Quantifying Digestion in California Yellowtail ( lalandi dorsalis), Technological Advances Towards the Development of the Sector

The Graduate Division The University of Hawai‘i at Hilo In Partial Fulfillment of the Requirements for the Degree of

Master of Science: Tropical Conservation Biology and Environmental Science

Hilo, Hawai’i December 2016

By: George Rod Parish IV

Thesis Committee: Armando Garciaa Charles Farwellbc Luke Gardnerbd Kevin Hopkinsa Barbara Blockbd

a Pacific Aquaculture & Coastal Resources Center, College of Agriculture, Forestry and Natural Resource Management, University of at Hilo, 1079 Kalanianaole Ave., Hilo, HI 96720, United States b Research and Conservation Center, 886 Cannery Row, Monterey, CA 93940, USA c Monterey Bay Aquarium, 886 Cannery Row, Monterey, CA 93940, USA d Biology Department, Hopkins Marine Station, Stanford University, 120 Ocean View Blvd, Pacific Grove, CA 93950, USA

Table of Contents Acknowledgements ...... iv List Of Figures ...... vi List Of Tables ...... vii Chapter 1: Introduction ...... 1 I. Yellowtail Biology and Aquaculture ...... 1 A. Yellowtail Physical Traits and ...... 1 B. Distribution and Movement Patterns in a Natural Setting ...... 2 C. Yellowtail Aquaculture ...... 4 II. The Necessity For A New Source Of ...... 7 III. A Movement Towards The Ocean ...... 8 IV. Respirometry: A Tool for Aquaculture ...... 10 V. Primary Objectives for this Study ...... 11 Chapter 2: Postprandial Metabolic Response Of California Yellowtail (S. lalandi dorsalis) Abstract ...... 13 VI. Introduction ...... 14 A. Metabolism in Aquatic ...... 14 B. Calorimetry as a Means to Calculate Energy Budget ...... 15 C. Specific Dynamic Action ...... 17 VII. Materials and Methods ...... 19 A. Experimental Fish ...... 19 B. Swim Tunnel Characteristics ...... 20 C. Swim Tunnel Calculations ...... 24 D. Experimental Design ...... 25 E. Nutritional Analysis of a Flesh Meal...... 27 F. Calculation of Meal and Respiration Energy ...... 28 VIII. Results ...... 29 A. SDA Metrics ...... 29 B. Measured Respirometry Values ...... 31 IX. Discussion...... 34 A. Baseline Metabolic Rate ...... 34

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B. Overall Postprandial Response ...... 37

C. SDA and SDACoefficient ...... 38 X. Conclusions ...... 40 Chapter 3: Influence of Feed Source On The Postprandial Metabolic Response Of California Yellowtail (S. lalandi dorsalis) In A 1,237 L Static Swim Respirometer ...... 42 I. Abstract ...... 42 II. Introduction ...... 44 A. Advances in Aquaculture Practices: Natural Feed items and Formulated Pellets ...... 44 B. Reduced Costs Associated with Feed Development: Defining the Mechanical Digestion of a Meal ...... 45 C. Swim Speed and its Influence on Energy Expenditure...... 48 D. Importance of Fecal Output Collection and Characterization ...... 51 III. Materials and Methods ...... 53 A. Experimental Fish ...... 53 B. Primary Components of the Static Respirometer ...... 53 C. Experimental Design Of The Study ...... 56 D. Nutritional Analysis: Calculating the Proximate Composition of Meal Types and Fecal Output ...... 59 E. Fecal Collection Procedures and Storage ...... 61 F. Statistical Analyses ...... 63 IV. Results ...... 65 A. Dietary Composition Analyses ...... 65 B. Postprandial Metabolic Response ...... 65 C. Visual Documentation of Movement During Respirometry ...... 67 D. Fecal Proximate Composition as a Function of Meals Type ...... 68 V. Discussion...... 69 A. Metabolism ...... 69 B. Baseline Routine Metabolic Rate ...... 70

C. SDAMax, SDADuration, and Factorial Scope ...... 72 D. Protein and Lipid Contribution to Metabolic Response ...... 73 E. Variability in Metabolic Response from Meal Size and Structure ...... 74 F. A Description of Routine Movement for Different Feed Types ...... 76

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G. Digestibility of Various Feed Types ...... 79 VI. Conclusions ...... 80 Chapter 4: Summary and Conclusions ...... 82 Bibliography ...... 87

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Acknowledgements

I would like to sincerely thank everyone who has shown their support and encouragement towards my education I’ve had the opportunity to work with some amazing people in a range of classroom, laboratory, and field settings. Each person has brought such a unique personality and wealth of expertise, I truly value everyone’s contribution to my studies.

I would like to thank my committee members for taking the time to share their knowledge, resources, and personal experiences towards my education. I greatly appreciate all their support and value the opportunities that have been made available to me during this time. Luke Gardner, for all the long hours of calculations and tackling system design issues, as well as constantly providing daily advice on my project. Chuck Farwell, for your continued support and advice in and out of the lab, thanks for ensuring an interesting and multidimensional thesis experience. Barbara Block, for encouraging me to explore the world of both in a lab and natural setting. Kevin Hopkins, for sparking my interest in aquaculture from day one as well as helping me see my project through. Armando Garcia, for taking the time to have me as a student, discussions in your office on life and aquaculture, and the constant support over the years.

I couldn’t have completed this work without all the help from everyone at the Tuna

Research and Conservation Center. Special thanks to Ian Rowbotham and Nickolas Mendoza for enduring months of hard work to see this project completed. You both contributed greatly to the study design and direction of this research. I greatly appreciate all the intellectual support, direction, and project advice from Alex Norton, John Dale, and Dane Klinger. And

iv much appreciation to both Luke Hiserman and Tor Mowatt-Larssen for the long hours of data analysis.

And to all my family and friends not listed here, I can’t thank you enough for all the help and support.

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List Of Figures

Figure 1. California yellowtail (adult) S. lalandi dorsalis ©Diane Rome Peebles ...... 7

Figure 2. Partitioning of input and output energy throughout the digestive process (Values described in Cho and Bureau 1995)...... 15

Figure 3. Side view of 30 L respirometer including temperature probe (grey), dissolved oxygen probe (black) and variable speed motor (far right)...... 22

Figure 4. Top view of 30 L respirometer including temperature probe (grey), dissolved oxygen probe (black), and flow straighteners (orange and white)...... 23

Figure 5. Experimental flume trials measure oxygen consumption in a “fasted” state...... 26

Figure 6. Representative model of a specific dynamic action event overlapped with a baseline routine metabolic rate for yellowtail (S. lalandi dorsalis) at 20.6±0.1⁰C ...... 31

Figure 7. Relationship between BaselineRMR values (open bars) and SDAMAX (shaded bars) for all fish...... 33

Figure 8. Raw values for baseline routine metabolic rate before determining estimated baselineRMR...... 33

Figure 9. Specific dynamic action (solid line) for yellowtail, including the estimated baselineRMR (Matching dotted line) for each event...... 34

Figure 10. Comparison of the heat increment of feeding from previous works with the calculated values in the present study. Values from prior studies represent the mean value for the stated range ...... 39

Figure 11. Side view of the static respirometer (Volume: 1,237 L) ...... 55

Figure 12. External reservoir (Volume: 1,146 L) ...... 55

Figure 13. Sardine and formulated feed treatments. Black (solid) lines correspond with the postprandial response of the commercial feed treatment while the black (dotted) lines estimate the range for the baseline routine metabolic rate. Red (solid and dotted) lines indicate metabolic range for the sardine fillet treatment...... 67

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List Of Tables

Table 1. Taxonomic classification for California yellowtail (S. lalandi dorsalis) (Gill 1863) ...... 1

Table 2. Dimensions for yellowtail used during feed calculations and adjusting for solid blocking effect...... 20

Table 3. Proximate composition and energy content of sardine fillets...... 28

Table 4. Calculated specific dynamic action values for yellowtail fed an equivalent of 5% of their body weight in sardine fillets...... 32

Table 5. Comparison of baseline metabolic rate values for three of yellowtail (S. quinqueradiata, S. dumerili, and S. lalandi)...... 36

Table 6. Proximate composition and energy content of sardine fillet and commercial pellet. .. 60

Table 7. All measured variables pre and post specific dynamic action based on a dietary source of commercial feed and sardine flesh for yellowtail...... 66

Table 8. Analysis of meal energy load and fecal composition remaining after 24 and 48 h...... 69

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Chapter 1: Introduction

I. Yellowtail Biology and Aquaculture

A. Yellowtail Physical Traits and Taxonomy

Yellowtail kingfish is a warm water species belonging to the family , comprised of

four distinct subpopulations within the Seriola (Table 1) including S. quinqueradiata, S

dumerili, S. lalandi, and S. rivoliana (Heine and Kolkovski 2004; Tetreault and Peet 2008). The

body morphology of S. lalandi has been described as streamlined, elongated, and laterally

compressed (Nakada 2008). The Yellowtail’s body coloration is -brown with a golden

brown being prominent on top with a yellow stripe along the side and yellowish fins (Figure 1).

They possess two separate dorsal fins, the first with 4 to 7 splines, the second with one spline

and 31 to 39 soft rays. In addition, they also have two anal splines the first one with 2 splines

and the second with 19 to 23 soft rays. There are between 7 and 8 gill rakers on the upper limb

and 18 to 22 rakers on the lower limb totaling 26 to 30 rakers. Their body structure is made of

25 vertebrae including the hypural (Miller 1972). Larger yellowtail reach lengths of more than

1.5-2 meters and weigh as much as 60 kg (Nakada 2008; Gillanders et al. 2001).

Table 1. Taxonomic classification for California yellowtail (S. lalandi dorsalis) (Gill 1863) Common Name Great , yellowtail, yellowtail jack Kingdom Animalia Subkingdom Bilateria Infrakingdom Deuterostomia Phylum Chordata Subphylum Vertebrata Infraphylum Gnathostomata Superclass Class Subclass Neopterygii

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Infraclass Teleostei Superorder Acanthopterygii Order Suborder Family Carangidae Genus Seriola Species Seriola lalandi

B. Distribution and Movement Patterns in a Natural Setting

Yellowtail are found in warm temperate and subtropical coastal and oceanic regions of the

Atlantic, Pacific, and Western Indian Oceans (Eschmeyer et al. 1983; Smith-Vaniz 1995), primarily between 54°N and 43°S latitude (Ottolenghi et al 2004). Seriola populations found within the western Pacific waters off of Japan are considered genetically divergent to Seriola populations residing in the south Pacific waters off and (Nugroha et al

2001). Furthermore, evidence based on phylogenetic and morphological data indicate a regional separation of lineages into the Northwest Pacific, Northeast Pacific, and Southern

Hemisphere (Martinez-Takeshita 2015). Within the eastern Pacific, California yellowtail are known to have a distribution in the California Current and coastal waters with reported occurrences from the southern part of Washington to Mazatlán Mexico and as far north as Los

Angeles Bay within the Gulf of California (Baxter 1960; Collins 1973). This diverse species can thrive in an open ocean setting while also possessing the ability to exploit estuary habitats (May and Maxwell 1986). Regional abundance patterns have been shown to correlate well with increases in SST. Thus, as the ocean warms, the population shifts its distribution from Mexico waters to further north into California during warmer years (Radovich 1962). In the California fishery, large numbers are caught during their summertime after the northward migration from

Baja California. Similar studies in the Sea of Japan have documented decadal-scale variation of

2 catch rates seen in migration, distribution, and overwintering being primarily driven by sea surface temperatures (Tian et al. 2012).

California yellowtail is known to migrate seasonally into California waters (Dotson 2003), and originates from a central population off Baja California (Baxter 1960). Populations residing in California have thermal preference ranging from 18 to 24°C (Smith-Vaniz 1995) although they have also been observed in cooler regions (Starling 1988). Tagging data indicates smaller yellowtail (>600 mm TL) have a more compact range than larger yellowtail (>750 mm), most likely due to restricted temperature distribution (Gillanders et al. 2001). The majority of size classes of yellowtail remain within 50 km of the initial conventional tagging location while some venture as much as 3000 km from their site of origin (Gillanders et al. 2001). Larger fish have been shown to have greater variability in their distance traveled compared to smaller fish

(Gillanders et al. 2001). Juveniles less than 1-year-old primarily remain in coastal areas above

10 meters of sea surface depth with minimal horizontal movement. Older fish expand their habitat into slightly deeper waters between 10 to 30 meters (Kasai et al. 2000). Diurnal patterns thought to be associated with foraging (Baxter 1960) have been noted with preferences to coastal regions during the night and horizontal transiting between dawn and dusk within frontal zones (Kasai et al. 2000). It is thought the cooperative foraging tactics are exclusively used during daytime when this method of prey exploitation is possible, while nighttime feeding activities are limited due to the ineffectiveness of this strategy (Schmitt and

Strand 1982). Prey items consist of , , jack , pacific mackerel, , and invertebrates (Robins et al. 1991; Smith-Vaniz 1984; Bianchi et al. 1993; Smith-Vaniz 1995).

Schooling organisms found in the stomach of yellowtail indicates their hunting tactics are

3 driven towards their prey’s defense mechanism rather than from a particular species of interest

(Baxter 1960).

Spawning off California and Baja California, Mexico occurs from July to October, with peak activity taking place during July (Collins 1973; Smith and Paul 1960). Larval distributions are limited to warmer regions indicated by seasonal dispersals ranging from Point Conception in the north and extend to the southern tip of Baja California with maximum concentrations occurring in central Baja (Sumida 1985). Areas thought to be prime habitat for reproduction range from nearshore to as far as 200 miles offshore (Sumida 1985). 100% maturity has been documented for both males and females in the range of 925-1275 mm FL, while the first onset of maturity occurs between 750-775 mm FL (Poortenaar et al. 2001).

C. Yellowtail Aquaculture

The yellowtail fishery in California has suffered from drastic fluctuations over the last century, resulting in an end shortly after the 1950s (Collins 1973). Irrespective of historical declines, their popularity as both a sport and commercial fishing resource has continually increased (Collins 1973; Squire 1987). The rise in consumer demand for yellowtail as a fresh fish product has resulted in the rapid expansion of the aquaculture industry. Both from an economic and logistical standpoint, yellowtail continually stands out as a top choice for aquaculture operations. The recent global resurgence for this species has been attributed due to its rapid growth (Thompson et al 1999), high market value (Stuart and Drawbridge 2013), high flesh quality (Nakada 2008), and rising global demand for sushi (Nakada 2000). In addition, their adaptation and resilience to captive conditions has been noted in numerous studies with a

4 proven ability to thrive in both land based structures and ocean pens (Mazzola 2000; Fowler et al. 2003; Sims 2013). From an economic standpoint, yellowtail culture in sea pens presents a relatively low level of investment costs (Mazzola 2000), as well as a high resale value in the world marketplace (Nakada 2008, Mcleod 2010). Within captivity, these fish have exhibited excellent survival rates and rapid growth throughout their lifespan (Thompson 1999; Kolkovski and Sakakura 2004; Roo et al. 2014). Biological development in the larval stages of yellowtail show superior growth as compared with similar fin fish species (Chen 2006), and with an accelerated development, fish can begin feeding on a manufactured diet earlier in their life. In addition, their acceptance of numerous types of formulated feed has been shown from early life phases throughout the culture process and significantly reduces the costs accrued from wild caught fish (Kubitza and Lovshin 1999; Naylor 2000).

In Japan, three of the four species of yellowtail including S. lalandi, S. dumerili, and S. quinqueradiata are actively cultured from egg to adult (Nakada 2002). Japanese yellowtail aquaculture contributes the majority of the global production of yellowtail which currently is approximately 150,000 tons/year since 1979 (FAO 2016). Within the United States, S. dumerili has been raised for research purposes in California (Jirsa 2011) and S. rivoliana for consumption in Hawaii (Laidley 2004). Recent figures indicate total production of yellowtail in Hawaii in the range of 500 tons annually (Sims 2013). S. lalandii has also been cultured in Australia (Kolkovski and Sakakura 2004) with roughly 1000 tons produced annually (Econsearch 2014).

Yellowtail aquaculture in a number of regions have traditionally relied on the initial capture of juveniles from drifting seaweed (Anraku and Azeta 1965; Sakakura and Tsukamoto

1997). Current methods for Japanese yellowtail aquaculture are not considered sustainable,

5 with such a considerable reliance on wild broodstock. However, within Australia, New Zealand

(Kolkovski and Sakakura 2004; Fowler et al 2003), and the United States (Verner-Jeffreys et al.

2006), juvenile broodstocks (S. lalandi and S. rivoliana) have been obtained from hatchery reared animals. With the success of spawning and larval rearing in captivity (Stuart and

Drawbridge 2013), focus has begun to shift towards the nutritional inputs necessary for a marketable high quality fish. Historically, feed for yellowtail culture was primarily supported through the use of sand eel, , mackerel, sardine, and saury, however, the rising demand in farming could not compete with the available production from wild stocks (Nakada 2002).

Additionally, various nutritional disorders arose from unbalanced diets comprised solely of sardines, often resulting in insufficient protein and energy ratios for the yellowtail (Nakada

2000; Nakada 2002). Efforts eventually concentrated towards feed development, as the associated monetary costs were major contributors to the success of commercial facilities.

Formulated feeds not only reduced the impacts of marine aquaculture on existing fish stocks, but allowed managers to implement low-cost ingredients as well as supplement vitamins and minerals which were previously lacking in traditional feed practices.

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Figure 1. California yellowtail (adult) S. lalandi dorsalis ©Diane Rome Peebles

II. The Necessity For A New Source Of Fish

Population growth and urbanization has created a more efficient international

distribution network while bolstering the global consumption rates of finfish (Delgado 2003).

Over the last 5 decades, global food fish supply has increased at an annual rate of 3.2%,

amounting to an overall fishery production of nearly 80 million tons in the year 2012 (FAO

2014b). As a result, per capita apparent fish consumption has soared from an average of 9.9 kg

in the 1960s to 19.2 kg in 2012 (FAO 2014b). Projections for the growth of this industry are

encouraging from an economic standpoint, however the expectations of maintaining this type

of expansion are unrealistic. It is estimated that 28.8% of fish stocks are being captured at

biologically unstable levels (FAO 2014b) and therefore developing alternatives to wild fish

capture is essential in order to alleviate the pressures on wild stocks (Ye and Beddington 1996).

Aquaculture is an ever-expanding industry with the potential to relieve some of the impacts

due to overfishing (Naylor 2000).

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While the aquaculture industry has gained much attention as a significant component

towards food security, the need to further improve production methods is of the utmost

importance. Over the last decade, the aquaculture industry has increased production from 32.4

to 66.6 million tons (FOA 2014b), and is forecasted to reach 73.9 million tons by the 2014-year

end (FAO 2014a). Although marine farming may be the only alternative to fishing wild stocks, it

unfortunately does not come without a cost (Naylor and Burke 2005). Aquaculture operations

have historically relied on low value fish to create formulated feeds rich in based

proteins (FOA 2014b). The demand for protein in pelleted feeds, particularly for carnivorous

finfish, is highly dependent on low valued marine fishmeal and fish oil (Edwards 2004; Tacon

2008). In 2012, more than 16 million tons of the total fish production had been partitioned for

fishmeal and fish oil (FAO 2014b). Market value prices for low value fish will continue to

escalate with increased pressures on existing stocks (Delgado et al. 2003; FOA/GLOBEFISH

2007), and may lead to the eventual collapse of forage fisheries. Aquaculture management

decisions must not be based solely on maximizing profits, but should be guided by natural and

social ethics towards environmental conservation (Frankic and Hershner 2003). Continued

research must focus on alternative feed techniques that will mimic the natural dietary

requirements for these fish.

III. A Movement Towards The Ocean

While fish have been shown to have considerably higher dietary protein requirements

than terrestrial farmed vertebrates, their ability to convert this energy is far superior (Bowen

1987; Tacon and Cowey 1985). Their efficiency is evident in respect to their basal energy use as

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ectotherms and ammonotelic organisms. Ectothermic efficiencies, in comparison with those found in endotherms, is thought to be attributed to reduced energy use in homeostasis both from body composition and temperature regulation (Calow 1977). Ectotherms do not expend energy on body temperature regulation and are controlled by their surrounding thermal environment (Brett and Higgs 1970; Windell et al. 1978a). In ectothermic fish, their neutrally buoyant nature enables less energy expenditure for movement and structural support (Williams

1999). Additionally, energy absorption and conversion efficiencies are thought to be far greater in non-endotherms (Calow 1975; Calow 1977). A number of theories have been suggested to explain higher rates of protein conversion in ectotherms. One theory is centered on their ability to metabolize nitrogen into ammonia rather than consume energy to convert it to urea and uric acid (Maetz 1972; Steffens 1989). A second hypothesis examines their capacity to directly oxidize amino acids, which would otherwise require energy for the breakdown of complex storage molecules (Ballantyne 2001).

This unequivocal discrepancy in efficiency is apparent when comparing their associated

SDA’s or heat increment of feeding, with fish’s dissipation of digestive energy ranging from 8% to 12% while terrestrial animals nearly double this value (Farrell 1974). The ability of fish to convert energy at more efficient rates than terrestrial organisms is an important component for management of the global food supply. While the energy intake for fish per unit body weight is roughly the same as for terrestrial creatures, they convert feed into tissue in a much more economic fashion (Talbot 1993). The reduction in energy necessary for digestion eliminates a substantial load on costs and materials for grow outs in comparison to those seen in terrestrial operations.

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Fishmeal and fish oil are not only excellent sources of protein but also an ideal supply of

essential amino acids and fatty acids. Fish have high rates of protein utilization, resulting in a

rapid accumulation of healthy, nutrient-rich muscle containing long-chain omega-3

polyunsaturated fatty acids (Sargent 1997; Kaushik and Seiliez 2010). The quality of fuel

produced from this type of food production suppresses the likelihood of cardiovascular disease,

cancer, and a number of other deleterious disorders in humans (Simopoulos 2002). A

movement toward marine aquaculture would facilitate a higher yield output in terms of feed

conversion while supporting the development of high quality and nutrient-rich food sources.

IV. Respirometry: A Tool for Aquaculture

Technological advances in aquaculture, such as the ability to measure energy use and

conversions with flume respirometers, can aid in the process of culture optimization.

Respirometers allows researchers to calculate respiration rates, observe the geometry and

kinematics of fish in motion, as well as identify various sources of both internal and external

influence. For this reason, they have gained attention from fishery managers and are

considered an effective tool to maximize the biological output for a species (Grottum and

Sigholt 1998; Secor 2009). Traditionally, respirometry studies have been focused on fish with

commercial, aquaculture, and recreational value (Secor 2009) including quantifying stress in

broodstock (Barton 1987), exercise influence (Fu et al. 2009), temperature effects (Blank et al.

2007a, 2007b; Pang et al. 2011), feed efficiency (Kumar 2010), and energy expenditure (Clark et

al. 2010b; Whitlock et al. 2013, 2015). A number of other studies have explored digestion in

fish as a means to predict ideal growth conditions (Tang 1995; Alsop and Wood 1997; Tang

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2000). Respirometers have the ability to quantitatively model the physiological response of a

species during a feeding event. The information gathered from this type of analysis can be used

to streamline growing operations, providing a wealth of information such as the timing of

nutrient absorption, acceptance of unnatural feed sources, or even variability in responsive

behavior. Mapping the pathways of nutrient incorporation assists aquaculture managers by

emphasizing areas of concern as well as methods to improve existing operating procedures.

V. Primary Objectives for this Study

The following chapters explore the metabolic response of yellowtail to a number of feed

sources, and is the first truly detailed approach at examining yellowtails digestive response in a

controlled system. This work attempts to provide a quantitative description of the digestive

nature of yellowtail in two unique settings, one being a controlled swim unit and the other a

static system. A number of nutritional sources were used in order to quantitatively evaluate

their efficiency and overall acceptance. Initially, a pre-absorptive state for yellowtail was

measured as a means to assess baseline metabolic values. Fish were then exposed to two

separate respirometer types as well as two different dietary sources and the change in

respiration from a pre-absorptive to post-absorptive state was examined. The difference in

energy expenditure between the two metabolic states was used to describe the total energy

required for digestion as a function of presented meal type. The results from this study provide

a descriptive metric for the efficiency of various meal types.

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A. Postprandial Metabolic Response Of California Yellowtail (S. lalandi dorsalis) In A 30 L Swim Respirometer

1. Define Yellowtail’s metabolic response to a sardine flesh ration in a controlled swim respirometer.

2. Characterize the pre and post absorptive metabolic states in terms of the following metrics: a. Baseline Routine Metabolic Rate (BaselineRMR) b. Specific Dynamic Action Maximum (SDAMax) c. Factorial Scope (SDAMax/BaselineRMR) d. Duration of postprandial metabolic response e. Energy Representation of the SDA Event f. SDA Coefficient (SDA Energy/ Total Meal Energy)

B. Influence of Feed Source On The Postprandial Metabolic Response Of California Yellowtail (S. lalandi dorsalis) In A 1,237 L Static Swim Respirometer

1. Define Yellowtails metabolic response to two ratio based caloric equivalent meal types (sardine flesh versus a commercially available pelleted yellowtail feed) within a static respirometer system.

2. Characterize the pre and post absorptive metabolic states in terms of the following metrics: a. Baseline Routine Metabolic Rate (BaselineRMR) b. Specific Dynamic Action Maximum (SDAMax) c. Factorial Scope (SDAMax/ BaselineRMR) d. Duration of postprandial metabolic response e. Energy Representation of the SDA Event f. SDA Coefficient (SDA Energy/ Total Meal Energy)

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Chapter 2: Postprandial Metabolic Response Of California Yellowtail (S. lalandi dorsalis) Abstract

The term specific dynamic action (SDA) encompasses the energy expenditures during ingestion, digestion, absorption, and assimilation of a meal. Respirometry has been a valuable tool to measure changes in the metabolic rate following consumption of a meal and offers a quantitative approach to measure the digestive capacity for S. lalandi dorsalis. Five individual

yellowtail (700.8 ± 30.2 g) were placed in a Loligo 30L flume at a regulated velocity of 1 body

length per second (BL s-1) to determine their “fasted” baseline metabolic rate over a period of

48 h. After the fish were fed a meal of sardine at a mass equivalent to 5% of their body weight,

their postprandial metabolic response (SDA) was calculated based on the difference in oxygen consumption between their “fasted” and “digestive” states. The baseline values for five fish

-1 -1 were determined to be on average 221.8 ± 3.4 mg O2 kg h at 20.6 ± 0.1⁰C, and post-prandial

-1 1 metabolic rate increased to an average maximal rate of 430.3 ± 1.1 mg O2 kg h . The factorial scope, or ratio of peak oxygen consumption to baseline RMR was determined to be 1.9 ± 0.0.

The duration of SDA event from the moment of feeding until values fell within a range of the initial baseline rates lasted 36:31 ± 3:36 h. The caloric equivalent of the entire SDA event was

calculated to be 33.5 ± 1.9 kJ. The SDACoefficient, or the percent of energy expended on digestion

in relation to the total energy available in a meal, averaged 34.5 ± 2.0%. The results of this

work indicate a considerably large percentage of the total energy of a meal placed towards the digestion in yellowtail, a common theme for a number of other active pelagic fish of similar

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proportion. The baseline data for energy use in yellowtail is reflective of a fish with an

expansive home range and broad choice of dietary needs.

VI. Introduction

A. Metabolism in Aquatic Animals

Metabolic theory as described by Brown et al. (2004) links biological metabolism to the

intricacies occurring within both the spatial and temporal structures of ecological systems. The

mechanics driving metabolism cannot be viewed solely in a general manner as species specific

events, however a more accurate description involves a complex set of biochemical reactions of

mass and energy flow within and around an organism (Sousa 2008). Temperature (Dickson et

al. 2002; Blank et al. 2007a, 2007b; Whitlock et al. 2013; Sievert 2013) and salinity (Yagi et al.

1990; Blanco Garcia 2015) are known to have significant influence on the metabolic response of

various aquatic species, particularly ectotherms. While other studies have concentrated on

aerobic exercise (Clark and Seymour 2006, Hill et al. 2007) and swim performance impacts on

routine energetic costs (Beamish 1984; Kieffer et al. 1998). Cho and Bureau (1995) and

Whitlock et al. (2013), specifically examined the physiological costs of diet on the bioenergetics

of fish. Cho and Bureau (1995), describe the process in a broad sense as the total metabolic

energy as a consequence of both absorbed and dissipated energy. In a more detailed

breakdown of the process, the gross energy of a diet conforms to a strict regimen concluding to

various pathways of dissipation. Roughly 30% of the total intake will be discarded as feces,

urine, or gill excretion. The remaining 70% is supplied to the body as metabolizable energy. It

is important to note that diets present themselves with varying degrees of digestible matter,

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and at present, digestibility was estimated in the range of 70%. The remaining energy available

can be broken down into recovered energy (40%), and routine maintenance (20%), and the

heat increment of feeding (10%) (Figure 2). The heat increment of feeding, or specific dynamic

action (SDA), is the metric of interest for calculating the energetic cost of digestion. This value can shift rapidly and must be carefully evaluated based on the test subject as well as nutritional choice and temperature.

Figure 2. Partitioning of input and output energy throughout the digestive process (Values described in Cho and Bureau 1995).

B. Calorimetry as a Means to Calculate Energy Budget

Calorimetry examines the heat combustion of a fuel source resulting in the consumption

of oxygen as well as production of carbon dioxide, ammonia, and lactic acid by-products (Webb

1975). While easy to perform on a mammal, it would be nearly impossible to do on a fish living

in water. Respirometry is referred to as indirect calorimetry as its main purpose is related to

measuring oxygen utilization as oxycaloric equivalents rather than heat (Cho and Bureau 1995).

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Oxygen consumption has become a common standard of measurement for determining the

metabolic output during a feeding event or SDA (Secor 2009; Clark et al. 2010a; Whitlock et al.

2013).

Aerobic metabolism is a measurement of oxygen consumption by an organism, and

normally divided into various levels of metabolic rates, each of which is defined by the amount

of activity in relation to respiration rates. The first level of this metabolic activity is standard

metabolic rate (SMR), the process that describes the relationship during which a fish is

completely at rest (Webb 1975). Because in fish, remaining completely still can be difficult,

routine metabolic rate (RMR) has been established to describe the lowest mean oxygen

consumption rates for fasted, swimming fish (Blank et al. 2007a,2007b; Clark et al. 2010a;

Fitzgibbon et al. 2007). As a note, RMR does not take into account any additional movement

considered routine behavior. Accurately interpreting this value is somewhat difficult, as the

natural behavior of some fish, such as tuna, requires continuous movement and therefore

tends to present with these methodologies a higher SMR (Brill 1987). Fish can be immobilized by neuromuscular or spinal blocks to more precisely estimate this value (Clark and Seymour

2006; Dewar and Graham 1994), however, many studies use the lowest mass specific rate of oxygen for a given time period while the fish is moving at its slowest possible velocity (Brown et al. 2011; Clark et al. 2010a). In teleost fish, high SMRs have been correlated with high maximum aerobic rates (Brill 1987) and aerobic scope (Priede 1985).

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C. Specific Dynamic Action

Many studies have sought out to describe precisely the process of food intake and the body’s reaction to incorporation. The term “specific dynamic action” encompasses this process of energy expenditure during ingestion, digestion, absorption, and assimilation of a meal (Secor

2009). This term has also been referred to as the heat increment of feeding, calorigenic effect, and diet induced thermogenesis (Fitzgibbon et al. 2007; Cho and Bureau 1995). With SDA, the landscape for energy assimilation is drawn in a sense, providing a value that can be used in reference to previously determined inactive or routine rates. Continuous or intermittent values for oxygen consumption are recorded following ingestion of a meal until values fall to the SMR

(Secor 2009). A quantitative caloric measurement of energy consumption can therefore be

calculated from the resulting change in metabolic response. The derived product of this

formula will in essence estimate the cost of digestion after a meal (Mccue 2006). The SDA process has been synthesized into a three-part series of events including pre-absorptive

(energetic costs of meal heating, enzyme secretion, and protein catabolism), absorptive

(energetic costs related to intestinal absorption and nutrient transport), and post-absorptive

(protein synthesis and general costs of growth) (McCue (2006). The entire process is an extremely complex series of events involving both internal and external stimulus on the body.

SDA is primarily a result of post-absorptive biochemical and cellular processes, highly influenced from the source of protein (James 1992). It represents a measure of the energy freed or lost through the deamination of protein catabolism and anabolism (Pirozzi and Booth

2009; Elliott and Davidson 1975). Protein uptake can increase metabolic activity by as much as

40%, while carbohydrates generally will escalate this effect 5-10%, and fat consumption has

17

marginal effects (Belokopytin 2004). Therefore, various nutrient combinations will produce a

culmination of by-products with a signature mixture of elements being released and absorbed

(Webb 1975). In terms of metabolizable energy, fat and carbohydrate catabolism results in the formation of carbon dioxide and water while the processing of amino acids produces ammonia, carbon dioxide, and water (Cho and Bureau 1995). Dietary protein has been documented to

have a greater effect on postprandial metabolic response than lipids or carbohydrates, however

nutrient sources with reduced protein levels can exhibit increased metabolic responses in some

cases.

Fish raised on an optimal diet will exhibit maximal absorption of nutrients while

minimizing waste byproducts. Therefore, feed optimization implies matching specific fuel

proportions during formulation in order to maximize nutrient uptake (Fu et al. 2005a, 2005b;

Moran 2007). These fuels are either stored for somatic growth (Weatherley 1979) or directly

utilized for metabolic processes related to the breakdown of ATP (Mckenzie 2001).

Respirometry provides a useful tool to evaluate the energy use during a digestive state,

however does not fully define all aspects of energy integration. In this study, a more accurate

description of feed optimization would be minimizing the total respiration during a feeding

event and although respiration does not equate to growth, it does provide a metric for

comparison in which a descriptive statistic can be used to understand how much energy was

allocated towards digestion. The exact channels of incorporation would require a further

analysis, though the information from an investigation based on respiration can lead to an overall understanding of the efficiency of a diet.

18

This study was specifically focused at quantifying the digestive efficiency for yellowtail

exposed to a diet of sardine. The choice for feed source in this study is a common nutritional

constituent used in feed formulation, however as a standalone product, it has demonstrated

adverse effects on broodstock (Nakada 2000; Nakada 2002). The results of this work may

introduce a further component to the ineffectiveness of diets composed primarily of bait fish.

As yellowtail migrate over a wide range of microcosms, each of which exposing them to

numerous prey items, it should be assumed their nutritional requirements will be equally

diverse.

VII. Materials and Methods

A. Experimental Fish

Pacific yellowtail kingfish, S. lalandi dorsalis were captured off Catalina Island California

at a temperature of 20⁰C using barbless circle hooks during December 2014. Aerated wells

aboard the fishing vessel Shogun were used as a temporary holding platform during the three-

day sea excursion. The fish were then transported by truck within a 10,600 L tank to the Tuna

Research and Conservation Center (TRCC) in Pacific Grove, CA, USA. All fish were held in a

25,790 L circular seawater tank and fed a diet consisting of sardine, squid, enriched gelatin,

night , and etouffee shrimp three times per week as previously described (Farwell et al.

2001). Fish were maintained within the facility for 90 days prior to experimentation at an

acclimation temperature of 21.5°C. Fish were individually fitted with uniquely colored external

tags (Hallprint tags, Victor Harbor, SA, Australia) through the dorsal musculature for

19 identification during trial. Prior to implantation, each tag was soaked in betadine to reduce the risk of infection. Mean curved fork length of fish used in the swim respirometer was 38.4 ± 0.4 cm (range 37-39 cm), width 4.8 ± 0.1 cm (range 4.5-5.2 cm), depth 9.0 ± 0.2 cm (range 8.2-9.5 cm), and mass 700.8 ± 30.2 g (range 618-806 g) (Table 2). All fish were fed within the holding tank and were fasted 48 h prior to introduction to the swim tunnel. All procedures related to fish handling and maintenance were approved according to the guidelines and procedures set out by the Stanford University Institutional Animal Care and Use Committee (protocol 7297).

Table 2. Dimensions for yellowtail used during feed calculations and adjusting for solid blocking effect. Subject CFL (cm) Width (cm) Depth (cm) Mass (g) Fish 1 37.0 4.9 9.5 684.0 Fish 2 39.0 4.5 8.6 696.0 Fish 3 38.0 4.5 8.2 618.0 Fish 4 39.0 4.8 9.3 700.0 Fish 5 39.0 5.2 9.2 806.0 Mean 38.4 ± 0.4 4.8 ± 0.1 9.0 ± 0.2 700.8 ± 30.2 CFL, curved fork length; values given in bottom row expressed as means ± S.E. B. Swim Tunnel Characteristics

The respirometer used for this study was an unpressurized Loligo 30 L swim tunnel with chamber dimensions of 55 x 14 x 14 cm (Loligo Systems, Denmark) (Figure 3 & 4). The 30 L chamber was constructed within an overflow water reservoir, which acted as a thermal buffer from the outside environment. This unit had a removable lid on top for placement and extraction of fish into the chamber. Within the respirometer, swim speed was maintained at 1

BL s-1 and temperature was set at 20°C. Black tape was placed vertically along the tunnel of the flume every 2-4” for depth perception while within the chamber. Water flow was circulated by a 0.37 kW variable speed motor (1,700 rpm) in series with a 0.5 HP SEW Eurodrive digital motor

20

control center. The design of this unit integrates honeycomb water straighteners as well as

radial and vertical vanes to redistribute flow, reducing variable turbulence among test sections.

Water velocity was calibrated using a Hontzsch vane wheel flow sensor and corrected for solid

blocking effect according to individual fish (Bell and Turhune 1970). Experiments were

conducted at 20.6±0.1⁰C throughout the entire trial.

Water flow velocity within the swim tunnel was corrected for interference from the

solid blocking effect (Equations 1 & 2). This effect must be accounted for, as an increase in velocity and dynamic pressure surrounding the subject will occur (Bell and Turhune 1970).

= ( ) . (1) 𝑨𝑨𝒐𝒐 𝟏𝟏 𝟓𝟓 𝑬𝑬𝒔𝒔 𝝉𝝉ƛ 𝑨𝑨𝒕𝒕

• Es = Fractional error in test space velocity due to blockage • τ = Dimensionless factor depending on cross sectional shape (0.8) • ƛ = Shape factor for the test object • AO = Cross sectional area of the fish • AT = Cross sectional area of swim tunnel

= ( + ) (2)

𝑽𝑽𝒇𝒇 𝑽𝑽𝒕𝒕 𝟏𝟏 𝑬𝑬𝒔𝒔

• VF = Free stream velocity around the fish • VT = Average velocity in the swim tunnel

A 1,146 L external closed seawater reservoir was utilized as a sump for water

replacement within the system. Water within the external sump was fitted with aeration

stones and temperature was regulated by a 1,500 W WT-15A22 Gloquartz electric heater in

conjunction with an 828 W TLC4 Cyclone Pro drop in water coil chiller. Water was transferred

21 from the external sump directly into the swim flume during the flush cycle with an EHEIM 65 watt 40 L min-1 submersible water pump. Oxygen and temperature were monitored by a Fibox

3 fiber-optic oxygen meter (Precision Sensing GmbH, Regensburg, Germany) at a sampling rate of 1 sec intervals and resolution of 0.01 mg L-1 and 0.1°C. Measurements were recorded through the Oxyview program in conjunction with the Fibox 3 sensors. A conventional two- point calibration was performed on the oxygen minisensor (DP-PSt3) before experimentation

(rangehigh: air-saturated water (cal100); rangelow: 1 g of Na2SO3 to 100mL water (cal0)). The entire swim respirometer was surrounded by black plastic to block off adverse effects brought on by external visual stimuli. A camera was positioned directly above the unit and connected to a computer monitor with real time footage to assess the behavior of fish within the flume. The bottom of the swim chamber was coated in a reflective white plastic, providing an ideal optical contrast for identifying any unusual behavior.

Figure 3. Side view of 30 L respirometer including temperature probe (grey), dissolved oxygen probe (black) and variable speed motor (far right).

22

Figure 4. Top view of 30 L respirometer including temperature probe (grey), dissolved oxygen probe (black), and flow straighteners (orange and white).

The protocol involved a training or “trial” period during which the fish was introduced to

the flume (Figure 5). Prior work with has shown that training the fish reduces stress that

can artificially raise metabolic rates (Blank et al. 2007a, 2007b). During the experimental

period, the yellowtail were transferred to a 5867 L circular fiberglass tank within the tuna

research and conservation center. Fish were held within a smaller tank during their acclimation

period in order to reduce stress during capture and improve the capacity to individually feed

fish. The water level within this system was reduced to approximately 0.5 m in depth and a

vinyl-dividing curtain was used to corral the fish within the tank. Fish were collected with a soft

mesh net and placed into a sling for morphometric measurements including length, width, and

depth. Exact body measurements were used for adjusting the swim flume blocking effect. Fish

were then placed into a 7.5 L plastic bag, which helped direct the fish head-first into the swim

chamber of the flume as well as minimize the risk of escape during transfer. Water velocity

within the chamber was set to 1 BL s-1. A multipoint water velocity calibration was performed

across the chamber space with a Hontzsch HFA vane wheel flow sensor. Immediately after

23 placement into the flume, the lid was covered over the entry portion and sealed with wing nuts.

The initial 30 to 60 min within the flume was considered the acclimation period during which fish were constantly monitored for tail beat frequency, unbalanced motion, and positioning in the flume. During this period, the water cycle was set to continuously flush fresh water into the system, ensuring exposure to elevated dissolved oxygen levels. All monitoring of fish behavior was done in close proximity to the swim flume via camera and television. Any fish with irregular movement or considered at risk of injury was removed and returned to the holding tank. Upon completion of a period of 30 min of normal motion within the flume, the flush cycle was activated to a 5 min open cycle, followed by a 5 min closed cycle. During the closed cycle, oxygen saturation was measured relative to fish mass, while during the open cycle, fresh oxygenated water was pumped into the system from an external tank to replenish dissolved oxygen levels within the tunnel chamber. A cycle period of 5 min open followed by 5 min closed was selected to ensure dissolved oxygen levels remained above 80% for the entire closed cycle.

C. Swim Tunnel Calculations

Swim flume operation was set at alternating open and closed cycles. Open cycles lasted 5 min in duration and flushed the system with fresh water, followed by 5 min of closed cycles during which measurements were taken to monitor the drop in oxygen concentration within the flume. The following steps were used to calculate the change in oxygen concentration

-1 within the flume during a closed cycle (Equation 3). Units were presented in mg O2 s , therefore the equation factors in a time conversion.

24

× × ( ) = (3) 𝒎𝒎𝒎𝒎𝑶𝑶𝟐𝟐 𝑹𝑹𝑶𝑶 𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑 𝑽𝑽𝑹𝑹 − 𝑾𝑾𝑭𝑭 𝑶𝑶𝑶𝑶𝑶𝑶𝑶𝑶𝑶𝑶𝑶𝑶 𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪 � � 𝒌𝒌𝒌𝒌𝒌𝒌 𝑾𝑾𝑭𝑭

• RO ( ) = Resulting slope for the regression of the change in oxygen over a 5-min closed 𝐦𝐦𝐦𝐦 cycle.𝑶𝑶𝟐𝟐 • VR (L) = Volume of the swim respirometer (30 L). • WF (kg) = Weight of individual fish.

D. Experimental Design

Each fish underwent a two-step respirometry protocol (Step 1: Measurement of routine metabolic rate (RMR) in a fasted state; Step 2: Calculation of postprandial response to feeding event) for determining the oxygen consumption requirement during a feeding event. Unfed fish for this experiment are referred to as “fasted” while fed fish were categorized as

“digesting”. Food was withheld for 48 h prior to experimentation in both fasted and digesting fish. Upon entry into the respirometer, Mo2 was usually elevated due to the stress of handling during transfer (Blank et al. 2007a). The initial step of this study was focused on calculating the

RMR for a fasted fish. Routine oxygen consumption as previously described encompasses measuring a state of routine behavior involving spontaneous movement (Beamish 1964; Pirozzi and Booth 2009). After placing a fasted fish in the respirometer for a period of 48 hours, the lowest three-hour mean oxygen consumption (Mo2) was therefore selected as the ideal range for RMR (Clark et al. 2010a). The RMR from fasted fish would therefore set the baseline oxygen consumption as a comparison against the rates for digesting fish.

25

Figure 5. Experimental flume trials measure oxygen consumption in a “fasted” state.

The protocol for digesting fish was as follows: sardines were removed from the freezer

(-20C), and allowed 24 h to defrost prior to feeding to room temperature. Sardines were filleted to remove all bones, belly, and innards from the available feed samples. Total mass for each sample fillet was recorded and individually labeled at room temperature prior to feeding.

Fish (2-3) were partitioned with curtains from the other fish in the tank and allowed 1 h to acclimate to isolation. Individual fish mass was documented in order to calculate rations for the feeding protocol. Two researchers presented sardine fillets one by one and recorded total consumption for each fish until it ingested 5% of its body weight in sardines. Fish that consumed 5% BW were visually identified by tag color and remained untouched in the confinement area for approximately 30 min to prevent regurgitation of food. There was no sign of regurgitation for any of the selected fish for respirometry. Fish were then transferred into the swim flume and remained unaltered for a 48 h period at a rate of 1 BL s-1. Total time from

26 the moment of ingestion of food to placement into the flume and initiation of the pump cycle took approximately 0.5 to 1 h depending on fish behavior.

E. Nutritional Analysis of a Sardine Flesh Meal

A total of five sardine fillets were sent to Nestle Purina Analytical Laboratories (St. Louis,

MO, USA) for proximate composition analysis (Table 3). Bomb calorimetry was used to determine the protein, lipid, moisture, and caloric content of the sardine fillets used during the digesting portion of the swim protocol. The following methods describe the procedure taken for proximate analysis. For determining protein content, nitrogen compounds within the sample were boiled using sulfuric acid in conjunction with a catalyst (potassium sulfate, titanium dioxide, and cupric sulfate) to produce ammonium sulfate. The resulting ammonia sulfate can be made alkaline with a sodium hydroxide solution, liberating ammonia into a known portion of standard acid. The solution was then titrated and nitrogen was determined from the known amount of standard acid solution used in the process. The crude protein was then calculated from a predetermined value of 6.25 for total nitrogen using protein factors

(OMA AOAC International 988.05, 920.87, 991.20). Fat was determined by a method known as acid hydrolysis. The portion of fillet was hydrolyzed with hydrochloric acid to liberate heat- bound fats and oils (OMA AOAC International 954.02, 922.06, 933.05, 925.32, 950.54, 948.15;

LI-75.204). Using ethyl and petroleum ether, the fat was extracted and volatilized. The resulting fat was dried and weighed to determine the percentage within the sample. For moisture content of the sample, a forced draft oven method was selected at a temperature of

133°C, and a measurement of moisture lost could be calculated from the change in mass (NP

27

Analytical Laboratories 2012). Total calorie value for the sample was calculated by freeze- drying to remove all moisture, then burning the remaining material in a calorimeter. The sample was ignited in an oxygen filled metal bomb and placed in a bath of water. Heat generated was determined from the burning of elements in the sample and therefore calculated from an increase in temperature of the bath water (No. 463M).

Table 3. Proximate composition and energy content of sardine fillets. Date Protein (%) Fat (%) Energy (kcal g-1) Sardine Fillet 1 21.5 10.7 2.21 Sardine Fillet 2 19.7 8.5 2.31 Sardine Fillet 3 22.3 6.3 1.87 Sardine Fillet 4 22.1 4.7 1.67 Sardine Fillet 5 20.6 11 2.12 Average 21.2 ± 0.5 8.3 ± 1.2 2.03 ± 0.12 Protein, Kjeldahl (N x 6.25). Fat, acid hydrolysis. Energy, bomb calorimetry. Values listed on bottom row given in means ± S.E. 1kcal = 4.1868kJ (Brett and Groves 1979).

F. Calculation of Meal and Respiration Energy

The nutritional value of each meal used during the digesting protocol was derived from the results of the bomb calorimetry analysis in conjunction with the total mass ingested.

Equation 4 describes the steps taken to calculate meal energy content. The difference in oxygen consumption between the baseline RMR and SDA response could be interpreted as energy lost due to digestion. The increase in respiration was translated from a unit of oxygen consumption into an energy equivalent value. Oxygen consumption could be converted to an

-1 oxy-caloric value equal to 14.32 J mg O2 (Beamish and Trippel 1990). Equation 5 describes the previously mentioned series of steps to convert oxygen consumption into kJ of energy. By comparing the meal energy with the calculated respiration energy estimate, the resulting

28

SDACoefficient of the meal can be determined. The calculations involved in defining this term are

seen in Equation 6.

( ) = × × (4)

𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪 𝒌𝒌𝒌𝒌 𝑴𝑴𝑴𝑴 𝑪𝑪𝑽𝑽 𝑴𝑴

• MM (g) = Meal Mass • CV (kJ/g)= Caloric Value of Sample Meal • M (%) = Fillet without Moisture or (100- %Moisture)/100

( ) = (( × × ) × × (5) ) 𝟏𝟏𝟏𝟏𝟏𝟏 𝟏𝟏𝟏𝟏𝟏𝟏 • 𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 𝒌𝒌𝒌𝒌 𝚺𝚺 𝑾𝑾𝑭𝑭 ∆𝑶𝑶 𝜷𝜷 𝟔𝟔 𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄 𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏 • WF (kg) = Fish Weight -1 -1 • ΔO (mg O2 kg h ) = Increase in Oxygen Consumption above BaselineRMR -1 • β (J mg O2) = 14.32 J of Energy per 1 mg of Oxygen Consumed

(%) = × (6) 𝑹𝑹𝑬𝑬 𝑺𝑺𝑺𝑺𝑺𝑺𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪 𝟏𝟏𝟏𝟏𝟏𝟏 𝑴𝑴𝑬𝑬 • RE (kJ) = Respiration Energy • ME (kJ) = Meal Energy

VIII. Results

A. SDA Metrics

SDA in yellowtail at 20⁰C was estimated using respirometry. The postprandial increment in

respiration was described (Figure 6) in terms of baseline RMR, SDAMax, SDADuration, factorial

scope, SDA, and SDACoefficient. The limits set by this protocol therefore would encompass the

change in oxygen levels from a routine metabolic state to those reflective of a digestive period.

RMR as described previously was determined from preliminary fasted trials estimating the

29 point at which the postprandial increment returned to their original fasted state of oxygen consumption. Peak oxygen consumption, or SDAMax was the point at which oxygen levels reached a maximal value post-feeding event. The length of the SDA is noted as SDADuration and calculated as the time from the feeding event through maximum consumption and ended when the decline in oxygen reached the range of RMR values. To account for the initial period of high stress and acclimation to the flume, a linear increase in oxygen consumption from the moment of feeding through the beginning of the SDA event was used as an estimate of this time period.

As the fish were fed in a separate holding tank than the respirometer, this addition was used to account for elevated oxygen consumption during the transfer process and its value was added to the SDA. Factorial scope was calculated as the SDAMax divided by RMR. SDA was calculated as the change in oxygen over the entire event with respiration rates being converted to an energy equivalent assuming 14.32 J of energy per 1 mg of oxygen consumed (Beamish and

Trippel 1990). SDACoefficient was calculated by dividing the SDA by the meal energy, and it represents the percent of energy expended on digestion in relation to the total energy available in a meal (Secor 2009).

30

Figure 6. Representative model of a specific dynamic action event overlapped with a baseline routine metabolic rate for yellowtail (S. lalandi dorsalis) at 20.6±0.1⁰C .

B. Measured Respirometry Values

In total, 53 days of fasting and digesting trials were conducted with the aim of modeling the specific dynamic action associated with S. lalandi dorsalis within a 30 L swim respirometer

(Table 4). Fish used for this portion of the study ranged in size from 618 g to 806 g, averaging

700.8 ± 30.2 g. Body dimensions were measured in terms of curved fork length, girth, and depth and averaged 38.4 ± 0.4 cm, 4.8 ± 0.1 cm, 9.0 ± 0.2 cm respectively. All fish fed readily when presented sardine fillets and consumed an equivalent of 5% of their total body weight

(avg. 4.9 ± 0.0 g). On average, total energy content of the 5% sardine body weight equivalent was calculated at 97.4 ± 4.3 kJ, ranging from 86.9 to 112.9 kJ. At a rate of 1 BL s-1, the routine

-1 -1 metabolic rate value of yellowtail in five fish was determined to be 221.8 ± 3.4 mgO2 kg h

(Figure 8). The maximum value of SDA was relatively consistent across all fish and averaged

-1 -1 430.3 ± 1.1 mgO2 kg h . The factorial scope represented as the ratio of peak oxygen 31

consumption to baselineRMR was determined to be 1.9 ± 0.0 (Figure 7). The duration of the SDA

event was estimated at 36:31 ± 3:36 h with 33:35 h being the earliest the SDA values returned

to baseline while the longest period resulted in a duration of 38:45 h. The entire SDA event was

calculated to represent an energy use of 33.5 ± 1.9 kJ (Figure 9). The SDACOEFFICIENT ranged from

32.0% to 42.3%, averaging 34.5 ± 2.0% for all five fish.

Table 4. Calculated specific dynamic action values for yellowtail fed an equivalent of 5% of their body weight in sardine fillets. Parameter Unit Fish 1 Fish 2 Fish 3 Fish 4 Fish 5 Mean ± S.E. Fish Mass g 684.0 696.0 618.0 700.0 806.0 700.8 ± 30.2 Meal Mass (Wet) g 33.5 34.8 30.7 33.3 39.9 34.4 ± 1.5 Percent Meal Size % 4.9 5.0 5.0 4.8 5.0 4.9 ± 0.0 Energy Content kJ 94.7 98.4 86.9 94.2 112.9 97.4 ± 4.3 -1 -1 BaselineRMR mgO2 kg h 216.9 216.0 217.1 233.4 225.7 221.8 ± 3.4 -1 -1 SDAMax mgO2 kg h 429.4 434.3 428.0 429.8 429.7 430.3 ± 1.1

SDADURATION h 38:45 33:35 38:45 34:35 36:55 36:31 ± 1:03 -1 Factorial Scope SDAMax BaselineRMR 2.0 2.0 2.0 1.8 1.9 1.9 ± 0.0 SDA kJ 40.1 31.5 28.9 32.4 34.6 33.5 ± 1.9

SDACOEFFICIENT % 42.3 32.0 33.2 34.4 30.7 34.5 ± 2.0 SDARMR, routine metabolic rate; SDAMax, specific dynamic action maximum. Values given in final column are listed as means ± S.E.

32

450 429 434 428 430 430

400

) 350 1 - h 1 -

kg 300 2

250 233 226 217 216 217 200

150

Oxygen consumption (mgOOxygen consumption 100

50

0 Fish 1 Fish 2 Fish 3 Fish 4 Fish 5

Figure 7. Relationship between BaselineRMR values (open bars) and SDAMAX (shaded bars) for all fish.

Figure 8. Raw values for baseline routine metabolic rate before determining estimated baselineRMR.

33

Figure 9. Specific dynamic action (solid line) for yellowtail, including the estimated baselineRMR (Matching dotted line) for each event.

IX. Discussion

A. Baseline Metabolic Rate

Yellowtail are a highly active and mobile pelagic species, with a uniquely designed

musculature and morphology that results in high efficiency for swimming. Indeed, these fish

are well known for their athleticism amongst sport fishers. Their elevated routine metabolic

rate is a reflection of their lifestyle as a high-energy demand species commonly inhabiting a

wide array of oceanic conditions and feed sources (Baxter 1960; Pirozzi and Booth 2009; Clark

and Seymour 2006). A number of studies have explored the limits of both RMR and SMR in

yellowtail in static and swimming respirometers with a great degree of variability among

34

findings (Table 5). Yamamoto et al. (1981) has estimated the SMR for S. quinqueradiata to be

-1 -1 roughly 147 mgO2 kg h , which is relatively similar to results for S. lalandi by Yanase et al.

-1 -1 -1 -1 -1 -1 (2012) of 159 mgO2 kg h , 172 mgO2 kg h by Brown et al. (2011), 137 mgO2 kg h by De la

-1 -1 Gandara (2002), and 93-198 mgO2 kg h by Clark and Seymour (2006). Furukawa et al. (1992)

discussed an initial low routine metabolic rate in pre-feeding yellowtail in the range of 141-185

-1 -1 mgO2 kg h while post feeding routine metabolic rate was 1.1 to 2.4 times higher than the initial rate. Yamamoto et al (1981), Furukawa et al. (1992), and a portion of the study methods by Brown et al. (2011) described the experimental system as a static environment whereas

Yanase et al. (1981), Clark and Seymour (2006), and the latter half of Brown et al. (2011) utilized a swim flume.

In this study a flume respirometer maintained fish swimming at a rate of 1 BL s-. 1,

therefore these measurements must be placed into a separate category then static respirometry conducted previously. Measurements of baselineRMR from this study were in the

-1 -1 range of 216 to 233 mgO2 kg h , which is significantly higher than static respirometry

measurements cited above. The routine metabolic rates at 1 BL s-1 are a scenario that may be

increasing the activity of these fish in comparison to their tank, pen or natural behavior as the

fish are often seen to remain calm and not swimming within captive environments. Thus our

measurements may be an overestimate of RMR in this species.

35

Table 5. Comparison of baseline metabolic rate values for three species of yellowtail (S. quinqueradiata, S. dumerili, and S. lalandi). RMR/SMR Source Species Flume Temp (⁰C) (mgO2 kg-1 h-1) Yamamoto et al. (1981) S. quinqueradiata 2.44mm sec-1 147 19 Furukawa et al. (1992) S. quinqueradiata Static 141-185 23-24 De la Gandara et al. (2002) S. dumerili Static 137 19 Clark and Seymour (2006) S. lalandi 0 & 2.3 Bl sec-1 93 & 198 20 & 25 Brown et al. (2011) S. lalandi 0 & 0.75 Bl sec-1 172 & 159 22 Yanase et al. (2012) S. lalandi 1 Bl sec-1 129 18-19 Current Study S. lalandi 1 Bl sec-1 221 20

Thermal influence on SMR for S. lalandi indicates significant fluctuations between 93 mgO2

-1 -1 -1 -1 kg h at 20°C to 198 mgO2 kg h at 25°C (Clark and Seymour 2006). Later studies on routine

-0.8 -1 metabolic rate for juvenile S. lalandi at 10°C was 85 mgO2 kg h while higher temperatures

-0.8 -1 around 32.5°C were 382 mgO2 kg h (Pirozzi and Booth 2009). While thermosensitivity was

not explored as a major predictive variable in this study, it is interesting to consider as a means

to understand rates at which the body performs at an optimal level, with numerous works

documenting the postprandial metabolic rate as a function of temperature (Whiteley et al.

2001, Blank et al. 2007, Luo 2008, Vanella et al. 2010). Throughout the world, yellowtail are

known to experience a wide range of climates, with both spatial and temporal preference

within each region (Jung 2014; Tian et al. 2012). In addition, most yellowtail aquaculture

facilities operate over multiple seasons and can experience a range of temperatures from 12°C

to 27°C (Kofuji et al. 2005). Within each thermal niche, digestive capacity is influenced by the

body’s reaction and acceptance of a dietary source through enzyme expression and digestive

evacuation. Thus knowledge of the species metabolic optimum temperature are important for

improving metabolic efficiency while in captivity. Cooler temperatures during winter seasons

36

seem to restrict growth for fish residing in sea cage cultures in concert with the studies above

that show cooler temperatures result in lower metabolism (Kolkovski and Sakakura 2004).

Temperature not only influences the metabolic rate but the enzymatic capacity vital for

digestive processes. Yellowtail have been shown to have higher enzyme activity levels,

specifically protease and lipase, as well as slower gut transit times in the range of 36-48 h in

cooler winter temperatures (12.6°C) in comparison to 12 to 16 h transit time in warmer

summer water (20.8°C) (Miegel et al. 2010). Yellowtail exhibit a parabolic response and has the

lowest Q10 occurring between the temperature ranges of 20°C and 25°C, with the asymptote

centered on 22.8°C (Pirozzi and Booth 2009). Based on previous studies, the temperatures used in this study are within the preferred range for testing dietary sources and their influence on digestion. As development of yellowtail facilities continue to expand into the global ocean, temperatures and dietary source selection must be reflective of effects brought on from seasonal variations. Efforts concentrated towards better understanding the narrow window for maximal production will be difficult, as facilities will need to evolve with the diverse nature of the ocean environment.

B. Overall Postprandial Response

In this study the postprandial response in yellowtail was measured at 20⁰C in a swim respirometer. The factorial scope for yellowtail in this study averaged 1.9, ranging from a

-1 -1 -1 -1 baselineRMR rate of 221.8 ± 3.4 mgO2 kg h to a maximal rate of 430.3 ± 1.1 mgO2 kg h .

Previous yellowtail studies conducted in static respirometers indicate the factorial scope in the

-1 -1 range of 1.3 with maximal rates of 250 mgO2 kg h (19⁰C) for fish fed to satiation (De la

37

Gandara 2002). Yellowtail fed at a rate of three times per day resulted in a mean oxygen

-1 -1 consumption of 215 mgO2 kg h while reducing the feed period to twice daily caused the O2

-1 -1 consumption to drop to 146 mgO2 kg h (De la Gándara 2002). The aforementioned work was followed up with an investigation on the effect of continuous food supply versus twice daily feeding on yellowtail oxygen consumption, resulting in a similar average oxygen consumption

-1 -1 rates for both treatments ranging from 193-195 mgO2 kg h (De la Gandara 2002). Furukawa

et al. (1992) looked at metabolism in yellowtail fed commercial eel feed mash to satiety, and

concluded oxygen consumption rate increases between 2.2 and 4.1 times baseline values, with

-1 -1 maximal rates reaching 443-638 mgO2 kg h . The consistency seen across all fish in this study in terms of baselineRMR, SDAMax, and SDAduration indicate a somewhat predictive relationship between the energetic value of a set weight of sardine feed and subsequent metabolic curve.

The findings from this study show the baseline values are slightly higher than previous works, although the SDAMax was determined to be nearly identical to other studies. Differences in metabolic rate are likely a result of study design, age of fish, holding temperatures, and the

source of feed components.

C. SDA and SDACoefficient

A number of yellowtail studies have focused on a representative diagram of the flow of

nutrients from the initial intake of energy until somatic incorporation. Accurately measuring

energy expenditure has been somewhat of a difficult task considering the numerous routes

involved in the process of nutrient assimilation. In addition, there are countless factors

contributing to poor dietary acceptance and nutrient loss. Masumoto et al. (1997) conducted a

study on yellowtail under a controlled diet of 52% crude protein and 16% crude lipid, and

38

concluded that energy budgets were partitioned as 33% for growth, 17% for maintenance and

activity, and 36% for the heat increment of feeding. Watanabe et al. (2000a) estimated energy

budgets for yellowtail fed 45-49% crude protein and 16-20% crude lipid diets, concluding 39-

44% of the gross intake was used for productive energy while 7-19% was partitioned for

maintenance energy, and 17-34% as the heat increment of feeding. In a similar study on

yellowtail, energy was distributed as 23-36% productive energy, 8-25% maintenance, and 31-

40% heat increment of feeding (Watanabe et al. 2000b).

45 40 41.5 40.5 35 36 34.5 30 33 29.5 25 25.5 20 15 17 16.5 10 13 5 Percent of EnergyBudget (%) 0 Growth Maintenance Heat Increment

Masumoto et al. (1997) Watanabe et al. (2000a) Watanabe et al. (2000b) Current Study (Swim Flume) Sardine

Figure 10. Comparison of the heat increment of feeding from previous works with the calculated values in the present study. Values from prior studies represent the mean value for the stated range

The findings from Masumoto et al. (1997) and Watanabe et al. (2000a; 2000b) report

SDACoefficient at 36%, 17-34%, and 31-40%, respectively. It is important to note the techniques used for calculating heat increment of feeding used in the previous studies were based on estimates of energy transfer between energy intake and daily energy retention, with calculations centered on fluctuations in whole body proximate composition. The SDA results

39

from the present experiment however are based on an energy value calculated from respiration

versus the total amount of energy from an ingested meal. Interestingly, the SDACoefficient

calculated in the current study fell in the range of 30 to 42%, averaging 34.5%, relatively

consistent with the results from the other studies (Figure 10). The magnitude of this value

validates the conclusive remarks of previous works, as such, a considerably large portion of the

total amount of ingested energy is allocated towards digestion. It would be worthwhile to

explore additional dietary sources as a means to illustrate fuel consumption and incorporation,

building on the existing knowledge of yellowtail biology.

X. Conclusions

This work provides the first measurements of SDA in a flume respirometer for California

yellowtail, S. lalandi, at 20⁰C. The RMR, SDA, and SDA coefficient were similar to other works,

however the experimental approach varied greatly across studies. This work set out to provide

a detailed description of a pre and post digestive state based off of respiration rates. RMR

-1 -1 under conditions used in this study were 221.8 ± 3.4 mgO2 kg h , while post feeding oxygen

-1 -1 consumption rates doubled in value to 430.3 ± 1.1 mgO2 kg h . Digestion determined from

respirometry had a duration of 36:31 ± 1:03 hours. The total cost of this digestion was

considerably large, amounting to approximately one third of the value of their consumed meal.

The proportion of this diet partitioned for digestion may indicate a degree of inefficiency of this

fuel source as a sole dietary item. The calculated values for SDA do fall in a range comparable

to previous yellowtail studies though, validating this technique as a means to quantify energy

partitioning during feeding events.

40

The results of this study have provided a model for the range of metabolic performance for

S. lalandi dorsalis in a controlled swim respirometer. Based on the relatively high SDA coefficients for yellowtail there likely exists significant scope to reduce this energetic cost and improve diet utilization for growth. This will be an important consideration as the yellowtail aquaculture industry continues to develops. It is recommended further studies should be conducted specifically aimed at various size classes, temperature ranges, diets, and swim speeds.

41

Chapter 3: Influence of Feed Source On The Postprandial Metabolic Response Of California Yellowtail (S. lalandi dorsalis) In A 1,237 L Static Swim Respirometer

I. Abstract

Feed nutrition is critical for captive populations of yellowtail that can attain rapid

growth in short periods of captivity. Nutrient incorporation into feeds has gained much

attention as managers are faced with increasing expenditures due to the rising demand for fish

meal, and an understanding towards ideal energy sources will lead to better management

choices. One way to test feed efficiency is with the use of respirometry, which offers a

technique capable of defining the mechanisms and pathways controlling nutritional assimilation

during ingestion of a meal. This type of analysis produces an estimate of both the baseline

metabolic rate and post-absorptive state resulting from various diets. Specific dynamic action

refers to the metabolic processes of digestion, and can be measured. This provides an

energetic measurement for the overall cost of digestion for a meal. Energy transformation has

increasingly become a major theme in aquaculture as feed sourcing accounts for a substantially

large percent of all operating expenses and by further understanding the total amount of

energy required for digestion, managers are able to calculate the efficiency of alternative diets

as well as the future direction for feed formulation.

This research set out to compare the digestive efficiency for two feed treatments provided

to captive populations of wild caught yellowtail; sardine flesh versus a commercially available

pelleted yellowtail feed (Ewos Dyna Yellowtail 400NN). Both meals were fed at isoenergetic

equivalent values, however the mass, physical structure, and nutritional composition were

42

substantially different. As a result of the noted inconsistencies, the postprandial response was

found to vary between the two treatments. The mass of the commercial diet was calculated at

nearly one-third of the size of the sardine fillets

To utilize SDA as an energetic tool, a fasted and fed metabolic rate must be obtained. The

baseline routine metabolic rate (BaselineRMR) for the commercial fed yellowtail was found to be

-1 -1 120.2 ± 3.7 mgO2 kg h , significantly lower than that associated with the sardine fillet meal of

-1 -1 150.8 ± 3.9 mgO2 kg h , at 20.6 ± 0.1⁰C. The SDAMax for the commercial feed (203.7 ± 22.5

-1 -1 -1 -1 mgO2 kg h ) was statistically lower than the sardine fed fish (279 ± 27.3 mgO2 kg h ).

Factorial scope for the two treatments was found to be significantly different with 1.7 ± 0.01 for

the commercial feed in comparison with the sardine flesh of 1.8 ± 0.01. Although there was no

effect noted in the total duration of the SDA event, the total energy represented in the

postprandial response for commercial feed (18.2 ± 1.4 kJ) was more than half the value

calculated for the sardine fillets (42.9 ± 0.7 kJ). Therefore, the percent of energy expended on

digestion in relation to the total energy available in a meal for the commercial feed (12.3 ±

1.0%) was significantly lower than that of the sardine feed (24.9 ± 0.2%). Fish were observed

and categorized according to level of motion during the digestive period. The results of the

movement analysis indicated there was 12.3% greater activity during the sardine fillet digestive state compared to that of the commercial feed digestive state.

After 24 hours, the total amount of protein and lipid remaining in the feces from the commercial feed (10.8 ± 0.9 kJ, 5.3 ± 1.3 kJ respectively) was found to be significantly lower than the remaining protein and lipid in the sardine feed treatment (25.4 ± 1.0 kJ, 13.4 ± 3.1 kJ respectively), however no effect was noted after 48 h. Both diets were almost completely 43

processed by the fish, however there were differences noted in the digestibility with the

commercial feed being slightly higher than the sardine feed at 97.7 ± 0.2% as compared with

95.3 ± 0.5%. The reduction in energy required for digestion of the commercial feed makes it an

attractive alternative to a raw fish diet.

II. Introduction

A. Advances in Aquaculture Practices: Natural Feed items and Formulated Pellets

It is widely accepted that captive yellowtail have a feeding preference for raw fish over a

formulated diet and therefore bait fish was traditionally used as a primary constituent for

aquaculture production systems (Watanabe 1991). The use of bait fish as a sole dietary source

had been shown to cause nutritional deficiencies and diseases and therefore the development

of alternative diets became a necessity (Nakada 2000; Nakada 2002). Initial yellowtail feed

formulations were expensive, time consuming, and often resulted in poor palatability

(Watanabe 1991; Hardy 1995; Watanabe 1998). In addition, the demand for fishmeal and fish

oil was expected to increase with global population growth, and projections indicated the

current holding stocks of bait fish would no longer be able to meet the demand for production

(Hardy and Tacon 2002). With fishmeal being a finite feed source, growing concern escalated

over the sustainability of this resource.

Advances in the production of yellowtail fish feeds have resulted in a soft dry pellet,

superior in nutritional content and palatability to both moist pellets and raw fish (Watanabe

1992). Further developments in feed formulation began to shift the focus from animal to plant-

44

based ingredients as a means to reduce costs. A number of studies have explored the use of

different plantstuffs as protein and lipid sources. These alternative ingredients include barley

(Molina-Poveda 2004), canola (Drew et al. 2007), corn (Robaina et al. 1997), cottonseed (Lim

and Lee 2009), pea seed (Gouveia and Davies 2000), wheat (Torstensen et al. 2008), soy

(Samocha et al. 2004), and algae (Kissinger et al. 2016). Formulated pellets are able to supplement essential vitamins and minerals by providing a combination of nutrient sources required for optimal development as well as accelerating growth in comparison with raw fish

(Watanabe 1991). The integration of formulated feeds to the aquaculture industry is positive in

terms of realizing economic benefits as well as superior growth results. Despite the potential

advantages gained from formulated feed development, there has been scant attention directed

towards their effects on the digestive capabilities of yellowtail.

B. Reduced Costs Associated with Feed Development: Defining the Mechanical Digestion of a Meal

Dietary influence has been the topic of a growing number of metabolic studies focused on the effects of feed level and satiation (Beamish 1974; Kiorboe 1985), meal caloric value (Tandler

and Beamish 1979), age specific catabolism (Tandler and Beamish 1981; Moran 2007), mechanical digestion (Krogdahl 2005; He and Wurtsbaugh 1993), as well as ratios of chemical components in diets (Belokopytin 2004). Fish growth performance is highly regulated by their ability to properly catabolize molecules supplied through feeds and further anabolize this material for somatic deposition (Booth et al. 2013). Secor (2009) summarized a number of studies on metabolism in a wide variety of vertebrates, and concluded SDA increases as a function of meal energy, independent of texture, however the influence of meal type has been

45

shown to have innumerable effects on digestibility. While the physical nature of consumed

meals will vary in size and composition, so too will the energy required to properly assimilate

these items. Larger items with more complex structures will therefore require increased

energy to break down, resulting in less of the ingested product directed to body stores.

Although the rise in apparent SDA following a meal has been noted in numerous studies

(Tandler and Beamish 1979; 1981), minimal attention has been focused on the substantially large portion of mechanical energy required from the total operating budget (Mcgaw and

Penney 2014). The energy necessary to physically breakdown meals by mastication, swallowing, and breaking apart matter are technically different from the apparent SDA however most studies have not attempted to differentiate between the two (Beamish and

Trippel 1990). Tandler and Beamish (1979) explored the mechanic aspects associated with digestion and reported energy values accounting for 10 to 30% of the total apparent SDA. The additional energy required for mechanical digestion has been the source of variability across numerous studies and indicates a higher amount of energy required for a natural meal source in comparison with a processed and more malleable version. Mechanical SDA in fish is hypothesized to represent a measurable portion, not exceeding 25% of the energy ingested

(Tandler and Beamish 1979).

The physiological processes influencing an SDA event has been explored as a means to differentiate their respective allocation of total presented fuels. Differentiating between each digestive phase is rather difficult however observations of the total energy required based on meal presentation has been a useful tool. This process is best illustrated in species which allocate substantially large budgets towards digestion. Burmese pythons fed fuel sources 46

ground, liquefied, and intestinally-infused experienced reductions in SDA by as much as one- third of the total cost of the original unaltered state of the diet. Compared with the untreated meal, a significant reduction in the coefficient was seen in ground matter, lower values for liquefied portions, and an even further reduction noted for infused treatments provided to the pythons (Secor 2003). These results illustrate how varying degrees of meal processing directly relate to the duration of mechanical processing required from the body. Likewise, trout are able to bypass specific routes related to mechanical digestion as a result of intestinally infused meal treatments (Seth 2009). Salmon raised on a formulated feed yield a SDA coefficient of

5.6%, nearly a third of that of chopped whole fish (Oliva-Teles and Kaushik 1987). Green shore experienced a 35% decrease in SDA and a 50% reduction in SDA coefficient when substituting whole flesh with a homogenized diet (Mcgaw and Penney 2014). Gila monsters had similar results allocating a significantly less percentage of energy for digesting with a softer egg meal (13%) versus a whole rodent (18%) (Christel et al. 2007). These studies highlight a similarity across numerous species in which there is a consistent decrease in energy required for mechanical digestion coinciding with an increase in meal processing.

It is important to note many organisms exhibit drastic differences in feeding behaviors and digestive functioning over the course of a lifetime and should be a major consideration when comparing across studies. It can often be difficult to relate species in this manner as SDA response does not always scale linearly with meal size and the relationship between the size of an organism and the SDA response is not a consistent factor (McCue 2006). SDACOEFFICIENT has been considered an acceptable metric for comparisons across species and size classes, as its value accounts for both the meal size and metabolic response on a per weight basis. However,

47

experiments need to be compared at similar temperatures when ectotherms are involved.

Natural food sources are known to have an SDACOEFFICIENT in the range of 25%, however a drastic

drop to nearly 5% has been noted in formulated feeds (Secor 2009). Other cumulative works

have reported a wider range of values from 3 to 41% for fish fed natural diets while formulated

meals falling lower in the range of 11 to 29% (Beamish and Trippel 1990).

The production of enzymes within the stomach contributes to the overall digestive

functioning of an organism and directly impacts the total amount of available energy for

somatic growth. Secretion of these enzymes occurs from the onset of organ development through all phases of life, and while a substantial portion is produced from the body, exogenous components have been shown to support this activity by aiding in the utilization of nutrients

(Lauff and Hofer 1984; Kolkovski 2001). Periods of reduced nutritional availability can be offset by slower gut motility and an increase in enzyme secretion as a means to maximize absorption of matter in the stomach (Kofuji et al. 2005; Miegel et al. 2010). Nutritional status influences both the enzyme expression and protein synthesis rates and has been correlated with changes in oxygen consumption (Jobling 1982). The concept of mechanical digestion from both meal size and structure were explored as a means to explain differences in metabolic rates between various feeds.

C. Swim Speed and its Influence on Energy Expenditure

Quantifying energy expenditure can be a complex undertaking factoring in the effects of

fluid dynamics and the overall impacts on nutrient allocation. Swimming costs are known to

largely affect the energy budgets of fish (Ware 1975; Beamish and Trippel 1990) and therefore

48

can alter the body’s ability to properly metabolize nutrients for somatic growth (Billerbeck

2001). Sustained swimming during respirometry has often been employed as a method to

stabilize pre-feeding metabolic rates and increase the accuracy and precision of specific dynamic action studies (Tandler and Beamish 1981). Unfortunately, continuous movement is considered an unnatural representation of behavior for many fish, and though the benefits from the consistency and repeatability are apparent, unconfined motion is considered the

optimal choice for understanding normal behavioral (Ellerby and Herskin 2013). Beamish

(1974) previously demonstrated SDA does not fluctuate across a range of swimming speeds and

therefore it would be assumed the results from fish within a forced swim environment would

be similar to those found in a static system. Other studies have reported contrasting evidence

citing activity level effects on SDA (Blaikie and Kerr 1996), therefore difficulties can arise when

attempting to separate the factors driving various levels of swim efficiency from those

associated with energy metabolism.

The type of locomotion seen in Carangidae fish such as yellowtail is primarily designed for a

specific range of cruising speeds. Optimal swimming velocities established for three size classes

of yellowtail measuring 145 mm, 206 mm, and 311 mm were observed to be 4.83 BL s-1, 3.25 BL

s-1, and 2.73 BL s-1 respectively (Palstra et al. 2014). Applying constraints set by previous works

(Clark and Seymour 2006; Brown et al. 2011), 2.46 BL s-1 was calculated as the optimal velocity by Palstra et al (2014), in contrast to proposed speeds of 0.75 BL s-1 (Brown et al. 2011), 1.2 BL

s-1 to 1.7 BL s-1 (Clark and Seymour 2006), and 1.5 to 2.0 BL s -1 (Yogata 2000). The value

established by Palstra et al (2014), however, is relatively similar to that suggested by Yanase et al. (2012) of 2.25 BLs-1. Swimming activity is thought to play a critical role in the circulation of

49

fish whereas increased motion can assist processes such as oxygen uptake (Muir and Niimi

1972). Two distinct phases of respiration have been noted in yellowtail between 400 to 500 g, whereas their active use of the opercula for respiration ceases above a threshold of 43 cm s-1

and fish are able to ram ventilate with the assistance of the elevated water velocity (Hanyu et

al. 1979). This information alludes to an ideal hydrodynamic corridor for yellowtail promoting greater efficiency, whereas respiration rates are greatly reduced with an increase in water movement over the gills (Hanyu et al. 1979). By reducing the percent of energy necessary during respiration, a greater proportion of ingested nutrients can be allocated towards somatic growth and energy reserves.

Above normal cruising speeds, the body experiences a range of physiological changes, whereas this increase in motion redirects not only their muscle form, but also alters the pathways which provide ATP to muscle groups (Gibb and Dickson 2002). Their muscle groups have set functional roles, as demonstrated by the arrangement of their slow twitch (red) and

fast twitch (pink and white muscle) groups being 65% posterior and 35% anterior, respectively

(Yanase et al. 2012). Sustained swimming relies almost exclusively on aerobic metabolism in

red musculature with minimal assistance from white muscle (Coughlin 2002). This type of

swimming refers to the slower motion primarily utilized by fish in nature and therefore can be

maintained for extended periods of time (Sfakiotakis 1999). The activation point of white

muscles is estimated to occur between 1.69 and 1.81 BL s-1, and is considered the moment

yellowtail recruit the power from anaerobic metabolism in ordinary (pink) muscle groups to

meet added physical demands (Tsukamoto 1981; Yogata 2000). Recruitment of distinct muscle fiber type orchestrates the utilization of energy sources during the ATP process, therefore diet

50

optimization will directly relate to a degree of importance for digestible fuels and their

respective storage depots (Moyes and West 1995; Kaushik and Seiliez 2010). Richards et al.

(2002) describes the use of substrates at sustainable swimming speeds in a two-step series comprised of an initial acclimation period involving carbohydrate oxidation followed by an

extended duration of lipid oxidation. Substrate preferences have been noted in both muscle

types, showing red muscles dependent on ATP generation from mitochondrial oxidative

phosphorylation (Moyes and West 1995), in contrast to white muscles, which are primarily

driven by creatine phosphate hydrolysis and glycolysis (Moyes et al. 1992; Richards et al. 2002).

The concepts outlined may help to build a better understanding of variability across studies relating to both unconstrained and constrained motion. In an uncontrolled arena, fish have the ability to alter the course of nutrient assimilation from increased motion or extended periods of stagnant behavior. This information enforces the increased difficulty associated with attempting to measure fish metabolism in an uncontrolled environment, however visual observation of movement can be used to compare consistencies between each treatment.

D. Importance of Fecal Output Collection and Characterization

Monitoring changes in oxygen consumption during a feeding event is a common

technique used to assess energy use, however some studies have also incorporated concurrent

observations of the organic output produced (Lauff and Wood 1996; Kieffer et al. 1998;

Richards et al. 2002). The relationship between SDA and waste excretion can account for the total energy allocated during digestion relative to the available nutritional components present in a diet (Rolland et al. 2014). The dynamic interactions between feed input, matter exchange,

51

and waste products establish guidelines for energy sourcing and integration. This multifaceted

approach results in a respirometric estimate of the magnitude and duration of an event while

simultaneously providing a breakdown of the exact nutrients being absorbed as well as the

timing of use.

The energy released in the form of waste is termed fecal loss or fecal energy, and the ratio

of this discarded matter to meal energy will provide the apparent digestibility of a given meal

(Cho et al. 1982; Blaxter 1989). The overall digestibility of feedstuffs determines their usefulness as an energy source (Blaxter 1989) however maintenance costs can add to the complexities of interpretation and extensive studies are necessary to accurately depict an event. Compared with terrestrial species, waste collection from fish can be problematic as nutrient leaching is a common occurrence in aquatic systems (Windell et al. 1978b; Fagbenro and Jauncey 1995). A number of techniques have been used during fecal analysis with considerable variation in the estimates of nutrient digestibility (Cho et al. 1982; Kaushik et al.

2002).

A component of this study was directed at examining the total settleable solids present post-feeding and later quantifying the digestibility of the two feeding treatments. Observing the total fecal energy content, as well as ratios of protein and lipid expelled, offers yet another perspective to interpret the postprandial response and overall metabolic nature of yellowtail.

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III. Materials and Methods

A. Experimental Fish

Pacific yellowtail kingfish, S. lalandi dorsalis (Valenciennes 1833) were captured off

Catalina Island, California at temperature of 20⁰C using barbless circle hooks during a voyage on

a sport fishing vessel (F/V Shogun) in December 2014. Aerated wells aboard the fishing vessel

Shogun were used as a temporary holding platform during the 3-day sea excursion. The fish

were then transported by truck within a 10,600 L tank to the Tuna Research and Conservation

Center (TRCC) in Pacific Grove, CA, USA. Fish were capturd in waters of X C, and held in

captivity in X volume tanks (T4) at 20C + 1C. Get the thermal data right. Fish were individually

fitted with uniquely colored external tags (Hallprint tags, Victor Harbor, SA, Australia) through

the dorsal musculature for identification during trial. Prior to implantation, each tag was

soaked in betadine to reduce the risk of infection. The mean individual weights of fish (6 fish in

total) during the initial commercial feed trial was 1017.6 ± 75.6 g (range 850-1112 g), and the

values for fish (6 fish in total) during the post sardine feed trial were 1072.2 ± 84.4 g (range 884-

1206g). All procedures were conducted according to the guidelines and procedures set out by

the Stanford University Animal Care and Use Committee (protocol 7297).

B. Primary Components of the Static Respirometer

The “static” respirometer for this portion of the study was constructed previously at the

Tuna Research and Conservation Center as described by Freund (1999). The tank (1237 L) was

constructed with insulated fiberglass (A/J Fiberglass, Salinas CA) and ovular in shape measuring

53

215 cm long x 135 cm wide x 60 cm deep (Figure 11). The inside of the tank was colored sky blue with vertical lines set at roughly 10 cm to provide a reference for fish within the system.

The top of the respirometer was sealed with a 1.25 cm thick acrylic lid and gasket secured watertight with 14 pipe clamps. The center of the lid had a 60 cm x 40 cm square opening to allow access for insertion and extraction of fish from the unit. An acrylic lid with gasket was placed over the square opening and secured with 22 bolts. Before sealing the system with fresh seawater, a squeegee was used to remove any bubbles remaining under the lid.

There were two 10 cm openings on either end of the respirometer, allowing for inflow and outflow of water during recurrent cycles. Both ports were connected in series with a 1,146 L external reservoir designed for water exchange during open cycles. The outflow of water from the respirometer traveled through a 6.5 cm flexible tube into the external reservoir (Figure 12).

A 10 cm inflow tube into the respirometer was powered by a Rule 1,800 110 L min-1 submersible pump and connected with a 3.5 cm plastic tube from the external reservoir. Water within the external sump was fitted with aeration stones in an effort to maintain elevated levels of oxygen during flush cycles.

54

Figure 11. Side view of the static respirometer (Volume: 1,237 L)

Figure 12. External reservoir (Volume: 1,146 L)

To ensure water quality within the system remained at safe levels for the fish, the addition of a flow through component from a larger external reservoir was added to the system. This reservoir was a 338,000 L external tank that was fitted with an EHEIM 40 L min-1 submersible water pump and transferred fresh water to the 1,146 L external reservoir. Within the 1,146 L external reservoir, a Eurobuilt 1/6hp submersible utility pump with float valve was used to

55 control the exchange of reservoir water. The Eurobuilt pump removed 25% (260 L) of the reservoir water in 20 min and was replenished from the 338,000 L tank in 8 min. Water temperature was set based on the temperature of the inflow to the external reservoir and remained at 20.4° ± 0.2°C during both the commercial feed and sardine feed portion of the trials. A one-way valve was placed on the inflow tube to the respirometer to prevent a siphoning effect caused by water levels within the external reservoir falling below the height of the respirometer.

Oxygen and temperature were monitored by a Fibox 3 fiber-optic oxygen meter

(Precision Sensing GmbH, Regensburg, Germany) at a sampling rate of 1 sec intervals and resolution of 0.01 mg L-1 and 0.1°C. Measurements were recorded through the custom

Oxyview program in conjunction with the Fibox 3 sensors. A conventional two-point calibration was performed on the oxygen minisensor (DP-PSt3) before experimentation (rangehigh: air- saturated water (cal100); rangelow: 1 g of Na2SO3 to 100mL water (cal0)). The entire swim respirometer was surrounded by black plastic to block off adverse effects brought on by external visual stimuli. A camera was positioned directly above the unit and connected to a computer monitor with real time footage to assess the behavior of fish within the flume.

C. Experimental Design of the Study

The yellowtail experimental animals were held in a 5,223 L circular fiberglass tank within the tuna research and conservation center. Fish were weighed to the nearest gram during the morning of the respirometry trial and returned back to the holding tank for several hours until feeding commenced. All fish were fasted for 48 h prior to feeding trials. To capture a fish for

56 the experiment, the water level within the tank was reduced to approximately 0.5 m in depth and a plastic curtain was used to individually partition fish from the remaining subjects. Sardine fillets were cut and measured to the nearest gram and placed on a labeled sheet for use during the feeding. For the commercial feed portion of the trial, isocaloric portions of pellets equivalent to 5.2% BW in sardine fillets were used as a nutritional source. Sardine fillets or pellets were offered to the partitioned fish until the desired percent body weight or isocaloric equivalent had been consumed. This procedure was repeated until all fish had consumed their respective feed portions. Commercial feed (Brand: EWOS, Canada; Type: Dyna yellowtail; Size:

XL, 13mm), Sardine Fillet (Brand: Seawave, USA; Type: Pacific sardines).

Using a dividing curtain, fish were corralled to a small area on one side of the tank.

Concentrating the fish allowed researchers to capture all fish while minimizing stress. Fish were relocated from the feeding tank into the 1,237 L respirometer using a mesh net. All monitoring of fish behavior was done in close proximity to the swim flume via live video. During the closed cycle, oxygen consumption was measured relative to fish mass while during the open cycle fresh oxygenated water was pumped into the system from an external tank to replenish dissolved oxygen levels within the tunnel chamber. A cycle period of 25 min open followed by

25 min closed was selected to ensure dissolved oxygen levels remained above 80% for the entire closed cycle. Fish generally appeared stressed with irregular movement immediately after placement into the tank. The initial entry period included rapid movement, constant direction change, and schooling behavior, therefore oxygen consumption readings were not used until values fell within the range consistent with the slope of the remaining period. After

57 completion of an SDA trial, fish were removed with the use of a mesh net and returned to the original 1,380 L holding tank at 20⁰C.

The fish in each treatment underwent a two-step respirometry protocol (Step 1:

Measurement of routine metabolic rate (RMR); Step 2: Calculation of postprandial response to feeding event) for determining the oxygen consumption requirement during a feeding event.

Unfed fish for this experiment are referred to as ’fasted‘, while fed fish were categorized as

’digesting‘ (Clark et al. 2010a; Whitlock et al. 2012) . Food was withheld for 48 h prior to experimentation in both fasted and digesting fish. Upon entry into the respirometer, oxygen consumption was usually elevated due to the stress of handling during transfer. The initial step of this study was focused on calculating the RMR for a fasted fish. Routine oxygen consumption as previously described encompasses measuring a state of routine behavior involving spontaneous movement (Beamish 1964; Pirozzi and Booth 2009). After placing a fasted fish in the respirometer for a period of 48 hours, the lowest 3 h mean oxygen consumption was therefore selected as the RMR (Clark et al. 2010a). The RMR from fasted fish would therefore set the baseline oxygen consumption as a comparison against the rates for digesting fish. Each fish underwent 2-3 ‘fasted’ trials at a temperature of 20.4±.02⁰C in order to determine their baselineRMR state.

The protocol for digesting fish was as follows: sardines were removed from the freezer and allowed sufficient time to defrost to room temperature prior to feeding. Sardines were filleted to remove all bones and innards from the available feed samples. Fillets were divided into sections ranging from 3 to 10 g (wet weight) and weighed to the nearest 0.1 g. Total mass for each sample fillet was recorded and individually labeled. Fish selected for respirometry 58 were partitioned with curtains from the rest of population and allowed to acclimate to solitary confinement conditions. Two researchers presented sardine fillets one by one and recorded total consumption for each fish until 5.2% of their body weight had been consumed. Fish were visually identified by tag color and remained untouched in the confinement area for approximately 30 min to one hour to prevent regurgitation of food. There was no sign of regurgitation for any of the selected fish for respirometry.

D. Nutritional Analysis: Calculating the Proximate Composition of Meal Types and Fecal Output

A total of five sardine fillets and 10 g of the formulated commercial feed were sent to Nestle

Purina Analytical Laboratories (St. Louis, MO, USA) for analysis (Table 6). Bomb calorimetry was used to determine the protein, lipid, moisture, and caloric content of the sardine fillets used during the digesting portion of the swim protocol. The procedure used by the Nestle Lab for r determining protein content, nitrogen compounds within the sample was as follows: The samples were boiled using sulfuric acid in conjunction with a catalyst (potassium sulfate, titanium dioxide, and cupric sulfate) to produce ammonium sulfate. The resulting ammonia sulfate can be made alkaline with a sodium hydroxide solution, liberating ammonia into a known portion of standard acid. The solution was then titrated and nitrogen was determined from the known amount of standard acid solution used in the process. The crude protein was then calculated from a predetermined value of 6.25 for total nitrogen using protein factors

(OMA AOAC International 988.05, 920.87, 991.20). Fat was determined by a method known as acid hydrolysis. The portion of fillet was hydrolyzed with hydrochloric acid to liberate heat- bound fats and oils (OMA AOAC International 954.02, 922.06, 933.05, 925.32, 950.54, 948.15;

59

LI-75.204). Using ethyl and petroleum ether, the fat was extracted and volatilized. The

resulting product was the fat, and was dried and weighed to determine the percentage within

the sample. For moisture content of the sample, a forced draft oven method was selected at a

temperature of 133°C, and a measurement of moisture lost could be calculated from the

change in mass (NP Analytical Laboratories 2012). Total calorie value for the sample was

calculated by freeze drying to remove all moisture, then burning the remaining material in a

calorimeter. The sample was ignited in an oxygen filled metal bomb and placed in a bath of

water. Heat generated was determined from the burning of elements in the sample and

therefore calculated from an increase in temperature of the bath water (No. 463M).

Table 6. Proximate composition and energy content of sardine fillet and commercial pellet. Protein (%) Fat (%) Moisture (%) Calories (kcal g-1) Sardine Fillet1 20.6 8.36 66.5 2.11 Commercial Feed1 47.6 20 4.35 5.46 Protein, Kjeldahl (N x 6.25). Fat, acid hydrolysis. Energy, bomb calorimetry. 1kcal = 4.1868kJ (Brett and Groves 1979). 1Wet weight basis. A BPM commercial pellet feed (47.6% protein, 22% lipid, 4.35% moisture, 5.46 kcal/g) was

used as a comparison against the previous sardine fillet diet. Isoenergetic rations of the

commercial diet were used based on an equivalent value for a diet consisting of 5.2% sardines.

While the nutritional constituents within a sardine fillet and the commercial diet are different, by standardizing their energy value, an effect based on digestive efficiency of the total presented fuel could be assessed. Using the total mass of the fish, a 5.2% calculation of body weight can be made to determine the sardine fillet meal size. The energetic value of the 5.2% sardine feed was recorded in terms of dry weight, therefore a specific weight of filet would be reflective of the energy without moisture. Calculations were made to determine the

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isoenergetic value of the commercial feed relative to the dry weight of the sardine feed

(Equation 7 & 8).

= ( ×. ) (7) 𝑺𝑺𝑺𝑺𝑹𝑹 𝒀𝒀𝑾𝑾 𝟎𝟎𝟎𝟎𝟎𝟎

( × ) × = × × (8) 𝑺𝑺𝑺𝑺𝑹𝑹 𝑺𝑺𝑫𝑫 𝑺𝑺𝑪𝑪 𝑪𝑪𝑪𝑪𝑹𝑹 𝑪𝑪𝑪𝑪𝑪𝑪 𝑪𝑪𝑪𝑪𝑫𝑫

• SFR = Sample reference ration equivalent to 5.2% of the total body weight of one fish (g)

• YW = Total weight of one yellowtail (g)

• CFR = BPM commercial pellet treatment (kcal/g)

• SD = Percent dry weight of a sample reference ration

• SC = Caloric value of 1 gram of sample reference ration

• CFC = Caloric value of 1 gram of BPM commercial pellet treatment

• CFD = Percent dry weight of BPM commercial pellet treatment

E. Fecal Collection Procedures and Storage

Three replicate trials for each feed treatment were used in order to quantify the total

amount of feces accumulated over a period of 48 h. The intestine of yellowtail has been

documented to evacuate all excrement as early as 16 h within the range of 20°C to as much as

48 h for cooler temperatures of 12°C (Miegel et al. 2010), therefore the maximal time period

was chosen to ensure no feces were lost. At 24 and 48 h post-feeding, all settled material was

drained from each tank and collected with a 100 µm mesh net. Net size of 180 µm has been used in previous trials analyzing feces in yellowtail (Moran et al. 2007), therefore the use of a finer grade mesh was considered acceptable for this analysis. In salmonids, nutrient

61 digestibility estimates determined from feces collection is documented to have no significant differences between 40 min, 24 h, and 48 h (Lee et al. 2001), however for comparison purposes, a sampling time of 24 h was chosen. Each of the samples were weighed to the nearest 0.01 g and stored at a temperature of -20°C immediately after collection and then later placed in an -80°C refrigerator until ready for analysis (Lee et al. 2001; Blythe 2015). Samples were sent to Nestle Purina Analytical Laboratories (St. Louis, MO, USA). Variables analyzed included protein, lipid, moisture, and caloric content, and followed the same guidelines as described in the nutritional analysis section of Chapter 2. Digestibility calculations for the diets used in this study were determined accordingly with equations 9-13. Protein and lipid caloric equivalents of 23.87 kJ g-1 and 36.43 kJ g-1 were used to estimate the total energy value for all fecal samples (Brett and Groves 1979; Estess et al. 2014).

( ) (9) (%) = ( × ) ( ) 𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹 𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 𝒌𝒌𝒌𝒌 𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴 𝑫𝑫𝑫𝑫𝑫𝑫𝒆𝒆𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔 𝟏𝟏𝟏𝟏𝟏𝟏 − � � 𝟏𝟏𝟏𝟏𝟏𝟏 𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻 𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 𝒌𝒌𝒌𝒌 ( ) = + + + (10) 𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹 𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭 𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴 𝒌𝒌𝒌𝒌 𝑷𝑷𝟏𝟏 𝑷𝑷𝟐𝟐 𝑳𝑳𝟏𝟏 𝑳𝑳𝟐𝟐 ( ) = × (11) 𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 𝒌𝒌𝒌𝒌 𝑷𝑷𝑾𝑾 𝑷𝑷𝑬𝑬 ( ) = × (12) 𝑳𝑳𝑳𝑳𝑳𝑳𝑳𝑳𝑳𝑳 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 𝒌𝒌𝒌𝒌 𝑳𝑳𝑾𝑾 𝑳𝑳𝑬𝑬 ( ) = × (13) 𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻 𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 𝒌𝒌𝒌𝒌 𝑴𝑴𝑾𝑾 𝑴𝑴𝑬𝑬

• P1 (kJ) = Total protein energy remaining at the end of 24 h

• P2 (kJ) = Total protein energy remaining at the end of 48 h

• L1 (kJ) = Total lipid energy remaining at the end of 24 h

• L2 (kJ) = Total lipid energy remaining at the end of 48 h

• PW (g) = Total weight of proteins in feces 62

• LW (g) = Total weight of lipids in feces -1 • PE (kJ g ) = Protein caloric value (23.87) -1 • LE (kJ g ) = Lipid caloric value (36.43)

• MW (g) = Dry meal mass

• ME (kJ) = Meal energy value determined from bomb calorimetry

F. Statistical Analyses

The postprandial increment in Mo2 was described at 20.4±.02⁰C, in terms of baseline RMR,

SDAMax, SDADuration, factorial scope, SDA, and SDACoefficient. The limits set by this protocol therefore would encompass the change in oxygen levels from a routine metabolic state to those reflective of a digestive period. RMR as described previously was determined from preliminary fasted trials and estimated to the point at which the postprandial increment returned to their original fasted state of oxygen consumption. Peak oxygen consumption, or

SDAMax was the point at which oxygen levels reached a maximal level post-feeding event. The length of the SDA is noted as SDADuration and calculated as the time from the feeding event through maximum consumption and ended when the decline in MO2 reached within range of

RMR. To account for the initial period of high stress and acclimation to the flume, a linear increase in oxygen consumption from the moment of feeding through the beginning of the SDA event was used as an estimate of this time period. SDA was calculated as the change in oxygen over the entire event with MO2 being converted to an energy equivalent assuming 14.32 J of energy per 1 mg of oxygen consumed (Beamish and Trippel 1990). SDAcoefficient was calculated by dividing the SDA by the meal energy and represents the percent of energy expended on digestion in relation to the total energy available in a meal (Secor 2009). Factorial scope was calculated as the SDAMax divided by RMR.

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Monitoring fish movement within the respirometer was done by visual observation by

randomly selecting 3 minutes within each open cycle to determine various levels of activity. A

total of 318 min were analyzed for motion, 153 min during the sardine fillet trials and 165 min

during the commercial feed portion. Four separate levels of motion were assigned for the

group of fish within the respirometer based on previously determined criteria (0- none/little

activity, 5– medium activity, 10– high activity, 15– very high activity). Data was later

transformed into two separate categories for analysis (None/little activity versus medium to

very high activity).

Statistical analyses were performed with the use of Minitab 17.2.1 (Minitab 17 Statistical

Software (2010). [Computer software]. State College, PA: Minitab, Inc. (www.minitab.com). All

data were analyzed for normality and transformed when appropriate. Two-sample t-tests were

used to compare the differences in meal mass and energy presented in each treatment. Paired

t-tests were performed to determine significant differences (p<0.05) of the postprandial metabolic variables and digestive efficiencies between the two nutritional sources (sardine fillet versus commercial feed). Data for each of the feed treatments was pooled and Fisher’s exact

test was used to compare significant differences (p<0.05) in the ratios of movement types

associated with each feed.

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IV. Results

A. Dietary Composition Analyses

The fish in this study were fed two meal treatments that were the isoenergetic equivalent

of 5.2% of their body weight. The total wet meal mass presented to the fish was a major factor

in this trial as the commercial feed consumed by fish averaged 40.7 ± 0.21 g in comparison to

the sardine fillets which were on average 349.3 ± 3.3 g. Although a larger portion of the sardine

fillet was moisture as compared with the commercial feed, 66.5% and 4.35% respectively, it

was determined the sardine fillets had three times greater mass after removing all moisture.

Average dry feed weight for the commercial diet was 38.9 ± 0.2 g, and found to be significantly

lower (p = 0.000) than 117.0 ± 1.1 grams for the sardine fillets.

B. Postprandial Metabolic Response

The results from the 1237 L static respirometer demonstrate a level of consistency in the

metabolic response to various feed treatments (Table 7, Figure 13). The mean yellowtail

-1 -1 baselineRMR value for the three commercial feed treatments was 120.2 ± 3.7 mgO2 kg h ,

-1 statistically lower (p=0.036) than that of the mean baseline RMR values of 150.8 ± 3.9 mgO2 kg

-1 h (20.4 ± 0.2⁰C) for the three sardine trials. SDAMAX was also found to be statistically different

-1 -1 (p=0.04) between the two diets, with commercial feed mean values of 203.7 ±22.5 mgO2 kg h

-1 -1 and the sardine feed at 279 ± 27.3 mgO2 kg h . In terms of the factorial scope, RMR and

SDAMAX did reveal close symmetry however were found to be significantly different (p=0.037)

between the commercial and sardine feed, 1.7 ± 0.1 and 1.8 ± 0.1 respectively. There was no

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significant difference in the duration of the SDA event with the commercial feed and the

sardine fillet lasting 27:46 ± 1:32 h and 33:36 ± 00:16 h, respectively.

A significantly greater (p=0.003) amount of energy was allocated towards the digestion of

the sardine fillet treatment with an average SDA value of 42.9 ± 0.7 kJ in comparison with the

commercial feed which required roughly half the energy to digest at 18.2 ± 1.4 kJ. In terms of

SDAcoefficient, digestion of the sardine fillet was found to be less efficient (p=0.005) and required

24.9 ± 0.2% of energy presented to digest an equivalent meal in comparison to the commercial feed values which required half the energy to be allocated towards digestion at 12.3 ± 1.0%.

Table 7. All measured variables pre and post specific dynamic action based on a dietary source of commercial feed and sardine flesh for yellowtail. Commercial Sardine Fillet Feed Parameter 47P: 20L 20P: 8L p value N 3 3 Temp °C 20.5 ± 0.1 20.4 ± 0.1 0.797 Total Fish Mass# g 6084.0 ± 39.0a 6682.0 ± 64.2b 0.004* Meal Mass (wet) g 40.7 ± 0.2a 349.3 ± 3.3b 0.000** Meal Mass (dry) g 38.9 ± 0.2a 117.03 ± 1.1b 0.000** Meal Energy Content kJ 889.6 ± 4.6a 1033.1 ± 9.9b 0.006* -1 -1 a b BaselineRMR mgO2 kg h 120.2 ± 3.7 150.8 ± 3.9 0.036* -1 -1 a b SDAMax mgO2 kg h 203.7 ± 22.5 279.6 ± 27.3 0.040*

SDADuration h 27:46 ± 1:32 33:36 ± 00:16 0.070 a b Factorial Scope SDAMax/BaselineRMR 1.7 ± 0.1 1.8 ± 0.1 0.037* SDA kJ 18.2 ± 1.4a 42.9 ± 0.7b 0.003** a b SDACoefficient % 12.3 ± 1.0 24.9 ± 0.2 0.005** # All trials and values refer to 6 fish. Data are presented as means ± S.E. Means in the same row with different superscripts indicate significantly different values (p<0.05). * represents a p value of <.05; ** is a p value of less than 0.01.

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Figure 13. Sardine and formulated feed treatments. Black (solid) lines correspond with the postprandial response of the commercial feed treatment while the black (dotted) lines estimate the range for the baseline routine metabolic rate. Red (solid and dotted) lines indicate metabolic range for the sardine fillet treatment.

C. Visual Documentation of Movement during Respirometry

Movement was categorized into various levels of activity (None/little activity versus

medium to very high activity) in an effort to evaluate the degree of motion within the tank.

During the sardine fillet treatment, significant differences in movement (p=0.027) during the digestive periods were observed between the two feed treatments. Fish had 12.3% greater movement during the sardine fillet digestive state, as compared to that of the commercial feed digestive state.

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D. Fecal Proximate Composition as a Function of Meals Type

The total calorie intake for each treatment was equal in terms of total energy as a ratio of

body weight. The fish increased in size throughout the trials and therefore total energy

consumed was reflective of the growing differences in body weight. There were significant

differences in the total meal energy presented as the trials were done in succession and test

subjects experienced growth over this time period (Table 8).

The mass of the sardine treatment was calculated at nearly double the size of the commercial feed, however there were no differences detected (p=0.644) in the total mass of excrement remaining between the two treatments. The feces remaining from the commercial feed trial contained on average 4.0 ± 0.3 g as compared with the sardine feed which was measured at 4.2 ± 0.2 g. Although the mass of feces was consistent for both treatments, the total energy remaining in this matter significantly differed with 20.5 ± 1.6 kJ contained in the commercial feed whereas the sardine feed had 45.2 ± 4.2 kJ. Differences in the total amount of protein and lipids in the excrement at 24 h was seen in both meal treatments however no effect was detected at 48 h (P=0.102 and P=0.423 respectively). After 24 hours, the total amount of protein remaining in the feces from the commercial feed was on average 10.8 ± 0.9 kJ and found to be significantly lower (P=0.007) than the remaining protein in the sardine feed treatment of 25.4 ± 1.0 kJ. At 24 h, the lipid levels of the feces were found to be significantly different (P=0.049) with 5.3 ± 1.3 kJ in the commercial feed, roughly half of which remained from the sardine treatment at 13.4 ± 3.1 kJ. Both diets were almost completely processed however the amount of matter remaining did reveal differences in digestibility (P= 0.020) with

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the commercial feed calculated at 97.7 ± 0.2% as compared with 95.3 ± 0.5% for the sardine

feed.

Table 8. Analysis of meal energy load and fecal composition remaining after 24 and 48 h. Commercial Feed Feces Sardine Feed Constituent Value Constituent Value p Value Meal Energy (kJ) 898.3 ± .7a Meal Energy (kJ) 972.3 ± 11.0b 0.023* Meal Weight (g) (dry) 39.3 ± 0.0a Meal Weight (g) (dry) 110.1 ± 1.3b 0.000** Meal Weight (g) (wet) 41.11 ± 0.03a Meal Weight (g) (wet) 328.77 ± 3.8b 0.000** Protein (kJ) 446.8 ± 0.3a Protein (kJ) 541.6 ± 6.3b 0.004** Lipid (kJ) 315.2 ± 0.2a Lipid (kJ) 417.3 ± 4.9b 0.002** Feces (24 h) Feces (24 h) Protein (kJ) 10.8 ± 0.9a Protein (kJ) 25.4 ± 1.0b 0.007* Lipid (kJ) 5.3 ± 1.3 a Lipid (kJ) 13.4 ± 3.1b 0.049* Feces (48 h) Feces (48 h) Protein (kJ) 3.8 ± 0.7 Protein (kJ) 6.3 ± 0.2 0.102 Lipid (kJ) 0.5 ± 0.5 Lipid (kJ) 0.1 ± 0.1 0.423 Feces Energy (kJ) 20.5 ± 1.6a Feces Energy (kJ) 45.2 ± 4.2b 0.013* Feces Weight (g) 4.0 ± 0.3 Feces Weight (g) 4.2 ± 0.2 0.644 Digestibility (%) 97.7 ±0.2a Digestibility (%) 95.3 ± 0.5b 0.020* Data presented as means ± S.E. Means in the same row with different superscripts are significantly different (P<0.05). Values for mass listed as dry weight (g). Energy content for meal mass based off NPAL chemical composition analysis. Energy content values for feces calculated from a protein energy equivalent of 23.6 kJ g-1 and 39.5 kJ g-1 for lipid (Brett and Groves 1979).

V. Discussion

A. Metabolism The physiological processes of fish are highly dependent on a number of biotic and abiotic

factors. In a natural setting, fish must select nutrient sources that maximize their fitness while

factoring in the energy to locate and consume them. Although some items are rich in energy,

their natural physical structure may prohibit efficient digestion and prove costlier than the

energy required acquire them. As migratory fish cover a wide expanse and variety of prey

items, their choice for nutrition must be highly selective and focused, avoiding sources

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requiring more energy than the added benefits. In a similar fashion, aquaculturists must carefully choose between nutrient sources that will minimize the impacts of poor acceptance while simultaneously maximizing returns. Feed preparation including pellet rigidity, size, and shape are all factors presenting a degree of variability during nutrient assimilation, and must not be overlooked when evaluating the effectiveness of a diet. The physical structure of a feed source influences an animal’s ability to properly digest, directly impacting acceptance and integration into the system. Formulated feed utilization has grown over the years as an effective means to provide essential vitamins and minerals, improve storage times, and replace costly ingredients. Formulated feed does not necessarily guarantee superior performance to an alternative diet of whole fish however greater acceptance due to its structural presentation and rapid breakdown is promising.

Meal energy has become a widely accepted metric for SDA trials, although caution should be taken as two identical energy rations may have drastic inconsistencies in their energetic value (Brown and Cameron 1991a; Rolland et al. 2014), or ratios of dietary components (Fu et al. 2005; Eliason et al. 2007), and the overall outcome of the trial may appear skewed. The question arises as to whether the energetic value of a meal, nutritional constituents, or the physical structure controls digestion and what proportion of each will influence the overall cellular mechanisms promoting growth.

B. Baseline Routine Metabolic Rate

Routine metabolic rate and SDA with two different diets was measured in a static

respirometer at 20.4 ± 0.2⁰C. After an acclimation period for each of the meal trials, the

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baseline values were observed after 48 h of fasting and found to be significantly different

between the two treatments. Baseline values for the commercial feed treatment were 120.2 ±

-1 -1 3.7 mgO2 kg h , whereas the sardine feed treatment baseline values were significantly higher,

-1 -1 in the range of 150.8 ± 3.9 mgO2 kg h conducted at 20.4 ± 0.2⁰C. The baseline values for the

sardine treatment are nearly identical to those results found by De la Gándara (2002) who

-1 -1 reported reductions in yellowtail respiration post-fasting to 140-150 mgO2 kg h , however the

commercial feed routine metabolic rate was recorded to be 20% lower. Baseline values for fasted salmon acclimated to a manufactured diet composed of 100% fish oil have demonstrated significantly higher baseline oxygen consumption rates in relation to those values associated with a diet derived from 50% soy oil and 50% fish oil (Grisdale-Helland et al. 2002). While this study is focused on a different diet and species of fish, it does support the theory that baseline metabolism may be affected by the source of energy. Other works have cited similar findings in terms of changes in baselineRMR as a result of dietary sources (Jobling and Davis 1980; Helland

et al. 2006).

One potential source of the discrepancy here could be attributed to a nutritional

limitation from dietary source. Digestive inefficiencies have been noted in formulated artificial feed over a meal of meat fed to Octopus demonstrating variability in the expression of

digestive enzymes as well as energetic costs associated with insufficient nutritional components

of a diet (Aguila et al. 2007). The differences between the two baseline values may have been

caused from a combination of muscle and fat reserve differences as well as changes in enzyme

response to diet acclimation (Yang and Somero 1993). While some species lack the ability to

adjust their metabolism during periods of fasting and rely on lipid stores for general

71

maintenance (Jensen 2010), the lower metabolic levels recorded for the commercial feed could

potentially be a result of the body receiving insufficient energy stores and reacting in a

preservative manner towards a state of energy depletion.

Differences in SMR have been shown to be correlated with growth rate in salmonids with

increases in ration level resulting in the elevation of SMR (Van Leeuwen et al. 2012), somewhat similar to the results found in this experiment. Other works have had contrasting views however and indicate no such oxygen consumption differences between fed and fasted

treatments from nutritional source (Aragao et al. 2003; Kumar et al. 2012). Further studies

would be necessary to understand the complex interactions involved in the metabolic response

to a number of feed sources.

C. SDAMax, SDADuration, and Factorial Scope

In this study measurements of RMR, and SDA were determined for California Yellowtail,

S. lalandi, at 20.4 ± 0.2⁰C. Maximum respiration rates for the two diet treatments differed

significantly, and higher values were found for the sardine diet treatment over the commercial

-1 -1 -1 -1 feed diet at 279.6 ± 27.3 mgO2 kg h and 203.7 ± 22.5 mgO2 kg h , respectively. The added

metabolic costs accrued from the sardine meal may have been caused from a greater

requirement of energy towards the initial breakdown of matter, as the mass of the sardine flesh

was significantly greater than that of the formulated pellet. Although the factorial scope was

rather similar amongst the treatments, with sardine feed being 1.8 ± 0.1 and commercial feed

at 1.7 ± 0.1, their differences were shown to be statistically significant, however poorly

correlated (p=.037). The values for factorial scope recorded in this experiment were similar to a

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number of other studies which measured values between 1.44 and 1.81 for fish presented both formulated and fish meals at a comparable rate of 5-6% BW (Du Preez 1986; Lucas and Priede

1992; Furnell 1987). Interestingly, there was no difference in the duration of the SDA event

between the two treatments and although cumulative works have pointed out a successively

larger duration relative to meal size (Secor 2009), the results from this work did not indicate

differences in the amount of time it would take to process various meal types.

D. Protein and Lipid Contribution to Metabolic Response

The total energy for both treatments was an equal ratio of calories relative to 5.2% of the body weight of the subject, however, the nutritional constituents were found to be different.

Protein and lipid composition varied between each feed type with the sardine treatment containing 21% and 32% greater loads than the commercial feed, respectively. The differences in postprandial response could be viewed in terms of protein assimilation in which the fish are reacting to a level of nourishment, directly influencing their immediate metabolic response.

Brown and Cameron (1991b) demonstrated a correlation between essential amino acid infusion and an increase in oxygen consumption rates post treatment. These findings were reinforced after protein synthesis inhibitors were added to the existing trial, decreasing both protein synthesis and oxygen consumption rates. Therefore, the role of protein as an influential element toward SDA cannot be undermined as insignificant when analyzing the postprandial response for these diets. For example, the energetic cost of digestion in tortoises fed a number of diets including plants, fungi, and invertebrates resulted in SDA coefficients of 16%, 21%, and

30% respectively (Hailey 1998). While each of the meal types fed to the tortoises contained a

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different physical and chemical composition, differences in the cost of SDA were determined to be directly related to the total digestible protein intake (Hailey 1998). Likewise, SDA coefficients in common carp showed an increasing relationship with dietary protein concentrations (Chakraborty et al. 1992), similar to the findings of Ross et al. (1992). This however was not in complete agreement with Watanabe et al. (2001) who conducted a study on yellowtail and noted no remarkable differences between the heat increment of feeding and gross energy intake values as a result of either protein or lipid contents.

E. Variability in Metabolic Response from Meal Size and Structure

Treatments used in this study were designed to be representative of a fish based meal;

the first being a complete filet from a sardine, the second being a processed, ground, and

enriched version with fish meal and oil as the primary constituents. Although the two meal

treatments were nearly an identical ratio of energy, the relative differences in size and

structure cannot be overlooked when interpreting the postprandial metabolic effects. On

average the total weight of sardine fillet rations fed to all six fish was 117.0 ± 1.1 g, compared

with a 5.2% BW isocaloric equivalent of formulated feed amounting to 38.9 ± 0.2 g. A threefold

increase in mass between the two meal treatments is speculated to have affected their

digestive efficiency. While such variables as meal size have been shown to cause a proportional

SDA response, higher maximal rates, and an increase in the factorial increments (Andrade

2005), only a select few studies have explored the added benefits of feed structure and size

towards digestion and acceptability.

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SDA for both meal types were found to be significantly different, with sardine fillets in a

range of 41.7 kJ and 44.1 kJ, while the commercial feed values were between 16.2 kJ and 20.8 kJ. Thus more energy must be digested in the fresh sardine feed, then the dry diet, partially accounting for the increase in metabolism with SDA. Expressing the SDA values relative to the total meal energy consumed, the associated coefficient for the sardine fillet was between

24.7% and 25.2%, higher than that of the commercial feed of 10.8% to 14.2%. The increased

SDA associated with larger meal sizes is considered a result of up-regulation of intestinal nutrient transporters occurring directly after ingestion (Secor and Diamond 1997; Secor and

Faulkner 2002; Clark et al. 2010; Whitlock et al. 2013). Such that, as the meal size increases, the body must respond to excess matter by producing additional enzymes, increasing the energy required to breakdown excess matter in the stomach and intestines. Whitlock et al.

(2013) reported tuna demand elevated rates of ATP, lipid oxidation, and protein metabolism as meal size increases. In the same way, marine toads fed a diet of hard bodied superworms and crickets as compared with soft bodied earthworms and juvenile rats will experience nearly double the cost of digestion for the harder items, with an added increase in enzymatic activity, acids, and mechanical blending (Secor and Faulkner 2002).

Similarly, a number of studies specifically aimed at yellowtail have noted a unique metabolic response centered on feed structure. Watanabe et al. (1998) explored yellowtail digestive responses to various diets prepared with either a small-sized twin screw extruder or large-screw extruder. They noted differences in stomach content evacuation times for the two diet compositions in unison with variations in plasma free amino acid profiles (Watanabe et al.

1998). As the results from the previous work indicated a potential source of variation

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attributed to the structure of meal type, a further study was undergone on yellowtail to focus

on the physical attributes of dietary sources utilizing three distinct pellet designs. Significant

differences in digesta evacuation time and plasma amino acid levels were detected, with

responsibility for variability placed on the physical properties of the diets (Watanabe et al.

2001). Gastric evacuation time related to the structure of the feed resulted in a delay in meal

disintegration for the large-sized twin-screw extruder shape whereas the small-sized twin

extruder and the single moist pellets were physically broken down in nearly a quarter of the

time (Watanabe et al. 2001). The results from these studies suggest an increased effort may be

allotted towards digestion of more complex and heavily structured meal items.

The differences arising in SDA and SDAcoefficient between the two treatments are

interesting from a physiological standpoint as well as a management perspective. The lower coefficient seen in the commercial feed suggests the fish are able to more efficiently digest the processed material and allocate less energy towards the mechanical aspect thereof. As the coefficients indicated a greater amount of energy was directed towards digestion for the sardine feed, it would be expected that commercial feed would be the obvious choice for production. Less energy would be placed towards the breakdown of the formulated feed both from a size and rigidity perspective and could be allocated towards increased somatic growth.

F. A Description of Routine Movement for Different Feed Types

Energy expenditure is closely linked with the life history of a given species, with specific

limits setting the level of sustainable function (Bacigalupe et al. 2002). The importance of

monitoring motion during metabolic studies is critical when properly interpreting results, as

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changes in activity due to the presence of food can potentially skew data and misrepresent a

metabolic event (Du Preez et al. 1986). Actively monitoring the spontaneous swim behavior of fish has been explored as a means to demonstrate the source of variability in metabolic rates

(Forstner and Wieser 1990). For example, temperature has been a major predictive variable in the behavioral responses of salmon, with reactionary movement described as a means to mitigate physiological imbalances arising from metabolic requirements (Breau et al. 2011).

Similarly, the influence of diet has been a predictive variable in terms of behavior for Thunnus maccoyii which have exhibited increased swim velocity post feeding events, a behavioral trait directed at increasing ventilation volume in response to elevated metabolic rates (Fitzgibbon et al. 2007). High oxygen demand post feeding can potentially reduce the scope for activity, an effect that severely impacts the expansive abilities for a given organism (Soofiani and Hawkins

1982).

While little is known about the behavior of yellowtail during feeding events, the results of this study have provided valuable information concerning yellowtail movements.

Measurements of swim speed by video analyses were taken throughout this study and indicated a 12.3% increase in movement during the sardine flesh treatment as compared with

the formulated diet. This information provides a possible source of the significant differences in routine metabolism seen in the two meal types. In a previous study by Palstra et al. (2014), yellowtail motion within a static resting compartment was described as spontaneous with preference to swim speeds below 1 BL s-1. And although an exact measure of swim velocity was not calculated in this trial, it was speculated that the average amount of movement while within the static respirometer was similar to the results found by Palstra et al. (2014). The first

77

portion of this study estimated the baseline metabolic rate during a period of fasting at a swim

-1 -1 -1 velocity of 1 BL s to be in the range of 216-233 mgO2 kg h , while the baselineRMR for both diets in the static trials were largely below this estimate, with the commercial feed baseline

-1 -1 -1 -1 values between 115-127 mgO2 kg h and 143-157 mgO2 kg h for the sardine feed. Baseline

metabolic rates for the fish held in a static environment was calculated to be 1.4 to 1.8 times less than at a controlled swim speed of 1 BL s-1, and although the study design was considerably

different between each system, it is reasonable to propose the routine metabolic rate for yellowtail in this trial was descriptive of swim movement below 1 BL s-1. A possible strategy for the future would be to allocate time spent swimming with telemetry devices. Allocation of energy for swim speeds in this range show preference for growth in specific muscle regions

(Moyes et al. 1992; Moyes and West 1995; Richards et al. 2002. As fish swim, they select the slow-twitch red muscle fiber types thus activity within the respirometer could be associated with the selection for these fibers within the diet type. Alternatively running a transcriptomic screen on the red muscle in each diet type could potentially show more aerobic training with the sardine diet. In addition, directing a proportion of focus towards the biochemical interactions during fuel utilization in respect to swim speed provides an effective means to determine fuel use and regulation of associated enzymes during a number of hydrodynamic settings (Richards et al. 2002). As aquaculture facilities experience a wide range of environmental influences, factoring in the synergistic effects of water motion and nutrition will aid in the development of feeds.

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G. Digestibility of Various Feed Types

The mass of remaining expelled fecal matter indicated the feeds were accepted well and almost entirely digested. Although the input from the sardine treatment was nearly three times the total mass as the pelleted form, the output from both treatments were nearly identical. Hence, the sardine treatment was reduced nearly 26 times its original mass whereas the commercial feed was only reduced by 9 times its original size. Therefore, the difference in digestive responses is partially attributed to the mechanical energy associated with the breakdown of larger meal size. However, the difference in metabolic response is not entirely a result of meal mass. As both the feed preparation and nutritional constituents within the diets

were different, it would be expected the composition of the expelled matter would follow suit.

Yellowtail feces has been estimated to compose roughly 7-9% of the total feed input when

given a raw fish diet as compared with 12-15% on moist pellet, and nearly 27% for dry pellets

(Tanaka 1977; Yokoyama 2010). The results from this study however were not in agreement with the previously mentioned works. The sardine fillets were found to have a lower digestibility estimate than that of the commercial feed, with the total remaining undigested matter being 4.7% and 2.3% respectively.

Understanding the influence of various dietary interactions and their role towards the growth and physiology of an organism is a difficult task. Fish are accustomed to a specific ratio of nutritional components, and assimilate feeds at different rates depending on how well the item matches their ideal digestive configuration. Differences in absorption rates for various feeds show changes in the form of the postprandial metabolic curves (Thomson 2008) with

79

specific components influencing digestion at various timing intervals (Murray 1994). The

composition of the feces was found to be significantly different after 24 h, though no effect was

detected at 48 h, likely a result of nutrient leaching. The remaining protein and lipid content

within the feces at 24 h was nearly double in the sardine feed (25kJ and 13kJ) over the

commercial feed (10kJ and 5kJ) and a slightly higher degree of digestibility for the latter was

therefore noted. The higher remaining proportion of protein and lipid content in the sardine

feces is interesting as this diet originally contained greater proportions of both. Leaching rate

may be a major source of variability as other works have documented significant differences in

total feed waste and feces for various compositions of fish feed (Fernandes and Tanner 2008;

Lee et al. 2009). This may also indicate a lack of digestibility or an increased cost to digestion

for sardine versus the formulated diet, reflective in the higher metabolic rate seen for sardine.

While the proportion of feces being evaluated comprises only a small percentage of the total

amount of feed energy, it is something to consider, as excess waste for large-scale operations

can have detrimental effects on the surrounding (Xu et al 2007).

VI. Conclusions

The process of feed formulation is likely the most critical step towards developing the

aquaculture industry, accounting for one of the principal expenses facing all operations. The

results of this study indicate that by introducing a formulated feed into the diet of yellowtail,

significant energy costs of digestion can be reduced. The digestive efficiency, as indicated by

the SDACoefficent, was reduced from nearly one-third to one-tenth of the total value of the

presented meal. As both of the meals consumed in this study were equal in terms of total

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calories as a percent of the subject’s mass, the differences found are thought to have been

caused from the size, structure, and to some degree the composition of each. Information from

this type of analysis can be used by aquaculture managers towards making informed decisions

on the effectiveness of particular feeds from a biological perspective, timing of feeding events, and aid in the direction of future operating conditions. This information is valuable not only for choices faced by aquaculture managers but also ecosystem modelers. Understanding the value of forage items in a natural setting indicates preference for key habitat use areas as well as movement patterns. Wild stock assessments should explore the physiological expenditures associated with each prey item and how the influence of dietary intake controls their distribution.

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Chapter 4: Summary and Conclusions

The success of any aquaculture facility relies heavily upon the ability of managers to streamline the grow-out process, ensuring the input costs are continually less than the return

they are receiving for an end product. These input costs vary with each type of system and

choice of organism, however the most influential input for any operation will likely be the feed

costs. Tracing the path of feed from the moment of ingestion to later phases of growth and

excretion has proven extremely difficult due to the nature of marine species. Numerous

studies have attempted to quantitatively describe dietary efficiency however no method more

accurately embodies the digestive process as respirometry. Respirometry is a method that

produces a computation for respiration with end products capable of being expressed in either

a gaseous value or caloric equivalent. The results from this type of analysis can be compared

with the initial meal energy to provide an estimate towards the efficiency of digestion.

Reducing the total energy necessary for digestion will lower operating costs for facility

managers by partitioning a greater percent of the input product towards growth.

The results from the 30 L flume-type swim respirometer indicate a relatively consistent

range of values to describe yellowtail metabolism. Within this scenario (1 BL s-1), baseline

respiration values were used against post-ingestion estimates to describe the digestive

efficiency of a sardine meal. Baseline and postprandial metabolic values fell slightly above the

range determined in other yellowtail studies, however differences were most likely to have

arisen from variability in methodology and equipment, and temperatures of prior

measurements. Specific dynamic action within the methodologies of this study did fall within a

82

similar range as a number of other works, with energy allocation of specific dynamic action in

the range of 30 to 42%. The results of this work indicate a considerably large percent of the

total energy of a meal placed towards digestion in yellowtail, a common theme for a number of

other active pelagic fish of similar proportion. This information is valuable as a tool for

comparison against future nutritional studies based on dietary source. The results indicate the

swim respirometry environment produced consistent values for respiration and while this may

inaccurately describe the routine behavior for this species, the design of this system ensures a

high level of consistency in respect to motion. Overall there is a higher cost of both RMR and

SDA that can, in part, be attributed to the combined costs of digestion and locomotor metabolic inputs. Therefore, the fish potentially cannot devote as much energy towards digestion while swimming.

The second experiment placed the yellowtail within a static flow system in which movement was not controlled or forced and therefore explored the differences in digestive efficiency arising from a meal of sardine flesh versus that of a commercially available formulated pellet. Meal energy between the two feeds was a consistent ratio in terms of 5.2% of the body weight of the subject however the results indicated significant differences in respiration reflecting distinct energetic costs related to diets. By introducing a formulated feed to the diet of yellowtail routine metabolic rate was lowered. In addition, the cost of digestion, or SDACoefficient, was reduced from one-third to nearly one-tenth of the total meal value. The

greater mass and structural integrity of the sardine fillet was thought to contribute to an

increase in the up-regulation of digestive enzymes as well as added mechanical digestion,

further escalating the respiration rate. The commercial pelleted diet had been manufactured

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and processed prior to feeding and therefore was structurally less bound. In essence, the pellet

had gone through an extensive treatment, breaking down the building blocks constituting its

assembly, and allowing for a more efficient digestion within the body. In addition, the sardine

treatment contained 21% and 32% greater loads of protein and lipids, respectively which energetically take longer to digest and cost more energy. These noted differences in metabolism between treatments could be viewed in terms of protein assimilation in which the fish are reacting to a level of nourishment, as seen in the resulting postprandial curve. Routine motion was monitored throughout the postprandial period and indicated the sardine treatment did have a slightly higher rate of motion and was perhaps another source of the difference in metabolism.

There were substantial differences in the design of the two studies, with fish placed in a forced swim system (1 BL s-1) in the preceding 30 L trial, while the second trial was within a

1,237 L static open water respirometer. In addition, the fish in the 30 L trial were fed at a rate of 5% BW while the 1,237 L at a rate of 5.2% BW. There is still value in looking at these trials for a rough comparison, as the study subjects were both in a similar size range and fed nearly identical ration sizes relative to their total BW. While a statistical analysis between the two trials was not undergone, exploring the sardine feed results offered a rather interesting comparison. The total amount of energy required for digestion relative to the presented meal energy (SDACoefficient) was approximately 30% greater for the sardine meal in the forced swim scenario as compared with the static system. The larger amount of fuel allocated towards digestion could potentially be a result of additional costs associated with increased motion.

Also, the time required for digestion (SDADuration) was nearly 3 hours longer for the forced swim

84

system, adding yet another factor that may have contributed towards the differences in respiration. Both the baselineRMR and SDAMAX values had a considerably large span as compared

with the forced swim scenario, producing respiration rates nearly 1.5 times as great as within the static system. The substantially lower operating cost in the static system indicates the fish likely operate at a slightly lower metabolic rate than within the forced scenario of 1 BL s-1 in a

contained system, as indicated by the factorial scope of 1.8 and 1.9, respectively.

From an aquaculture perspective, the static system is thought to more accurately describe a

land-based facility with little to no water movement, whereas the forced swim scenario would

likely mimic conditions in an open ocean arena, or penned fish, with increased movement of

water due to currents and winds, and potentially require additional energy allocation towards muscular maintenance, buoyancy constraints, and other added costs of motion. Further

studies on exact current rates within ocean pens would be valuable for project design, as

energy allocation will vary with added maintenance costs from elevated activity. Likewise, energy storage in the body has been shown to fluctuate with movement and therefore a

detailed analysis of muscle development across various levels of water motion would be

advantageous for both site selection and fuel sourcing.

The data collected from this type of analysis stresses the importance of further integrating

marine aquaculture into the global food production system. Ocean farming offers an attractive

alternative to existing terrestrial farming methods, whereas aquatic species are able to convert

fuel sources into flesh in a more economic fashion. While the digestive efficiency of a natural

sardine diet offers minimal advantages as compared with estimates of terrestrial SDA

efficiencies, the formulated diet did prove to substantially reduce the total cost of digestion. 85

Sustainable and affordable are somewhat synonymous in light of rising costs of aquafeed and

the economics of limited resources, therefore assigning a value to continuing farming practices

at existing rates. As farm managers learn more about the physiology of their subjects, the overall efficiency of their facilities should simultaneously improve and optimize. For example,

information related to the duration of digestion can aid in determining appropriate feeding

schedules, leading to a reduction in excess waste byproducts from overindulgence.

The use of physiological analyses such as respirometry provides an added vantage point to describe the change in matter during a grow operation and estimates of SDA can offer a useful metric for evaluating feed types. The cost of digestion can significantly impact the overall growth of an organism, efficiency of a feed type, and expenditures associated with a grow operation. Aquaculture managers can benefit from the findings of this type of analysis as a means to interpret growth models and matter exchange for their broodstock. Additional studies concentrated on feed type, pellet structure, and protein substitutes would facilitate a better understanding of an ideal nutritional source for yellowtail. The future of sustainable aquaculture will heavily depend on strategy to overcome existing operational downfalls, innovations to promote long-term solutions, and realistic planning for future industry growth.

86

Bibliography

Aguila, J., Cuzon, G., Pascual, C., Domingues, P. M., Gaxiola, G., Sánchez, A., & Rosas, C. (2007). The effects of fish hydrolysate (CPSP) level on Octopus maya (Voss and Solis) diet: digestive enzyme activity, blood metabolites, and energy balance. Aquaculture, 273(4), 641-655. Alsop, D., & Wood, C. (1997). The interactive effects of feeding and exercise on oxygen consumption, swimming performance and protein usage in juvenile rainbow trout (Oncorhynchus mykiss). The Journal of experimental biology, 200(17), 2337-2346. Andrade, D. V., Cruz-Neto, A. P., Abe, A. S., & Wang, T. (2005). Specific dynamic action in ectothermic vertebrates: A review of the determinants of postprandial metabolic response in , amphibians, and reptiles. Physiological and Ecological Adaptations to Feeding in Vertebrates, 305-324. Anraku, M., & Azeta, M. (1965). The feeding habits of larvae and juveniles of the yellowtail, Seriola quinqueradiata Temminck et. Schlegel, associated with floating seaweeds. Bull. Seikai Reg. Fish. Res. Lab, 33, 13-45. Aragão, C., Conceição, L. E., Dias, J., Marques, A. C., Gomes, E., & Dinis, M. T. (2003). Soy protein concentrate as a protein source for Senegalese sole (Solea senegalensis Kaup 1858) diets: effects on growth and amino acid metabolism of postlarvae. Aquaculture Research, 34(15), 1443-1452. Averett, R. C. (1968). Influence of temperature on energy and material utilization by juvenile coho salmon. (Published doctoral dissertation). Oregon State University. Bacigalupe, L. D., & Bozinovic, F. (2002). Design, limitations and sustained metabolic rate: lessons from small mammals. Journal of Experimental Biology, 205(19), 2963-2970. Ballantyne, J. S. (2001). Amino acid metabolism. Fish physiology 20, 77-107. Barton, B. A., & Schreck, C. B. (1987). Metabolic cost of acute physical stress in juvenile steelhead. Transactions of the American Fisheries Society, 116(2), 257-263. Baxter, J. L. (1960). A study of the yellowtail, Calif. Dep. Fish and Game, Fish Bull. (110), 1-91. Blaxter, K. L. (1989). Energy metabolism in animals and man. CUP Archive. Blyth, D., Tabrett, S., Bourne, N., & Glencross, B. (2015). Comparison of faecal collection methods and diet acclimation times for the measurement of digestibility coefficients in barramundi (Lates calcarifer). Aquaculture Nutrition, 21(2), 248-255. Beamish, F. W. H. (1964). Respiration of fishes with special emphasis on standard oxygen consumption: II. Influence of weight and temperature on respiration of several species. Canadian Journal of , 42(2), 177-188.

87

Beamish, F. W. H. (1974). Apparent specific dynamic action of largemouth bass, Micropterus salmoides. Journal of the Fisheries Board of Canada 31(11), 1763-1769. Beamish, F. W. H. (1984). Swimming performance of three southwest Pacific fishes. Marine biology 79(3), 311-313. Beamish, F. W. H., & Trippel, E. A. (1990). Heat increment: a static or dynamic dimension in bioenergetic models? Transactions of the American Fisheries Society, 119(4), 649-661. Belokopytin, Y.S., (2004). Specific dynamic action of food and energy metabolism of fishes under experimental and natural conditions. Hydrobiology. J. 40, 68–75. Bell, W. H. & Turhune, L. D. B. (1970). Water tunnel design for fisheries research. Can. Fish. Res. Board Tech. Rep. 195, 1-69. Bianchi, G., K. E. Carpenter, J.P. Roux, F. J. Molloy, D. Boyer, and H. J. Boyer. (1993). FAO species identification field guide for fishery purposes. The living marine resources of Namibia. FAO, Rome. Billerbeck, J. M., Lankford, T. E., & Conover, D. O. (2001). Evolution of intrinsic growth and energy acquisition rates. I. Trade‐offs with swimming performance in Menidia. Evolution, 55(9), 1863-1872. Blaikie, H. B., & Kerr, S. R. (1996). Effect of activity level on apparent heat increment in Atlantic cod, Gadus morhua. Canadian Journal of Fisheries and Aquatic Sciences, 53(9), 2093-2099. Blanco Garcia, A., Partridge, G. J., Flik, G., Roques, J. A., & Abbink, W. (2015). Ambient salinity and osmoregulation, energy metabolism and growth in juvenile yellowtail kingfish (Seriola lalandi Valenciennes 1833) in a recirculating aquaculture system. Aquaculture Research, 46(11), 2789-2797. Booth, M. A., M. D. Moses, & G. L. Allan. (2013). Utilization of carbohydrate by yellowtail kingfish Seriola lalandi. Aquaculture 376, 151-161. Bowen, S. H. (1987). "Dietary protein requirements of fishes-a reassessment. “Canadian Journal of Fisheries and Aquatic Sciences 44(11), 1995-2001. Breau, C., Cunjak, R. A., & Peake, S. J. (2011). Behavior during elevated water temperatures: can physiology explain movement of juvenile Atlantic salmon to cool water? Journal of Animal Ecology, 80(4), 844-853. Brett, J. R., & D. A. Higgs. (1970). Effect of temperature on the rate of gastric digestion in finger ling sockeye salmon, Oncorhynchus nerka. Journal of the Fisheries Board of Canada 27(10), 1767-1779. Brett, J. R., & Groves, T. D. D. (1979). Physiological energetics. Fish physiology, 8(6), 280- 352.

88

Brill, R. W. (1987). On the standard metabolic rate of tropical tunas, including the effect of body size and acute temperature change. Fishery Bull. Fish. Wildl. Serv. U.S. 85, 25– 35. Brown, C. R., & Cameron, J. N. (1991a). The induction of specific dynamic action in channel catfish by infusion of essential amino acids. Physiological zoology, 276-297. Brown, C. R., & Cameron, J. N. (1991b). The relationship between specific dynamic action (SDA) and protein synthesis rates in the channel catfish. Physiological Zoology, 298- 309. Brown, J. H., Gillooly, J. F., Allen, A. P., Savage, V. M., & West, G. B. (2004). Toward a metabolic theory of ecology. Ecology, 85(7), 1771-1789. Brown, Elliot J., et al. (2011). Do swimming fish always grow fast? Investigating the magnitude and physiological basis of exercise-induced growth in juvenile New Zealand yellowtail kingfish, Seriola lalandi. Fish physiology and biochemistry 37(2), 327-336. Calow, P. T. (1975). The feeding strategies of two freshwater gastropods, Ancylus fluviatilis Müll. and Planorbis contortus Linn. (Pulmonata), in terms of ingestion rates and absorption efficiencies. Oecologia 20(1), 33-49. Calow, P. (1977). Conversion efficiencies in heterotrophic organisms. Biological Reviews 52(3), 385-409. Chakraborty, S. C., Ross, L. G., & Ross, B. (1992). Specific dynamic action and feeding metabolism in common carp, Cyprinus carpio L. Comparative Biochemistry and Physiology Part A: Physiology, 103(4), 809-815. Chen, B. N., Qin, J. G., Kumar, M. S., Hutchinson, W., & Clarke, S. (2006). Ontogenetic development of the digestive system in yellowtail kingfish Seriola lalandi larvae. Aquaculture, 256(1), 489-501. Cho, C. Y., S. J. Slinger, & H. S. Bayley. (1982). Bioenergetics of salmonid fishes: energy intake, expenditure and productivity. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 73(1), 25-41. Cho, C. Young, & D. P. Bureau. (1995). Determination of the energy requirements of fish with particular reference to salmonids. Journal of Applied Ichthyology 11(3‐4), 141-163. Christel, C. M., DeNardo, D. F., & Secor, S. M. (2007). Metabolic and digestive response to food ingestion in a binge-feeding lizard, the Gila monster (Heloderma suspectum). Journal of Experimental Biology, 210(19), 3430-3439. Clark, T. D., & R. S. Seymour. (2006). Cardiorespiratory physiology and swimming energetics of a high-energy-demand teleost, the yellowtail kingfish (Seriola lalandi). Journal of Experimental Biology 209(19), 3940-3951. Clark, T. D., Brandt, W. T., Nogueira, J., Rodriguez, L. E., Price, M., Farwell, C. J., & Block, B. A. (2010a). Postprandial metabolism of Pacific Bluefin tuna (Thunnus orientalis). The Journal of experimental biology, 213(14), 2379-2385. 89

Clark, T. D., Sandblom, E., Hinch, S. G., Patterson, D. A., Frappell, P. B., & Farrell, A. P. (2010b). Simultaneous biologging of heart rate and acceleration, and their relationships with energy expenditure in free-swimming sockeye salmon (Oncorhynchus nerka). Journal of Comparative Physiology B, 180(5), 673-684. Collins, R. A. (1973). The status of the California yellowtail resource and its management. California Department of Fish and Game. MRTR (16), 1-17. Coughlin, David J. (2002). Aerobic muscle function during steady swimming in fish. Fish and Fisheries 3(2), 63-78. De la Gándara, F., A. Garcıa-Gómez, & M. Jover. (2002). Effect of feeding frequency on the daily oxygen consumption rhythms in young Mediterranean yellowtails (Seriola dumerili). Aquacultural engineering 26(1), 27-39. Delgado, C. L., Wada, N., Rosegrant, M. W., Meijer, S., & Ahmed, M. (2003). Outlook for fish to 2020: meeting global demand (Vol. 15). Intl Food Policy Res Inst. Dewar, H., & J. Graham. (1994). Studies of tropical tuna swimming performance in a large water tunnel-energetics. Journal of Experimental Biology 192(1), 13-31. Dickson, K. A., Donley, J. M., Sepulveda, C., & Bhoopat, L. (2002). Effects of temperature on sustained swimming performance and swimming kinematics of the chub mackerel Scomber japonicus. Journal of experimental biology, 205(7), 969-980. Dotson, R., & Charter, R. (2003). Trends in the Southern California sport fishery. CALIFORNIA COOPERATIVE OCEANIC FISHERIES INVESTIGATIONS REPORT, 94- 106. Drew, M. D., Ogunkoya, A. E., Janz, D. M., & Van Kessel, A. G. (2007). Dietary influence of replacing fish meal and oil with canola protein concentrate and vegetable oils on growth performance, fatty acid composition and organochlorine residues in rainbow trout (Oncorhynchus mykiss). Aquaculture, 267(1), 260-268. du Preez, H. H., Strydom, W., & Winter, P. E. D. (1986). Oxygen consumption of two marine teleosts, Lithognathus mormyrus (linnaeus, 1758) and Lihognathus lithognathus (cuvier, 1830) (Teleosti: Sparidae). Comparative Biochemistry and Physiology Part A: Physiology, 85(2), 313-331. Econsearch (2014) The economic impact of aquaculture on the South Australian state and regional economies, 2012/13. Econsearch, Adelaide. Available at www.pir.sa. gov.au/__data/assets/ Edwards, P., L. Tuan, & G. L. Allan. (2004). A survey of marine trash fish and fishmeal as aquaculture feed ingredients in Vietnam. Canberra, Australia, ACIAR© Australian Centre for International Agricultural Research 56. Eliason, E. J., Higgs, D. A., & Farrell, A. P. (2007). Effect of isoenergetic diets with different protein and lipid content on the growth performance and heat increment of rainbow trout. Aquaculture, 272(1), 723-736.

90

Ellerby, D. J., & Herskin, J. (2013). Swimming Flumes as a Tool for Studying Swimming Behavior and Physiology: Current Applications and Future Developments. Swimming Physiology of Fish. 345-375. Elliott, J. M., & Davison, W. (1975). Energy equivalents of oxygen consumption in animal energetics. Oecologia, 19(3), 195-201. Eschmeyer, W.N., E.S. Herald & H. Hammann, (1983). A field guide to Pacific coast fishes of North America. Houghton Mifflin Company, Boston, U.S.A. 1-336. Estess, E. E., Coffey, D. M., Shimose, T., Seitz, A. C., Rodriguez, L., Norton, A., Block, B. & Farwell, C. (2014). Bioenergetics of captive Pacific Bluefin tuna (Thunnus Orientalis). Aquaculture, 434, 137-144. Fagbenro, O., & Jauncey, K. (1995). Water stability, nutrient leaching and nutritional properties of moist fermented fish silage diets. Aquacultural Engineering, 14(2), 143- 153. Farrell, D. J. (1974). "General principles and assumptions of calorimetry." Energy requirements of poultry 1-24. Farwell, C. J., B. A. Block, & E. D. Stevens. (2001). "Tunas in captivity." Fish Physiology 19, 391-412. Fernandes, M., & Tanner, J. (2008). Modeling of nitrogen loads from the farming of yellowtail kingfish Seriola lalandi (Valenciennes, 1833). Aquaculture Research, 39(12), 1328-1338. Fitzgibbon, Q. P., A. Strawbridge, & R. S. Seymour. (2007). "Metabolic scope, swimming performance and the effects of hypoxia in the mulloway, japonicus (Pisces: )." Aquaculture 270(1), 358-368. FOA/GLOBEFISH, (2007). Ever-growing demands and extreme supply constraints pushed fishmeal/oil prices to a record high in 2014; Fishmeal and Fish Oil November 2014. GLOBEFISH Market Reports 2014, (http://www.globefish.org/ever-growing-demands- and-extreme-supply-constraints-pushed-fishmeal-oil-prices-to-a-record-high-in- 2014.html). FOA (2014a). Food Outlook– Biannual Report on Global Food Markets. October 2014. FOA, Rome. 1-143. FAO, (2014b). The State of World Fisheries and Aquaculture 2014. FOA Fisheries Technical Paper, FOA, Rome. 1-110. FAO (2016). Fisheries and aquaculture software. FishStatJ - software for fishery statistical time series. In: FAO Fisheries and Aquaculture Department [online]. Rome. [Cited 12 April 2016]. Forstner, H., & Wieser, W. (1990). Patterns of routine swimming and metabolic rate in juvenile cyyprinids at three temperatures: analysis with a respirometer-activity-

91

monitoring system. Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology, 160(1), 71-76. Fowler A.J., Ham J.M. & Jennings P.R. (2003). Discriminating between Cultured and Wild Yellowtail Kingfish (Seriola lalandi) in South Australia, SARDI Aquatic Sciences Publication. South Australian Research and Development Institute (Aquatic Sciences), Adelaide, Australia. 03, 159. Frankic, A., and C. Hershner. (2003). Sustainable aquaculture: developing the promise of aquaculture. Aquaculture International 11.6: 517-530. Freund, E. V. (1999). Comparisons of metabolic and cardiac performance in scombrid fishes: insights into the evolution of endothermy (Doctoral dissertation, Stanford University). Fu, S. J., Xie, X. J., & Cao, Z. D. (2005a). Effect of dietary composition on specific dynamic action in southern catfish Silurus meridionalis Chen. Aquaculture Research, 36(14), 1384-1390. Fu, S. J., Xie, X. J., & Cao, Z. D. (2005b). Effect of meal size on postprandial metabolic response in southern catfish (Silurus meridionalis). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 140(4), 445-451. Fu, S. J., Zeng, L. Q., Li, X. M., Pang, X., Cao, Z. D., Peng, J. L., & Wang, Y. X. (2009). Effect of meal size on excess post-exercise oxygen consumption in fishes with different locomotive and digestive performance. Journal of Comparative Physiology B, 179(4), 509-517. Furnell DJ (1987) Partitioning of locomotor and feeding metabolism in sablefish (Anoplopoma fimbria). Canadian Journal of Zoology (65), 486–489. Furukawa, K., Yamamori, K., Satoh, H., & Nimura, Y. (1992). Changes in oxygen consumption of the yellowtail Seriola quinqueradiata due to feeding. Nippon Suisan Gakkaishi, 58(7), 1383-1383. Gibb, A. C., & Dickson, K. A. (2002). Functional morphology and biochemical indices of performance: is there a correlation between metabolic enzyme activity and swimming performance? Integrative and Comparative Biology, 42(2), 199-207. Gill, T. (1863). Catalogue of the fishes of Lower California, in the Smithsonian Institution, collected by Mr. J. Xantus. Phila. Acad. Nat. Sci., 2, 465-465. Gillanders, B. M., Ferrell, D. J., & Andrew, N. L. (2001). Estimates of movement and life history parameters of yellowtail kingfish (Seriola lalandi): how useful are data from a cooperative tagging programme? Marine and Freshwater Research, 52(2), 179-192. Gouveia, A., & Davies, S. J. (2000). Inclusion of an extruded dehulled pea seed meal in diets for juvenile European sea bass (Dicentrarchus labrax). Aquaculture, 182(1), 183- 193.

92

Grisdale-Helland, B., Ruyter, B., Rosenlund, G., Obach, A., Helland, S. J., Sandberg, M. G., & Røsjø, C. (2002). Influence of high contents of dietary soybean oil on growth, feed utilization, tissue fatty acid composition, heart histology and standard oxygen consumption of Atlantic salmon (Salmo salar) raised at two temperatures. Aquaculture, 207(3), 311-329. Grottum, J. A., & Sigholt, T. (1998). A model for oxygen consumption of Atlantic salmon (Salmo salar) based on measurements of individual fish in a tunnel respirometer. Aquacultural engineering, 17(4), 241-251. Hailey, A. (1998). The specific dynamic action of the omnivorous tortoise Kinixys spekii in relation to diet, feeding pattern, and gut passage. Physiological and Biochemical Zoology, 71(1), 57-66. Hanyu, I., K. Tsukamoto, K. Yamamori, P. Van Ngan, K. Furukawa, & T. Hibiya. (1979). Simultaneous recording of physiological information from swimming fish. Bulletin of the Japanese Society of Scientific Fisheries. 45(10), 1261-1265. Hardy, R. W. (1995). Current issues in salmonid nutrition. Nutrition and utilization technology in aquaculture, 26-35. Hardy, R. W., & Tacon, A. G. (2002). Fish meal: historical uses, production trends and future outlook for sustainable supplies. Responsible marine aquaculture, 311-325. He, E., & Wurtsbaugh, W. A. (1993). An empirical model of gastric evacuation rates for fish and an analysis of digestion in piscivorous brown trout. Transactions of the American Fisheries Society, 122(5), 717-730. Helland, S. J., Hatlen, B., & Grisdale-Helland, B. (2006). Changes in the utilization of energy and amino acids caused by feed intake level–implications for amino acid requirement studies. In XII Int. Symp. Fish Nutrition and Feeding. Heine, J. & Kolkovski, S. (2004). Potential aquaculture species for Western Australia, yellowtail kingfish Seriola lalandi. Fish Res. Rep., department of Fisheries, Western Australia. Hill, A. M., Buckley, J. D., Murphy, K. J., & Howe, P. R. (2007). Combining fish-oil supplements with regular aerobic exercise improves body composition and cardiovascular disease risk factors. The American journal of clinical nutrition, 85(5), 1267-1274. James WPT. (1992). In: Kinney JM and Tucker HN, eds. Energy metabolism: tissue determinants and cellular corollaries. New York: Raven Press, 163-183. Jensen, K., Mayntz, D., Wang, T., Simpson, S. J., & Overgaard, J. (2010). Metabolic consequences of feeding and fasting on nutritionally different diets in the wolf spider Pardosa prativaga. Journal of physiology, 56(9), 1095-1100. Jirsa, D., Davis, A., Stuart, K., & Drawbridge, M. (2011). Development of a practical soy‐ based diet for California yellowtail, Seriola lalandi. Aquaculture Nutrition, 17(4), 869-874.

93

Jobling, M., & Davies, P. S. (1980). Effects of feeding on metabolic rate, and the specific dynamic action in plaice, Pleuronectes platessa L. Journal of Fish Biology, 16(6), 629- 638. Jobling, M. (1983). Towards an explanation of specific dynamic action (SDA). Journal of Fish Biology, 23(5), 549-555. Jung, S., Pang, I. C., Lee, J. H., Choi, I., & Cha, H. K. (2014). Latitudinal shifts in the distribution of exploited fishes in Korean waters during the last 30 years: a consequence of climate change. Reviews in Fish Biology and Fisheries, 24(2), 443-462. Kasai, A., Sakamoto, W., Mitsunaga, Y., & Yamamoto, S. (2000). Behavior of immature yellowtails (Seriola quinqueradiata) observed by electronic data‐recording tags. Fisheries Oceanography, 9(3), 259-270. Kaushik, S. J., & Cho, C. Y. (2002). Dominique P. Bureau. Fish Nutrition, 1, 1-59. Kaushik, S. J., & Seiliez, I. (2010). Protein and amino acid nutrition and metabolism in fish: current knowledge and future needs. Aquaculture Research, 41(3), 322-332. Kieffer, J. D., Alsop, D. E. R. E. K., & Wood, C. M. (1998). A respirometric analysis of fuel use during aerobic swimming at different temperatures in rainbow trout (Oncorhynchus mykiss). Journal of Experimental Biology, 201(22), 3123-3133. Kiorboe, T., Møhlenberg, F., & Hamburger, K. (1985). Bioenergetics of the planktonic copepod Acartia tonsa: relation between feeding, egg production and respiration, and composition of specific dynamic action. Mar. Ecol. Prog. Ser, 26(1-2), 85-97. Kissinger, K. R., García-Ortega, A., & Trushenski, J. T. (2016). Partial fishmeal replacement by soy protein concentrate, squid and algal meals in low fish-oil diets containing Schizochytrium limacinum for Seriola rivoliana. Aquaculture, 452, 37- 44. Kofuji, P. Y. M., Akimoto, A., Hosokawa, H., & Masumoto, T. (2005). Seasonal changes in proteolytic enzymes of yellowtail Seriola quinqueradiata (Temminck & Schlegel; Carangidae) fed extruded diets containing different protein and energy levels. Aquaculture Research, 36(7), 696-703. Kolkovski, S. (2001). Digestive enzymes in fish larvae and juveniles—implications and applications to formulated diets. Aquaculture, 200(1), 181-201. Kolkovski, S., & Sakakura, Y. (2004). Yellowtail kingfish, from larvae to mature fish– problems and opportunities. Avances en Nutricíon Acuícola VII., Hermosillo, Sonora, México. Krogdahl, Å, Hemre, G. I., & Mommsen, T. P. (2005). Carbohydrates in fish nutrition: digestion and absorption in postlarval stages. Aquaculture nutrition, 11(2), 103-122. Kubitza, F., & Lovshin, L. L. (1999). Formulated diets, feeding strategies, and cannibalism control during intensive culture of juvenile carnivorous fishes. Reviews in Fisheries Science, 7(1), 1-22. 94

Kumar, V., Makkar, H. P. S., & Becker, K. (2010). Dietary inclusion of detoxified Jatropha curcas kernel meal: effects on growth performance and metabolic efficiency in common carp, Cyprinus carpio L. Fish physiology and biochemistry, 36(4), 1159-1170. Laidley C.W., Shields R.J. & Ostrowksi A.O. (2004) Progress in amberjack culture at The Oceanic Institute in Hawai‘i. Global Aquaculture Advocate, 7, 42–43. Lauff, M., & Hofer, R. (1984). Proteolytic enzymes in fish development and the importance of dietary enzymes. Aquaculture, 37(4), 335-346. Lauff, R. F., & Wood, C. H. (1996). Respiratory gas exchange, nitrogenous waste excretion, and fuel usage during aerobic swimming in juvenile rainbow trout. Journal of Comparative Physiology B, 166(8), 501-509. Lee, K. J., Dabrowski, K., Blom, J. H., & Bai, S. C. (2001). Replacement of fishmeal by a mixture of animal by-products in juvenile rainbow trout diets. North American Journal of Aquaculture, 63(2), 109-117. Lee, J. H., Jo, J. Y., Lee, S. M., Cho, S. H., Hutabarat, J., & Spj, N. T. (2009). Biological Estimation of Waste Products from Olive Flounder (Paralichthys olivaceus) Fed on Three Different Feed Types. Fisheries and aquatic sciences, 12(4), 317-323. LI-75.204 Fat by Acid Hydrolysis Gravimetric Method AOAC Procedure Lim, S. J., & Lee, K. J. (2009). Partial replacement of fishmeal by cottonseed meal and soybean meal with iron and phytase supplementation for parrot fish Oplegnathus fasciatus. Aquaculture, 290(3), 283-289. Lucas, M. C., & Priede, I. G. (1992). Utilization of metabolic scope in relation to feeding and activity by individual and grouped zebrafish, Brachydanio rerio (Hamilton‐ Buchanan). Journal of Fish Biology, 41(2), 175-190. Luo, Y., & Xie, X. (2008). Effects of temperature on the specific dynamic action of the southern catfish, Silurus meridionalis. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 149(2), 150-156. MacCall, A. D., Stauffer, G. D., & Troadec, J. P. (1976). Southern California recreational and commercial marine fisheries. Mar. Fish. Rev, 38(1), 1-32. Maetz, J. (1972). Branchial sodium exchange and ammonia excretion in the goldfish Carassius auratus. Effects of ammonia-loading and temperature changes. Journal of Experimental Biology, 56(3), 601-620. Martinez-Takeshita, N., Purcell, C. M., Chabot, C. L., Craig, M. T., Paterson, C. N., Hyde, J. R., & Allen, L. G. (2015). A tale of three tails: cryptic speciation in a globally distributed marine fish of the genus Seriola. Copeia, 103(2), 357-368. Masumoto, T. (2002). Yellowtail, Seriola quinqueradiata. Nutrient requirements and feeding of finfish for aquaculture, 131-146.

95

McCue, M. D. (2006). Specific dynamic action: a century of investigation. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 144(4), 381- 394. May, J.L. & Maxwell, J.G.H. (1986). Trawl fish from temperate waters of Australia. Tasmania, Australia, CSIRO Division of Fisheries Research. Mazzola, A., Favaloro, E., & Sarà, G. (2000). Cultivation of the Mediterranean amberjack, Seriola dumerili (Risso, 1810), in submerged cages in the Western . Aquaculture, 181(3), 257-268. McGaw, I. J., & Penney, C. M. (2014). Effect of meal type on specific dynamic action in the green shore crab, Carcinus maenas. Journal of Comparative Physiology B, 184(4), 425- 436. McKenzie, D. J. (2001). Effects of dietary fatty acids on the respiratory and cardiovascular physiology of fish. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 128(3), 605-619. McLeod, P., & McElroy, S. (2010). Does Large-Scale offshore Aquaculture Farming in Western Australia Make Economic Sense? The Case of Yellowtail Kingfish. Proceedings of the Fifteenth Biennial Conference of the International Institute of Fisheries Economics & Trade. Miller, D. J., & Lea, R. N. (1972). Guide to the coastal marine fishes of California (No. 154- 158). UCANR Publications. Miegel, R. P., Pain, S. J., Van Wettere, W. H. E. J., Howarth, G. S., & Stone, D. A. J. (2010). Effect of water temperature on gut transit time, digestive enzyme activity and nutrient digestibility in yellowtail kingfish (Seriola lalandi). Aquaculture, 308(3), 145-151. Molina‐Poveda, C., & Morales, M. E. (2004). Use of a mixture of barley‐based fermented grains and wheat gluten as an alternative protein source in practical diets for Litopenaeus vannamei (Boone). Aquaculture research, 35(12), 1158-1165. Moran, D., Gara, B., & Wells, R. M. (2007). Energetics and metabolism of yellowtail kingfish (Seriola lalandi Valenciennes 1833) during embryogenesis. Aquaculture, 265(1), 359- 369. Moyes, C. D., Schulte, P. M., & Hochachka, P. W. (1992). Recovery metabolism of trout white muscle: Role of mitochondria. American Journal of physiology, 262(2), 295-304. Moves, C. D., & West, T. G. (1995). Exercise metabolism of fish. Biochemistry and molecular biology of fishes, 4, 367-392. Muir, B. S., & Niimi, A. J. (1972). Oxygen consumption of the euryhaline fish aholehole (Kuhlia sandvicensis) with reference to salinity, swimming, and food consumption. Journal of the Fisheries Board of Canada, 29(1), 67-77. Murray, H. M., Wright, G. M., & Goff, G. P. (1994). A study of the posterior esophagus in the winter flounder, Pleuronectes americanus, and the yellowtail flounder, Pleuronectes 96

ferruginea: morphological evidence for pregastric digestion? Canadian Journal of Zoology, 72(7), 1191-1198. Nakada, M. (2000). Yellowtail and related species culture. In: Stickney, R.R (Ed.). 2000. Encyclopedia of Aquaculture. John Wiley & Sons, Inc. New York, NY. 1007-1036. Nakada, M. K. (2002). Yellowtail culture development and solutions for the future. Reviews in Fisheries Science, 10(3-4), 559-575. Nakada, M. (2008). Capture-based aquaculture of yellowtail. Capture-based aquaculture. Global overview. FAO Fisheries Technical Paper, 508, 199-215. Naylor, R. L., Goldburg, R. J., Primavera, J. H., Kautsky, N., Beveridge, M. C., Clay, J., Folke, C., Lubchenco, J., Mooney, H. & & Troell, M. (2000). Effect of aquaculture on world fish supplies. Nature, 405(6790), 1017-1024. Naylor, R., and M. Burke. (2005). Aquaculture and ocean resources: raising tigers of the sea. No. 463M: Operating Manual for the 6300 Isoperibol Calorimeter NP Analytical Laboratories (2012). Moisture, Force Draft Oven Methods. Test Codes: MOB, DMCP, MOA, MASB, MFDF, M100, M125, M130, MOWE. Nugroho, E., Ferrell, D. J., Smith, P., & Taniguchi, N. (2001). Genetic divergence of kingfish from Japan, Australia and New Zealand inferred by microsatellite DNA and mitochondrial DNA control region markers. Fisheries science, 67(5), 843-850. Oliva-Teles, A., & Kaushik, S. J. (1987). Metabolic utilization of diets by polyploid rainbow trout (Salmo gairdneri). Comparative Biochemistry and Physiology Part A: Physiology, 88(1), 45-47. Official Methods of Analysis of the AOAC International, Method 988.05; Method 920.87; Method 991.20. Official Methods of Analysis of the AOAC International Method 954.02, 922.06 (Modified), 933.05, 925.32, 950.54, 948.15 Ottolenghi, F., Silvestri, C., Giordano, P., Lovatelli, A., & New, M. B. (2004). Capture-based aquaculture: the fattening of eels, , tunas and yellowtails. FAO. Palstra, A. P., Mes, D., Kusters, K., Roques, J. A., Flik, G., Kloet, K., & Blonk, R. J. (2014). Forced sustained swimming exercise at optimal speed enhances growth of juvenile yellowtail kingfish (Seriola lalandi). Frontiers in physiology, 5. Pang, X., Cao, Z. D., & Fu, S. J. (2011). The effects of temperature on metabolic interaction between digestion and locomotion in juveniles of three cyprinid fish (Carassius auratus, Cyprinus carpio and Spinibarbus sinensis). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 159(3), 253-260. Pirozzi, I., & Booth, M. A. (2009). The routine metabolic rate of mulloway (Argyrosomus japonicus: Sciaenidae) and yellowtail kingfish (Seriola lalandi: Carangidae) acclimated 97

to six different temperatures. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 152(4), 586-592. Poortenaar, C. W., Hooker, S. H. and Sharp, N. (2001). Assessment of yellowtail kingfish (Seriola lalandi lalandi) reproductive physiology, as a basis for aquaculture development. Aquaculture 201, 271 -286. Priede, I. G. (1985). Metabolic scope in fishes. In Fish Energetics: New Perspectives (ed. P. Tytler and P. Calow), Baltimore: John Hopkins University Press. 33-67. Radovich, J. (1962). Effects of Water Temperature on the Distribution of Some Scombrid Fishes Along the Pacific Coast of North American. FAO of the United Nations. Fish. Rep. 3(6), 1459-1975. Richards, J. G., Mercado, A. J., Clayton, C. A., Heigenhauser, G. J., & Wood, C. M. (2002). Substrate utilization during graded aerobic exercise in rainbow trout. Journal of experimental biology, 205(14), 2067-2077. Robaina, L., Moyano, F. J., Izquierdo, M. S., Socorro, J., Vergara, J. M., & Montero, D. (1997). Corn gluten and meat and bone meals as protein sources in diets for gilthead seabream (Sparus aurata): nutritional and histological implications. Aquaculture, 157(3), 347-359. Robins, C. R., R. M. Bailey, C. E. Bond, J. R. Brooker, E. A. Lacher, R. N. Lea, and W. B. Scott. (1991). World fishes important to North Americans, exclusive of species from the continental waters of the United States and Canada. American Fisheries Society Special Publication 21, 1-243. Rolland, M., Larsen, B. K., Holm, J., Dalsgaard, J., & Skov, P. V. (2014). Effect of plant proteins and crystalline amino acid supplementation on postprandial plasma amino acid profiles and metabolic response in rainbow trout (Oncorhynchus mykiss). Aquaculture International, 1-17. Roo, J., Fernández‐Palacios, H., Hernández‐Cruz, C. M., Mesa‐Rodriguez, A., Schuchardt, D., & Izquierdo, M. (2014). First results of spawning and larval rearing of longfin yellowtail Seriola rivoliana as a fast‐growing candidate for European marine finfish aquaculture diversification. Aquaculture Research, 45(4), 689-700. Ross, L. G., McKinney, R. W., Cardwell, S. K., Fullarton, J. G., Roberts, S. E. J., & Ross, B. (1992). The effects of dietary protein content, lipid content and ration level on oxygen consumption and specific dynamic action in Oreochromis niloticus L. Comparative Biochemistry and Physiology Part A: Physiology, 103(3), 573-578. Sakakura, Y., & Tsukamoto, K. (1997). Age Composition in the Schools of Juvenile Yellowtail Seriola quinqueradiata Associated with Drifting Seaweeds in the East China Sea. Fisheries science, 63(1), 37-41. Samocha, T. M., Davis, D. A., Saoud, I. P., & DeBault, K. (2004). Substitution of fishmeal by co-extruded soybean poultry by-product meal in practical diets for the Pacific white shrimp, Litopenaeus vannamei. Aquaculture, 231(1), 197-203.

98

Sargent J.R. (1997). Fish oils and human diet. British Journal of Nutrition, 78, 5-13. Schmitt, R. J., & Strand, S. W. (1982). Cooperative foraging by yellowtail, Seriola lalandei (Carangidae), on two species of fish prey. Copeia, 3, 714-717. Secor, S. M., & Diamond, J. (1997). Determinants of the postfeeding metabolic response of Burmese pythons, Python molurus. Physiological zoology, 202-212. Secor, S. M., & Faulkner, A. C. (2002). Effects of meal size, meal type, body temperature, and body size on the specific dynamic action of the marine toad, Bufo marinus. Physiological and Biochemical Zoology, 75(6), 557-571. Secor, S. M. (2003). Gastric function and its contribution to the postprandial metabolic response of the Burmese python Python molurus. Journal of Experimental Biology, 206(10), 1621-1630. Secor, S. M. (2009). Specific dynamic action: a review of the postprandial metabolic response. Journal of Comparative Physiology B, 179(1), 1-56. Seth, H., Sandblom, E., & Axelsson, M. (2009). Nutrient-induced gastrointestinal hyperemia and specific dynamic action in rainbow trout (Oncorhynchus mykiss)—importance of proteins and lipids. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 296(2), 345-352. Sfakiotakis, M., Lane, D. M., & Davies, J. B. C. (1999). Review of fish swimming modes for aquatic locomotion. IEEE Journal of Oceanic Engineering, 24(2), 237-252. Sievert, L. M., Suita, Y., Mayes, D. M., Bontrager, L. R., Tuttle, M., & Everly, A. (2013). Specific dynamic action, postprandial thermophily, and the impact of temperature on gastric digestion in the cornsnake Pantherophis guttatus. Zoological Studies, 52(1), 1-9. Sims, N.A. 2013. Kona Blue Water Farms case study: permiting, operations, marketing, environmental impacts, and impediments to expansion of global open ocean mariculture. In A. Lovatelli, J. Aguilar-Manjarrez & D. Soto, eds. Expanding mariculture farther offshore: Technical, environmental, spatial and governance challenges. FAO Fisheries and Aquaculture Proceedings. Rome, FAO. 24, 263–296.

Simopoulos, A. P. (2002). The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomedicine & pharmacotherapy, 56(8), 365-379. Smith, E.J., Jr., and C.V. Paul. (1960). Maturity and fecundity. In J.L. Baxter (ed.), A study of the yellowtail (Gill), Calif. Dept. Fish Game Fish Bull. 110, 46-52. Smith-Vaniz, W. F. (1984). Carangidae. In W. Fischer and G. Bianchi, editors. FAO species identification sheets for fishery purposes. Western fishing area. FAO, Rome. 51(1). Smith-Vaniz, W. F., Kaufman, L. S., & Glowacki, J. (1995). Species-specific patterns of hyperostosis in marine teleost fishes. Marine Biology, 121(4), 573-580.

99

Soofiani, N. M., & Hawkins, A. D. (1982). Energetic costs at different levels of feeding in juvenile cod, Gadus morhua L. Journal of Fish Biology, 21(5), 577-592. Sousa, T., Domingos, T., & Kooijman, S. A. L. M. (2008). From empirical patterns to theory: a formal metabolic theory of life. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 363(1502), 2453-2464. Squire, J. L. (1987). Relation of sea surface temperature changes during the 1983 El Nino to the geographical distribution of some important recreational pelagic species and their catch temperature parameters. Marine Fisheries Review, 49(2), 44-57. Starling, S., (1988). The fisherman's handbook. How to find, identify and catch the top Australian angling fish. Angus and Robertson Publishers, Sydney. 1-263. Steffens, W. (1989). Principles of fish nutrition. Ellis Horwood Limited, 1-384. Stuart, K. R., & Drawbridge, M. A. (2013). Captive spawning and larval rearing of California yellowtail (Seriola lalandi). Aquaculture Research, 44(5), 728-737. Sumida, B. Y., Moser, H. G., & Ahlstrom, E. H. (1985). Descriptions of larvae of California yellowtail, Seriola lalandi, and three other carangids from the eastern tropical Pacific: Chloroscombrus orqueta, Caranx caballus, and Caranx sexfasciatus. Calif. Coop. Ocean. Fish. Investig. Rep, 26, 139-159. Tacon, A. G. J., & Cowey, C. B. (1985). Protein and amino acid requirements. Fish Energetics, 155-183. Tacon, A. G., & Metian, M. (2008). Global overview on the use of fishmeal and fish oil in industrially compounded aquafeeds: trends and future prospects. Aquaculture, 285(1), 146-158. Talbot, C. (1993). Some aspects of the biology of feeding and growth in fish. Proceedings of the Nutrition Society, 52(03), 403-416. Tanaka, Y (1977). Deposition processes of organic matter. In: The Japanese Society of Fisheries Science (ed.). Mariculture and self-induced pollution, Koseisha-Koseikaku, Tokyo. 42–51. Tandler, A., & Beamish, F. W. H. (1979). Mechanical and biochemical components of apparent specific dynamic action in largemouth bass, Micropterm salmoides Lacéepède. Journal of Fish Biology, 14(4), 343-350. Tandler, A., & Beamish, F. W. H. (1981). Apparent specific dynamic action (SDA), fish weight and level of caloric intake in largemouth bass, Micropterus salmoides Lacepede. Aquaculture, 23(1), 231-242. Tang, M., & Boisclair, D. (1995). Relationship between respiration rate of juvenile brook trout (Salvelinus fontinalis), water temperature, and swimming characteristics. Canadian Journal of Fisheries and Aquatic Sciences, 52(10), 2138-2145.

100

Tang, M., Boisclair, D., Ménard, C., & Downing, J. A. (2000). Influence of body weight, swimming characteristics, and water temperature on the cost of swimming in brook trout (Salvelinus fontinalis). Canadian Journal of Fisheries and Aquatic Sciences, 57(7), 1482-1488. Tetreault, I., & Peet, C. (2008). Farmed Yellowtail Seriola spp. Japan & Australia, Final Report Aquaculture Research Manager, Monterey Bay Aquarium. Thompson, B. A., Beasley, M., & Wilson, C. A. (1999). Age distribution and growth of , Seriola dumerili, from the north-central . Fishery Bulletin, 97(2), 362-371. Thomson, I. S. I. (2008). Shi-Jian Fu, Zhen-Dong Cao and Jiang-Lan Peng. Growth performance and postprandial metabolic responses of southern catfish (Silurus meridionalis Chen) fed formulated feed and loach flesh. Journal of Fisheries and Aquatic Science, 3(4), 261-267. Tian, Y., Kidokoro, H., Watanabe, T., Igeta, Y., Sakaji, H., & Ino, S. (2012). Response of yellowtail, Seriola quinqueradiata, a key large predatory fish in the Japan Sea, to seawater temperature over the last century and potential effects of global warming. Journal of Marine Systems, 91(1), 1-10. Torstensen, B. E., Espe, M., Sanden, M., Stubhaug, I., Waagbø, R., Hemre, G. I., Fontanillas R., Nordgarden, U., Hevroy, E.M., Olsvik, P., & Berntssen, M. H. G. (2008). Novel production of Atlantic salmon (Salmo salar) protein based on combined replacement of fishmeal and fish oil with plant meal and vegetable oil blends. Aquaculture, 285(1), 193-200. Tsukamoto K. (1981). Direct evidence for functional and metabolic differences between dark and ordinary muscles in free-swimming yellowtail, Seriola quinqueradiata. Nippon Suisan Gakkaishi, 47, 573-575. Van Leeuwen, T. E., Rosenfeld, J. S., & Richards, J. G. (2012). Effects of food ration on SMR: influence of food consumption on individual variation in metabolic rate in juvenile coho salmon (Onchorhynchus kisutch). Journal of Animal Ecology, 81(2), 395-402. Vanella, F. A., Boy, C. C., Lattuca, M. E., & Calvo, J. (2010). Temperature influence on post-prandial metabolic rate of sub-Antarctic teleost fish. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 156(2), 247-254. Verner-Jeffreys, D. W., Nakamura, I., & Shields, R. J. (2006). Egg-associated microflora of Pacific , sexfilis and amberjack, Seriola rivoliana, eggs. Characterization and properties. Aquaculture, 253(1), 184-196. Ware, D. M. (1975). Growth, metabolism, and optimal swimming speed of a pelagic fish. Journal of the Fisheries Board of Canada, 32(1), 33-41. Watanabe, T., Sakamoto, H., Abiru, M., & Yamashita, J. (1991). Development of a new type of dry pellet for yellowtail. Nippon Suisan Gakkaishi, 57(5), 891-897.

101

Watanabe, T., V. Viyakarn, H. Kimura, K. Ogawa, N. Iso. (1992). Utilization of soybean meal as a protein source in a newly developed soft-dry pellet for yellowtail. 58(9), 1761- 1773. Watanabe, T., Aoki, H., Shimamoto, K., Hadzuma, M., Maita, M., Yamagata, Y. & Satoh, S. (1998). A Trial to Culture Yellowtail with Non-fishmeal Diets. Fisheries science, 64(4), 505-512. Watanabe, K., Hara, Y., Ura, K., Yada, T., Kiron, V., Satoh, S., & Watanabe, T. (2000a). Energy and protein requirements for maximum growth and maintenance of bodyweight of yellowtail. Fisheries science, 66(5), 884-893. Watanabe, K., Ura, K., Yada, T., Kiron, V., Satoh, S., & Watanabe, T. (2000b). Energy and protein requirements of yellowtail for maximum growth and maintenance of body weight. Fisheries science, 66(6), 1053-1061. Watanabe, T., Akimoto, A., Aoki, H., & Shimeno, S. (2001). Effects of physical properties of diets on evacuation time of digesta and plasma free amino acid patterns in yellowtail. Fisheries science, 67(3), 456-460. Weatherley, A. H., Gill, H. S., & Rogers, S. C. (1979). Growth dynamics of muscle fibers, dry weight, and condition in relation to somatic growth rate in yearling rainbow trout (Salmo gairdneri). Canadian Journal of Zoology, 57(12), 2385-2392. Webb, P. W. (1975). Hydrodynamics and energetics of fish propulsion. Bulletin of the Fisheries Research Board of Canada, 190(1), 1-156. Williams, T. M. (1999). The evolution of cost efficient swimming in marine mammals: limits to energetic optimization. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 354(1380), 193-201. Whiteley, N. M., Robertson, R. F., Meagor, J., El Haj, A. J., & Taylor, E. W. (2001). Protein synthesis and specific dynamic action in : effects of temperature. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 128(3), 593-604. Whitlock, R. E., Walli, A., Cermeño, P., Rodriguez, L. E., Farwell, C., & Block, B. A. (2013). Quantifying energy intake in Pacific Bluefin tuna (Thunnus orientalis) using the heat increment of feeding. Journal of Experimental Biology, 216(21), 4109-4123. Windell, J. T., Foltz, J. W., & Sarokon, J. A. (1978a). Effect of fish size, temperature, and amount fed on nutrient digestibility of a pelleted diet by rainbow trout, Salmo gairdneri. Transactions of the American Fisheries Society, 107(4), 613-616. Windell, J. T., Foltz, J. W., & Sarokon, J. A. (1978b). Methods of fecal collection and nutrient leaching in digestibility studies. The Progressive fish-culturist, 40(2), 51-55. Yamamoto, K. I., Itazawa, Y., & Kobayashi, H. (1981). Gas exchange in the gills of yellowtail, Seriola quinqueradiata under resting and normoxic condition. Bulletin of the Japanese Society of Scientific Fisheries, 47, 447-451.

102

Yang, T. H., & Somero, G. N. (1993). Effects of feeding and food deprivation on oxygen consumption, muscle protein concentration and activities of energy metabolism enzymes in muscle and brain of shallow-living (Scorpaena guttata) and deep-living (Sebastolobus alascanus) scorpaenid fishes. The Journal of Experimental Biology, 181(1), 213-232. Yokoyama, H. (2010). Monitoring, assessment and management of fish farm environments in Japan. Reviews in Aquaculture, 2(3), 154-165. Xu, Z., Lin, X., Lin, Q., Yang, Y., & Wang, Y. (2007). Nitrogen, phosphorus, and energy waste outputs of four marine cage-cultured fish fed with trash fish. Aquaculture, 263(1), 130-141. Yagi, H., Ceccaldi, H. J., & Gaudy, R. (1990). Combined influence of temperature and salinity on oxygen consumption of the larvae of the pink shrimp, Palaemon serratus (Pennant) (Crustacea, , Palaemonidae). Aquaculture, 86(1), 77-92. Yanase, K., Herbert, N. A., & Montgomery, J. C. (2012). Disrupted flow sensing impairs hydrodynamic performance and increases the metabolic cost of swimming in the yellowtail kingfish, Seriola lalandi. The Journal of experimental biology, 215(22), 3944- 3954. Ye, Y., & Beddington, J. R. (1996). Bioeconomic interactions between the capture fishery and aquaculture. Marine Resource Economics, 11, 105-123. Yogata, H. (2000). The effects of swimming exercise on growth and whole‐body protein and fat contents of fed and unfed fingerling yellowtail. Fisheries science, 66(6), 1100-1105.

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