Improvement of Yellow Perch Larvae Culture Via Live Food Enrichment with Polyunsaturated Fatty Acids THESIS Presented in Partial

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

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

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

ii 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.

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

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 vi

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

vii

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 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 ix

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

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 xi

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

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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).

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

2 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

3 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.

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

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.

D D E E

G A A

F C

C E 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),D 100 µm mesh outlets (C), air stones (D), and automatedA belt feeders (E). A

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.

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

Phase I

Day 1 Day 3 Day 5 Day 10 - Rotifers - Artemia - Otohime A Phase II

Day 11 Day 14 Day 20 Day 24

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,

9 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.

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

11 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).

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

14 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

15 understanding the influence of the total and relative fatty acid composition of live feeds used in Yellow Perch intensive larvae culture.

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

17 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

18 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

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

Phase I

Day 1 Day 3 Day 5 Day 10 - Rotifers - Artemia - Otohime A Phase II

Day 11 Day 14 Day 18

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

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.

22 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

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.

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

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

TG TG

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

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

27 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.

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

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

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

31 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

32 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

33 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.

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 35 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)

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 36 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

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)

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.

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

39 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.

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 41 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)

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 42 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

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)

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 44 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

45 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%.

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

47 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

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

49 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.

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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.

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”).

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

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

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

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

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

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

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

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

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*

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

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

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 69

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*

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

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 72

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*