fed up to 10% tank bio
measurement process were made during data analysis (see Appendix A).
Statistical Analysis
Statistical analyses of the data were carried out using JMP 10® statistical software.
Data are represented as mean ± standard deviation, with individual tanks considered
as experimental units (n=3 tanks/treatment). The average weight per tank was
calculated as the sum of the weight of the swim bladder inflated and uninflated
groups multiplied by the proportion of the final harvest that they represented. Prior
to statistical comparison, daily growth rate, survival, and swim bladder inflation
rate were Arcsin transformed and all data were first tested for normality using the
Shapiro-Wilk W test. Additionally, Levine’s tests and Brown-Forsythe tests were
used to test for unequal variance in the data. This Levene’s test was failed for Phase
I swim bladder inflation rates (P=0.017), but the removal of an outlier within the
control group (tank 5) resulted in equal variance across treatments. Final weight,
daily growth rate, survival, and swim bladder inflation data for both phases were
analyzed for significance using one way ANOVA (2 df). Two way ANOVA tests
were used to compare the interaction of dietary treatment and swim bladder
inflation on the mean weight of fish at the end of both phases. Tukey-Kramer tests
were conducted to identify differences between means when ANOVA tests were
significant. Significance was accepted at P-values less than 0.05. Two-way and
one-way ANOVA tables and Tukey-Kramer outputs for significant results are
shown in Appendix B.
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Results
In Phase I, statistically significant differences among treatments were seen in
survival and swim bladder inflation rate (Figure 4). These data showed that survival
was significantly lower in the DHA group than the control group and that swim
bladder inflation rates were significantly higher in both the DHA and ARA groups
than the control group. Phase II data showed that growth rate (Figure 4) and mean
weight (Table 2) were significantly higher in both the DHA and ARA groups than
the control group. No significant differences were found between the ARA and
DHA treatments. The critical period for swim bladder inflation is presumed to be
complete for all fish by the end of the first phase (15 dph), thus the differences in
percentage of swim bladder inflation between Phase I and Phase II fish were most
likely the result of differential mortality in the second phase.
Discussion
Culture System
High rates of survival and growth in this experiment are partially the result of the
specialized larviculture system used, which included sprinkler head inlets as well
as elevated salinity and turbidity. Slightly elevated salinity has been shown to
reduce osmotic stress on freshwater larvae, as well as promote the survival and
vitality of rotifers and Artemia nauplii within the culture tanks (Bein and Ribi 1994;
Ribi 1992). Water turbidity above 5 nephelometric turbidity units (NTU) helps to
reduce clinging tendencies and improves larval feeding (Bristow et al. 1996), as
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well as reducing rates of cannibalism (Clayton et al. 2009). Most larval fish rely on
sight to identify and capture prey (Blaxter 1986). Additions of microalgae in larvae
tanks is thought to improve prey contrast, and to release compounds known to
stimulate feeding (Rocha et al. 2008). There is also evidence that microalgae helps
to preserve the nutritional quality of live feeds within culture tanks (Reitan et al.
1997). Non-inflation of the swim bladder in percid larvae occurs when fish are
unable to ingest an air bubble from the surface during the developmental period
when a pneumatic duct is present between the esophagus and the uninflated swim
bladder (Reiger and Summerfelt 1998; Clayton and Summerfelt 2010). Sprinkler
heads are essential for swim bladder inflation in tank cultures because they help
reduce surface tension, making it easier for larvae to penetrate the water’s surface
(Clayton and Summerfelt 2010; Moore et al. 1994).
Stocking densities and the feeding regime used in the DHA-ARA experiment also
played an important role in the high growth and survival observed in this
experiment. Baras et al. (2003) found reduced cannibalism and increased survival
of Eurasian Perch Perca fluviatilis in intensive larvae culture when initially stocked
at 31-100 larvae/L. Considering the close similarities of Eurasian and Yellow
Perch, the stocking density of Phase I tanks in the DHA-ARA experiment was
likely within the optimal range for Yellow Perch survival. The optimal stocking
density of Yellow Perch juveniles during the formulated diet weaning stage (Phase
II) is likely different than that of the larval stage (Phase I), and a previous study has
found reduced cannibalism at 13.7 fish/L when compared to 37.4 fish/L (Malison
and Held 1992).
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Other investigations have examined the first feeds of larval Yellow Perch. Oetker
(1998) attempted to provide Artemia nauplii as a first feed for larval Yellow Perch,
but this diet was only accepted by ≤20% of larvae and survival through the first 15
days of feeding was 0-0.15%. A second study compared the use of Artemia, vinegar
eels, and a commercial plankton preserve as first feeds for Yellow Perch, and found
no survival after 8-9 days of feeding (Amberg 2001). Both of these studies suggest
that Artemia nauplii cannot be used as a first feed for Yellow Perch larvae, and thus
justify the initial use of rotifers until larvae reach an appropriate size to capture and
ingest Artemia.
Fish Performance
After 24 days of feeding on experimental and formulated diets, the enriched diet
groups showed improvement in growth (Phase II), mean weight (Phase II), and
swim bladder inflation (Phase I) when compared to the control group. While no
significant differences were found between the DHA and ARA treatments, other
Table 2. Mean (±SD) weight (mg) of Yellow Perch juveniles at the end of Phase I
(10 days feeding) and Phase II (24 days feeding) with unenriched (Control), DHA
enriched, and ARA enriched live feeds. Larvae/juveniles are further stratified into
those with inflated swim bladders (SB+) and those without inflated swim bladders
(SB-). Thirty inflated and 30 non-inflated fish were measured per tank for both
phases. The mean weight was calculated as the average of three tanks per treatment.
Superscript a,b, and c indicate statistical differences between dietary treatment
groups (P≤0.05), while superscript x and y indicate statistical differences between
fish with inflated and uninflated swim bladders (P≤0.05).
Phase I Phase II
Treatment SB+ SB- Total SB+ SB- Total
Control 7.2±0.7x 5.8±0.5y 6.5±0.2 60.1±8.3xb 33.1±1.2yb 50.2±5.8b
DHA 8.0±0.5x 6.6±0.8y 7.4±0.4 82.0±9.4xa 46.5±2.9ya 74.5±7.0a
ARA 7.4±0.6x 5.9±0.6y 6.8±0.6 76.6±4.6xab 46.0±3.2ya 69.7±4.5a
14
Figure 4. Mean (±SD) daily growth rate, survival and percent swim bladder
inflation of Yellow Perch after 10 days of feeding (Phase I) and 24 days of feeding
(Phase II) with unenriched (Control), DHA enriched and ARA enriched rotifers and
Artemia (n=3 tanks/trea
15
studies support the theory that DHA is a more important limiting nutrient for larval
growth than ARA. Copeman et al. performed a similar enrichment study with
Yellowtail Flounder larvae and found that DHA based enrichments supported
higher growth and survival rates than DHA+ARA and olive oil-based enrichments.
The authors suggest that there is a competitive interaction between DHA, ARA,
and EPA that result in lower DHA utility when ARA levels are high (Copeman et
al. 2002). While this may be true for a marine species, other evidence suggests that
percids are capable of synthesizing necessary amounts of DHA from linolenic acid
(C18:3[n-3]; Henrotti et al. 2011) possibly explaining the lack of significant
improvements in Yellow Perch culture when enriched with DHA, compared to
ARA. Other studies have tied high levels of dietary ARA to cranial bone
deformities, malpigmentation, and reduced growth, but only when ARA comprises
≥7% of the total fatty acids in live foods (Boglino et al. 2012; Boglino et al. 2013;
Copeman et al. 2002). On the other hand, depressed growth was associated with
ARA concentrations <1% in Senegalese Sole Solea senegalensis (Boglino et al.
2012). Lipid analysis of diets (Chapter 3) suggest that ARA only exceeded 7% of
the fatty acids in ARA enriched rotifers, which were only consumed for the first 2-
4 days of Phase II. The short duration of feeding on ARA enriched rotifers may
explain why survival was not significantly impacted in this dietary treatment. The
positive association of PUFA content and swim bladder inflation has also been
documented in larval Amberjack Seriola dumerili, and is presumably the result of
differences in activity level during the critical period for initial swim bladder
inflation (Matsunari et al. 2013). This study was an important first step toward
16
understanding the influence of the total and relative fatty acid composition of live
feeds used in Yellow Perch intensive larvae culture.
17
Chapter 2: Comparison of Enrichments in TG and EE forms
Introduction
While extensive research has focused on relative concentrations of fatty acids
within enrichment formulas (Bell et al. 1991; Copeman et al. 2002; Lund et al.
2012), relatively little attention has been given to the molecular form of lipid
emulsions. Fatty acids can be provided in isolated forms (free fatty acids), but more
commonly are attached to a carrier molecule such as glycerol, ethanol, or fatty
alcohols (Hardy et al. 2011). The most common chemical form of fatty acid used
for storage and transfer is the triglyceride (TG), comprised of three fatty acids
attached to a glycerol backbone (Hardy et al. 2011). Fatty acids are also stored in
the form of wax esters, consisting of a fatty acid attached to a long chain fatty
alcohol (Olsen et al. 2004). Although not common in nature, fatty acids can be
attached to methanol (methyl esters; ME) and ethanol (ethyl esters; EE) via
chemical processing (Nordoy et al. 1991). These chemical forms are more stable
against oxidation and are easier to purify into highly concentrated fatty acid
solutions (Hardy et al. 2011).
Upon ingestion, fatty acids are severed from transport molecules and reassembled
into triglycerides for transfer to the liver. Fatty acids are stored in triglyceride form
in the liver and other body tissues before circulation to body cells (Wallaert and
Babin 1994). Previous studies have shown that the chemical form of lipids have a
18
significant effect on their biological availability (Castell et al. 1972; Ibeas et al.
2000; Lochmann and Gatlin 1993b). While Sea Bream Spartus aurata larvae had
depressed growth when fed ME enriched rotifers (Izquierdo et al. 1989), juveniles
utilize ME and TG equally well (Ibeas et al. 2000). Rainbow Trout Oncorhynchus
mykiss and Red Drum Sciaenops ocellatus showed reduced growth when provided
with PUFA in EE form, as opposed to TG (Castell et al. 1972; Lochmann and Gatlin
1993a). Previous studies on rotifers and Artemia have had conflicting results as to
which chemical form is preferred (Coutteau and Mourente 1997; Izquierdo et al.
1992; Rainuzzo et al. 1994; Takeuchi et al. 1992b).
A second live food enrichment experiment was carried out in 2014 that compared
Yellow Perch performance when enrichment lipids were offered in different
chemical forms. While both experimental enrichment solutions had similar PUFA
concentrations, fatty acids were provided in either triglyceride (TG) or ethyl ester
forms (EE).
Methods
Facilities and Fish
Yellow Perch larvae used in this experiment were bred from two 5-6 year old
females from the OSU aquaculture facility and one male from Millcreek Perch
Farm (Marysville, Ohio). Egg ribbons were deposited and fertilized within a
broodstock tank on April 23rd and 25th, 2014. Limited broodstock diversity
reduced the influence of genetics on production outcomes. Eggs were placed in 15
L flow-through troughs for incubation. Embryos were force-hatched using strong
19
agitation (syphoning through 0.8 cm tube) on May 4th, 2014, then transferred to
the Phase I experimental system in the OSU aquaculture greenhouse facility on
May 6th, 2014.
For Phase I, nine 50-L conical tanks were initially stocked with 1628 larvae/tank.
Larvae were initially held in a 19 L bucket that was placed under strong aeration
and repeatedly sampled for larval density. The density within the bucket was
multiplied by the volume of water distributed to each tank in order to estimate initial
stocking density of larvae in Phase I tanks. Phase I began with the first feeding of
larvae at 3 dph and continued throughout the first 10 days of exogenous feeding.
Fish were reared in the system equipped with a constant inflow of evaporated sea
salt (100 ppt Instant Ocean®) and Nannochloropsis algae paste (Nanno 3600 Instant
Algae®). Each tank was set up with a sprinkler head inlet providing 300 mL/min
inflow. Individual tanks also had removable screen outlets that were exchanged
daily for cleaning. The size of the outlet mesh was changed sequentially depending
on food type (50 µm for rotifers and 100 µm for Artemia nauplii). Water quality
was monitored daily to ensure acceptable conditions were maintained (Table 3).
After 10 days of feeding in the greenhouse facility, 300 larvae were randomly
sampled from each tank and moved to the indoor laboratory facility for the second
phase of the experiment. Fish from
Phase I tanks were moved to Phase II tanks of the same tank number and
corresponding dietary treatment. Phase II lasted from days 11-18 of exogenous
feeding. During this phase, fish were reared in nine 60 L cylindrical tanks with
constant inflow of treated tap water. Tanks were outfitted with 100 µm mesh outlets
20
and no additional salt or algae inputs were used. Temperature remained at 17.2±0.2
˚C throughout this phase. In both phases, tanks were cleaned daily to remove solid
waste and dead individuals. The number of dead perch per tank were counted and
removed at this time.
Feeding Regimes
The rotifers used in this experiment came from a continuous culture maintained at
the OSU aquaculture facility, and were fed a diet of yeast and Nannochloropsis
algae paste before enrichment. Artemia nauplii were hatched from cysts 6-15 hours
before enrichment. Argentemia Platinum® and Argentemia Silver® Artemia cysts
(Argent Chemical Laboratories) were used for the first and second phase,
respectively. At the onset of Phase I, larvae were provided with rotifers up to a
density of 10/mL. After two days of feeding with rotifers, fish were transitioned to
Artemia nauplii at a density of 5 nauplii/mL (Figure 5). The density of live food
was monitored regularly and additional food was added 3-4 times a day. During the
second phase, fish were initially provided with Artemia nauplii, then gradually
transitioned to Otohime A® formulated starter diet (Figure 5). Fish were manually
fed 4-6 times throughout the day during this phase. The occurance of Flexibacter
columnaris infection within experimental tanks dictated the collection of final
samples after seven days of feeding, prior to the complete transition to Otohime A®
Table 3. Mean (±SD) water chemistry parameters within culture tanks during the
first 10 days of feeding in EE-TG enrichment experiment (Phase I).
Temperature (°C) Dissolved oxygen (%) pH NH4 (mg/L) Turbidity (NTU) Salinity (‰)
23.2±1.7 106.7±4.4 7.6±0.1 3.8±0.7 8.9±1.8 3.3±0.5
21
Figure 5. Feeding regime of larval/juvenile Yellow Perch during both phases of the
EE-TG enrichment experiment. Rotifers were provided at a density of 10/mL, while
Artemia were provided at a density of 4 nauplii/mL, and Otohime A diet was fed
up to 10% tank biom
diet. Infection was first observed on May 25th, 2014 (after 6 days of feeding), and
symptoms included erratic swimming behavior and cotton-like growths on fins and
gills. In both phases, three tanks were randomly assigned to each dietary treatment,
and dietary enrichments were the only differences between treatments.
Enrichment Procedure.
Rotifers and Artemia nauplii used as live feeds in the experiment were enriched
using a similar procedure to that used in the DHA-ARA enrichment experiment.
Initially, 1 mL of lipid concentrate, 0.4 mL chicken egg yolk, and 0.5 mL
Nannochloropsis algae paste were homogenized in 50 mL salt water (20 ppt) for 2
min. For this experiment, oleic acid methyl ester® (MP Biomedicals) was used for
the control group solution, while AlaskOmega EE600200M® and AlaskOmega
TG530200M® (Organic Technologies, Coshocton, Ohio) were used for the EE and
Phase I
Day
1
Day
3
Day
5
Day
10
- Rotifers
- Artemia
- Otohime A
Phase II
Day
11
Day
14
Day
18
22
TG group solutions, respectively. Unlike the DHA-ARA enrichment experiment,
oleic acid enrichment was used for the control group. Oleic acid has been used as a
control in pervious enrichment experiments so that control and PUFA-enriched
zooplankton are exposed to the same environmental conditions prior to entry into
culture tanks, and due to oleic acid’s non-involvement in PUFA synthesis pathways
(Watanabe 1993). Solutions were then added to 5-L McDonald jars containing
4,500,000 rotifers or 2,250,000 Artemia nauplii (Figure 6). Live feeds were
maintained in the enrichment solution for 13-23 hours before removal to culture
tanks.
Sample Collection and Measurement.
At the end of the first phase approximately 100 fish per tank were sampled for
analysis. At the end of the second phase the entire experimental population was
sampled for analysis. Samples were initially preserved in a 10% neutral formalin
solution, and transferred to 70% ethyl alcohol solution after 24 hours. After
collection in both phases, fish from each tank were sorted into inflated and
uninflated groups. Thirty fish from each group were randomly selected for weight
and length measurement.
Statistical Analysis.
Statistical analyses of the data were carried out using JMP 10® statistical software.
Data are represented as mean ± standard deviation, with individual tanks considered
as experimental units (n=3 tanks/treatment). The average weight per tank was
calculated as the sum of the weight of the swim bladder inflated and uninflated
groups multiplied by the proportion of the final harvest that they represented. Prior
23
to statistical comparison, daily growth rate, survival, and swim bladder inflation
rate were Arcsin transformed and all data were first tested for normality using the
Shapiro-Wilk W test. Phase II swim bladder inflation and survival failed this test
(Prob<W=0.045 and 0.033, respectively), but the removal of two outliers within
the EE group (tank 9) swim bladder inflation and control group (tank 7) survival
resulted in acceptable normality (Prob<W=0.82 and 0.14, respectively).
Additionally, Levine’s tests and Brown-Forsythe tests were used to test for unequal
variance in the data. The Levene’s test was failed for Phase I swim bladder inflation
rates (P=0.017) and Brown-Forsythe test was failed for Phase I survival. Mean
weight, daily growth rate, survival (Phase II only), and swim bladder inflation
(Phase II only) data for both phases were analyzed for significance using one way
ANOVA (2 df). Phase I swim bladder inflation and survival were tested using
Welch’s ANOVA (2 df), which did not rely on the assumption of equal variation.
Two way ANOVA tests were used to compare the interaction of dietary treatment
and swim bladder inflation on the mean weight of fish at the end of both phases.
Tukey-Kramer tests were conducted to identify differences between means when
ANOVA tests were significant. Significance was accepted at P-values less than
0.05. Two-way and one-way ANOVA tables and Tukey-Kramer outputs for
significant results are shown in Appendix B.
24
Figure 6. Enrichment station, including 50 mL tubes of enrichment solution (A)
and 5 L McDonald jars (B) where rotifers and Artemia were placed in lipid
emulsion for ≥12 h prior to entry into larvae culture tanks (EE-TG enrichment
experiment).
Results
In the first phase, the EE group had significant greater growth rate (Figure 7) and
mean weight (Table 4) than the TG group. No significant differences were
calculated in Phase II data, primarily due to the high standard deviation of these
data.
Discussion
Fish Performance
Takeuchi et al. (1992b) is one of relatively few investigations that examined lipid
forms in live food enrichment for larval fish. This study compared the performance
of Striped Knifejaw Oplegnathus fasciatus and Red Sea Bream Pagrus major when
A
B
25
Table 4. Mean (±SD) weight (mg) of Yellow Perch juveniles at the end of Phase I
(10 days feeding) and Phase II (17 days feeding) with oleic acid enriched (Control),
EE enriched, and TG enriched live feeds. Juveniles are further stratified into those
with inflated swim bladders (SB+) and those without inflated swim bladders (SB-
). Thirty inflated and 30 non-inflated fish were measured per tank for both phases.
The mean weight was calculated as the average of three tanks per treatment.
Superscript a,b, and c indicate statistical differences between dietary treatment
groups (P≤0.05), while superscript x and y indicate statistical differences between
fish with inflated and uninflated swim bladders (P≤0.05).
Phase I Phase II
Treatment SB+ SB- Total SB+ SB- Total
Control 10.0±0.5x 7.0±1.3y 8.0±0.7ab
35.3±3.7x 15.2±1.9y 20.0±1.1
EE 11.1±1.2x 7.3±1.8y 9.0±0.6a 38.3±3.4x 20.2±1.7y 28.6±2.8
TG 9.6±0.7x 5.5±1.1y 6.8±0.9b 34.3±3.5x 18.9±3.2y 24.2±6.1
26
Figure 7. Mean (±SD) daily growth rate, survival and percent swim bladder
inflation of Yellow Perch after 10 days of feeding (Phase I) and 17 days of feeding
(Phase II) with OE enriched (Control), EE enriched and TG enriched rotifers and
Artemia nauplii (n=3 tanks/treatment). Different letters indicate statistical
differences between treatment groups (P≤0.05).
fed Artemia enrichment with 43% PUFA solutions in triglyceride, methyl ester,
ethyl ester, and free fatty acid (FFA) forms. Greater growth and survival occurred
in the TG enriched, EE enriched, and ME enriched groups when compared to the
TG TG
27
FFA enriched and unenriched groups (Takeuchi et al. 1992b). While these authors
found no significant differences in growth or survival among the TG and EE
groups, incorporations of specific fatty acids and total lipid content were the
greatest in the EE group. Unlike Takeuchi et al. (1992b), the 2014 study found a
significant improvement in average weight and growth rate associated with EE-
based enrichments.
Rainuzzo et al. (1994) examined the chemical form of PUFA enrichments of
rotifers and Artemia and found that EE-based enrichments had higher PUFA
assimilation into zooplankton, but this did not result in significantly improved
growth or survival of larval Turbot Scophthalmus maximus. In this study, results in
growth and survival may also be driven by the vastly different DHA:EPA within
EE and TG-based enrichments (3.8 and 1.3, respectively).
Izquierdo et al. (1992) examined the chemical form of PUFA enrichments and its
influence on the performance of larval Japanese Flounder Paralichthys olivaceus.
This investigation found lower grow and equivalent survival of larvae fed EE-
enriched rotifers and Artemia, when compared to
TG and ME groups. The EE-TG experiment found better growth in Yellow Perch
larvae fed EE-enriched rotifers and Artemia, contradictory to the Izquierdo et al.
(1992) study. This is likely due to differences in the purity of enrichment oils used
in these two studies, as well as metabolic differences between fish species.
While several studies show higher incorporation of fatty acids by zooplankton
enriched with EE-based PUFA, the EE-TG enrichment experiment was the first
28
recorded evidence of increased fish growth associated with PUFA enrichments in
EE form.
Results in Phase I growth, survival, and swim bladder inflation in the EE-TG
enrichment experiment were similar to those from the DHA-ARA enrichment
experiment. Unlike the DHA-ARA enrichment study, swim bladder inflation rates
did not increase between the first and second phase. It was observed that the
Flexibacter columnaris infection in 2014 differentially targeted large, healthy
individuals, balancing survival disadvantages associated with uninflation of the
swim bladder. This would also explain why Phase II survival was similar for both
experiments, despite the phase lasting half as long in the EE-TG enrichment
experiment as in the DHA-ARA enrichment experiment. Despite this, the Phase II
daily growth rates for both years were very similar.
29
Chapter 3: Lipid Analysis of Yellow Perch and Enriched Live Feeds
Introduction
While differences in growth, survival, and swim bladder inflation were associated
with experimental live feed enrichments, further analysis is needed to substantiate
the nutritional basis of these trends. The indirect means by which enrichment oils
influence larval performance make quantitative analysis of lipid contents especially
important. Even within a short window of time between exposure to oil emulsions
and consumption by fish larvae, live zooplankton are known to transfer and
metabolize fatty acids (Coutteau and Mourente 1997; Estevez et al. 1998; Takeuchi
et al. 1992a). While temperatures within enrichment jars and culture tanks remained
relatively high (25°C in enrichment jars and 17-26°C in culture tanks) throughout
the experiments, food density measurements within the tanks suggest that rotifers
and Artemia were consumed within 3-4 hours of removal from enrichment
solutions. Current evidence suggests that PUFA contents of zooplankton do not
begin to decline until 12 hours of starvation, even at temperatures of 20°C (Estevez
et al. 1998), and thus fatty acid loss is not a major concern for this experiment.
While the DHA-ARA and EE-TG enrichment experiments are novel investigations
into the manipulation of lipids in Yellow Perch larvae, inferences about specific
fatty requirements can be drawn based on the body composition of pond-reared
larvae. Dabrowski et al. (1993) characterized the protein and lipid composition of
30
Yellow Perch larvae from 6 mg to 858 mg in size. Larvae were reared in outdoor
ponds on a diet of wild zooplankton. Larvae/juveniles in the 6-13 mg range had
similar fatty acid contents for neutral and polar lipid fractions, consisting of 9-10%
DHA, 4-5% EPA, and 4-5% ARA. Juveniles at 110 mg had similar ARA and EPA
compositions, but DHA was reduced to 4-5% of total fatty acids (Dabrowski et al.
1993). While this study provides an estimate of the fatty acid composition of fish
in ‘wild’ conditions, it is important to note that availability of prey items was
affected by larval size and behavior and a dynamic zooplankton community within
culture ponds. Dietary limitations may discredit the idea that ‘wild’ fish represent
the ideal nutritional status of cultured Yellow Perch. This chapter addresses the
lipid composition of live feeds and Yellow Perch from both enrichment
experiments, and thus attempt to characterize the diets that best support fish
performance.
Methods
Lipid Separation and Analysis
Samples of rotifers, Artemia, and Yellow Perch larvae/juveniles were collected
during both experiments for lipid analysis. Initials samples were collected in 3 mL
cryotubes and placed immediately into liquid nitrogen, before long term storage at
-80°C. Lipids were prepared and analyzed in four stages. In the first stage, total
lipids were extracted from whole samples following Folch et al. (1957). After
thawing, 0.8-0.9g of samples were homogenized in 20 mL of 2:1 chloroform-
methanol and dissolved lipids were filtered through Whatmans #1® filter paper (GE
31
Healthcare UK Limited, Buckinghamshire, UK). This method has been shown to
be >97% accurate in separating total lipids from whole body samples (Folch et al.
1957). In the second stage, neutral (NL) and polar (PL) lipid fractions were
separated using a simple technique first described by Juaneda and Rocquelin
(1985). Total lipids were inserted into a Sep-pak® Classic Silica Cartridge (Waters
Corporation, Milford, Massachusetts) and neutral lipids were rinsed through the
filter with 20 mL of chloroform. Following that, 20 mL of methanol was used as a
solvent to remove the polar lipid fraction from the filter. In the third stage, neutral
and polar lipid fractions were converted into methyl ester form with a variation of
the transmethylation procedure described by Metcalfe and Schmitz (1961). Two
milliliters of boron trifluoride-methanol solution were added to each sample and
tubes were heated to 80°C for 30 minutes. Methyl esters were diluted into hexane
and removed from the previous solution. Finally, methylated fatty acid samples
were analyzed for relative fatty acid concentration with an electronic gas
chromatograph (Varian 3900®; Varian Analytical Instruments, Walnut Creek, CA).
Statistical Analysis
The calculation of relative fatty acid contents is based on chromatograph data.
Percent composition is calculated as the area of the identified fatty acid peak over
the cumulative area of all peaks. Statistical analysis of the data were carried out
using JMP 10® statistical software. Samples of rotifers, Argentemia Platinum®
(hereafter Artemia Platinum), Argentemia Silver® (hereafter Artemia Silver), Phase
I Yellow Perch, and Phase II Yellow Perch were run for each experimental
treatment. Prior to statistical comparison, all data were Arcsin transformed tested
32
for normality using the Shapiro-Wilk W test. Additionally, Levine’s tests and
Brown-Forsythe tests were used to test for unequal variance in the data. This
Levene’s test was failed for EPA content in the neutral lipids (P=0.01) and DHA
content in the polar lipids (P=0.03) of rotifers from the EE-TG enrichment
experiment. Percent total lipids, neutral lipids, and polar lipids, as well as the
relative proportions of individual fatty acids and fatty acid groups were analyzed
for significance using one way ANOVA (1-2 df), with the exception of the
parameters mentioned above that failed the Levene’s test. A Welch’s ANOVA was
run for these two parameters. Tukey-Kramer tests were conducted to identify
differences between means. Significance was accepted at P-values less than 0.05.
One-way ANOVA tables and Tukey-Kramer outputs for significant results in
linoleic acid, linolenic acid, ARA, EPA, and DHA composition are shown in
Appendix B.
Results
DHA-ARA Enrichment Experiment
Both enriched and unenriched rotifers and Artemia had approximately 3% total
lipid content (wet wt.), but NL fractions in enriched rotifers were significantly
larger than those in the unenriched rotifers (Table 5). The n-3:n-6 varied widely
among all treatments, and was generally highest in DHA-enriched zooplankton.
The incorporation of DHA also varied widely among treatments in both the neutral
and polar lipid fractions, but was consistently highest in the DHA enriched
treatment. Arachidonic acid was readily incorporated into rotifers, especially within
33
the NL fraction of the ARA enriched group (25.6%). Artemia incorporated ARA
poorly, never exceeding 1% of the NL or PL fractions. It is important to note that
EPA was found in much higher concentrations in the high-grade Artemia than the
other two live feeds. Also, rotifers tended to incorporate DHA and ARA better than
Artemia, and high-grade Artemia incorporated DHA better than low-grade Artemia.
Total lipids and NL within Phase II Yellow Perch juveniles were significantly
greater in the enriched groups, particularly with the DHA enrichment (Table 6).
Unsaturated fatty acids represented a larger proportion of the NL than the PL in
both size classes of Yellow Perch (74.6% and 54.2%, respectively). The
incorporation of DHA into the NL of perch larvae/juveniles did not reflect that of
the live feeds used in this experiment. Polar lipid fractions showed better
incorporation of DHA, which increased markedly between Phase I and Phase II
sample collections (11.8% to 26.6%, respectively). Arachidonic acid was
predominantly found in the NL fraction, and was between 1-2.5% of neutral lipids
in all groups.
EE-TG Enrichment Experiment
Total lipids were consistently greater in the EE group than the TG group in rotifers
and Artemia platinum (Table 7). Levels of NL and PL were relatively constant
among zooplankton. The relative proportion of individual fatty acids varied widely
among zooplankton species and enrichments. In general, enriched groups had
higher concentrations of PUFA and lower concentrations of palmitoleic acid
(C16:1) and oleic acid than the control groups, in addition to greater n-3:n-6. With
34
the exception of ARA, PUFA in EE form were assimilated better by rotifers and
worse by Artemia, and this trend was more pronounced in the NL than the PL.
Total lipid and NL content were similar in all Yellow Perch sampled (Table 8).
Overall, the PUFA enriched groups had higher PUFA contents than the control
group, especially in the NL fraction. The only significant differences found between
the PUFA enriched groups were a higher concentration of EPA in the polar lipid
fraction of fish in the EE group than those in the TG group. The n-3:n-6 varied
greatly among Phase I and Phase II fish, and was largely driven by concentrations
of linoleic (C18:2[n-6]) and linolenic acid (C18:3[n-3]). Ethyl ester enrichment
supported the highest n-3:n-6 in both lipid fractions of Phase II Yellow Perch.
35
Table 5. Lipid and fatty acid composition of neutral lipid (NL) and polar lipid (PL) fractions of rotifers and Artemia enriched for four
hours with DHA-based and ARA-based lipid emulsions (mean±SD, n=2). Marked letters represent significant differences within the
zooplankton type (P≤0.05). Rotifers Artemia Platinum Artemia Silver
Control DHA ARA Control DHA ARA Control DHA ARA
Total Sample
% lipids (wet wt.) 2.6±1.0 3.1±0.0 3.2±0.0 3.4±0.2 3.2±0.2 3.1±0.1 3.1±0.1 2.9±0.2 3.5±0.2
% NL 63.8±2.0b 79.9±1.5a 79.7±2.9a 70.4±0.9 72.6±0.1 72.3±0.2 69.7±1.2 71.2±0.8 73.3±0.9
% PL 36.2±2.0a 20.1±1.5b 20.3±2.9b 29.6±0.9 27.4±0.1 27.7±0.2 30.3±1.2 28.8±0.8 26.7±0.9
Neutral Lipids
Fatty Acids (%)
C16:0 15.3±13.8 11.6±2.3 8.7±0.2 20.6±1.2 18.0±0.4 22.7±0.8 16.7±4.4 15.1±0.1 15.0±4.5
C16:1[n-9] 3.6±2.2 1.9±0.2 0.4±0.0 22.0±0.7a 15.6±0.3b 19.1±0.6a 4.1±0.9 3.3±0.1 1.1±0.3
C18:0 5.7±5.5 1.3±0.2 6.3±0.1 18.4±0.5 19.7±0.0 22.4±0.1 15.4±9.2 22.8±0.5 20.8±5.7
C18:1[n-9] 0.4±0.6 0.2±0.1 0.1±0.0 11.4±0.4 7.4±0.1 9.6±0.3 2.4±3.4 5.0±0.2 5.7±1.5
C18:2[n-6] 25.1±14.2 25.1±5.2 24.8±1.8 0.8±0.7 0.9±0.0 1.2±0.0 0.3±0.1 0.1±0.0 0.2±0.2
C18:3[n-3] 0.3±0.4 0.0±0.0 1.2±1.0 0.2±0.1 0.0±0.0 0.0±0.0 44.0±11.3 32.7±0.6 31.8±8.4
C20:1[n-9] 0.9±0.6 0.2±0.1 0.3±0.0 0.3±0.0 0.2±0.0 0.3±0.0 5.1±1.3 3.8±0.5 3.7±1.0
C20:4[n-6] 2.2±0.7b 0.6±0.1c 25.6±0.4a 0.7±0.1 0.5±0.1 0.7±0.0 1.0±0.3 0.6±0.0 0.7±0.2
C20:5[n-3] 2.5±0.3a 0.5±0.0b 0.4±0.0b 15.7±0.2a 10.5±0.1c 13.2±0.1b 0.0±0.0 0.0±0.0 0.0±0.0
C22:6[n-3] 0.8±0.7b 20.8±4.3a 0.3±0.2b 0.2±0.1b 15.4±0.3a 0.5±0.3b 0.1±0.0b 8.4±0.6a 0.4±0.3b
DHA:ARA 0.3±0.2b 33.9±2.4a 0.0±0.0b 0.3±0.0b 32.4±4.8a 0.8±0.5b 0.0±0.0b 13.1±1.0a 0.8±0.7b
Fatty Acid Class
Saturated 54.4±2.8 33.4±4.1 35.8±0.1 44.5±1.3 46.8±0.8 50.3±0.6 40.0±13.0 43.0±0.1 53.8±12.0
Unsaturated 45.6±2.8 66.6±4.1 64.2±0.1 55.5±1.3 53.2±0.8 49.7±0.6 60.0±13.0 57.0±0.1 46.2±12.0
n-3 6.9±5.6 22.0±3.8 2.9±1.3 16.1±0.2 26.0±0.2 13.8±0.2 45.3±13.2 42.8±0.1 33.8±8.5
n-6 28.6±12.6b 25.9±5.0b 50.8±1.4a 1.9±0.5 1.6±0.1 3.7±0.1 1.3±0.5 0.8±0.0 1.5±0.6
n-3:n-6 0.3±0.3 0.9±0.3 0.1±0.0 8.9±2.5b 16.3±1.1a 3.7±0.1b 36.2±3.0b 55.6±0.2a 22.6±3.6c
(continued)
35
36
Table 5. Cont.
Rotifers Artemia Platinum Artemia Silver
Control DHA ARA Control DHA ARA Control DHA ARA
Polar Lipids
Fatty Acid (%)
C16:0 21.0±8.8 16.9±0.0 19.0±0.0 11.3±0.1 12.3±0.2 12.8±0.2 12.6±0.5 16.4±3.8 15.6±2.6
C16:1[n-9] 3.2±0.3a 2.2±0.1ab 1.6±0.0b 9.9±0.3a 8.9±0.1b 9.0±0.0ab 1.0±0.0 2.1±0.7 1.3±0.5
C18:0 7.8±5.5 4.5±0.4 10.7±0.1 22.6±0.3 23.0±0.6 23.0±0.8 28.5±0.3 22.4±8.2 24.0±7.6
C18:1[n-9] 5.4±4.2 10.3±0.4 8.8±0.1 19.9±1.0 18.5±0.7 19.4±0.4 13.4±0.0 9.9±2.4 11.8±3.1
C18:2[n-6] 33.8±28.7 27.4±1.3 29.1±0.3 0.8±0.0 0.7±0.0 1.1±0.0 0.1±0.0 0.1±0.0 0.1±0.0
C18:3[n-3] 3.1±4.3 0.0±0.0 3.5±0.0 1.9±0.8 1.1±0.1 1.1±0.0 29.2±0.9 31.7±7.5 30.5±4.4
C20:1[n-9] 1.4±0.9 0.8±0.0 1.1±0.0 0.9±0.1 0.7±0.0 0.6±0.0 7.3±0.0 7.8±1.9 7.6±1.1
C20:4[n-6] 2.5±0.8b 2.0±0.2b 14.4±0.0a 0.3±0.0 0.3±0.0 0.3±0.0 0.8±0.0 0.9±0.2 0.7±0.1
C20:5[n-3] 4.9±0.9 2.8±0.1 2.9±0.1 17.8±0.8 17.6±0.3 16.6±0.2 4.9±0.2 2.4±3.4 5.3±0.8
C22:6[n-3] 0.9±0.8b 14.6±0.2a 0.5±0.2b 0.1±0.0 2.5±0.1 0.1±0.0 0.0±0.0 2.5±0.4 0.3±0.3
DHA/ARA 0.2±0.1b 5.3±0.1a 0.2±0.1b 0.2±0.1b 9.9±0.0a 0.4±0.1b 0.1±0.0b 3.0±0.3a 0.4±0.4b
Fatty Acid Class
Saturated 38.3±15.8 35.4±0.4 33.5±0.1 35.3±0.4 38.1±0.3 37.4±0.6 41.3±2.7 42.0±3.8 41.3±4.5
Unsaturated 61.7±15.8 64.6±0.4 66.5±0.1 64.8±0.4 61.9±0.3 62.6±0.6 58.7±2.7 58.0±3.8 58.7±4.5
n-3 10.5±5.8 18.6±0.2 7.9±0.1 19.8±0.0 21.3±0.3 17.8±0.2 36.8±4.4 36.7±4.5 36.2±5.7
n-6 36.9±27.8 30.0±1.0 44.0±0.3 1.4±0.0 1.2±0.0 2.0±0.0 1.1±0.2 1.0±0.3 1.2±0.2
n-3:n-6 0.5±0.5 0.6±0.0 0.2±0.0 14.0±0.2ab 17.4±0.1a 9.0±0.2b 32.7±0.8 35.0±4.1 30.0±0.6
36
37
Table 6. Lipid and fatty acid composition of neutral lipid (NL) and polar lipid (PL)
fractions of whole Yellow Perch after 10 days (Phase I) and 24 days (Phase II) of
feeding with DHA and ARA enriched zooplankton (mean±SD, n=3). Marked
letters represent significant differences within the experimental phase (P≤0.05).
Phase I Yellow Perch Phase II Yellow Perch
DHA ARA Control DHA ARA
Total Sample
% lipids (wet wt.) 2.6±0.1 2.4±0.1 2.4±0.4b 3.3±0.2a 3.1±0.3ab
% NL 54.0±4.0 48.4±0.3 45.2±4.0b 53.4±0.9a 51.6±0.8ab
% PL 46.0±4.0 51.6±0.5 54.8±4.0a 46.6±0.9b 48.4±0.8a
Neutral Lipids
Fatty Acids (%)
C16:0 14.1±0.7 13.0±1.5 20.2±0.3 21.9±0.2 21.9±0.5
C16:1[n-9] 18.6±0.8 17.9±0.3 7.5±1.0 7.4±0.5 7.7±0.6
C18:0 1.2±0.1 1.0±0.2 0.2±0.0 0.2±0.0 0.2±0.1
C18:1[n-9c] 26.0±1.6 24.2±1.5 18.0±0.3 18.5±0.3 19.1±0.6
C18:2[n-6] 1.6±0.6 1.7±0.1 2.1±1.4 2.0±1.4 1.2±0.7
C18:3[n-3] 1.7±0.4 1.6±0.1 2.7±0.4b 4.2±0.6a 4.4±0.5a
C20:1[n-9] 0.6±0.6 0.7±0.1 2.7±0.2b 3.2±0.2a 3.2±0.1a
C20:4[n-6] 1.0±0.4 2.1±1.5 2.4±1.4 1.2±0.2 2.5±0.1
C20:5[n-3] 11.6±0.1 11.1±1.1 11.5±1.2 9.8±1.1 9.3±0.2
C22:6[n-3] 3.5±0.5b 5.5±0.4a 14.7±1.6 13.7±0.9 12.7±0.1
DHA:ARA 4.9±3.5 3.3±1.6 7.7±4.1 11.2±1.3 5.1±0.2
Fatty Acid Class
Saturated 18.0±1.6 17.4±1.3 29.4±2.6 31.2±2.0 31.3±1.3
Unsaturated 82.0±1.6 82.6±1.3 70.6±2.6 68.7±2.0 68.7±1.3
n-3 17.6±0.6 19.2±1.3 16.9±13.0 28.7±1.6 27.2±0.5
n-6 3.3±0.4 4.4±1.3 2.6±2.0 3.4±1.6 4.0±0.6
n-3:n-6 5.4±0.8 4.6±1.0 6.6±1.4 10.0±5.1 7.0±1.1
(continued)
38
Table 6. Cont.
Phase I Yellow Perch Phase II Yellow Perch
DHA ARA Control DHA ARA
Polar Lipids
Fatty Acid (%)
C16:0 25.4±0.2b 27.9±0.7a 28.6±0.7b 28.8±0.2b 31.0±0.5a
C16:1[n-9] 6.1±0.0 6.1±0.1 2.3±0.1a 1.8±0.1b 1.9±0.1b
C18:0 17.3±0.1 17.8±0.2 15.1±0.8a 12.7±0.1b 13.3±0.1b
C18:1[n-9c] 12.8±0.1 12.2±0.1 6.3±0.4a 5.2±0.1b 5.4±0.1b
C18:2[n-6] 0.6±0.0b 0.7±0.0a 0.3±0.0 0.2±0.0 0.3±0.0
C18:3[n-3] 0.7±0.1 0.8±0.0 3.1±0.4a 2.4±0.2b 2.5±0.2b
C20:1[n-9] 0.2±0.0 0.2±0.0 1.1±0.1 0.8±0.1 0.8±0.1
C20:4[n-6] 0.4±0.0 0.2±0.2 0.1±0.0 0.1±0.0 0.1±0.0
C20:5[n-3] 18.6±0.6 19.5±0.5 15.3±0.3a 14.5±0.2ab 13.6±0.1b
C22:6[n-3] 14.3±0.8a 9.3±0.1b 23.1±0.5c 30.0±0.3a 27.1±0.6b
DHA/ARA 348.7.±27.7 127.6±88.2 514.0±348.1 429.2±115.0 291.3±12.9
Fatty Acid Class
Saturated 44.6±0.1b 47.5±0.6a 46.1±0.3a 43.9±0.3b 47.0±0.5a
Unsaturated 55.4±0.1a 52.5±0.6b 54.0±0.3b 56.1±0.3a 53.1±0.5b
n-3 33.9±0.2a 30.0±0.5b 42.6±0.4b 47.0±0.5a 43.4±0.5b
n-6 1.2±0.0b 1.7±0.2a 0.9±0.1a 0.6±0.0b 0.8±0.0a
n-3:n-6 27.5±1.0 17.6±2.0 50.0±4.7 85.1±2.6 51.6±1.7
Discussion
DHA-ARA Experiment
While no significant differences in total lipid content was noted between
treatments, the neutral lipid content of rotifers and Phase II Yellow Perch larvae
was significantly greater in both PUFA enrichments when compared to the control
group. This trend has been observed in other studies (Castell et al. 1972; Copeman
et al. 2002; Coutteau and Mourente 1997), and suggests that the additional lipids
found in these groups are stored in the neutral lipid fraction.
39
The results of this experiment show a clear transfer of DHA from the enrichment
oil to zooplankton and on to perch larvae/juveniles, but this trend is not as apparent
for ARA. Dietary requirements of DHA vary by species, as fish have varying
capacities to elongate and desaturate linolenic acid and octadecatetraenoic acid
(C18:4[n-3]) into DHA. Rainbow Trout are incapable of converting sufficient
DHA, and thus show significantly depressed growth when dietary DHA is limited
(Wirth et al. 1997). Henrotte et al. (2011) investigated this characteristic in Eurasian
Perch and found that juveniles and adults are capable of synthesizing DHA and
EPA from linolenic acid, and did not show depressed growth when DHA was
limited. This was not true for ARA, and thus dietary sources were necessary
(Henrotte et al. 2011). The results of the DHA-ARA enrichment experiment suggest
that Yellow Perch are also capable of synthesizing DHA, as DHA was found in
greater abundance in perch than in live feeds for the control and ARA-enriched
groups. Another possible explanation for this trend is that the high concentrations
of DHA present in perch embryos (~18%; Dabrowski et al. 1993) are retained
throughout larval stages. The relatively high concentration of DHA in the PL
fraction suggests that Yellow Perch conserve DHA within cell membranes, as has
been seen with other species (Koven et al. 1989; Rainuzzo et al. 1994). Preferential
retention of n-3 fatty acids may also help explain the high n-3:n-6 within PL of
Yellow Perch.
Aside from limiting growth and survival, DHA deficiency is associated with other
developmental disadvantages. Lund et al. (2014) found impaired threat avoidance
40
behavior and spatial learning ability in juvenile Pikeperch when dietary DHA levels
were below 3% of total fatty acids. Another investigation found that dietary
deficiencies in DHA led to impaired vision in Atlantic Herring Clupea harengus
(Bell et al. 1995). While neurological development in larval/juvenile Yellow Perch
was not measured in either of the enrichment experiments, this offers a possible
avenue of future research.
Harel and Place (2003) examined DHA:ARA in Artemia enrichments and their
influence on Morone spp. These authors found no significant influence on larval
survival, but generally improved growth in ratios >1:1. The influence of DHA:ARA
in Yellowtail Flounder culture was less clear, as growth and survival shared a much
more direct correlation to DHA:EPA (Copeman et al. 2002). Arachidonic acid in
excess of 5% in enriched rotifers resulted in high rates of malpigmentation in
Yellowtail Flounder larvae (Copeman et al. 2002). Boglino et al. (2014) also found
increased rates of malpigmentation and cranial deformities in Senegalese Sole
when ARA content in enriched rotifers and Artemia was high (>7%). However,
growth of Senegalese Sole was depressed when dietary ARA was low (<1.0%;
Boglino et al. 2012). In the DHA-ARA enrichment experiment DHA:ARA in both
rotifers and Artemia only exceeded 1:1 in DHA enriched treatments, which also
supported the highest growth rate. Arachidonic acid was relatively high in rotifers,
especially in the ARA-enriched group, but was never recorded above 1.0% in any
of the Artemia groups.
41
Table 7. Lipid and fatty acid composition of neutral lipid (NL) and polar lipid (PL) fractions of rotifers and Artemia enriched for ≥13
hours with PUFA emulsions in EE and TG form (mean±SD, n=3). Marked letters represent significant differences within the
zooplankton type (P≤0.05). Rotifers Artemia Platinum Artemia Silver
Control EE TG Control EE TG Control EE TG
Total Sample
% lipids (wet wt.) 5.6±0.3*a 3.4±0.2b 2.2±0.2c 2.9±0.4b 4.1±0.1a 3.2±0.1b 2.8±0.2 3.2±0.2 2.6±0.1
% NL 88.6±3.1*a 78.8±2.9b 78.8±4.3b 80.5±1.2 80.6±2.2 81.2±0.9 74.8±2.9 79.6±2.1 74.6±2.7
% PL 11.4±3.1*b 21.2±2.9a 21.2±4.3a 19.5±1.2 19.4±2.2 18.8±0.9 25.2±2.9a 20.4±2.1b 25.4±2.7a Neutral Lipids
Fatty Acids (%)
C16:0 6.6±0.0 5.5±0.2 6.2±0.4 12.7±0.1a 12.8±0.4a 11.3±0.1b 9.4±0.8a 8.0±0.5b 7.4±0.6b
C16:1[n-9] 5.6±0.1a 2.8±0.1b 3.9±0.3c 12.8±0.2a 13.4±0.5a 11.5±0.6b 3.0±0.3a 2.1±0.1b 1.9±0.2b
C18:0 0.0±0.0b 1.5±0.1a 1.7±0.1a 3.2±0.1a 2.7±0.3b 2.8±0.2ab 3.3±0.2a 2.3±0.1b 2.3±0.2b
C18:1[n-9] 2.4±0.2b 7.3±0.3a 7.5±0.5a 32.9±0.4a 11.6±0.3b 11.4±0.7b 26.6±2.5a 12.0±0.6b 11.1±1.1b
C18:2[n-6] 14.7±0.2a 1.5±0.0b 2.0±0.1b 8.3±0.9 7.3±1.3 10.0±1.9 8.6±0.9 9.7±0.6 10.9±0.9
C18:3[n-3] 1.1±0.0 1.4±0.0 1.5±0.1 1.4±0.0 1.4±0.0 1.3±0.1 20.3±1.8 20.8±1.3 18.0±1.8
C20:1[n-9] 0.4±0.0 0.4±0.0 0.5±0.0 0.7±0.0 0.2±0.0 0.5±0.2 2.5±0.5 2.7±0.2 2.5±0.2
C20:4[n-6] 0.5±0.0c 1.9±0.0b 2.2±0.1a 2.3±0.1c 3.2±0.0a 2.8±0.2b 0.7±0.1 0.9±0.1 0.9±0.1
C20:5[n-3] 0.6±0.1b 44.9±0.5a 42.5±2.9a 8.1±0.3b 26.8±0.3a 29.9±1.4a 0.9±0.1c 13.9±1.1b 18.6±1.4a
C22:6[n-3] 0.3±0.0c 20.6±0.4a 16.8±0.9b 0.2±0.1c 5.5±0.1b 8.1±0.1a 0.1±0.0c 3.9±0.3b 6.5±0.5a
DHA:EPA 0.4±0.1 0.5±0.0 0.4±0.0 0.0±0.0b 0.2±0.0a 0.3±0.0a 0.1±0.0b 0.3±0.0a 0.4±0.0a
Fatty Acid Class
Saturated 11.0±0.2 7.8±0.2 8.8±0.5 21.1±0.2 19.1±1.5 14.7±0.2 29.9±5.3 27.7±4.7 24.7±6.7
Unsaturated 89.0±0.2 92.2±0.2 91.2±0.5 78.9±0.2 80.8±1.5 85.3±0.2 70.1±5.3 72.3±4.7 75.3±6.7
n-3 2.0±0.0b 67.2±0.9a 61.0±3.8a 10.0±0.3b 34.5±0.5a 40.4±1.6a 21.4±1.9b 38.9±0.7a 43.4±3.7a
n-6 15.7±0.2a 5.1±0.0b 5.7±0.3b 11.1±0.9 11.0±1.3 13.2±1.7 9.5±0.9 10.7±0.6 11.9±1.0
n-3:n-6 0.1±0.0c 13.2±0.1a 10.7±0.1b 0.9±0.1b 3.2±0.4a 3.1±0.5a 2.3±0.3b 3.6±0.1a 3.6±0.0a
(continued)
41
42
Table 7. Cont.
Rotifers Artemia Platinum Artemia Silver
Control EE TG Control EE TG Control EE TG
Polar Lipids
Fatty Acid (%)
C16:0 12.2±0.2 11.4±1.5 11.5±1.4 11.8±0.4 11.4±0.5 11.6±0.6 9.1±0.1 9.1±0.3 9.4±0.2
C16:1[n-9] 4.6±0.1a 3.2±0.5b 4.3±0.6a 8.0±0.1ab 9.0±0.4a 8.0±0.1b 2.4±0.0 2.0±0.1 1.9±0.1
C18:0 2.2±1.9 3.2±0.5 3.2±0.7 6.5±0.3 8.5±0.1 8.2±0.2 0.1±0.0 0.1±0.0 0.1±0.0
C18:1[n-9] 36.6±1.0a 6.6±1.2b 6.9±1.3b 26.1±0.5a 18.5±0.3b 18.2±0.1b 20.8±0.3a 17.3±0.2b 18.9±2.2ab
C18:2[n-6] 30.3±2.9 27.8±11.0 39.3±8.9 6.8±0.3 2.9±0.1 3.6±0.0 31.0±2.3 29.7±2.1 23.5±9.0
C18:3[n-3] 0.1±0.0b 2.9±0.6a 3.6±0.7a 1.1±0.0 1.0±0.0 1.0±0.0 15.4±0.2 14.4±0.6 17.5±2.6
C20:1[n-9] 1.3±0.1a 0.4±0.1b 0.5±0.1b 0.5±0.1 0.5±0.0 0.5±0.0 4.0±0.1a 3.4±0.0b 3.6±0.4ab
C20:4[n-6] 0.6±0.2b 1.5±0.3a 1.4±0.4ab 3.9±0.2 4.1±0.3 3.6±0.4 1.4±0.0 1.6±0.0 1.9±0.4
C20:5[n-3] 1.3±0.4c 22.3±3.1a 12.9±1.4b 13.1±0.4b 20.1±0.5a 22.0±0.4a 3.0±0.2b 9.9±0.5a 9.2±1.1a
C22:6[n-3] 0.6±0.1c 9.9±1.4a 5.2±0.5b 0.0±0.0 0.5±0.0 0.9±0.1 0.0±0.0 0.5±0.3 0.7±0.1
DHA:EPA 0.4±0.0 0.4±0.0 0.4±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0b 0.0±0.0ab 0.1±0.0a
Fatty Acid Class
Saturated 16.8±1.8 16.6±2.5 17.0±2.5 19.5±0.7 20.8±0.4 20.7±0.4 10.5±0.6 10.5±1.0 10.5±0.3
Unsaturated 83.2±1.8 83.3±2.5 83.0±2.5 80.5±0.7 79.1±0.4 79.3±0.4 89.5±0.6 89.5±1.0 89.5±0.4
n-3 2.4±0.5c 37.6±5.5a 24.6±3.0b 14.6±0.5b 21.9±0.6ab 24.4±0.5a 18.8±0.7b 25.1±1.5ab 27.7±3.9a
n-6 31.3±2.7 30.0±10.7 41.1±8.5 11.0±0.1 7.3±0.4 7.5±0.4 32.6±2.3 31.6±2.1 25.7±8.6
n-3:n-6 0.1±0.0b 1.5±0.9a 0.6±0.2ab 1.3±0.1b 3.0±0.2a 3.2±0.2a 0.6±0.0 0.8±0.1 1.2±0.5
*Contamination of whole algal cell within separations likely affecting measurement
42
43
Table 8. Lipid and fatty acid composition of neutral lipid (NL) and polar lipid (PL)
fractions of whole Yellow Perch after 10 days (Phase I) and 17 days (Phase II) of
feeding with EE and TG enriched zooplankton (mean±SD, n=3). Marked letters
represent significant differences within the phase (P≤0.05).
Phase I Yellow Perch Phase II Yellow Perch
Control EE TG Control EE TG
Total Sample
% lipids (wet
wt.) 2.1±0.3 2.3±0.2 2.4±0.4 2.1±0.2 2.6±0.1 2.5±0.3
% NL 41.7±8.7 37.9±6.9 48.8±5.7 53.1* 52.7±4.4 49.7±4.3
% PL 58.3±8.7 62.1±6.9 51.2±5.7 46.9* 47.3±4.4 50.3±4.3
Neutral Lipids
Fatty Acids (%)
C16:0 0.7±0.1 0.6±0.1 0.5±0.1 6.5±2.0 5.1±0.7 4.9±0.5
C16:1[n-9] 5.7±1.1 7.3±1.2 6.5±2.0 2.7±1.0 2.1±0.3 1.1±0.6
C18:0 3.0±0.5 2.8±0.7 2.6±0.7 3.1±1.0 2.7±0.4 2.7±0.8
C18:1[n-9] 11.8±2.2 9.8±1.9 8.9±2.5 16.0±5.8 10.4±1.3 10.3±2.0
C18:2[n-6] 30.1±6.9 30.4±3.1 30.1±5.6 4.6±1.7 3.8±0.4 3.5±1.0
C18:3[n-3] 0.6±0.1 0.8±0.1 0.7±0.2 6.1±2.5 9.6±0.6 7.5±2.1
C20:1[n-9] 0.2±0.0 0.6±0.1 0.5±0.2 1.5±1.2 2.7±0.3 2.5±0.6
C20:4[n-6] 2.0±0.4 2.6±0.5 2.1±0.6 0.9±0.3 0.9±0.1 0.9±0.2
C20:5[n-3] 4.6±1.6b 11.9±1.7a 9.3±3.0a 1.6±0.6 4.9±0.5 3.1±0.9
C22:6[n-3] 1.3±0.2 2.1±0.5 2.1±0.8 0.5±0.2 1.2±0.0 1.1±0.1
DHA/EPA 0.9±0.1 0.2±0.0 0.2±0.0 0.3±0.1 0.3±0.0 0.4±0.1
Fatty Acid Class
Saturated 29.5±13.8 19.9±12.0 26.5±17.2 58.3±14.9 58.6±4.3 64.3±7.2
Unsaturated 70.5±13.8 80.1±12.0 76.5±17.2 41.7±14.9 41.4±4.3 35.7±7.2
n-3 6.8±2.0b 15.1±2.3a 12.3±4.1ab 9.0±3.3b 16.4±1.1a 12.5±2.8ab
n-6 32.3±7.2 33.4±3.6 32.4±5.9 5.6±2.1 4.8±0.5 4.5±1.1
n-3:n-6 0.2±0.0 0.5±0.0 0.4±0.1 1.6±0.0c 3.4±0.2a 2.8±0.1b
(continued)
44
Table 8. Cont.
Phase I Yellow Perch Phase II Yellow Perch
Control EE TG Control EE TG
Polar Lipids
Fatty Acid (%)
C16:0 17.7±1.1 18.0±1.3 16.6±0.7 21.9±0.7 20.8±0.3 20.4±1.1
C16:1[n-9] 3.2±0.1 3.3±0.1 3.4±0.1 2.2±0.3a 1.6±0.5ab 1.3±0.1b
C18:0 6.9±1.4 6.8±0.5 6.5±1.3 7.3±0.6 8.1±0.3 7.8±0.5
C18:1[n-9] 13.0±0.2a 9.6±0.3b 9.4±0.2b 19.2±0.3a 13.2±0.2c 14.4±0.4b
C18:2[n-6] 18.1±4.3 16.8±1.0 20.4±4.5 6.2±0.6 3.5±0.1 4.2±0.1
C18:3[n-3] 0.6±0.0 0.4±0.1 0.4±0.1 8.4±0.3a 7.4±0.2b 8.4±0.3a
C20:1[n-9] 0.3±0.0 0.3±0.0 0.3±0.0 0.5±0.0a 0.4±0.0b 0.5±0.0b
C20:4[n-6] 6.4±0.8a 5.0±0.1b 4.9±0.5b 4.4±0.2 3.6±0.1 4.0±0.4
C20:5[n-3] 11.8±0.3c 15.5±0.5a 14.3±0.6b 8.4±0.1c 15.1±0.2a 13.4±0.4b
C22:6[n-3] 7.3±1.5 9.7±0.7 9.7±1.6 4.7±0.4b 10.0±0.4a 8.9±0.1ab
DHA/EPA 0.6±0.1 0.6±0.1 0.7±0.1 0.6±0.0 0.7±0.0 0.7±0.0
Fatty Acid Class
Saturated 25.8±0.3 25.9±0.8 24.2±1.2 34.8±0.3 35.9±0.2 34.8±0.5
Unsaturated 74.2±0.3 74.1±0.8 75.8±1.2 65.2±0.3 64.1±0.2 65.2±0.5
n-3 19.8±1.8b 25.6±0.3a 24.5±1.9a 23.5±0.4b 33.9±0.7a 32.3±0.7a
n-6 25.0±3.4 22.3±1.1 25.7±4.0 11.0±0.5 7.4±0.0 8.5±0.4
n-3:n-6 0.8±0.2 1.1±0.1 1.0±0.2 2.1±0.1c 4.6±0.1a 3.8±0.1b
*Two of three replicates compromised during separation and eliminated from table.
EE-TG Experiment
The results of the EE-TG enrichment experiment suggest that preferential
assimilation of PUFA in EE or TG form varies with species of zooplankton
enriched. Rotifers assimilated higher levels of DHA and EPA from the EE
enrichments, while both types of Artemia nauplii had higher concentrations of these
two fatty acids in the TG group. Preferential incorporation of EE by rotifers was
also observed in Rainuzzo et al. (1994), although the TG emulsions in this study
were only 43% triglycerides. The influence of chemical form on Artemia
45
assimilation is less clear. Takeuchi et al. (1992a) and Rainuzzo et al. (1994) both
observed greater assimilation of PUFA in EE enrichments, while Izquierdo et al.
(1992) achieved approximately equal rates of incorporation. Coutteau and
Mourente (1997), on the other hand, found higher assimilation rates associated with
TG based PUFA emulsions. One explanation of variation may be the strain of
Artemia used in the experiment. It has been shown that PUFA levels in unenriched
Artemia vary widely with geographic range (Navarro et al. 1992; Oetker 1998). It
is also likely that different species and strains of Artemia have varying capacities
of fatty acid incorporation when in EE form. While Takeuchi et al (1992a),
Rainuzzo et al (1994), and Coutteau and Mourente (1997) used Artemia harvested
from the Great Salt Lake, Izquierdo et al. (1992) used Artemia from Tein-Tsin,
China. The current studies used Artemia from San Francisco, CA, which have not
been used in previous investigations of this nature.
Arachidonic acid was relatively high in enriched rotifers and Artemia (≥1%), but
never exceeded the detrimental limits reported by Copeman et al. (2002) and
Boglino et al. (2014). Eicosapentaenoic acid was also found in high abundance in
enriched rotifers and Artemia, with DHA:EPA never exceeding 0.5. Enrichment
oils had DHA:EPA ranging from 2.6-3.0, but the Nannochloropsis algae added to
enrichment jars in this experiment is reported to have a high EPA content (19.0%;
Reitan et al. 1997). While several studies associated low DHA:EPA with depressed
growth and survival (Copeman et al. 2002; Rodriguez et al. 1997), this association
largely dependents on fish species (Estevez et al. 1999). This ratio does not seem
46
to be as critical to Yellow Perch, as similar rates of growth, survival, and swim
bladder inflation were observed in the EE-TG and DHA-ARA enrichment
experiments (almost no EPA in DHA-ARA enrichmented diets). Both enrichment
groups had consistently higher levels of DHA, EPA, ARA, and n-3 fatty acids than
the control groups, which likely explains the depressed growth and swim bladder
inflation rates in Yellow Perch fed the control diet.
Fatty acid composition of experimental larvae/juveniles reflects that of their live
prey. Again, higher concentration of DHA, EPA, and ARA are seen in the polar
lipid fractions of samples. Levels of these fatty acids are higher within control
group Yellow Perch than within their live prey, suggesting either retention or
synthesis of PUFA within these fish. Despite the noted differences in assimilation
within live feeds, differences in the PUFA content of Yellow Perch were rarely
observed.
Culture Success
Both of the live food enrichment experiments mark some of most successful
recorded attempts at the intensive culture of Yellow Perch larvae. The DHA
enrichment resulted in a mean daily growth rate of 24.1±1.2% through the entire
experimental period, with a high proportion of swim bladder inflation among
surviving juveniles (79.0±5.9%). The average juvenile weight after 24 days of
feeding on this diet was 74.5±7.0 mg in this enrichment group. In the ARA enriched
group the average rate of survival through both phases was 36.0±5.0%.
47
Fish weight at 15 dph was similar to that found by Hinshaw (1985) when culturing
Yellow Perch larvae in tanks (14 dph). The DHA-ARA enrichment experiment,
however, had a much higher survival rate during this period (45-60%) than the 1985
investigation (0.0-44.2%;Hinshaw 1985).
Oetker (1998) examined the use of different Artemia types as first feeds for
intensively cultured Yellow Perch larvae. This study cultured fish in 50 L tanks
initially stocked at 160 larvae/L, and did not utilize sprinkler inlets or elevated
salinity and turbidity. Survival after 15 days of feeding was 0.0-0.15%, depending
on the type of Artemia used. A second series of experiments by Amberg (2001)
compared the growth and survival of intensively cultured Yellow Perch larvae
when offered Artemia nauplii, vinegar eels, or a commercial plankton product as
first feeds. Eighteen liter tanks were equipped with sprinkler inlets and initially
stocked at 6 larvae/L. No survival was recorded after 13 days of feeding with any
of the diets (Amberg 2001).
Several other studies of prey selectivity in Yellow Perch larvae have also attempted
rearing perch larvae in tank-based systems. Raisanen and Applegate (1983) offered
newly hatched larvae a variety of wild zooplankton throughout the first 20 days of
exogenous feeding. This study recorded larval size of ~8 mm after 10 days of
feeding. To compare, the average recorded lengths for larvae at the end of the first
phase for both the DHA-ARA and EE-TG enrichment experiments were 11.0 mm.
Fulford et al. (2006) raised Yellow Perch larvae up to 15 dph on a feeding regime
of rotifers and Artemia nauplii, and recorded an average length of 8.6 mm at this
48
age. Neither of the previous two studies included survival of larvae as a parameter
of interest. Graeb et al. (2004) examined survival and growth of Yellow Perch
larvae when offered different zooplankton live feeds. This investigation had 0-5%
survival of newly hatched larvae after six days of feeding and 0-15% survival of 7-
12 mm larvae after nine days of feeding. Growth rates were recorded as mm*day-
1, and the maximum achieved were 0.1 and 0.25 mm/day for newly hatched and 7-
12 mm larvae (Graeb et al. 2004). The DHA-ARA and EE-TG enrichment
experiments recorded growth rates of 0.49±0.03 mm/day and 0.57±0.6 mm/day,
respectively.
The development of a specialized feeding regime and culture system for Yellow
Perch larvae in the DHA-ARA and EE-TG enrichment experiments can be used as
an important reference for the commercial production of this species, as well as an
excellent platform for future research on Yellow Perch.
Future Research
While the current studies on live feed enrichments have provided important insights
into the nutritional requirements of Yellow Perch in the early life stages, additional
research is necessary to truly optimize larvae culture success. One direction for
future research would be to investigate different methods of live food enrichment.
The DHA-ARA and EE-TG experiments have both utilized the “direct method”
(Takeuchi et al. 1992a) of enrichment, in which batches of live zooplankton are
placed in lipid emulsions for 4-24 hours prior to larval feeding. Dhert et al. (2014)
investigated a method of continuous enrichment with rotifers and found greater
49
DHA and n-3 fatty acid assimilation than when the “direct method” was used. The
continuous enrichment method involves the addition of enrichments as part of
zooplankton culture diets over a longer periods of time. This method also has
advantages in consistency, handling stress, and low labor demands (Dhert et al.
2014). Considering the significantly lower proportion of DHA in the neutral lipid
fraction than in the polar lipid fraction of perch samples, it is reasonable to expect
that the optimal concentration of DHA in Yellow Perch live feeds has yet to be
reached. Continuous enrichment techniques could potentially increase the DHA
content of rotifers and Artemia further, and thus further improve larval
performance. Additionally, the physical form of enrichments can significantly
influence incorporation of nutrients into rotifers and Artemia. For instance, the
microencapsulation of amino acids in liposomes provides a more direct route of
ingestion and assimilation into rotifers and prevents nutrient leakage (Pinto et al.
2013). While liposomes may not be an appropriate vessel for PUFA, other options
have yet to be explored.
Another potential topic for further investigation with Yellow Perch larvae culture
would be to enrich live feeds with vitamins and other nutrients. Culture exposure
to chemoattractants such as arginine, alanine, and glycine have been shown to
increase the consumption rates of larval Gilthead Seabream by 35% (Kolkovski et
al. 1997). The addition of specific proteins and amino acids in first feeds could also
potentially increase the feeding rates of larval perch, but this technique has yet to
be studied. Enrichment of Artemia with vitamin C and PUFA has also been
50
investigated in larval fish, and significantly increased the growth and survival of
larval Rainbow Trout (Akbary et al. 2011). Other studies suggest that there is a
strong link between dietary vitamin C and stress resistance in larval fish (Merchie
et al. 1997), but again, this enrichment has yet to be tested on Yellow Perch.
Overall, this thesis represents one of the first recorded successful attempts at
Yellow Perch larvae culture in indoor-intensive conditions. Live food enrichment
with PUFA can be a useful technique for increasing larvae culture success,
especially when enrichment oils are high in DHA and contained in ethyl ester form.
This, in turn, represents an additional advantage of the intensive culture technique,
as opposed to the use of fertilized ponds, and can help to optimize the aquaculture
production of this species.
51
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Wallaert, C., and P. J. Babin. 1994. Effects of temperature variations of dietary
lipid absorption and plasma lipoprotein concentrations in trout
(Oncorhynchus mykiss). Comparative Biochemistry and Physiology B-
Biochemistry & Molecular Biology 109(2-3):473-487.
Watanabe, T. 1993. Importance of docosahexaenoic acid in marine larval fish.
Journal of the World Aquaculture Society 24(2):152-161.
56
57
Appendix A: Weight Corrections for DHA-ARA Enrichment Experiment
The original measurements of Yellow Perch weight and length for both phases in
2013 were made in sets of ten. Fish were removed from 70% ethyl alcohol solution
and placed on a paper towel, where total length was measured with digital calipers.
Following length measurement, fish were weighed with an analytical balance and
returned to the alcohol solution. Upon later review, it became apparent that this
technique left samples exposed for 5.45±0.26 minutes, resulting in significant
evaporative weight loss prior to measurement.
In order to help correct for evaporative loss, an additional measurement trial was
carried out using experimental fish. A random sample of 20 fish from Phase I and
20 fish from Phase II were weighed using the original method as well as a technique
that minimized evaporative weight loss. When plotted, a linear relationship
between “semi-dry” and wet weight was apparent for both Phase I and Phase II fish.
The relationships for both size classes are shown in Figure 8. Linear equations were
used to transform the original weight datum, as to account for evaporative losses.
58
Figure 8. Relationship between the measured weight of fish samples when weighed
immediately after removal from 70% ethyl alcohol (wet) and after 5.5±0.3 minutes
of air exposure (“semi-dry”).
59
Appendix B: T Tests and ANOVA Tables
Two-Way ANOVA of DHA-ARA enrichment experiment mean weights based on dietary
treatment (treatment) and swim bladder inflation/noninflation (SB).
Phase I
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio
Model 3 11.909988 3.97000 11.3288
Error 14 4.906054 0.35043 Prob > F
C. Total 17 16.816042 0.0005*
Effect Tests
Source Nparm DF Sum of Squares F Ratio Prob > F
treatment 2 2 2.1788077 3.1087 0.0763
sb 1 1 9.7311805 27.7691 0.0001*
Phase II
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio
Model 3 5395.6752 1798.56 55.9069
Error 14 450.3880 32.17 Prob > F
C. Total 17 5846.0632 <.0001*
Effect Tests
Source Nparm DF Sum of Squares F Ratio Prob > F
treatment 2 2 1067.9901 16.5989 0.0002*
SB 1 1 4327.6851 134.5231 <.0001*
ANOVA tables and Tukey-Kramer test results for significant effects on mean weight,
growth, survival, and swim bladder inflation rates in DHA-ARA experiment.
Phase I
Swim Bladder Inflation (%)
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
treatment 2 0.07913716 0.039569 49.3922 0.0005*
Error 5 0.00400555 0.000801
C. Total 7 0.08314271
60
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
ARA Control 0.2528173 0.0258378 0.168745 0.3368900 0.0004*
DHA Control 0.1877439 0.0258378 0.103671 0.2718166 0.0018*
ARA DHA 0.0650734 0.0231100 -0.010123 0.1402703 0.0809
Survival (%)
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
treatment 2 0.06388738 0.031944 11.1064 0.0096*
Error 6 0.01725689 0.002876
C. Total 8 0.08114426
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
Control DHA 0.2063762 0.0437885 0.072027 0.3407258 0.0078*
Control ARA 0.1037762 0.0437885 -0.030573 0.2381258 0.1205
ARA DHA 0.1026000 0.0437885 -0.031750 0.2369496 0.1247
Phase II
Mean Weight
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Treatment 2 988.1934 494.097 14.5618 0.0050*
Error 6 203.5859 33.931
C. Total 8 1191.7793
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
DHA Control 24.23011 4.756117 9.63764 38.82259 0.0054*
ARA Control 19.44806 4.756117 4.85558 34.04054 0.0152*
DHA ARA 4.78206 4.756117 -9.81042 19.37454 0.6004
Growth Rate (%*day-1)
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Treatment 2 0.00109862 0.000549 7.7744 0.0216*
Error 6 0.00042394 0.000071
C. Total 8 0.00152255
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
ARA Control 0.0243285 0.0068632 0.003271 0.0453860 0.0282*
DHA Control 0.0224307 0.0068632 0.001373 0.0434882 0.0392*
ARA DHA 0.0018978 0.0068632 -0.019160 0.0229552 0.9590
61
Two-Way ANOVA of EE-TG Experiment mean weights based on dietary treatment
(treatment) and swim bladder inflation/noninflation (SB).
Phase I
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio
Model 3 70.252314 23.4174 18.5845
Error 14 17.640716 1.2601 Prob > F
C. Total 17 87.893030 <.0001*
Effect Tests
Source Nparm DF Sum of Squares F Ratio Prob > F
treatment 2 2 8.729509 3.4640 0.0600
sb 1 1 61.522805 48.8256 <.0001*
Phase II
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio
Model 3 1489.0969 496.366 55.7641
Error 14 124.6163 8.901 Prob > F
C. Total 17 1613.7132 <.0001*
Effect Tests
Source Nparm DF Sum of Squares F Ratio Prob > F
treatment 2 2 50.3132 2.8262 0.0931
SB 1 1 1438.7837 161.6399 <.0001*
ANOVA tables and Tukey-Kramer test results for significant effects on mean weight,
growth, survival, and swim bladder inflation rates in DHA-ARA experiment.
Phase I
Mean Weight Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
treatment 2 7.035929 3.51796 6.2288 0.0343*
Error 6 3.388721 0.56479
C. Total 8 10.424650
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
EE TRI 2.164329 0.6136160 0.281664 4.046995 0.0288*
Control TRI 1.150898 0.6136160 -0.731768 3.033563 0.2256
EE Control 1.013431 0.6136160 -0.869234 2.896097 0.2970
62
Growth Rate (%*day-1)
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
treatment 2 0.00264776 0.001324 6.0525 0.0364*
Error 6 0.00131240 0.000219
C. Total 8 0.00396016
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
EE TRI 0.0419057 0.0120757 0.004856 0.0789557 0.0308*
Control TRI 0.0235632 0.0120757 -0.013487 0.0606133 0.2052
EE Control 0.0183424 0.0120757 -0.018708 0.0553925 0.3477
ANOVA tables and Tukey-Kramer test results for significant effects on linoleic, linolenic,
arachidonic, eicosapentaenoic, and docosahexaenoic acid content in rotifers and Artemia
used in the DHA-ARA experiment.
Rotifers
Neutral Lipids
Arachidonic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Sample description 2 786.26615 393.133 1706.679 <.0001*
Error 3 0.69105 0.230
C. Total 5 786.95720
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
R ARA R DHA 25.02592 0.4799476 23.0204 27.03148 <.0001*
R ARA R cont 23.46637 0.4799476 21.4608 25.47193 <.0001*
R cont R DHA 1.55954 0.4799476 -0.4460 3.56511 0.0935
Eicosapentaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Sample description 2 5.2717835 2.63589 121.5907 0.0013*
Error 3 0.0650352 0.02168
C. Total 5 5.3368187
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
R cont R ARA 2.023217 0.1472359 1.40796 2.638473 0.0017*
R cont R DHA 1.951706 0.1472359 1.33645 2.566962 0.0019*
R DHA R ARA 0.071511 0.1472359 -0.54374 0.686768 0.8828
63
Docosahexaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Sample description 2 545.66122 272.831 42.0565 0.0064*
Error 3 19.46171 6.487
C. Total 5 565.12293
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
R DHA R ARA 20.43561 2.547005 9.7924 31.07881 0.0083*
R DHA R cont 20.01756 2.547005 9.3744 30.66076 0.0088*
R cont R ARA 0.41805 2.547005 -10.2252 11.06125 0.9853
Polar Lipids
Arachidonic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Sample description 2 196.05926 98.0296 430.9489 0.0002*
Error 3 0.68242 0.2275
C. Total 5 196.74169
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
R ARA R DHA 12.38945 0.4769423 10.3964 14.38245 0.0003*
R ARA R cont 11.84456 0.4769423 9.8516 13.83756 0.0003*
R cont R DHA 0.54489 0.4769423 -1.4481 2.53789 0.5564
Docosahexaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Sample description 2 259.24932 129.625 494.9572 0.0002*
Error 3 0.78567 0.262
C. Total 5 260.03499
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
R DHA R ARA 14.13772 0.5117525 11.9993 16.27619 0.0002*
R DHA R cont 13.74199 0.5117525 11.6035 15.88045 0.0002*
R cont R ARA 0.39573 0.5117525 -1.7427 2.53420 0.7426
Artemia Platinum
Neutral Lipids
Eicosapentaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Sample description 2 26.986518 13.4933 1022.446 <.0001*
Error 3 0.039591 0.0132
C. Total 5 27.026109
64
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
Plat Cont Plat DHA 5.192639 0.1148784 4.712595 5.672683 <.0001*
Plat ARA Plat DHA 2.727709 0.1148784 2.247665 3.207753 0.0003*
Plat Cont Plat ARA 2.464930 0.1148784 1.984887 2.944974 0.0005*
Docosahexaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Sample description 2 300.50578 150.253 2535.321 <.0001*
Error 3 0.17779 0.059
C. Total 5 300.68358
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
Plat DHA Plat Cont 15.16781 0.2434417 14.1505 16.18508 <.0001*
Plat DHA Plat ARA 14.85250 0.2434417 13.8352 15.86977 <.0001*
Plat ARA Plat Cont 0.31531 0.2434417 -0.7020 1.33258 0.4875
Artemia Silver
Neutral Lipids
Docosahexaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Sample description 2 88.192296 44.0961 330.5606 0.0003*
Error 3 0.400194 0.1334
C. Total 5 88.592490
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
Silv DHA Silv Cont 8.312683 0.3652370 6.78646 9.838903 0.0004*
Silv DHA Silv ARA 7.940347 0.3652370 6.41413 9.466567 0.0004*
Silv ARA Silv Cont 0.372336 0.3652370 -1.15388 1.898556 0.6162
65
T tests (Phase I), ANOVA tables and Tukey-Kramer test results (Phase II) for significant
effects on linoleic, linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acid content
in Yellow Perch from the DHA-ARA experiment.
Phase I Yellow Perch
Neutral Lipids
Docosahexaenoic acid content
t Test
Difference -1.9584 t Ratio -7.43181
Std Err Dif 0.2635 DF 2.41207
Upper CL Dif -0.9914 Prob > |t| 0.0102*
Lower CL Dif -2.9254 Prob > t 0.9949
Confidence 0.95 Prob < t 0.0051*
Polar Lipids
Linoleic acid content
t Test
Difference -0.15269 t Ratio -9.0771
Std Err Dif 0.01682 DF 3.480362
Upper CL Dif -0.10311 Prob > |t| 0.0015*
Lower CL Dif -0.20228 Prob > t 0.9992
Confidence 0.95 Prob < t 0.0008*
Docosahexaenoic acid content
t Test
Difference 4.97156 t Ratio 10.44913
Std Err Dif 0.47579 DF 2.034311
Upper CL Dif 6.98597 Prob > |t| 0.0085*
Lower CL Dif 2.95715 Prob > t 0.0043*
Confidence 0.95 Prob < t 0.9957
Phase II Yellow Perch
Neutral Lipids
Linolenic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
treatBS2:CU17ment 2 4.8385372 2.41927 9.0797 0.0153*
Error 6 1.5986891 0.26645
C. Total 8 6.4372262
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
ARA P2 Cont. P2 1.624735 0.4214643 0.33162 2.917850 0.0197*
DHA P2 Cont. P2 1.475274 0.4214643 0.18216 2.768389 0.0297*
ARA P2 DHA P2 0.149461 0.4214643 -1.14365 1.442576 0.9338
66
Polar Lipids
Linoleic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
PL 2 0.02819940 0.014100 12.3782 0.0074*
Error 6 0.00683447 0.001139
C. Total 8 0.03503387
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
Cont. P2 DHA P2 0.1325859 0.0275570 0.048037 0.2171348 0.0071*
ARA P2 DHA P2 0.0965490 0.0275570 0.012000 0.1810978 0.0296*
Cont. P2 ARA P2 0.0360369 0.0275570 -0.048512 0.1205858 0.4416
Linolenic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
PL 2 0.9330425 0.466521 5.9740 0.0374*
Error 6 0.4685503 0.078092
C. Total 8 1.4015928
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
Cont. P2 DHA P2 0.6959713 0.2281691 -0.004086 1.396028 0.0511
Cont. P2 ARA P2 0.6692942 0.2281691 -0.030763 1.369351 0.0591
ARA P2 DHA P2 0.0266771 0.2281691 -0.673380 0.726734 0.9925
Eicosapentaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
PL 2 3.8615158 1.93076 40.2496 0.0003*
Error 6 0.2878174 0.04797
C. Total 8 4.1493333
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
Cont. P2 ARA P2 1.604323 0.1788287 1.055650 2.152996 0.0003*
DHA P2 ARA P2 0.821369 0.1788287 0.272696 1.370042 0.0089*
Cont. P2 DHA P2 0.782954 0.1788287 0.234281 1.331627 0.0111*
Docosahexaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
PL 2 63.932088 31.9660 135.8824 <.0001*
Error 6 1.411488 0.2352
C. Total 8 65.343575
67
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
DHA P2 Cont. P2 6.475577 0.3960201 5.260528 7.690626 <.0001*
ARA P2 Cont. P2 3.956272 0.3960201 2.741223 5.171321 0.0001*
DHA P2 ARA P2 2.519305 0.3960201 1.304256 3.734354 0.0017*
ANOVA tables and Tukey-Kramer test results for significant effects on linoleic, linolenic,
arachidonic, eicosapentaenoic, and docosahexaenoic acid content in rotifers and Artemia
used in the EE-TG experiment.
Rotifers
Neutral Lipids
Linoleic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
NL 2 334.46284 167.231 6730.328 <.0001*
Error 6 0.14908 0.025
C. Total 8 334.61193
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
RotiCont RotiEE 13.16535 0.1287049 12.77046 13.56024 <.0001*
RotiCont RotiTAG 12.68486 0.1287049 12.28997 13.07974 <.0001*
RotiTAG RotiEE 0.48049 0.1287049 0.08561 0.87538 0.0226*
Arachidonic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
NL 2 4.8038106 2.40191 488.7984 <.0001*
Error 6 0.0294834 0.00491
C. Total 8 4.8332940
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
RotiTAG RotiCont 1.673051 0.0572358 1.497443 1.848658 <.0001*
RotiEE RotiCont 1.386600 0.0572358 1.210992 1.562207 <.0001*
RotiTAG RotiEE 0.286451 0.0572358 0.110843 0.462059 0.0058*
Eicosapentaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
NL 2 3719.2075 1859.60 658.6697 <.0001*
Error 6 16.9396 2.82
C. Total 8 3736.1471
68
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
RotiEE RotiCont 44.27847 1.371926 40.0692 48.48775 <.0001*
RotiTAG RotiCont 41.86654 1.371926 37.6573 46.07581 <.0001*
RotiEE RotiTAG 2.41194 1.371926 -1.7973 6.62121 0.2609
Welch's Test
F Ratio DFNum DFDen Prob > F
8143.0356 2 2.7191 <.0001*
Docosahexaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
NL 2 699.01603 349.508 987.5670 <.0001*
Error 6 2.12345 0.354
C. Total 8 701.13948
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
RotiEE RotiCont 20.29390 0.4857353 18.80359 21.78421 <.0001*
RotiTAG RotiCont 16.52082 0.4857353 15.03051 18.01113 <.0001*
RotiEE RotiTAG 3.77308 0.4857353 2.28278 5.26339 0.0006*
Polar Lipids
Linolenic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
PL 2 20.573429 10.2867 35.5218 0.0005*
Error 6 1.737533 0.2896
C. Total 8 22.310962
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
RotiTAG RotiCont 3.512318 0.4393850 2.16422 4.860416 0.0005*
RotiEE RotiCont 2.773230 0.4393850 1.42513 4.121329 0.0018*
RotiTAG RotiEE 0.739088 0.4393850 -0.60901 2.087187 0.2862
Arachidonic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
PL 2 1.4607218 0.730361 8.5900 0.0173*
Error 6 0.5101481 0.085025
C. Total 8 1.9708699
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
RotiEE RotiCont 0.8986405 0.2380822 0.168169 1.629112 0.0216*
RotiTAG RotiCont 0.8024418 0.2380822 0.071970 1.532913 0.0346*
RotiEE RotiTAG 0.0961987 0.2380822 -0.634273 0.826670 0.9152
69
Eicosapentaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
PL 2 662.81536 331.408 86.6086 <.0001*
Error 6 22.95900 3.827
C. Total 8 685.77437
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
RotiEE RotiCont 20.98332 1.597185 16.08291 25.88372 <.0001*
RotiTAG RotiCont 11.57931 1.597185 6.67890 16.47971 0.0009*
RotiEE RotiTAG 9.40401 1.597185 4.50361 14.30441 0.0026*
Docosahexaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
PL 2 131.65867 65.8293 82.9014 <.0001*
Error 6 4.76441 0.7941
C. Total 8 136.42308
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
RotiEE RotiCont 9.368625 0.7275840 7.136289 11.60096 <.0001*
RotiEE RotiTAG 4.715730 0.7275840 2.483394 6.94807 0.0016*
RotiTAG RotiCont 4.652894 0.7275840 2.420558 6.88523 0.0017*
Welch's Test
F Ratio DFNum DFDen Prob > F
135.4029 2 2.7743 0.0017*
Artemia Platinum
Neutral Lipids
Arachidonic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
NL 2 1.2820042 0.641002 39.1929 0.0004*
Error 6 0.0981304 0.016355
C. Total 8 1.3801346
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
ArtPEE ArtPCont 0.9241808 0.1044192 0.6038071 1.244555 0.0003*
ArtPTAG ArtPCont 0.4825711 0.1044192 0.1621973 0.802945 0.0086*
ArtPEE ArtPTAG 0.4416098 0.1044192 0.1212360 0.761984 0.0130*
Eicosapentaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
NL 2 835.17545 417.588 561.0968 <.0001*
Error 6 4.46541 0.744
C. Total 8 839.64086
70
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
ArtPTAG ArtPCont 21.83685 0.7043838 19.67570 23.99801 <.0001*
ArtPEE ArtPCont 18.66127 0.7043838 16.50012 20.82242 <.0001*
ArtPTAG ArtPEE 3.17558 0.7043838 1.01443 5.33674 0.0097*
Docosahexaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
NL 2 98.338346 49.1692 4376.760 <.0001*
Error 6 0.067405 0.0112
C. Total 8 98.405751
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
ArtPTAG ArtPCont 7.954057 0.0865415 7.688535 8.219579 <.0001*
ArtPEE ArtPCont 5.288100 0.0865415 5.022578 5.553622 <.0001*
ArtPTAG ArtPEE 2.665957 0.0865415 2.400435 2.931479 <.0001*
Polar Lipids
Eicosapentaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
PL 2 102.07000 51.0350 296.8673 <.0001*
Error 5 0.85956 0.1719
C. Total 7 102.92956
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
ArtPTAG ArtPCont 8.981596 0.3784969 7.750019 10.21317 <.0001*
ArtPEE ArtPCont 7.065006 0.3784969 5.833429 8.29658 <.0001*
ArtPTAG ArtPEE 1.916590 0.3385379 0.815034 3.01815 0.0055*
Artemia Silver
Neutral Lipids
Eicosapentaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
NL 2 503.12299 251.561 236.3805 <.0001*
Error 6 6.38534 1.064
C. Total 8 509.50832
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
ArtSTAG ArtSCont 17.68205 0.8423075 15.09772 20.26637 <.0001*
ArtSEE ArtSCont 12.97266 0.8423075 10.38833 15.55698 <.0001*
ArtSTAG ArtSEE 4.70939 0.8423075 2.12506 7.29371 0.0034*
71
Docosahexaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
NL 2 63.035876 31.5179 290.5357 <.0001*
Error 6 0.650893 0.1085
C. Total 8 63.686769
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
ArtSTAG ArtSCont 6.439847 0.2689265 5.614741 7.264954 <.0001*
ArtSEE ArtSCont 3.863519 0.2689265 3.038412 4.688625 <.0001*
ArtSTAG ArtSEE 2.576329 0.2689265 1.751222 3.401435 0.0002*
Polar Lipids
Eicosapentaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
PL 2 85.027740 42.5139 69.2562 <.0001*
Error 6 3.683185 0.6139
C. Total 8 88.710925
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
ArtSEE ArtSCont 6.855240 0.6397208 4.89248 8.817999 <.0001*
ArtSTAG ArtSCont 6.123567 0.6397208 4.16081 8.086326 0.0002*
ArtSEE ArtSTAG 0.731673 0.6397208 -1.23109 2.694432 0.5249
ANOVA tables and Tukey-Kramer test results for significant effects on linoleic, linolenic,
arachidonic, eicosapentaenoic, and docosahexaenoic acid content in Yellow Perch from the
EE-TG experiment.
Phase I Yellow Perch
Neutral Lipids
Eicosapentaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Treatment 2 81.43939 40.7197 8.3842 0.0183*
Error 6 29.14038 4.8567
C. Total 8 110.57978
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
PIEE PICont 7.266717 1.799394 1.74591 12.78753 0.0160*
PITAG PICont 4.689679 1.799394 -0.83113 10.21049 0.0892
PIEE PITAG 2.577038 1.799394 -2.94377 8.09785 0.3843
72
Polar Lipids
Arachidonic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Treatment 2 4.2625610 2.13128 6.4786 0.0317*
Error 6 1.9738391 0.32897
C. Total 8 6.2364000
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
PICont PITAG 1.522177 0.4683113 0.08533 2.959026 0.0400*
PICont PIEE 1.388395 0.4683113 -0.04845 2.825244 0.0568
PIEE PITAG 0.133783 0.4683113 -1.30307 1.570632 0.9563
Docosahexaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Treatment 2 20.624467 10.3122 41.9802 0.0003*
Error 6 1.473871 0.2456
C. Total 8 22.098338
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL p-Value
PIEE PICont 3.634546 0.4046769 2.39294 0.0003*
PITAG PICont 2.453502 0.4046769 1.21189 0.0022*
PIEE PITAG 1.181044 0.4046769 -0.06057 0.0602
Phase II Yellow Perch
Polar Lipids
Linolenic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Treatment 2 1.9401399 0.970070 14.4268 0.0051*
Error 6 0.4034449 0.067241
C. Total 8 2.3435848
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
PIITAG PIIEE 0.9963585 0.2117244 0.346757 1.645960 0.0079*
PIICont PIIEE 0.9730711 0.2117244 0.323469 1.622673 0.0088*
PIITAG PIICont 0.0232874 0.2117244 -0.626315 0.672889 0.9934
Eicosapentaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Treatment 2 73.056055 36.5280 430.6907 <.0001*
Error 6 0.508876 0.0848
C. Total 8 73.564931
73
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
PIIEE PIICont 6.728630 0.2377852 5.999070 7.458190 <.0001*
PIITAG PIICont 4.968117 0.2377852 4.238557 5.697678 <.0001*
PIIEE PIITAG 1.760512 0.2377852 1.030952 2.490073 0.0008*
Docosahexaenoic acid content
Analysis of Variance
Source DF Sum of Squares Mean Square F Ratio Prob > F
Treatment 2 46.944797 23.4724 175.1930 <.0001*
Error 6 0.803882 0.1340
C. Total 8 47.748679
Ordered Differences Report
Level - Level Difference Std Err Dif Lower CL Upper CL p-Value
PIIEE PIICont 5.287577 0.2988648 4.370615 6.204539 <.0001*
PIITAG PIICont 4.226048 0.2988648 3.309086 5.143009 <.0001*
PIIEE PIITAG 1.061530 0.2988648 0.144568 1.978492 0.0279*