Quantifying Digestion in California Yellowtail (Seriola lalandi dorsalis),
Technological Advances Towards the Development of the Aquaculture 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
Pacific Aquaculture & Coastal Resources Center, College of Agriculture, Forestry and Natural Resource
Management, University of Hawaii at Hilo, 1079 Kalanianaole Ave., Hilo, HI 96720, United States
b
Tuna 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
a
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 Taxonomy ......................................................................... 1
B.
Distribution and Movement Patterns in a Natural Setting .............................................. 2
C.
Yellowtail Aquaculture ..................................................................................................... 4
II.
The Necessity For A New Source Of Fish ............................................................................ 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 Animals ..................................................................................... 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 Sardine Flesh Meal.................................................................. 27
F.
Calculation of Meal and Respiration Energy .................................................................. 28
VIII.
Results ............................................................................................................................ 29
A.
SDA Metrics .................................................................................................................... 29
B.
Measured Respirometry Values ..................................................................................... 31
IX.
A.
Discussion.......................................................................................................................... 34
Baseline Metabolic Rate................................................................................................. 34
i
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
ii
G. Digestibility of Various Feed Types ................................................................................ 79
VI.
Conclusions ....................................................................................................................... 80
Chapter 4: Summary and Conclusions .......................................................................................... 82
Bibliography .................................................................................................................................. 87
iii
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
pelagic fish 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.
v
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
vi
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 species 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
vii
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 Carangidae, comprised of
four distinct subpopulations within the Seriola genus (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 olive-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
Kingdom
Subkingdom
Infrakingdom
Phylum
Subphylum
Infraphylum
Superclass
Class
Subclass
Great amberjack, yellowtail, yellowtail jack
Animalia
Bilateria
Deuterostomia
Chordata
Vertebrata
Gnathostomata
Osteichthyes
Actinopterygii
Neopterygii
1
Infraclass
Superorder
Order
Suborder
Family
Genus
Species
Teleostei
Acanthopterygii
Perciformes
Percoidei
Carangidae
Seriola
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 Australia and New Zealand (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 sardines, anchovies, jack mackerel, pacific mackerel, squid,
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, anchovy, 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.
6
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).
7
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 animal 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
8
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.
9
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
10
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.
11
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)
12
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
were determined to be on average 221.8 ± 3.4 mg O2 kg-1 h-1 at 20.6 ± 0.1⁰C, and post-prandial
metabolic rate increased to an average maximal rate of 430.3 ± 1.1 mg O2 kg-1 h1. 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
13
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,
14
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).
15
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).
16
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 threeday 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 smelt, 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
Fish 1
Fish 2
Fish 3
Fish 4
Fish 5
Mean
CFL (cm)
37.0
39.0
38.0
39.0
39.0
38.4 ± 0.4
Width (cm)
4.9
4.5
4.5
4.8
5.2
4.8 ± 0.1
Depth (cm)
9.5
8.6
8.2
9.3
9.2
9.0 ± 0.2
CFL, curved fork length; values given in bottom row expressed as means ± S.E.
Mass (g)
684.0
696.0
618.0
700.0
806.0
700.8 ± 30.2
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).
𝑨𝑨𝒐𝒐
𝑬𝑬𝒔𝒔 = 𝝉𝝉ƛ( )𝟏𝟏.𝟓𝟓
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
𝑽𝑽𝒇𝒇 = 𝑽𝑽𝒕𝒕 (𝟏𝟏 + 𝑬𝑬𝒔𝒔 )
•
•
(1)
(2)
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 twopoint 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 tunas 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
within the flume during a closed cycle (Equation 3). Units were presented in mg O2 s-1,
therefore the equation factors in a time conversion.
24
𝒎𝒎𝒎𝒎𝑶𝑶𝟐𝟐
𝑹𝑹𝑶𝑶 × 𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝟑 × (𝑽𝑽𝑹𝑹 − 𝑾𝑾𝑭𝑭 )
𝑶𝑶𝑶𝑶𝑶𝑶𝑶𝑶𝑶𝑶𝑶𝑶 𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪 �
�=
𝒌𝒌𝒌𝒌𝒌𝒌
𝑾𝑾𝑭𝑭
𝐦𝐦𝐦𝐦
)
𝑶𝑶𝟐𝟐
•
RO (
•
•
cycle.
VR (L) = Volume of the swim respirometer (30 L).
WF (kg) = Weight of individual fish.
(3)
= Resulting slope for the regression of the change in oxygen over a 5-min closed
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 heatbound 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 freezedrying 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
Sardine Fillet 1
Sardine Fillet 2
Sardine Fillet 3
Sardine Fillet 4
Sardine Fillet 5
Average
Protein (%)
21.5
19.7
22.3
22.1
20.6
21.2 ± 0.5
Fat (%)
10.7
8.5
6.3
4.7
11
8.3 ± 1.2
Energy (kcal g-1)
2.21
2.31
1.87
1.67
2.12
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
oxy-caloric value equal to 14.32 J mg-1O2 (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
𝟏𝟏𝟏𝟏𝟏𝟏
VIII.
(5)
WF (kg) = Fish Weight
ΔO (mg O2 kg-1 h-1) = Increase in Oxygen Consumption above BaselineRMR
β (J mg-1 O2) = 14.32 J of Energy per 1 mg of Oxygen Consumed
𝑺𝑺𝑺𝑺𝑺𝑺𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪𝑪 (%) =
•
•
𝟏𝟏𝟏𝟏𝟏𝟏
𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 (𝒌𝒌𝒌𝒌) = 𝚺𝚺((𝑾𝑾𝑭𝑭 × ∆𝑶𝑶 × 𝜷𝜷) × 𝟔𝟔 𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄 × 𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏)
RE (kJ) = Respiration Energy
ME (kJ) = Meal Energy
𝑹𝑹𝑬𝑬
× 𝟏𝟏𝟏𝟏𝟏𝟏
𝑴𝑴𝑬𝑬
(6)
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
metabolic rate value of yellowtail in five fish was determined to be 221.8 ± 3.4 mgO2 kg-1 h-1
(Figure 8). The maximum value of SDA was relatively consistent across all fish and averaged
430.3 ± 1.1 mgO2 kg-1 h-1. 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
Fish Mass
Meal Mass (Wet)
Percent Meal Size
Energy Content
BaselineRMR
SDAMax
SDADURATION
Factorial Scope
SDA
SDACOEFFICIENT
Unit
g
g
%
kJ
mgO2 kg-1 h-1
mgO2 kg-1 h-1
h
SDAMax BaselineRMR-1
kJ
%
Fish 1
684.0
33.5
4.9
94.7
216.9
429.4
38:45
2.0
40.1
42.3
Fish 2
696.0
34.8
5.0
98.4
216.0
434.3
33:35
2.0
31.5
32.0
Fish 3
618.0
30.7
5.0
86.9
217.1
428.0
38:45
2.0
28.9
33.2
Fish 4
700.0
33.3
4.8
94.2
233.4
429.8
34:35
1.8
32.4
34.4
Fish 5
806.0
39.9
5.0
112.9
225.7
429.7
36:55
1.9
34.6
30.7
Mean ± S.E.
700.8 ± 30.2
34.4 ± 1.5
4.9 ± 0.0
97.4 ± 4.3
221.8 ± 3.4
430.3 ± 1.1
36:31 ± 1:03
1.9 ± 0.0
33.5 ± 1.9
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
434
429
430
428
430
Oxygen consumption (mgO2 kg-1 h-1)
400
350
300
250
217
216
217
233
226
200
150
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
roughly 147 mgO2 kg-1 h-1, which is relatively similar to results for S. lalandi by Yanase et al.
(2012) of 159 mgO2 kg-1 h-1, 172 mgO2 kg-1 h-1 by Brown et al. (2011), 137 mgO2 kg-1 h-1 by De la
Gandara (2002), and 93-198 mgO2 kg-1 h-1 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
mgO2 kg-1 h-1 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
range of 216 to 233 mgO2 kg-1 h-1, 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).
Source
Species
Flume
Yamamoto et al. (1981)
Furukawa et al. (1992)
De la Gandara et al. (2002)
Clark and Seymour (2006)
Brown et al. (2011)
Yanase et al. (2012)
Current Study
S. quinqueradiata
S. quinqueradiata
S. dumerili
S. lalandi
S. lalandi
S. lalandi
S. lalandi
2.44mm sec-1
Static
Static
0 & 2.3 Bl sec-1
0 & 0.75 Bl sec-1
1 Bl sec-1
1 Bl sec-1
RMR/SMR
(mgO2 kg-1 h-1)
147
141-185
137
93 & 198
172 & 159
129
221
Temp (⁰C)
19
23-24
19
20 & 25
22
18-19
20
Thermal influence on SMR for S. lalandi indicates significant fluctuations between 93 mgO2
kg-1 h-1 at 20°C to 198 mgO2 kg-1 h-1 at 25°C (Clark and Seymour 2006). Later studies on routine
metabolic rate for juvenile S. lalandi at 10°C was 85 mgO2 kg-0.8 h-1 while higher temperatures
around 32.5°C were 382 mgO2 kg-0.8 h-1 (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
baselineRMR rate of 221.8 ± 3.4 mgO2 kg-1 h-1 to a maximal rate of 430.3 ± 1.1 mgO2 kg-1 h-1.
Previous yellowtail studies conducted in static respirometers indicate the factorial scope in the
range of 1.3 with maximal rates of 250 mgO2 kg-1 h-1 (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
consumption of 215 mgO2 kg-1 h-1 while reducing the feed period to twice daily caused the O2
consumption to drop to 146 mgO2 kg-1 h-1 (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
rates for both treatments ranging from 193-195 mgO2 kg-1 h-1 (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
maximal rates reaching 443-638 mgO2 kg-1 h-1. 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 3944% 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 3140% heat increment of feeding (Watanabe et al. 2000b).
Percent of Energy Budget (%)
45
40
41.5
35
30
40.5
36
33
34.5
29.5
25
25.5
20
15
17
10
13
16.5
5
0
Growth
Masumoto et al. (1997)
Watanabe et al. (2000a)
Maintenance
Watanabe et al. (2000b)
Heat Increment
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
under conditions used in this study were 221.8 ± 3.4 mgO2 kg-1 h-1, while post feeding oxygen
consumption rates doubled in value to 430.3 ± 1.1 mgO2 kg-1 h-1. 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
120.2 ± 3.7 mgO2 kg-1 h-1, significantly lower than that associated with the sardine fillet meal of
150.8 ± 3.9 mgO2 kg-1 h-1, at 20.6 ± 0.1⁰C. The SDAMax for the commercial feed (203.7 ± 22.5
mgO2 kg-1 h-1) was statistically lower than the sardine fed fish (279 ± 27.3 mgO2 kg-1 h-1).
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 onethird 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
crabs 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.
52
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 8841206g). 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: airsaturated 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 heatbound 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.
Sardine Fillet1
Commercial Feed1
Protein (%)
20.6
47.6
Fat (%)
8.36
20
Moisture (%)
66.5
4.35
Calories (kcal g-1)
2.11
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
60
isoenergetic value of the commercial feed relative to the dry weight of the sardine feed
(Equation 7 & 8).
𝑺𝑺𝑺𝑺𝑹𝑹 = (𝒀𝒀𝑾𝑾 ×. 𝟎𝟎𝟎𝟎𝟎𝟎)
𝑪𝑪𝑪𝑪𝑹𝑹 = ×
•
•
•
•
•
•
•
(𝑺𝑺𝑺𝑺𝑹𝑹 × 𝑺𝑺𝑫𝑫 ) × 𝑺𝑺𝑪𝑪
𝑪𝑪𝑪𝑪𝑪𝑪 × 𝑪𝑪𝑪𝑪𝑫𝑫
(7)
(8)
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).
𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴 𝑫𝑫𝑫𝑫𝑫𝑫𝒆𝒆𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔 (%) = 𝟏𝟏𝟏𝟏𝟏𝟏 − (�
•
•
•
•
•
𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹 𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 (𝒌𝒌𝒌𝒌)
� × 𝟏𝟏𝟏𝟏𝟏𝟏)
𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻 𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 (𝒌𝒌𝒌𝒌)
(9)
𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹𝑹 𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭 𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴 (𝒌𝒌𝒌𝒌) = 𝑷𝑷𝟏𝟏 + 𝑷𝑷𝟐𝟐 + 𝑳𝑳𝟏𝟏 + 𝑳𝑳𝟐𝟐
(10)
𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷𝑷 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 (𝒌𝒌𝒌𝒌) = 𝑷𝑷𝑾𝑾 × 𝑷𝑷𝑬𝑬
(11)
𝑳𝑳𝑳𝑳𝑳𝑳𝑳𝑳𝑳𝑳 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 (𝒌𝒌𝒌𝒌) = 𝑳𝑳𝑾𝑾 × 𝑳𝑳𝑬𝑬
(12)
𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻 𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴 𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬𝑬 (𝒌𝒌𝒌𝒌) = 𝑴𝑴𝑾𝑾 × 𝑴𝑴𝑬𝑬
(13)
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
PE (kJ g-1) = Protein caloric value (23.87)
LE (kJ g-1) = 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.
63
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.
64
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
baselineRMR value for the three commercial feed treatments was 120.2 ± 3.7 mgO2 kg-1 h-1,
statistically lower (p=0.036) than that of the mean baseline RMR values of 150.8 ± 3.9 mgO2 kg-1
h-1 (20.4 ± 0.2⁰C) for the three sardine trials. SDAMAX was also found to be statistically different
(p=0.04) between the two diets, with commercial feed mean values of 203.7 ±22.5 mgO2 kg-1 h-1
and the sardine feed at 279 ± 27.3 mgO2 kg-1 h-1. 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
65
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.
Parameter
N
Temp
Total Fish Mass#
Meal Mass (wet)
Meal Mass (dry)
Meal Energy Content
BaselineRMR
SDAMax
SDADuration
Factorial Scope
SDA
SDACoefficient
°C
g
g
g
kJ
mgO2 kg-1 h-1
mgO2 kg-1 h-1
h
SDAMax/BaselineRMR
kJ
%
Commercial
Feed
47P: 20L
3
20.5 ± 0.1
6084.0 ± 39.0a
40.7 ± 0.2a
38.9 ± 0.2a
889.6 ± 4.6a
120.2 ± 3.7a
203.7 ± 22.5a
27:46 ± 1:32
1.7 ± 0.1a
18.2 ± 1.4a
12.3 ± 1.0a
Sardine Fillet
20P: 8L
3
20.4 ± 0.1
6682.0 ± 64.2b
349.3 ± 3.3b
117.03 ± 1.1b
1033.1 ± 9.9b
150.8 ± 3.9b
279.6 ± 27.3b
33:36 ± 00:16
1.8 ± 0.1b
42.9 ± 0.7b
24.9 ± 0.2b
p value
0.797
0.004*
0.000**
0.000**
0.006*
0.036*
0.040*
0.070
0.037*
0.003**
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.
#
66
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.
67
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
68
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
a
b
Meal Energy (kJ)
898.3 ± .7
Meal Energy (kJ)
972.3 ± 11.0
0.023*
Meal Weight (g) (dry)
39.3 ± 0.0a
Meal Weight (g) (dry)
110.1 ± 1.3b
0.000**
a
b
Meal Weight (g) (wet)
41.11 ± 0.03
Meal Weight (g) (wet)
328.77 ± 3.8
0.000**
a
b
Protein (kJ)
446.8 ± 0.3
Protein (kJ)
541.6 ± 6.3
0.004**
a
b
Lipid (kJ)
315.2 ± 0.2
Lipid (kJ)
417.3 ± 4.9
0.002**
Feces (24 h)
Feces (24 h)
Protein (kJ)
10.8 ± 0.9a
Protein (kJ)
25.4 ± 1.0b
0.007*
a
b
Lipid (kJ)
5.3 ± 1.3
Lipid (kJ)
13.4 ± 3.1
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
a
b
Feces Energy (kJ)
20.5 ± 1.6
Feces Energy (kJ)
45.2 ± 4.2
0.013*
Feces Weight (g)
4.0 ± 0.3
Feces Weight (g)
4.2 ± 0.2
0.644
a
b
Digestibility (%)
97.7 ±0.2
Digestibility (%)
95.3 ± 0.5
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
69
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
70
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 ±
3.7 mgO2 kg-1 h-1, whereas the sardine feed treatment baseline values were significantly higher,
in the range of 150.8 ± 3.9 mgO2 kg-1 h-1 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
reported reductions in yellowtail respiration post-fasting to 140-150 mgO2 kg-1 h-1, 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 crab 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
feed diet at 279.6 ± 27.3 mgO2 kg-1 h-1 and 203.7 ± 22.5 mgO2 kg-1 h-1, 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
72
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
73
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.
74
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
75
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
76
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
velocity of 1 BL s-1 to be in the range of 216-233 mgO2 kg-1 h-1, while the baselineRMR for both
diets in the static trials were largely below this estimate, with the commercial feed baseline
values between 115-127 mgO2 kg-1 h-1 and 143-157 mgO2 kg-1 h-1 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.
78
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 ecosystem (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
80
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.
81
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
83
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
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