effects of natural foods on food selection and growth rates of cobia (rachycentron canadum) larvae

CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES





EFFECTS OF NATURAL FOODS ON FOOD SELECTION AND
GROWTH RATES OF COBIA (Rachycentron canadum) LARVAE

By

NGUYEN CHI




A thesis submitted in partial fulfillment of the requirements for
the degree of Bachelor of Aquaculture





Can Tho, December 2013

CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES

EFFECTS OF NATURAL FOODS ON FOOD SELECTION AND
GROWTH RATES OF COBIA (Rachycentron canadum) LARVAE

By

NGUYEN CHI

A thesis submitted in partial fulfillment of the requirements for
the degree of Bachelor of Aquaculture



Supervisor
Assoc. Prof. Dr. TRAN NGOC HAI
Dr. LY VAN KHANH






Can Tho, December 2013

APPROVEMENT

The thesis “Effects of natural foods on food selection and growth rates of cobia
(Rachycentron canadum) larvae” defended by Nguyen Chi, which was edited and

passed by the committee on 12-27-2013.
Sign of Supervisor 1 Sign of Student




Assoc. Prof. Dr. TRAN NGOC HAI NGUYEN CHI


Sign of Supervisor 2




Dr. Ly Van Khanh




i

Acknowledgements
First of all, I would like to express my honest thanks to the Rectorate of Cantho
University and the lecturers of College of Aquaculture and Fisheries for supporting me to
study after 4.5 years.
I would like to thank Assoc. Prof. Dr. Tran Ngoc Hai, Dr. Ly Van Khanh and Dr.
Le Quoc Viet who have instructed me enthusiastically to finish this graduating thesis. For
other valuable help and guide, thanks are extended to all my friends, Mr. Pho Van Nghi,
Mr. Nguyen Van Thang, Ms. Nguyen Thi Diem Chi, Mr. Nguyen Tuan Cuong and
students in AAP course 35.

I also send my gratefulness to my advisor Dr. Duong Thuy Yen for her constant
support and my beloved classmates in Advanced Aquaculture Program for great
encouragement during 4.5 years in CAF.
Finally, I thank my family and all my friends who have supported and encouraged
me to study and finish my course.
I honestly thank all of you!


NGUYEN CHI

ii

Abstract
The aim of study was to investigate the pattern of food selection and growth rates of
cobia (Rachycentron canadum) larvae reared in pond water. The study was based on the
stomach analysis of cobia larvae during the larval stage from hatching to 10 days old.
Stomach contents were compared to environment composition and electivity indices were
calculated. Natural pond water that contained various live organisms such as 3 taxons of
rotatoria, copepoda and nauplius were released to the cultured tanks. Four days after
hatching, the larvae commenced feeding and showed little selectivity on zooplankton for 10
days. Brachionus plicatilis, Brachionus pala and Nauplius, the main prey organisms were
found to be preferred by larvae during from day 4 to day 7, and subsequently replaced by
bigger size prey such as Schmakeria clubia and Microsetella norvegica during from day 8
to day 10. After 10 days of rearing, the fries reached the size of about 5.10 – 5.50mm. Daily
length gain (DLG) of larvae fluctuated from 0.09 to 0.16mm/day and specific growth rate
(SGR) ranged from 2.13 to 3.58%/day. Electivity indices on Nauplius and Schmakeria
clubia range from 0.27 to 0.40 and from 0.42 to 0.47, respectively, indicating that Nauplius
and Schmakeria clubia were found to be most preffered food for the cobia larvae during for
10 days old. The change on prey selectivity might be due to the mouth size, sluggish
movement of this fish at their initial stages.

iii

TABLE OF CONTENT

Acknowledgements i
Abstract ii
TABLE OF CONTENT iii
List of Tables v
List of Figures vi
List of abbreviations vii
CHAPTER I 1
1.1 Introduction 1
1.2 Objectives of the study 2
1.3 Contents of research 2
CHAPTER II 3
2.1 Biological features of Cobia (rachycentrum canadum) 3
2.1.1 Classification and taxonomy 3

2.1.2 Habitat and distribution 4
2.1.3 Food and Nutrition 4
2.2 Overview about the status of hatchery and farming production of cobia 6
2.2.1 Overview about the status of hatchery and farming production of Cobia in the
world 6
2.2.2 Overview about the status of hatchery and farming production of Cobia in
Vietnam 8
2.3 Food selection of fish 9
2.4 Types of natural food used for larval nusery 11
2.4.1 Rotifers 12
2.4.2 Copepods 13
CHAPTER III 15
3.1 Time and location 15
3.2 Materials 15
3.2.1 Equipment 15
iv

3.2.2 Water source 15
3.2.3 Feed 15
3.3 Research methodology 15
3.3.1 Experimental design 15
3.3.2 Sampling and data collection 16
3.4 Statistical analysis 19
Data will be analyzed for mean value, standard deviation by using Excel software. 19
CHAPTER IV 20
4.1 Water quality parameters 20
4.2 Growth rates 23
4.3 Food selection of cobia larvae 25
4.3.1 Planktons in rearing tank 25
4.3.1.1 Species composition of planktons in rearing tank 25

4.3.1.3 Percentage composition of zooplankton in rearing water 26
4.3.2 Planktons in stomach of cobia larvae 27
4.3.2.1 Species composition of planktons in stomach of cobia larvae 27
4.3.2.2 Density and amount of zooplankton in stomach of cobia larvae 28
4.3.2.3 Percentage composition of zooplankton in stomach of cobia larvae 30
4.3.3 Electivity index of cobia larvae 31
CHAPTER V 35
5.1 Conlusions 35
5.2 Recommendations 35
Appendix 46





v

List of Tables
Table 3.1 Physical and chemical parameter collection…………………………… 18
Table 4.1 Water quality parameters during culture period………………………… 20
Table 4.2 Growth rate of cobia larvae during 10 days……………………………….23
Table 4.3 Composition of zooplankton in rearing tank…………………………… 25
Table 4.4 Density of zooplankton in rearing tank………………………………… 26
Table 4.5 Composition of planktons in stomach of cobia larvae……………………28
Table 4.6 Density of zooplankton in stomach of cobia larvae………………………29
Table 4.7 Amount of zooplankton in stomach of cobia larvae………………………29
Table 4.8 Electivity index of cobia larvae……………………………………………32

vi

List of Figures
Figure 2.1 15 – 20 kg broodstock cobia……………………………………………….4
Figure 2.2 Global aquaculture production of Rachycentron canadum……………… 9
Figure 2.3 Euryhaline Brachionus species used in aquaculture as live food……… 13
Figure 4.1 Variation of temperature during the culture period…………………… 21
Figure 4.2 Variation of pH during the culture period……………………………… 22
Figure 4.3 Variation of water clarity during the culture period……………… 22
Figure 4.4 Variation of TAN during the culture period…………………………… 22
Figure 4.5 Variation of nitrite during the culture period…………………………….23
Figure 4.6 Illustration of cobia larvae growth in 10 days……………………………25
Figure 4.7 Density of zooplankton in rearing tank………………………………… 26
Figure 4.8 Percentage of zooplankton in rearing tank……………………………… 28
Figure 4.9 Density of zooplankton in stomach of cobia larvae………………………30
Figure 4.10 Amount of zooplankton in stomach of cobia larvae…………………….30
Figure 4.11 Percentage of zooplankton in stomach of cobia larvae………………….31

vii

List of abbreviations
CMFRI………………… Central Marine Fisheries Research Institute
HUFA………………………Highly unsaturated fatty acid
PUFA……………………….Polyunsaturated Fatty Acid
ARA……………………… Arachidonic acid
EPA……………………… Eicosapentaenoic acid
DHA……………………… Docosahexaenoic acid
PRC………………… Peoples Republic of China
HCG……………………… Human chorionic gonadotropin
DLG……………………… Daily length gain
SGR……………………… Specific growth rate
TAN……………………… Total ammonia – nitrogen
FAO……………………… Food and Agriculture Organization of the United Nations
dph…………………… Day post hatch

ppt…………………………Part Per Thousand
CFU/g…………………… Colony Former Unit
BL
i
…………………………Initial Body Length
BL
f
…………………………Final Body Length








1

CHAPTER I
INTRODUCTION

1.1 Introduction
Vietnam has great potential for aquaculture development. It has a 3,260km coastline,
12 lagoons, straits and bays, 112 estuaries, canals and thousands of small and big islands
scattering along the coast, more than 1 million km² exclusive economic zone and very
large water surface area of brackish water. In recent years, marine aquaculture in Vietnam
has been developing very fast and a large number of high quality juveniles is required.
Many research to produce sea bass (Lates calcarifer), grouper (Epinephelus spp), spotted
Scat (Scatophagus argus), Giant mottled eel (Anguilla marmorata), mudskipper
(Pseudapocryptes elongatus) and Cobia (Rachycentron canadum) juveniles in hatchery

have been being conducted to develop technology for seed production to apply into
practice.
Cobia (Rachycentron canadum), the only member of the family Rachycentridae in
North America, is a widely distributed species of pelagic fish found worldwide, except the
Eastern Pacific; in tropical, subtropical, and warm temperate waters (Shaffer and
Nakamura 1989). Cobia aquaculture has been expanding in many tropical countries during
the recent past mainly due to its fast growth rates and good meat quality. The success of
cobia farming in Taiwan (Yeh, 2000; Su et al., 2000; Liao and Leano, 2005) has led to the
rapid expansion of cobia farming throughout Southeast Asia, the Americas and Carribian
regions (Benetti and Orhun, 2002; Kaiser and Holt, 2004; Schawrz et al., 2006, 2007;
Benetti et al., 2008; Nhu et al., 2010; 2011). Realising the potential of cobia farming in
India, the Central Marine Fisheries Research Institute (CMFRI) focused research attention
on the broodstock development of cobia and the first successful spawning was obtained in
March 2010 (Gopakumar et al., 2011).Several studies have also reported successful larval
rearing of cobia in semistatic and recirculating aquaculture systems from both wild caught
(Hassler and Rainville, 1975) and captive spawned eggs (Faulk and Holt, 2003) with the
use of rotifers, Artemia, and/or wild zooplankton. However, little information is available
regarding the nutritional requirements of cobia larvae in recirculating aquaculture systems,
2

and such information is essential to maximize larval growth and survival and further the
successful commercial production of this species (Faulk and Holt, 2005).
In Vietnam, Cobia is considered the most popular species for culture in offshore
cages. This is because of its fast growth, high market value, good meat quality, the
established technology in mass production of larvae, the current innovation in intensive
and super intensive nursery rearing in ponds, and improved formulated feeds. However,
there are still many difficulties remained, such as environmental and diseases problems as
well as limitations when relying on natural seed sources, natural food sources, quantity and
quality of the seed. Therefore, the study of marine fish seed production in general and the
cobia seed production in particular is very necessary and urgent. Based on research and

practical demands, this study on “Effects of natural foods on food selection and growth
rates of cobia (Rachycentron canadum) larvae” was carried out to apply in practice.
1.2 Objectives of the study
To evaluate the effects of natural foods on food selection and growth of cobia
larvae at early stage in order to contribute to seed production of cobia fish.
1.3 Contents of research
- To evaluate water quality and natural food in tanks.
- To evaluate the growth rates of larvae.
- To evaluate food selection through food composition in stomach contents at
different stages of larvae.










3

CHAPTER II
LITERATURE REVIEW

2.1 Biological features of Cobia (rachycentrum canadum)
2.1.1 Classification and taxonomy
Cobia (Rachycentron canadum) is classified as the followed (Linnaeus, 1766):
Kingdom: Animalia
Phylum: Chrodata

Class: Actinopterygii
Order: Perciforms
Family: Rachycentridae
Genus: Rachycentron
Species: Rachycentron canadum
According to FAO (2009), the cobia is characterized by a dark brown dorsally,
paler brown laterally and white ventrally; black lateral band as wide as eye extends from
snout to base of caudal fin, bordered above and below by paler bands; below this is a
narrower dark band. Black lateral band very pronounced in juvenile, but tends to be
obscured in adult. The cobia has an elongated body that is strongly rounded with a broad
flat head and depressed head. Mouth large, terminal, with projecting lower jaw; villiform
teeth in jaws and on roof of mouth and tongue. First dorsal fin with 7 – 9 short but strong
isolated spines each depressed into a groove, not connected by a membrane, 28 – 33 rays.
Second dorsal fin long, anterior rays somewhat elevated in adults. Pectoral fins pointed,
becoming more falcate with age. Anal fin similar to dorsal, but shorter; 1 – 3 spines, 23 –
27 rays. Caudal fin lunate in adults, upper lobe longer than lower (caudal fin rounded in
young, the central rays much prolonged). Scales small, embedded in thick skin; lateral line
slightly wavy anteriorly.
4


Figure 2.1 15 – 20 kg broodstock cobia (FAO, 2009)
2.1.2 Habitat and distribution
Cobia, Rachycentron canadum, is considered one of the most promising candidates
for warm – water marine fish aquaculture in the world (Kaiser and Holt, 2004; Liao et al.,
2004; Benetti et al., 2007). Cobia, also known as lemonfish or ling, is the only member of
the family Rachycentridae, and is found in the warm – temperate to tropical waters of the
West and East Atlantic, throughout the Caribbean and in the Indo – Pacific off India,
Australia and Japan (Briggs, 1960; Hassler and Rainville, 1975; Shaffer and Nakamura,
1989; Ditty and Shaw, 1992). In the Eastern Pacific its occurrence has been reported as

marginal (Fowler, 1944; Briggs, 1960; Collette, 1999). Cobia are eurythermal and
euryhaline, tolerating ranges of temperature and salinity between 16.8 and 32.2°C and 5 –
44.5 ppt, respectively (Shaffer and Nakamura, 1989; Resley et al., 2006). They travel
alone or in small school and are often found near some kind of structure, whether floating
or in the water column (Kaiser and Holt, 2005).
2.1.3 Food and Nutrition
Cobia is a carnivorous species and widely distributed in tropical and subtropical
waters (Ditty and Shaw, 1992). Excellent flesh quality, rapid growth, and adaptability to
culture conditions, confer highly desirable characteristics for global commercial
aquaculture on cobia (Holt et al., 2007). The nutritional value of several plant protein
sources have been evaluated for potential use in cobia formulated feeds (Chou et al., 2004;
Lunger et al., 2006). Dietary requirements for macronutrients including crude protein
(Chou et al., 2001; Craig et al., 2006), lipid (Wang et al., 2005), methionine (Zhou et al.,
2006), and lysine (Zhou et al., 2007). They are opportunistic carnivores that eat many
5

species of fish, crab, shrimp and squid. Stomach content data show that they prefer
crustaceans, particularly portunids (swimming crabs). Cobia have elongated bodies and
grow to 6.5 feet (2 m) and 135 pounds (61 kg). Females grow both larger and faster than
males (Kaiser and Holt, 2005). Growth of cobia is rapid for the first two years, after which
it slows gradually. Females attain a larger size at age than males (Thompson et al., 1991,
Burns et al., 1998, Franks et al., 1999).
One important aspect of larval nutrition is providing adequate levels of highly
unsaturated fatty acids (HUFAs) including arachidonic acid (ARA, 20:4n-6),
eicosapentaenoic acid (EPA, 20:5n-3), and docosahexaenoic acid (DHA, 22:6n-3)
(Sargent et al., 1999a). HUFAs play an important role in maintaining cell membrane
structure and function, stress tolerance, and proper development and functioning of neural
and visual systems (Kanazawa, 1997; Rainuzzo et al., 1997; Sargent et al., 1997). Several
studies have shown that marine fishes are unable to convert shorter chain fatty acids such
as linolenic acid (18:3n-3) and linoleic acid (18:2n-6) to longer chain HUFAs due to low

activity of the necessary enzymes, thus making it necessary to provide these fatty acids
through the diet (Mourente and Tocher, 1993; Ghioni et al., 1999). Recently, researchers
have suggested that the biochemical composition of eggs and yolksac larvae reflect the
basic nutritional requirements of first feeding larvae (Rainuzzo et al., 1997; Sargent et al.,
1999b). Faulk and Holt (2003) examined the fatty acid composition of cobia Rachycentron
canadum eggs and yolksac larvae and reported high levels of HUFAs with DHA, EPA,
and ARA accounting for approximately 80% of the polyunsaturated fatty acids. This
suggests that cobia larvae may require high levels of these fatty acids in their diets
(Extracted from Faulk and Holt, 2005).
2.1.4 Reproduction
Females spawn multiple times during the season. The exact size and age at which
cobia are sexually mature varies with location; however, research has shown that males
are generally 1 to 2 years old and females are 2 to 3 years old at first spawning (Kaiser and
Holt, 2005). In the northwestern Gulf of Mexico, they arrive in the spring and can be
caught into the early fall, spawning multiple times from April to September, with activity
peaking in July. Spawning occurs in both nearshore and offshore waters where females
6

release several hundred thousand to several million eggs (1.4 mm diameter) which are
then fertilized by the attending males. The viable eggs begin development, are heavily
pigmented, buoyant, and hatch in approximately 24 hours. Cobia larvae grow rapidly and
are large in comparison to most marine species at 3.5 mm TL at hatching. Juvenile fish are
found in both nearshore and offshore waters, often among Sargassum patches or weedlines
where they seek shelter from predators and can feed (FAO, 2013). According to Patricia et
al., 1994 Protein was the major constituent of cobia ovaries and its contribution remained
fairly constant (49 - 55% of the dry weight) throughout all stages of development. Lipid
was the second most abundant component but the levels, ranging from 21 to 41%, changed
depending on the stage of ovarian development. Lipid concentration increased from stage
1 through 3 and decreased slightly in stage 4.
2.2 Overview about the status of hatchery and farming production of cobia

2.2.1 Overview about the status of hatchery and farming production of Cobia in the
world
As early as 1975, researchers in North Carolina collected cobia eggs from the wild
and reared them successfully. However, it was not until the early 1990s that Taiwan
reported captive spawning of cobia. Successful efforts in the U.S. followed in 1996.
Taiwan now has a commercial industry that produced nearly 5,000 tons in 2004, most of
which was cultured. An offshore cage project in Puerto Rico has successfully grown cobia
to market size since 2003. Research in Florida, Mississippi, South Carolina, Texas and
Virginia has resulted in many successful spawns, both natural and hormone induced.
Grow-out trials of juvenile and market-size cobia in ponds, recirculating systems and
cages will likely be conducted in the future (Kaiser and Holt, 2005).
The artificial breeding of cobia in Taiwan was first recorded in 1992. Mass seed
production technique was developed in 1997. It has fast became one of the most favorable
species in the domestic offshore cage culture. Later, sea farmers in Japan imported cobia
seed and started to culture in sea cages off Okinawa. The seed production of cobia was 3
million in 1999 from 4 hatcheries, as compared to 1.4 million in 1998. About 2 million
seed were exported to Japan, Peoples Republic of China (PRC), and Viet Nam. The rest, 1
million seed were stocked locally by 28 cage farmers. The present market price is US$ 0.5
7

per seed (10 cm) and US$ 6.0 per kg of adult (6 – 8 kg) (Yel et al., 2004). In the case of
cobia, brood stocks were usually collected from the wild before artificial propagation was
developed. Currently, cobia intended for broodstock are produced from hatcheries and
reared in open cages until they attain sexual maturity (about 1.5 – 2 years when fish
weighs about 10 kg). Handling and spawning of mature brooders have been described in
detail (Su et al., 2000; Liao et al., 2001; Liao, 2003). Maturing brooders are selected from
sea cages and transferred to land-based spawning ponds (400 – 600 m
2
area; 1.5 m depth)
with flow – through seawater, at a density of 100 fish per pond and a sex ratio of about 1:1

(male/female). Fish are fed to satiation with raw fish (e.g. sardines, mackerels, squids)
once or twice a day. Brooders spawn spontaneously year around, with a peak in spring and
autumn when water temperature is maintained at 23 – 27
o
C. The fertilized eggs are
collected using a seine net installed against the current created by paddlewheels. The eggs
are then transferred to outdoor larval rearing ponds (earthen ponds; < 5000 m
2
area ; 1 –
1.2 m water depth) with well – maintained ‘‘green water’’ (Chlorella sp.) and abundant
number of copepods. Water exchange is minimal or unnecessary in the early stage as long
as the ‘‘green water’’ is maintained. Eggs hatch 21 – 37 h after fertilization at temperature
of 31 – 22
o
C. Cobia larvae are vigorous and more resistant to some stressors compared to
other tropical marine fish (e.g. grouper). They open their mouth and starts feeding at day 3
after hatching. Rotifers and copepod nauplii are provided at this stage, with higher
preference to copepods during the first feeding stage. Larvae are reared up to day 20 with
survival rate of 5 – 10% (Liao et al., 2004).
Human chorionic gonadotropin (HCG) has been used successfully to induce
ovulation in a variety of marine fish (Lam, 1982; Donaldson and Hunter, 1983; Zohar,
1989), including Nassau grouper (Epinephelus striutus) (Tucker et al., 1991) and gag
grouper (Mycteroperca microlepis) (C. Koenig, personal communication, 1992).
Researchers in the U.S. have also used hormones to induce adult cobia caught during their
natural spawning season to produce eggs. Both HCG (human chorionic gonadotropin)
injected at 275 IU/kg and a slow – release pellet containing salmon GnRHa (gonadotropin
– releasing hormone analog) implanted in fish have resulted in spawns. Both of these
spawning methods have advantages and disadvantages, but the goal is the same –
8

consistent production of high – quality eggs and larvae for use in the aquaculture industry
and research. Cobia eggs are 1.20 to 1.40 mm in diameter, heavily pigmented, and hatch in
about 24 hours at 80 to 84°F (27 to 29°C). The fertilized eggs are buoyant and can be
gathered easily in a recirculating culture system using a side – looped egg collector with
an 800 – micrometer mesh bag. After collection, the eggs are counted (approximately 420
eggs/ml) volumetrically using graduated cylinders, which also allow separation of viable
(floating) and nonviable eggs. Eggs are usually stocked into rearing tanks at a density of 5
to 10 per L, although this phase of cobia production is still being researched in order to
optimize yield per tank (Kaiser and Holt, 2005).
Global aquaculture production of cobia has been increasing rapidly since 2002,
reaching approximately 30,000 metric tonnes (at a value of USD 60 million) in 2007 (Son,
2010). The three main producers of cobia in 2007 were China, Taiwan and Vietnam,
where annual production was approximately 26,000; 4,000 and 1,500 tonnes, respectively.
Production in Vietnam was estimated to be 2,600 tonnes in 2009 (Nhu et al., 2010). Of the
production reported to FAO in 2004, 80.6 percent was produced in China and all the rest
in Taiwan Province of China (FAO, 2013).








Figure 2.2 Global aquaculture production of Rachycentron canadum
(FAO Fishery Statistic, 2013)
2.2.2 Overview about the status of hatchery and farming production of Cobia in
Vietnam
Cobia culture in Vietnam began with the first successful intensive mass production
of fingerlings in 1999 (Nguyen, 2002; Nguyen et al., 2003). The industry has expanded

9

from protected areas into more exposed areas in the ocean with better water exchange.
Cobia is cultured in Vietnam by small to medium – scale family farms (approximately
1,000 tonnes, mostly for local consumption) as well as cooperative farms (approximately
1,600 tonnes/year, mostly for export) (Huy, 2008; Nhu et al., 2010). Production in
Vietnam is clustered in the north (from Ha Long Bay and Bai Tu Long Bay), north central
region (Nghe An), centre (Van Phong Bay, Khanh Hoa) and south (Ba Ria – Vung Tau,
Kien Giang); (Extracted from Petersen et al., 2011).
The main factor constraining development of cobia culture in Vietnam is a shortage
of quality fingerlings, although hatchery production in Vietnam is increasing at a rapid
rate (Nhu et al., 2010). For example, the Research Institute for Aquaculture No 1 in
Vietnam produced 400,000 fingerlings in 2007 and 900,000 in 2008 (Nhu et al., 2010).
The industry still relies on fingerling imports from Taiwan and China (Hainan) (Huy,
2008). Other constraints include disease outbreaks and lack of locally extruded feeds (Nhu
et al., 2010). Cobia are generally fed low – value fish (trash fish), although a small amount
of pelleted diets are used in Vietnam, by small to medium - sized farmers (Son, 2010). The
larger – scale cooperatives exclusively use pelleted feeds (Nhu et al., 2010). Problems
associated with low – value fish feed include a short storage life, rapid decline of
nutritional quality if stored for too long, unstable supply (depending on the season),
relatively low growth rates (compared with pelleted diets (although data is still scare)),
localised pollution and water quality degradation, and transmission of parasites and
diseases. The relative benefits of pelleted diets include faster growth rates (feed to biomass
conversion ratios are generally less than 2:1, compared with ratios of 8:1 and higher for
low – value fish diets), fewer parasites and diseases, fewer environmental problems, and
more stable water quality (Son, 2010); (Extracted from Petersen et al., 2011).
2.3 Food selection of fish
Most fish larvae are visual particulate feeders (Greene, 1985), able to feed
selectively on prey. Although factors such as prey colour and swimming behaviour can be
important in determining prey perception and recognition (Checkley, 1982; Govoni et al.,

1986), prey size is probably the major determinant of selectivity, and this is intimately
related to the mouth size of fish larvae (Shirota, 1970; Hunter, 1981). Several studies have
10

reported prey size dependent patterns of food selection in muskellunge (Esox
masquinongy) (Applegate, 1981); walleyes (Stizostedion vitreum vitreum); yellow perch
(Perca flavescens) ( Raisanen, 1982; Raisanen and Applegate, 1983) and other fish species
(e.g. Wong ang Ward, 1972). Fish food consumption might be influenced by many
environmental factors such as water temperature, food concentration, stocking density,
fish size and fish behaviour (Houlihan et al. 2001). The feeding rate relative to the body
weight decreases as fish size increases; however, the rate of food consumed increases per
individual (Wang et al. 1989).
Smelt Osmerus eperlanus (L.) is the main zooplanktivorous fish species in Lake
Peipsi and is so an important predator on zooplankton and large invertebrates in this lake.
Smelt is zooplanktivorous at younger ages, gradually shifting to larger invertebrates
during growth, and the oldest and largest smelts are piscivorous (Karjalainen et al., 1997;
Vinni et al., 2004). When smelt start feeding, the number of suitable food organisms
available is critical for fish survival. Rotifers and crustacean zooplankton (cladocerans and
copepods) are important food items during the first summer because the mouth width
seems to be the critical determinant of the ability of smelt to handle large food items
(Strelnikova & Ivanova, 1983; Næsje et al., 1987). Earlier research on smelt feeding in
Lake Peipsi and in Lake Pihkva (Tikhomirova, 1974) showed that smelt larvae feed
mainly on cladocerans (Bosmina, Chydorus, Daphnia cucullata, and Diaphanosoma
brachyurum, occasionally Leptodora and Sida) and copepods (Diaptomus, Cyclops,
Mesocyclops) during the spring.summer period (Extracted from Salujoe et al., 2008). Both
green sunfish fry (Lepomis cyanellus) and bluegill fry (Lepomis macrochirus) selected for
Cyclops vernalis and consistently selected against cladoceran egg cases and Potamocypris
spp. Moina brachiata was consistently selected for by bluegills but was initially consumed
by green sunfish in approximately the same proportion as they were available and later
preferentially selected for by larger green sunfish fry (Barkoh, 1984).

Among brachionid rotifers, Brachionus plicatilis Miiller, 1786 (Monogononta) is
probably one of the beststudied taxa because of its suitability as an initial live feed for
various finfish and shellfish larvae (Lubzens, 1987). In B. plicatilis, the size and shape of
lorica vary greatly according to the strain (Snell and Canillo, 1984). Rotifers are ideal as a
11

first exogenous food source due to their small size, slow swimming speed and ability to
stay suspended in the water column. They are also relatively easy to culture at high
densities and can be enriched with fatty acids and antibiotics (Lubzens et al., 1989).
Confer & O'Bryan (1989) demonstrated that size selectivities of planktivorous fish depend
on whether feeding is averaged over short or long time periods. The density of rotifers in
the water column has a significant effect on the feeding success of marine fish larvae by
influencing the probability of encounter (Hunter, 1980). It is therefore essential to
quantify the number of rotifers effectively available in the water column of the larval-
rearing tanks. Insufficient rotifer density decreases larval survival and growth because the
energetic requirements of the larvae are not satisfied (Dowd and Houde, 1980; Tandler
and Sherman, 1981). An excessive rotifer density can also decrease larval survival and
growth by promoting excessive ingestion of rotifers, hence decreased gut retention time
and a subsequent reduction in assimilation efficiency (Boehlert and Yoklavich, 1984;
Tandler and Mason, 1984). Overfeeding can also lead to accumulation of nutritionally
inadequate rotifers, and can cause decreased survival and growth of fish larvae (Lubzens
et al., 1989).
2.4 Types of natural food used for larval nusery
Nutritional requirements centre the research and hatchery management of larval
fish rearing and production (Cahu et al., 2003; Lee, 2003). Currently, the seed supply
of fish juveniles in commercial hatcheries relies on the successful supply of live
zooplankton species such as rotifers, copepods and Artemia nauplii, during the larval
stage (Hagiwara et al., 2001; Lee et al., 2005; Sorgeloos et al., 2001; Støttrup and
Norsker, 1997). To ensure a sufficient live food supply, the fish hatcheries need to
establish a food chain supply from algae to zooplankton. The high cost of infrastructure

and maintenance of live food culture have made researchers search for a replacement of
live food (Southgate and Partridge, 1998). In the past decade, intensive research has
focus on the replacements for live food organisms with compound diets (Kolkovski,
2001). Although significant improvement has been made in co – feeding live and
compound diets, especially at the Artemia feeding stage, compound diets alone have not
matched the growth and survival of fish fed with live feeds (Lee, 2003). The use of live
12

food organisms, especially at first feeding, is still obligatory in marine fish larvae mass –
culture. The fundamental reason for the reliance on live food is because the digestive
tract in most fish species at first feeding contains limited enzymes for digestion,
absorption and assimilation of large molecules of proteins, lipids and glycogen (Cara et
al., 2003; Chen et al., 2006b). The live food organisms consumed by the larvae assist the
digestion process by donating their digestive enzymes to the gut of fish larvae
(Dabrowski and Glogowski, 1977; Kolkovski et al., 1993). The nutritional requirements
of fish larvae change during the course of ontogenetic development during early life
history (Oozeki and Bailey, 1995). The variations of nutritional need depend on the
morphology, physiology and feeding behaviour of different fish larvae (Cited from Jian G.
Qin, 2008).

2.4.1 Rotifers
Euryhaline rotifers are an important food for rearing marine fish larvae. Their
availability to fish larvae in the water column may be reduced if they are transferred to
fish larval rearing tanks with different temperatures and salinities (Fielder et al., 2000).
Rotifers, which have been widely used in marine finfish hatcheries throughout the world,
are commonly referred to as one of three types: L – type, S – type, or SS – type, based on
their size initially B. plicatilis (130 – 340 μm in lorica length) is referred as an L – type
rotifer in cold water (18 – 25
o
C) and B. rotundiformis (100 – 210 μm) as an S – type in

warmwater (28 – 35
o
C, Figure 2.4.). The super small rotifers referred to as SS – type
rotifers (90 – 150 μm) are found in subtropical and tropical waters (Hagiwara et al.,
1995).
According to Jian G.
Qin (2008),
both species are common in brackish waters, but
show strong tolerance to high salinity. In Japan, these rotifers are previously known as
harmful organisms because they consume a large amount of oxygen and cause a rapid
change of water color in eel culture ponds. Before 1980, the major developments of
rotifer mass culture include: (1) introduction of Nannochloropsis oculata and baker’s
yeast as rotifer diets; and (2) establishment of an enrichment protocol before feeding
fish larvae (Watanabe et al., 1978). Since 1990, the major advances in rotifer culture are
(1) development of condensed freshwater Chlorella for rotifer food (Maruyama and
13

Hirayama, 1993), (2) the development of high density rotifer mass culture technology
using condensed phytoplankton products and (3) techniques for rotifer preservation
(Yoshimura et al., 1997).


Figure 2.3 Euryhaline Brachionus species used in aquaculture as live food: B.
p
licatilis
(left),
B. rotundiformis S - type (middle) and B. rotundiformis SS - type (right); (
from

Hagiwara

et al., 2001).
2.4.2 Copepods
A workshop was conducted at the Oceanic Institute, Hawaii with the focus of
copepods culture as a live food for aquaculture (Lee et al., 2005). Like rotifers and
brine shrimp naupplii, copepods could be additional desirable food for fish larvae.
Especially, nauplii of some copepod species can be used to raise fish larvae that require
starter food smaller than rotifers. At the workshop, a number of promising candidate
genera was identified for culture. Currently, mass production technology is not well
established. Among the calanoid copepods, the general Acartia, Pseudodiaptomus,
Sinocalanus, Eurytemora, Gladioferens, Parvocalanus, Bestiolina, Temora,
Centropages, and Labidocera were proposed for first feeding. Nauplii of some Acartia
spp. are as small as 100 μm in length and 50 – 60 μm in width, making them suitable
for first feeding as well. Harpacticoid copepod species tend to have high production
rates, are generally not cannibalistic, and are found all over the world. In addition, it is
likely that many genera among this group can be raised on formulated feeds. Two species
that can be mass cultured at high densities are Tisbe holothuriae and Nitokra lacustris.
Among the cyclopoid copepods, the genera Oithona and Dioithona were listed as
candidates for first – feeding larvae (Cited from Jian G.
Qin, 2008).

14

The use of copepods as live food for larval fish rearing is largely restricted to extensive
systems, where wild zooplankton are collected, using different filters to obtain specific size
– ranges, and fed directly or allowed proliferate prior to being fed as food for fish larvae
(Støttrup and Norsker, 1997). Attempts have been made to culture several species such as
Acartia spp., Tigriopus juponicus, Oithona spp., Paracalanus spp., and Euryternora spp., for
feeding fish larvae at stages in intensive systems (Støttrup et al., 1986). There are three main
groups of copepods, i.e., calanoids, cyclopoids and harpacticoids. Like rotifers, calanoid
copepods are planktonic and relatively easy to operate and to scale up. Calanoids, however,

can only be cultivated at very low densities. Harpacticoids, on the other hand, may be
produced in volumetrically much denser cultures, but being benthic, their proliferation
depends on the area of a solid substratum. Such an environment is not homogenous and hence
is considerably more difficult to manage and scale up. Cylopoids are pelagic, but
most species are carnivorous and are not easy to reach high biomass (Cited from Jian G.
Qin,
2008).



15

CHAPTER III
METHODOLOGY
3.1 Time and location
The study was carried out from June to December, 2013 at Vinh Chau
Experimental Station of Can Tho University at Soc Trang province.
3.2 Materials
3.2.1 Equipment
Blowers, pumps, lights, microscope, net, petri dishes, spoons, notes, coverage,
rackets, scale, valves, pipes, air stones, plastic bottles;
Thermometer, Salinometer, pH, NH
3
/NH
4
+, NO
2
/NO
3
+ and alkalinity test kits;

2 m
3
tank used for setting the experiments;
Chemicals: Na
2
S
2
O
3
, Chlorine, Formalin, Na
2
HPO
4
, NaH
2
PO
4
, distilled water.
3.2.2 Water source
Freshwater source: tap water;
Brine water (80-120 ppt): bought from Vinh Chau district, Soc Trang province;
Brackish water: mixing the freshwater with the brine water to achieve the
salinity (30‰) expected to use in experiment then disinfect the water by using chlorine.
3.2.3 Feed
Enriched rotifer (Brachionus plicatilis) was used to feed the larvae;
Vitamin C, DHA, products of Bayer Company, were used to enrich the rotifer;
Probiotics consisting of Bacillus subtilis, Nitrobacter and Nitrosomonas spp.
with the total bacteria count of more than 1.5*10
9
CFU/g was grinded and diluted with

water for about 30 minutes then applied into the tanks every 3 days to help maintain
the water quality. Usage dose of probiotics is 1 bags/tank (0.5 mg/L);
Chlorella algae, transported from Brackish water hatchery, College of
Aquaculture and Fisheries, Can Tho University to Vinh Chau district, which were used
to feed the larvae on 3 day-post-hatch (dph) to 10 dph at 10 inds/ mL.
3.3 Research methodology
3.3.1 Experimental design
The experiments were conducted in 2m
3
tank. Cobia larvae with initial length of
3.90 – 4.10mm were stocked at density of 20,000 inds/tank (at salinity 30‰). After