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.4 <<vT a

EFFECTS OF DIFFERENT SALINITY ON SURVIVAL RATE

OF MARBLE GOBY (Oxyeleotris marmoratus) LARVAE

001 PEY THENG

THIS DISSERTATION IS SUBMITTED IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR BACHELOR OF SCIENCE WITH HONOURS

AQUACULTURE PROGRAM

SCHOOL OF SCIENCE AND TECHNOLOGY

UNIVERSITI MALAYSIA SABAH

11

DECLARATION

I declare that this dissertation is the results of my own independent work, except where

otherwise stated.

March 2004

001 PEY THENG

HS2001-2243

iii

AUTHENTICATION

AUTHENTICATED BY MEMBERS OF DISSERTATION COMMITTEE

1. SUPERVISOR

(Associate Professor Dr. Shigeharu Senoo)

2. EXAMINER 1

(Mr. Kennedy Aaron Aguol)

3. EXAMINER 2

(Mrs. Rossita Shapawi)

4. DEAN

(Associate Professor Dr. Amran Ahmed)

iýiýý ý----=---------------------

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iv

ACKNOWLEDGEMENT

This dissertation has accumulated many debts of gratitude throughout its completion.

Thus I would like to extend my heartiest appreciation to a great number of people who

have contributed in my conquest of completing this dissertation. This dissertation would

not have been successful without their full commitment. Firstly, I would like to express

my sincere gratitude to Director of Borneo Marine Research Institute of Universiti

Malaysia Sabah, Prof Dr. Ridzwan Abdul Rahman for support in this study. Sincere

appreciation is also extended to Prof Madya Shigeharu Senoo who has been a great help

in constructive advices on my work, valuable suggestions, continuous encouragement at

various stages throughout the completion of dissertation, and providing me with a wide

range of references, also for his expert guidance and for his patience, without which this

work could not have been accomplished. I'm also greatly in debt for Mr. Kennedy Aaron

Agoul and Mr. Arun Prasad Baidya for their valuable advices, teaching and guidance. Last but not least, I would like to thank research assistants Ms. Sow Sok Hui, Ms. Nguang

Siew Ing, Mr. Ha Hou Chew and all staff of the hatchery of Borneo Marine Research

Institute, who had rendered assistance, support and cooperation in various ways-

V

ABSTRACT

Natural spawning of brood fish marble goby (Oxyeleotris marmoralus) was carried out in

960-liter square glass aquarium and 48 - 72 hours after spawning larvae were then

collected. Survivals of ten larvae in different salinities were determined at salinities from

0 to 10 (in 2 ppt increments) for 11 days in preliminary trial. Indication obtained when larvae in 10 ppt achieve greatest survival and health. Two experiments with higher

salinity were then done; from 0 to 28 ppt (0,10,20 and 28 ppt); and 0 to 25 ppt (0,10,20

and 25 ppt) in 40 days. One hundred larvae were used in 9-liter plastic aquarium with

aeration supplied. Desired salinities were increased 5 ppt within 1 hour. Water renewal,

survival rate and larval observation were conducted daily. Analyses of data revealed that

only survival in 10 ppt treatment was significant different between salinities (P<0.6 in

experiment I and P<0.3 in experiment II). No larvae could survive more than 12 days

after spawning in freshwater (0 ppt). Highest survival was achieved in 10 ppt. In 10 ppt

treatment, the greatest survival was obtained in one of the triplication with 33% of

survival rate (11% in mean) in experiment I; and 48% of survival rate (22.67% in mean) in experiment II. O. marmoratus larvae were able to survive and grow at salinities

ranging from 10 to 25 ppt. Analyses found no significant different (P>0.05) in the growth

rate for all treatments. The mortality occurred in the early larval stages 3-7 days after

spawning were much higher than those in the larval stages.

V1

KADAR KEMANI)IRIAN BAGI LARVA IKAN KETUTU

(Oxyeleotris marmoratus) DI BAWAH SALINITI YANG BERBEZA

ABSTRAK

Peneluran semulajadi bagi induk ikan ketutu (Oxyeleotris marmoralus) telah dijalankan

dalam akuarium segiempat berisipadu 960-liter dan anak ikan berumur lingkungan 48 -72 jam kemudian dikumpul. Kadar kemandirian untuk 10 anak ikan ditentukan dalam

kemasinan dari 0- 10 ppt (dengan kenaikan 2 ppt) selama 11 hari sebagai percubaan kajian. Keputusan kajian percubaan menunjukkan anak ikan dalam 10 ppt hidup paling lama dengan keadaan baik. Seterusnya 2 kajian dengan saliniti yang lebih tinggi

dijalankan iaitu 0- 28 ppt (0,10,20 dan 28 ppt); serta 0- 25 ppt (0,10,20 dan 25 ppt)

selama 40 hari. Seratus anak ikan diletakkan dalam akuarium berisipadu 9-liter dengan

kelengkapan pengudaraan. Kemasinan yang diingini dinaikkan 5 ppt dalam I jam.

Penukaran air, kadar kemandiran dan pemerhatian anak ikan dijalankan setiap hari.

Analisis data hanya menunjukkan 10 ppt mempunyai perbezaan bererti antara saliniti (P<0.6 bagi experiment I dan P<0.3 bagi experiment II). Tidak ada larva yang boleh

bertahan hidup lebih daripada 12 hari selepas menetas dalam air tawar (0 ppt). Kadar

kehidupan paling tinggi dicapai pada 10 ppt. Dalam rawatan 10 ppt, kadar kemandirian

tertinggi dicapai di salah satu daripada tiga replikasi dengan 33% kadar kehidupan

(dengan purata 11%) dalam kajian I; dan 48% kadar kemandirian (dengan purata 22.67%)

dalam kajian H. Anak ikan O. marmoratus berkebolehan hidup dan membesar dalam

lingkungan air masin 10 - 25 ppt. Analisis menunjukkan tiada perbezaan bererti (P>0.05)

dalam kadar pertumbuhan bagi semua rawatan. Kadar kematian berlaku pada peringkat 3

-7 hari selepas menetas adalah lebih tinggi daripada peringkat yang lain.

vii

LIST OF CONTENTS

DECLARATION

AUTHENTICATION

ACKNOWLEDGEMENT

ABSTRACT

ABSTRAK

LIST OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF PHOTOS

LIST OF ABBREVIATIONS

CHAPTER 1 INTRODUCTION

1.1 Malaysia Aquaculture Scenario

1.2 Marble Goby (O. marmoratus) 1.3 Objectives

CHAPTER 2 LITERATURE REVIEW

2.1 Spawning

2.2 Egg Hatching

2.3 Early Larvae Development

2.4 Mortality

2.5 Food Supply

2.6 Behavioral Changes

2.7 Salinity Effects

CHAPTER 3 MATERIALS AND METHODS

3.1 Brood Fish Selection

3.2 Hormone Treatment

3.3 Preparation of Spawning Aquarium

3.4 Fish Spawning

Page

ii

111

IV

V

vi

vii

ix

X

xi

xii

I

I

3

6

7

8

9

10

11

11

13

14

16

16

17

17

18

viii

3.5 Egg Developments and Hatching

3.6 Experimental System

3.61 Preliminary Trial

3.62 Experiment

3.7 Analysis Data

CHAPTER 4 RESULTS

4.1 Survival Rate in Preliminary Trial

4.2 Survival Rate in Experiment I

4.3 Survival Rate in Experiment II

4.4 Larvae Measurements in Experiment II

CHAPTER 5 DISCUSSION

5.1 Preliminary Trial

5.2 Comparison Survival in Experiment I and II

5.21 Freshwater -0 ppt Treatment

5.22 10 ppt Treatmant




5.3 Peak Mortality

5.4 Feeds and Feeding Regime

5.5 Analyses Data

CHAPTER 6 CONCLUSION

REFERENCES

APPENDIX -1

19

20

20

21

23

24

24

26

29

32

33

33

34

34

36

37

37

38

38

38

39

40

41

45

ix

LIST OF TABLES

Table No. Page

4.1 Water quality during preliminary trial 24

4.2 Water quality during experiment I 26

4.3 Water quality during experiment II 29

APPENDIX -1

Table No. Page

1.1 Brood measurement, fertilization rate, hatching rate and total hatched 45

larvae in the preliminary trial and experiment 1.2 Larvae measurement for preliminary trial and experiment used 46

1.3 Comparison of changes in survival rate (preliminary trial) 47

1.4 Comparison of larvae survival for every treatment in experiment I 48

1.5 Mean survival for different salinities in experiment I 49

1.6 Analyses oneway ANOVA and Post Hoc Tests for survival rate 50

in experiment I

1.7 Comparison of larvae survival for every treatment in experiment II 52

1.8 Mean survival for different salinities in experiment 11 53

1.9 Analyses oneway ANOVA and Post Hoc Tests for survival rate 54

1.10 Comparisons growth in different salinities for experiment II at 43 d AS 56

1.11 Analyses oneway ANOVA and Post Hoc Tests for larvae 57

measurement in experiment II at 43 d AS

x

LIST OF FIGURES

Figure No. Page

1.1 Comparison of 0. marmorulus production in Malaysia from 2

year 1988-1997

4.1 Comparison of changes in survival rate (preliminary trial) 25



4.4 Comparisons growth in difTerent salinities for experiment 11 at 43 d AS 32

xi

LIST OF PHOTOS

Photo No. Page

1.1 Marble Goby (Oxyeleotris marmoratus) with color in alcohol dark 3

brown above and pale brown below; body with a series of large,

dark blotches; fins with dark bands or dusky

3.1 Selected brood fish were given intramuscular injection 17

3.2 Spawning of O. marmoratus 18

A. 960-liter circular aquarium with filter system B. Substrate positioned as net house inside aquarium

3.3 48-72 hours hatching larvae with slightly pigmented eyes 19

3.4 Materials used for larvae rearing in preliminary trial 20

3.5 Materials used in experiment 21

A. 9- liter plastic aquarium with green water B. Control aeration were placed aside in aquarium

3.6 Brachionus sp. (<200 gm) feed as initial food for larvae 22

3.7 Management of water quality 23

A. Bottom cleaning by using siphoned tube

B. Collected and separated larvae by beaker after siphoned 5.1 Fungus was found attacked to alive larvae 35

xii

LIST OF ABBREVIATIONS

°C degree centigrade

% percentage

BW Body weight

cm centimeter d AF days after fertilization

d AS days after spawning

DO dissolved oxygen

E East

g gram

GDP Gross Domestic Product

H hour

HCG Human chorionic gonadotropin

IU International Unit

km kilometer

km2 kilometer square

1 liter

ml milliliter

mm millimeter

mt metric ton

N North

ppt part per thousand

Sig. Significance

sp. species

SPSS Statistical Package for Social Science

TL Total length

UMS Universiti Malaysia Sabah

1

CHAPTER 1

INTRODUCTION

1.1 Malaysia Aquaculture Scenario

Malaysia is blessed with numerous natural resources. Situated in the heart of South-East

Asia, between 1 °00' N to 6°30' N latitude and between 100 °E to 120 °E longitudes; it

lies at the crossroads of the major air and sea links within the region as well as the world.

It has a total area of about 329,758 km2 and 4,400 km of shorelines and has about 23

million populations in this country. The climate is generally warm throughout the year

with daily temperatures ranging around 30 °C. The total annual rainfall averages around

200 cm. The monsoon season weather months in the West Coast are between April to

August and from October to March in the East Coast. The abundance of sunshine and

rainfall account suitable for fish to spawn which rivers and swamps are naturally covered

by forests (Ang, 1990).

The fisheries sector in Malaysia plays an important role in providing fish as a

source of food and protein (Department of Fisheries Malaysia, 2000). In 2000, it

contributed about 1.6% to the National Gross Domestic Product (GDP) and provided

2

direct employment to 81,994 fishermen and 21,774 fish culturists. During the same

period, aquaculture sector recorded a production of 167,894 tons, which constituted about

11.6% of the total fish production. However, freshwater aquaculture production was

50,688 tons, contributing 30.2% of the total aquaculture production in Malaysia.

In 1997, freshwater fishes contributed the second highest after Mollusc

production. Of this, Marble Goby (Oxyeleotris marmoratus) contributed only 39 tones on

freshwater fishes (Fig 1.1). Although the production had increased, but the production of

0. marmoratus is still very low.

Marble Goby Production in Malaysia from Year 1991-1997

1991 1992 1993 1994

Year

1995 1996 1997

Fig 1.1: Comparison of 0. marmoratus production in Malaysia from year 1991-1997

Source: Department of Fisheries, 1997

1.2 Marble Goby (0. marmoratus)

Over the last several decades, people's increasing dependence on cultured O.

marmoratus, as opposed to wild fish, as a source of dietary protein has resulted in an

increasing level of scientific effort being directed toward propagation techniques

(Donaldson et al., 1983). That are indication that fish farmers wish to culture O.

marmoratus but their seed supply is inadequate. Presently, heavy mortality in the early

stages is one of the stumbling blocs especially after the yolk sac is completely absorbed

and the larvae switch to active natural feeding. Although artificial seed production can be

achieved now, the techniques have yet to be established (Senoo el al., 1994a).

Photo 1.1: Marble Goby (Oxyeleolris marmoralus) in light dark brown above and pale brown below; body with a series of large, dark blotches; fins with dark bands

or dusky

4

Presently, the natural resource of 0. marmoratus fisheries have be exhausted in

West Malaysia (Senoo et al., 1994a). Thus, fish middlemen in the Federal capital of

Kuala Lumpur are importing this fish from Indonesia, Thailand, Cambodia, and East

Malaysia. In Sabah, wild marble goby is harvested from its natural habitat mostly in

paddy field and freshwater reservoirs. In Peninsular Malaysia, it is cultured in ponds and

former mining pools either in through monoculture or polyculture with tilapias. Cage

culture in lakes and rivers has also been successful (Cheah et al, 1994).

Although local fish farmers success culture 0. marmoratus in ponds but there are

still some attempts. These attempts, however, are on a very limited scale. This is

primarily due to inadequate supply of seed for stocking in the ponds; the seed are not

readily available in great numbers from natural sources. Thus, in order to enhance the

culture of 0. marmoratus, which could be justifiable economically, it is necessary to

increase seed availability through controlled breeding (Tan and Lam, 1973).

However, even 0. marmoratus seeds have been produced artificially, the mass

seed production has remained a major constraint. This is mainly a result of low hatching

and larval survival rate (Senoo et. al., 1994b). Early and late hatching resulted in high

mortality. The optimum hatching stage was considered just before and after the

embryonic eyes had become slightly pigmented. All embryonic heads were located at the

basal part of the egg and this was taken as normal development for 0. marmoralus.

5

Synchronistic of food production and first feeding is critical for larval survival.

Starvation may account for high mortality and the ability to endure fasting is of major

importance for survival. Heavy mortality usually occurs early in the life history of fish if

suitable food is scarce, especially when the yolk sac is exhausted and the larvae must

begin active feeding. Instead of the mouth size is suitable feed with Brachionus sp.,

however the freshwater Brachionus sp. is hard to culture under the artificial condition.

In Thailand, Tavarutmaneegul et al. (1988) successfully produced the marble

goby seeds under a semi-natural breeding and rearing method. The newly developed

larvae were first fed with a combination of chicken-egg slurry and live rotifers

(Brachionus sp. ) and then older larvae were fed with live Moina sp., chironomid larvae,

and thrash fish. However, no information is available concerning culturing technique and

the survival rate. In Malaysia, the seeds were produced in an intensive artificial rearing

method (Senoo et al., 1994a and 1994b), but the survival rate was only 10.1% during the

first 5- 70 days after fertilization (Senoo et al., 1994a).

Koumans (1953) reported the occurrence of 0. marmoratus in estuaries as well as

rivers. It may occur in brackish habitat within its range, but all of the well-documented

locality records appear to be from freshwater. Studies of the behavior and ecology of

various stages of fish development related to the causes of mortality may provide much

needed information. The aquaculture of fish depends on an adequate knowledge of the

behavior of the early stages that cause the greatest technical difficulties in culture.

6

Therefore, it is important to know the condition and behavior of 0. marmoralus larvae in

different salinity, which could improve survival rate of cultured larvae.

1.3 Objectives

In order to overcome some of the constrains of 0. marmoratus seed production,

the following objectives were outlined for this study:

1. Determination of optimum salinity for a higher survival rate of larvae.

2. Behavioral observation of larvae under different salinity.

7

CHAPTER 2

LITERATURE REVIEW

The Eleotridae or sleepers comprise some genera and 150 species of mostly tropical,

marine, brackish, and freshwater fishes. Two genera and four species occur in the fresh

waters of western Borneo. Oxyeleotris is unusual among eleotrid genera in being almost

entirely restricted to fresh water species of the genus occur in Southeast Asia and others

in the Austrian region. Three species known from western in Borneo are Oxyeleotris

urophthalmus, O. urophthalmoides and O. marmoratus (Bleeker, 1874).

Instead of freshwater eleotrid, 0. marmoratus can grows to more than 50cm in

total length (TL) and 2 kg in weight (BW). 0. marmoratus, which is also known as

Marble goby, is found in Cambodia, Fiji, Indonesia, Malaysia, the Philippines, Singapore,

Thailand and Vietnam (Cheah et al., 1994). It is much relished as a delicacy in this part

of the world where it is known as "Sand Goby" in English, "Ikan Ketutu" or "Ikan

Hantu" in Malay and "Soon-Hock" in the Chinese Fukien and Cantonese dialect, and

nowadays it is also called "Bamboo Fish" (Senoo et al., 1994a).

8

Respectively 0. marmoratus is a popular freshwater fish of high market value in

Southeast Asia. A table-sized live fish weighing lkg commands an auction price ranging

from RM60/ kg to RM80/ kg in Malaysia, prices being higher during the festive season. It

has white fine-grained sweet flesh without small bones. Consumer can get live fresh 0.

marmoratus in the market. It has become a popular dish in the Asian region from China

to Indonesia. In Malaysia, a dish of 0. marmoratus is much more expensive than other

freshwater or seawater fish, such as Sea Bass (Lates calcarifer) and Red Snappers

(Lutjanus argentimaculatus). Besides being a food fish, 0. marmoratus is also popularly

kept as an ornamental fish among the Chinese.

2.1 Spawning

The availability of a good and healthy brood fish is a pre-requisite for any successful

artificial seed production programme of fish. 0. marmoratus can mature all year round

under proper rearing conditions in Malaysia. Under good weather condition and water

temperature between 27 - 32 °C, this fish can spawn naturally in ponds or tanks. Thus, the

brood fish should be reared separately in ponds or tanks for the planned egg collection in

the seed production (Senoo et al. 1994b).

Induced fish breeding programme using hormones is increasingly being practiced

in aquaculture. Human chorionic gonadotropin (HCG) is a hormone secreted by human

placenta during pregnancy that has often been used successfully. The first success of

induced spawning of 0. marmoratus in Malaysia was reported by Senoo el al. (1992) and

9

since then, several technical improvements have been achieved (Senoo et al., 1993).

However, natural spawning is easier if compare with induced breeding under artificial

condition.

2.2 Egg Hatching

According to Senoo et al. (1994b), there are not many developmental differences in eye

pigmentation or yolk sac absorption between unhatched embryos and hatched larvae of

the same time. The differences in hatching time were not dependent on the stage of

embryonic development. In all development stages, the embryonic head was located at

the basal part of the eggs.

During hatching, larvae broke free of the egg by pushing its tail against the side of

the egg membrane. Once a suture had been made, the tail pushes through the opening.

Then, by trashing movements of the tails, the larvae freed itself from the egg, which was

anchored down by its polar threads. The larvae were unable to free themselves completely

from the eggs membranes; they swam around for sometime with the eggs membrane

covering the head and trunk like a massive hood, and died within 1-2 hours (Tan and

Lam, 1973).

10

2.3 Early Larvae Development

At the time of yolk sac absorption, Glossogobius olivaceus larvae smaller than 0.

marmoratus larvae fed on Brachionus spp., but 0. marmoratus larvae did not, in spite of

having well-developed eyes, jaws, and intestinal tracts. This might be due to the smaller

mobility of 0. marmoratus. In many marine fish larvae, the eyes, jaws, and intestinal

tracts are functional just before yolk sac absorption and larvae have small mouths, but

their locomotory organs are not well developed. Therefore, the diets for the first feeding

of larvae are limited to small-plankton which are slow in locomotion (Senoo et al.,

1994b).

In newly hatched larvae at 49 h AF, the mouth and anus were not formed, and the

eyes were not pigmented. Most of the larvae hatched during 2-3d AF; newly hatched

larvae lay on the tank bottom and gradually exhibited the "swim up, sink down" behavior.

On 3d AF, the mouth was opened, and the pectoral fins were formed. On 4d AF, the

eyes were deeply pigmented, and the lower jaw and intestinal tracts began to move. At

this time, food supply is necessary (Senoo et al., 1994b).

During seed production of 0. marmoratus, deformed juveniles were found at a

very extremely low survival rate. The seeds had a deformation at the mouth, operculum,

caudal peduncle, pelvic fin, and occipital region. The inability of the fragile larvae to

consume enough live organisms in their early stages was attributed to their morphological

deformations due to poor conditions of the brood fish or as a result of artificial spawning.

11

These deformations were considered to be disadvantageous in feeding, and eventually

resulted in poor growth and survival (Senoo et al., 1994b).

2.4 Mortality

Very high percentages (90%) of fertilization and hatching were obtained although the

larvae did not survive for more than a few days. The problem of larvae mortality has also

been encountered in the induced breeding of other fishes, for example the grey mullet,

Mugil cephalus. It is possible that the 0. marmoratus larvae died from an attack by

ciliates, which was seen to occur frequently, especially when there was a concentration of

ciliates on dead larvae nearby (Tan and Lam, 1973). Mass mortality of larval and juvenile

fish might also occur if the food supply is inadequate (Houde, 1978).

2.5 Food Supply

The food supply during larval stage is an important factor to achieve high growth and

survival rates. In different species, the sequential food in early stages differs. In the early

larval stage, the freshwater fish are generally given Brachionus sp, Anemia or Moina as

an initial diet. At the later stage the larvae or juveniles are fed minced fish, shellfish and

shrimp or on artificial pellet. It is obvious that fish of different species require different

feeding techniques (Amornsakun et al., 2003).

41

REFERENCES

Amornsakum, T., Sriwatana, W and Chamnanwech, U., 2003. The culture of sand goby,

Oxyeleotris marmoratus I: Feed and feeding scheme of larvae and juveniles.

Songklanakarin J. Sci. Technol. 25 (3), 367-371.

Ang, K. J., 1990. Status of Aquaculture in Malaysia. Aquaculture in Malaysia, 265-279.

Baylon, J. C., Failaman, A. N. and Vengano, E. L., 2001. Effect of salinity on survival and

metamorphosis from zoea to megalopa of the mud crab Scylla serrala forskal

(Crustacea : Portunidae). Asian Fisheries Science 14,143-151.

Bleeker, P., 1874. Oxyeleotris Bleeker. In: Roberts, T. R. (editor) The Freshwater Fishes

of Western Borneo (Kalimantan Barat, Indonesia). California Academy of

Sciences, San Francisco, 166-167.

Bleeker, P., 1877. Oxyeleotris marmorata. In: Inger, R. F. and Chin, P. K. (editor) The

Fresh-water Fishes of North Borneo. Natural History Publications (Borneo), Kota

Kinabalu, Malaysia, 175.

Cheah, S. H., Senoo, S., Lam, S. Y. and Ang, K. J., 1994. Aquaculture of a high-value

freshwater fish in [Malaysia] : The Marble or Sand Goby (Oxyeleotris

marmoratus, Bleeker). Aquabyte Section, 22-25.

Department of Fisheries, 1999. Annual Fisheries Statistic 1997, Volume 1. Department of

Fisheries, Malaysia, 217.

42

Donaldson, E. M. and Hunter. G. A. 1983. Induced final maturation, ovulation and

spermiation in cultured fish. In fish Physiology (Hoar, W. S., Randall, D. J. and

Danaldson, E. M., eds. ). New York : Academic Press. Vol. IX (B), 351-403.

Forsgren, E., 1997. Mate sampling in a population of Sand goby. Animal Behaviour 53,

267-276.

Huang, W. B., Chiu, T. S. and Shih, C. T., 2000. Effects of salinity of egg and early larval

characteristics of the black porgy Acanthopargus schlegeli. The Israeli Journal of Aquaculture-Bamidgeh 52 (2), 61-69.

Houde, E. D., 1978. Crititical food concentrations for larvae of larvae of three species of

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