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 submit

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

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

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

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

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

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

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

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

BLi………Initial Body Length

BLf………Final Body Length

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,

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

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):

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

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

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

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

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 m2 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 – 27oC 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 m2 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 – 22oC 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 –

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

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

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

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

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 – 25oC) and B rotundiformis (100 – 210 μm) as an S – type in

warmwater (28 – 35oC, 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

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

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)

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)

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, NH3/NH4+, NO2/NO3+ and alkalinity test kits;

2 m3 tank used for setting the experiments;

Chemicals: Na2S2O3, Chlorine, Formalin, Na2HPO4, NaH2PO4, 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*109 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 2m3 tank Cobia larvae with initial length of 3.90 – 4.10mm were stocked at density of 20,000 inds/tank (at salinity 30‰) After

transported from Nam Du Island to Vinh Chau district, larvae were acclimated and screened carefully before stocking

Before stocking, the tank is renovated thoroughly with EP-01 (10mL/m3), Probiotic (2bags/tank) and Zimovac (1g/m3);

Aeration are supplied continuously and no water exchanged;

Duration for experiment: 10 days;

Rotifer enriched with DHA, vitamin C and probiotics Enriched rotifer were fed

to larvae to the end of the experiment Feeding frequency was maintained three times/day at 6:00a.m, 12:00a.m, and 6:00p.m

Natural food is filtered through zooplankton net and artemia net in pond After that zooplankton were supplied into the tank Feeding frequency was maintained five times/day at 6:00a.m, 12:00a.m, 2:00p.m, 5:00p.m and 6:00p.m

3.3.2 Sampling and data collection

Sampling

Tank water samples were collected to analyze the qualitative and quantitative composition of zooplanktons in the tank The water samples collected in 125ml plastic bottles were preserved by adding 5ml of 4% formalin

Fish samples were randomly collected 10 individual and fixed with 10% neutralized formalin solution to analyze the composition of feed in the stomach of the fish

Tank water samples and fish samples were collected daily from day 1 – 10

Plankton sampling and analysis: The plankton nets were used to collect water

samples for the qualitative and quantitative estimation of the plankton, by filtering a known volume of water (15 litres) through the net The sample was allowed to settle for 24 – 48 hours and was further concentrated to approximately 30 ml by decanting The concentration factor is used during the calculations Preserved samples in bottles are mixed uniformly by gentle inversion and then one drop of the sample is pipetted out from a calibrated pipette onto the glass slide for analysis A cover slip is carefully placed ensuring no air bubbles remain and the cover slip is ringed with a transparent nail enamel to prevent evaporation during the counting process A binocular compound microscope is used in the counting of plankton with different eyepieces such as 10X and 40X The microscope is calibrated using an ocular micrometer The following

outline of methods is based mainly on the reviews by T.V Ramachandra and Malvikaa Solanki, 2007) Plankton are classified to genus, species Plankton are classified to genus, species Concentrated water samples were shaken well and for to Sedgewick – Rafter counting chamber to analyze the plankton; plankton were counted randomly in

10 cells in the counting chamber and are classified to the genus (Mins et al, 1995)

Total plankton count per litre =

Where, T = Number of organisms counted per subsample

A = Area of one cell, mm2

N = Number of cells counted

L = Volume of concentrated sample (ml)

V = Volume of original sample (ml)

Fish samples analysis: The numerical methods (focus: Frequency of

Occurrence method) are used for the counts of constituent items in the gut contents

Stomach contents are examined and the individual food organisms sorted and identified The number of stomachs in which each item occurs is recorded and expressed as a percentage of the total number of stomachs examined

Frequency of Occurrence, Oi = Ji/P Where, Ji is number of fish containing prey i and P is the number of fish with food in their stomach

In addition, the points method is an improvement on the numerical method where consideration is given to the bulk of the food items The simple form of points method is the one in which the counts are computed falling a certain organisms as the unit In a more modified form, the food items are classified as ‘very common’,

‘common’, ‘frequent’, ‘rare’, etc., based on rough counts and judgments by the eye In this arbitrary classification the size of the individual organisms is also given due consideration The contents of all stomachs are then tabulated and as a further approximation, different categories are allotted a certain number of points and the summations of the points for each food item are reduced to percentages to show the percentage composition of the diet This method is essentially a numerical one; the volume being only a secondary consideration and it is only in the counts that a certain

amount of accuracy can be claimed (P.U Zacharia et al., 2011) Point number of each

type of foods depends on:

Frequency of occurrence: the food, mostly appeared, which would have the highest point while the least appeared one was the lowest point

Food size: larger food would have higher points than smaller food

Point of one food type was the percentage that was calculated base on total points of all food items

Food selection of fish: to determine if the fish are positively selecting prey of a certain size, a measure called an electivity index has been devised by several authors

We will use Ivlev's (1961):

E i = (r i - p i ) ÷ (r i + p i)

where r i = the percent that prey size i forms in the fish diet;

p i = the percent that prey size I forms in the environment (aquarium);

E i varies between -1 and +1;

If an item in the environment is always rejected by the fish, then r i = 0 and E i =

-p i /p i = -1 If an item is found in the diet that is so rare in the environment that our

measure does not detect it, then p i = 0 and E i = r i /r i = +1 If an item is exactly as

abundant in the diet as it is in the environment, then r i = p i and E i = 0 Thus, avoided

items have negative electivities, preferred items have positive electivities, and indifferent (not particularly selected) items have electivity of zero (Hairston, N jr and

M Stafford – Glase, 1993)

Water quality monitoring:

Table 3.1 Physical and chemical parameter collection

Water

parameters

Sampling frequency Equipment

pH 2 times/day (7:00a.m and 5:00p.m) Test kit

Temperature 2 times/day (7:00a.m and 5:00p.m) Thermometer

Transparency 2 times/day (7:00a.m and 5:00p.m) Test kit

Nitrite 3 days interval (7:00a.m) Test kit

Data collection

During larval rearing, larval activities are observed every day

Growth rate of the larvae:

When first stocking the fries into the pond, 20 samples were randomly measured for body length Then, 20 samples in tank is randomly collected and measured 4 times until the last sampling

Equation to calculate daily length gain (DLG) and specific growth rate (SGR) in body length:

DLG (mm/day) = (L final – L initial )/t SGR(length) (%/day)= (ln(L final ) – ln(L initial ))*100/t 3.4 Statistical analysis

Data did already be analyzed for mean value, standard deviation by using Excel software

CHAPTER IV RESULTS AND DISCUSSIONS 4.1 Water quality parameters

A summary of the water quality parameters during culture period is given in Table 4.1

Table 4.1 Water quality parameters during culture period

Water quality parameters Mean values for 10 days

28oC is suitable for fish, pH should be from 6.5 to 9.0 (Boyd, 1998) So, the temperature and pH in this experiment were quite suitable for cobia larvae