Transcript

i

Early Larval Development and Identification of Infectious

Microbes and Antibiotic Resistance in Hybrid Climbing

Perch, Anabas sp.

by

Normasarah bt Hj Ab Rahman

A thesis submitted in fulfillment of the requirements for the degree of

Master of Science

Faculty of Agro Based Industry

UNIVERSITI MALAYASIA KELANTAN

2016

ii

Early Larval Development and Identification of

Infectious Microbes and Antibiotic Resistance in

Hybrid Climbing Perch, Anabas sp.

NORMASARAH BT HJ AB RAHMAN

MASTER OF SCIENCE

2016

iii

THESIS DECLARATION

I hereby certify that the work embodied in this thesis is the result of the original

research and has not been submitted for a higher degree of any other University or

Institution.

OPEN ACCESS I agree that my thesis is to be made immediately

available as hardcopy or on-line open access (full text)

EMBARGOES I agree that my thesis is to be made available as hardcopy

or on-line (full text) for a period approved by the Post

Graduate Committee.

Dated from until .

CONFIDENTIAL (Contains confidential information under the Official

Secret Act 1972)*

RESTRICTED (Contains restricted information as specified by the

organization where research was done)*

I acknowledge that Universiti Malaysia Kelantan reserves the right as follows.

1. The thesis is the property of Universiti Malaysia Kelantan.

2. The library of Universiti Malaysia Kelantan has the right to make copies for the

purpose of research only.

3. The library has the right to make copies of the thesis for academic exchange.

SIGNATURE SIGNATURE OF SUPERVISOR

IC/ PASSPORT NO. DR LEE SEONG WEI

Date: Date:

iv

ACKNOWLEDGEMENT

Alhamdulillah, praise to Allah S.W.T, for His blessing and mercy that enabled

me complete this thesis.

Firstly, I would like to express my deepest gratitude and appreciation to my

supervisor, Dr. Lee Seong Wei for his guidance, constructive comments,

encouragement and support throughout the study on „Early larval development and

identification of infectious microbes and antibiotic resistance hybrid climbing perch,

Anabas sp.‟

Special thanks to Mr. Khalid for guidance in the laboratory and help throughout

this study. My appreciation to my friends especially all post graduate students for their

support. My deepest gratitude to my colleagues, Ms. Husna, Ms. Afiqah, Ms. Suaini,

Ms. Farisya and Mrs. Azwahanis for their support throughout this study. I am also

grateful to all my family members, especially my parents Haji Abdul Rahman Hamat

and Hajjah Che Hamidah Hassan, my uncle Adnan Hassan and my husband Asrul

Afandi Halim for their endless prayers and moral support.

Finally, I would like to thank Universiti Malaysia Kelantan especially Faculty

of Agro Based Industry for the research facilities and MyBrain for the scholarship

provided to me. My special thanks goes to Ministry of Higher Education, Malaysia

under Skill Niche Research Grant scheme (NRGS)

(R/NRGS/A07.00/00387A/006/2014/000152) for funding this research activity. Last

but not least, it is my pleasure to thank all others who have contributed either directly

or indirectly give their support and guidance.

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TABLE OF CONTENTS

PAGE

THESIS DECLARATION i

ACKNOWLEDGEMENT ii

TABLE OF CONTENTS Iii

LIST OF TABLES Viii

LIST OF FIGURES X

LIST OF ABBREVIATIONS Xiii

LIST OF SYMBOLS Xiv

ABSTRACT Xv

ABSTRAK Xvi

CHAPTER 1 INTRODUCTION

1.1 Background of the Study 1

1.2 Statement of the Problem 2

1.3 Objectives of the Study 3

1.4 Scope of the Study 4

1.5 Significances of the Study 4

1.6 Organisation of Thesis 5

CHAPTER 2 LITERATURE REVIEW

2.1 Introduction 6

2.2 General Characteristic and Taxonomy of Anabas testudineus 6

2.3 Early Development of Anabas testudineus 10

2.4 Water Parameter on Fish Larvae Development 12

2.4.1 Effect of Temperature on Early Development of Fish Larvae 12

2.4.2 Effect of Salinity on Early Development of Fish Larvae 14

2.4.3 Effect of pH on Early Development of Fish Larvae 16

2.4.4 Effect of Formalin on Early Development of Fish Larvae 18

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2.5 Bacterial Pathogens and Disease of Aquatic Organisms 19

2.6 Isolation and Identification of Bacterial from Aquaculture Sides 23

2.7 Characterization of Antibiogram for Salmonella sp. and Aeromonas sp. 24

2.8 Summary 29

CHAPTER 3 MATERIALS AND METHODS

3.1 Introduction 31

3.2 Material and Preparation of Sample 31

3.3 Process of Breeding Broodstock of Hybrid Anabas Species 32

3.4 Experiments on Environmental Effects During Early Development of

Hybrid Anabas Species

35

3.4.1 Effect of Temperature 36

3.4.2 Effect of pH 37

3.4.3 Effect of Salinity 38

3.4.4 Effect of Formalin 39

3.5 Method to Isolate and Identify Type of Bacteria on Infected Hybrid

Anabas species

39

3.5.1 Sampling and Isolation of Bacteria 40

3.5.2 Tests to Identify Type of Bacteria on Infected Hybrid Anabas 41

3.6

species

Methods to Characterize Antibiogram of Bacteria Isolated for Infected

43

Hybrid Anabas Species

3.6.1 Blood Haemolysis Test

43

3.6.2 Antibiotic Susceptibility Test 44

3.7 Statistical Analysis 45

3.8 Summary 46

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CHAPTER 4 RESULTS

4.1 Introduction 47

4.2 Fertilization and Hatching 47

4.3 Phases on Early Development of Hybrid Anabas Species 48

4.4 Results on Environmental Effects on Early Development of Hybrid

Anabas species

58

4.4.1 Effect of Temperature 59

4.4.2 Effect of pH 61

4.4.3 Effect of Salinity Concentration 62

4.4.4 Effect of Formalin Concentration 64

4.5 Isolation of Bacteria 66

4.6 Bacterial Identification 67

4.7 Blood Haemolysis Test 71

4.8 Antibiotics Susceptibility Test 72

4.8.1 Multiple Antibiotic Resistances (MAR) 75

4.9 Summary 75

CHAPTER 5 DISCUSSION

5.1 Introduction 77

5.2 Early Development of Hybrid Anabas species 77

5.2.1 Effect of Temperature 79

5.2.2 Effect of pH 81

5.2.3 Effect of Salinity Concentration 83

5.2.4 Effect of Formalin Concentration 85

5.3 Isolation and identification of Bacteria 88

5.4 Identification of Bacteria Salmonella sp. and Aeromonas sp. 89

5.5 Blood Haemolysis Test 90

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5.5 Antibiotic Susceptibility Test 91

5.5.1 Multiple Antibiotic Resistance (MAR) 96

5.7 Limitation of the study 97

5.8 Implication of the Study 98

5.9 Summary 98

CHAPTER 6 CONCLUSION AND FUTURE WORKS

6.1 Conclusions 99

6.2 Suggestions for Future Works 101

REFERENCES 102

APPENDICES 111

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LIST OF TABLES

NO. PAGE

4.1 Embryonic development stage of hybrid Anabas species 48

4.2 Larvae development stage of hybrid Anabas species 55

4.3 Number of suspected bacteria based on different isolation bacteria 66

media

4.4 Phenotypic characteristics of bacterial isolates, Salmonella sp. 68

4.5 Phenotypic characteristics of bacterial isolates, Aeromonas sp. 70

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LIST OF FIGURES

NO. PAGE

2.1 Thailand Anabas testudineus 5

2.2 Local Anabas testudineus 6

3.1 Process of breeding broodstock 34

4.1 Total times of hatching period, egg yolk absorption period and 59

starvation period emphasis on pH parameter

4.2 Total times of hatching period, egg yolk absorption period and 61

starvation period emphasis on temperature parameter

4.3 Total times of hatching period, egg yolk absorption period and 63

starvation period emphasis on salinity parameter

4.4 Total times of hatching period, egg yolk absorption period and 65

starvation period emphasis on formalin parameter

4.5 Total percentage (%) of antibiogram on isolate bacteria, 73

Salmonella sp.

4.6 Total percentage (%) of antibiogram on isolate bacteria, 74

Aeromonas sp.

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LIST OF ABBREVIATIONS

AML Amoxycilin

AMP Ampicilin

C Chloramphenicol

CT Colistin Sulphate

DO Doxycycline

E Erythromycin

F Florfenicol

FOS Fosfomycin

H(s) Hour(s)

Min(s) Minute(s)

MAR Multiple antibiotic resistance

MY Lincomycin

NA Nalidixic acid

NV Novobicin

OA Oxolinic acid

OL Oleondomycin

S3 Sulphonimides

SP Spiramycin

TE Tetracycline

TSA Tryptic soy agar

TSB Tryptic soy broth

GSP Glutamate starch pseudomonas

XLD Xylose lysine deoxycholate

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LIST OF SYMBOLS

cm Centimeter

% Percentage

g Gram

m Meter

mm Millimeter

mg Milligram

°C Degree Celsius

ml Milimeter

µm Micrometer

Nm Nanometer

dph Days post hatch

ppt Part per thousand

L Liter

µl Microliter

µg Microgram

(v/v) Volume over volume

(w/v) Weight over volume

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Perkembangan Awal Larva dan Pengenalpastian Jangkitan Mikrob dan

Ketahanan Antibiotik Terhadap Hibrid Ikan Puyu, Anabas sp.

ABSTRAK

Anabas spesis adalah ikan air tawar yang mempunyai potensi tinggi untuk

menjadi spesis komersial di Malaysia. Ia mempunyai permintaan pasaran yang tinggi

kerana kaya dengan kandungan protein. Tujuan utama kajian ini adalah untuk mengkaji

kesan faktor persekitaran terhadap masa penetasan, masa penyerapan kuning telur dan

masa kebuluran pada peringkat awal perkembangan puyu hibrid, Anabas spesis. Kedua,

untuk mengkaji pencilan dan mengenalpasti jenis bakteria yang hadir pada jangkitan

puyu hibrid, Anabas spesis. Ketiga, untuk mengenalpasti antibiogram yang sesuai

terhadap pencilan bakteria yang terdapat pada jangkitan puyu hibrid, Anabas spesis.

Dalam kajian ini, stok pasangan induk telah disuntik menggunakan hormon pituitari

untuk pembiakan. Peringkat perkembangan embrio dan larva direkodkan pada masa

persenyawaan telur (0 jam) sehingga 312 jam. Perkembangan larva dan embrio telah

dijalankan dalam empat faktor persekitaran yang berlainan seperti suhu, kemasinan, pH

dan formalin. Hasil kajian telah menunjukkan embrio terdiri daripada sebelas peringkat

bermula dari telur disenyawakan, belahan, morula, blastula, gastrula, pembentukan

badan embrio, vesikel optik dan pembentukan vesikel auditori, blastopore,

pembentukan ekor dan peringkat penetasan. Seterusnya, penetesan diteruskan dengan

proses organogenesis sehingga larva kelihatan seperti induk. Fasa larva terdiri daripada

dua peringkat iaitu pra-larva dan peringkat pembesaran larva. Data dianalisis dengan

menggunakan ANOVA sehala dan keputusan menunjukkan suhu adalah faktor penting

dalam tempoh penetasan dengan nilai signifikan (p<0.05). Keputusan faktor pH, suhu

dan kemasinan juga menunjukkan nilai signifikan pada penyerapan kuning telur.

Sementara itu, analisis data pada tempoh kebuluran menunjukkan nilai signifikan

(p<0.05) pada faktor suhu, formalin dan kemasinan. Seterusnya, kajian ke atas

antibiotik telah dilakukan ke atas jangkitan hibrid Anabas spesis. Antibiogram pencilan

bakteria telah diuji dengan 19 jenis antibiotik menggunakan kaedah cakera resapan.

Dalam eksperimen ini, Aeromonas sp. dan Salmonella sp. telah dipencilkan dengan

menggunakan medium kultur diikuti dengan ujian kimia untuk mengenal pasti jenis

bakteria yang hadir. Kepelbagaian Indeks Antibiotik Rintangan (KIAR) telah

digunakan untuk menentukan nilai indeks daripada jumlah kehadiran pencilan bakteria.

Dalam kajian ini, Aeromonas sp. menunjukkan indeks paling rendah (0.19). Sementara

itu, bakteria Salmonella sp. menunjukkan nilai indeks yang lebih tinggi (0.21 dan 0.22).

Keputusan pada antibiogram menunjukkan bahawa Salmonella sp. mempunyai nilai

sensitif yang lebih rendah (59.5%) berbanding Aeromonas sp. (67%). Keputusan hasil

kajian ini penting untuk memelihara dan meningkatkan pengurusan terhadap hybrid

Anabas spesis mengikut kekurangan bekalan ikan.

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Early Larval Development and Identification of Infectious Microbes and

Antibiotic Resistance in Hybrid Climbing Perch, Anabas sp.

ABSTRACT

Anabas species is a freshwater fish which has high potential of becoming a

commercial species in Malaysia. It has high market demand due to its rich protein

content. The main purpose of this study is to study the effect of environmental

parameters on hatching period, egg yolk absorption period and starvation period during

early development of hybrid Anabas species. Secondly, to isolate and identify type of

bacteria exist on infected hybrid Anabas species. Thirdly, to characterize antibiogram

of bacteria isolated from infected hybrid Anabas species. Matured broodstock were

injected using pituitary hormone to induce breeding. The development stages of

embryos and larvae were recorded over period of 0 hours (fertilization eggs) until 312

hours. The larvae and embryo development were studied in four different

environmental parameters, namely, water temperature, pH, salinity concentration and

formalin concentration. The early development was directly observed and documented

in embryonic phase and larvae phase. The growth during embryonic phase was

separated into eleven stages starting from a fertilized egg to cleavage, morula, blastula,

gastrula, embryonic body formation, optic vesicle and auditory vesicle formation,

blastopore closing, tail formation and hatching stages. The hatching further undergoes

organogenesis until appears like their parents. Larvae phase consists of two stages

which are pre-larvae and post larvae stage. Data analysis using one way ANOVA

revealed that temperature is an important and statistically significant factor on hatching

period (p<0.05). Result on pH, temperature and salinity concentration also gave

statistically significant effect on egg yolk absorption. Meanwhile, results on starvation

period indicated that temperature, formalin concentration and salinity concentrations

were significant. Investigation on antibiotic resistance on infected hybrid Anabas

species was done for 19 types of antibiotics using disc diffusion method. Aeromonas

sp. and Salmonella sp. were isolated by using medium differentiation followed by a

chemical test to identify the type of bacteria. The study reveals that isolates bacteria of

Aeromonas sp. has lower Multiple Antibiotic Resistance (MAR index = 0.19),

compared Salmonella sp. (MAR index = 0.21 and 0.22). The results on antibiogram

showed that Salmonella sp. has lowered sensitive cases (59.5%) compared to

Aeromonas sp. (67%). The findings in this study are important to conserve and

improve management of Anabas species due to limitation of fish supply.

1

CHAPTER 1

INTRODUCTION

1.1 Background of the Study

Freshwater fishes have attracted attention of many consumers due to

their diverse and high nutrient value. In Malaysia, about 300 species of

freshwater fishes have been estimated inhabit in the Malaysian rivers and lakes

area (Rahman et al., 2012). Among these, the freshwater fish of Anabas

testudineus is one of the most commonly found species. The Anabas

testudineus is popularly known as „Puyu‟ in Malaysia (Shinsuke et al., 2008). It

is popular among Malaysian due to its scrumptious taste, valuable diet for sick

and convalescent. It is a protein provider which contains high amounts of

nutrients such as iron and copper for haemoglobin synthesis. However, this

species suffers from slow growth for production and small size in culture

(Hussain, 2005). In contrast, in Thailand, the Anabas testudineus which is

commonly known as „Thai Koi‟ (Shinsuke et al., 2008) has faster growth rate

and shorter culture period. It also has larger size which is more suitable and

economical for industry and culture system (Rahman et al., 2015). Due to

technical inefficiencies of hatchery in Thailand, it has lost it appetizing taste

and high productivity characteristics (Rahman et al., 2015).

2

Sexually behaviour of this species was observed at approximately 70-

100 mm in size of fecundity and breeds in low lying water bodies with at least

10-25 cm depth (Binoy &Thomas, 2004). It matures about six months of age at

about 8.0 cm of length. This species also do not have parental care on their eggs

which is floating on the surface of water and do not make bubble nests for their

eggs (Marimuthu et al., 2009).

The habitat of this species were often found in lower land area such as

rice paddies, ditches, small pits, estuaries, and streams with dense vegetation

(Chandra et al., 2008). According to Zalina et al. (2012), this species is also

described as carnivorous or an insectivore. At the stage of early development of

larval and juvenile, they are feed on plankton. On the other hand, during the

adult stage they prefer both carnivorous and herbivores or known as

omnivorous feeding nature.

1.2 Statement of the Problem

In some Malaysian rural areas, people used to build their homes and

other infrastructure near paddy fields. As a result, the habitats for the freshwater

fish such as Anabas testudineus become under considerable pressure and are

nearly extinct in some areas. High market demand for fish as a protein source

(Baran et al., 2007) and insufficient freshwater supply have attracted

researchers in many countries in South East Asia to study on freshwater fish.

Unfortunately, very little work has been reported in Malaysia related to Anabas

3

testudineus species. Most of the previous researchers on the Anabas testudineus

were on breeding and spawning. There has been lack of research on hybrid

Anabas species focusing on their early developmental stage.

Besides changes in fresh water habitat, natural population of fresh water

fish is rapidly decreasing due to infection of bacteria and excessive use of

antibiotics during the past few years. If this situation continues, the freshwater

fish especially of Anabas species will be endangered in near future. The

freshwater fish has also been exposed to the toxicology and environmental

problem due to contamination and chemical used.

The bacterial contamination on freshwater fish is a main obstacle for its

economic development and also a health risk for human. The cases of

prohibited antibiotic residues and diseases contaminated aquaculture products

can lead to huge economic losses when such products are rejected by the

customers.

1.3 Objectives of the study

The objectives of this study are as the following:

i. To study the effect of environmental factors on hatching period, egg yolk

absorption period and starvation period on early development of hybrid

Anabas species.

ii. To isolate and identify type of bacteria exist on infected hybrid Anabas

species.

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iii. To characterize antibiogram of bacteria isolated from infected hybrid

Anabas species.

1.4 Scope of the Study

The scopes of this study are as the following:

i) This study focused on environmental factors on early development of

hybrid Anabas species only.

ii) Isolation and identification of gram negative bacteria only.

iii) Identification of antibiotic resistance only involved 19 types of antibiogram

1.5 Significance of the Study

In aquaculture industry, information on early development stages of

larvae such as egg yolk absorption, hatching period and starvation period are

important to implement an effective system on management of the larvae

stocks. The information from this study can serve as useful guidelines for the

fish farmers to find suitable period and critical period on culture larval rearing.

The findings from the experiments would provide useful baseline

information for optimization of large scale culture and ultimately for the culture

management of freshwater fish in the future. It is hypothesized that the growth

and survival rate of larval can be highly influenced by environmental conditions

including pH, temperature, salinity and formalin concentrations.

5

Extensive literature was unable to locate relevant information on

infections of bacteria Aeromonas sp. and Salmonella sp. in hybrid Anabas

species. Therefore, there is a need to collect information about the potential use

of antibiotics, antibiotic resistance and effectiveness of antibiotic to control

bacterial diseases in hybrid Anabas species. The study on bacterial resistance

could provide useful information to control bacterial infection in freshwater

fish.

The study on bacterial resistance could provide useful information to

control bacterial infection in freshwater fish. Besides that, it also used to point

out the safety level of consumption in addition to build public awareness,

minimize the risk and control the outbreak disease attributed by Aeromonas sp.

and Salmonella sp. bacteria. The antibiotics should be appropriately used in

aquaculture to minimize the risk of transferring side effect disease towards

human

1.6 Organisation of Thesis

This thesis is organised into six chapters. Chapter 1 serves as an essential

introduction to the research. Chapter 2 provides background information and

review of related literature. Chapter 3 describes the materials and methods.

Chapter 4 presents the results, Chapter 5 provides discussion and finally

Chapter 6 concludes the thesis.

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CHAPTER 2

LITERATURE REVIEWS

2.1 Introduction

This chapter provides background information to the field of study and

related previous researches. It covers the general characteristic and taxonomy of

Anabas testudineus in Malaysia and Thailand, early development of Anabas

testudineus, effect of water parameter on fish larvae development, bacterial

pathogens and disease of aquatic organisms and characterization of antibiogram

for Salmonella sp. and Aeromonas sp. Finally, a summary is provided to

conclude the literature review.

2.2 General Characteristic and Taxonomy of Anabas testudineus

Anabas testudineus is a type of fish with quite long in shape, thick and

can grow between 15cm to 20cm, and commonly much smaller size in the wild

(Webb et al., 2007). The taxonomy of Anabas testudineus belong to suborder

Anabantoidei and commonly known as labyrinth fish. Malaysian Anabas

testudineus has no body spot on the body surface (Zalina et al., 2012). It is a

type of fish having 15 strong spines at dorsal fin and 7 soft rays as shown in

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Figure 2.1. It pelvic fins are with one spine and five soft rays and caudal fin is

rounded. In matured size, adults females tend to have shorter dorsal fin compare

to males with a more elongate dorsal fin. It can grow within 5 until 6 months.

The size at sexual maturity for female was found around 12.70±0.4cm in total

length and 50.25±4.4g in body weight. In matured stage for male was

12.71±0.4cm in total length and 31.63±3.2g in body weight. Both male and

female are more darken in greyish with a silvery colour of varying intensity on

the mid lateral flank and on the caudal fin.

Figure 2.1: Malaysian Anabas testudineus

The Thailand Anabas testudineus have great variability in

morphological characters and body colouration with body spot on the body

surface (Mahmood et al., 2004). It is a type of fish having 18 strong spines at

dorsal fin and 9 soft rays as shown in Figure 2.2. In matured size, adults

Dorsal fin

Pelvic fin

Soft rays

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females tend to have shorter dorsal fin compare to males with a more elongate

dorsal fin. It also can grow very faster within 3 until 4 months. This species also

having higher resistant against disease and can be marketed in live condition.

The size at sexual maturity for female was around 15.20±1.24cm in total length

and 61.10±17.32g in body weight (Amornsakurn et al., 2005). Meanwhile size

at matured stage for male was 12.30±1.14cm in total length and 51.10±15.25g

in body weight (Kohinoor et al., 2007). Both male and female having colour

body in greyish with a pale silvery colour of varying intensity on the mid lateral

flank and on the caudal fin (Jacob, 2005).

Figure 2.2: Thailand Anabas testudineus

The paired ventral pelvic fins are filamentous in which the first ray is

elongated and the remainder are vestigial. The fins have a sensory function with

physical and chemoreceptors. It plays a role in feeding, courtship and mating,

Dorsal fin

Pelvic fin

Soft rays

9

and aggressive activities (Jacob, 2005). In the wild, the Anabas testudineus

occurs in two main colour morphs which are grey and dark grey, with several

variants. Many of this species are produced through commercial selective

breeding.

According to Fuiman & Werner (2009), the life history of a fish can be

divided into five primary periods: embryo, larva, juvenile, adult and senescent.

Each of these life history periods can be characterized by one or two dominant

physiological processes that largely determine the changes in morphological

structure, physiological capabilities, behavioural motivation, and ecological role

of an individual at that time of life. The embryonic periods is a time of

ontogeny, a complex set of changes that include rapid proliferation of cells,

differentiation of new tissues, and reorganization or loss of existing ones.

Anabas testudineus is a member of the anabantoid group of air-

breathing fishes occurring in freshwater and indigenous to southern Asia and

Africa (Nelson, 2006). According to Pal & Chaudhry (2010), Anabas species is

listed in all populations under Anabas testudineus. Below is scientific

classification of Anabas species (Bloch, 1792):

Kingdom : Animalia

Phylum : Chordata

Subphylum : Vertebrate

Class : Actinopterygii

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Order : Perciformes

Family : Anabantidae

Genus : Anabas

Species : Anabas testudineus

2.3 Early Development of Anabas testudineus

Early development of a fish can be divided into five primary periods

such as embryo, larva, juvenile, adult and senescent. The early life stages of

fishes are sometimes referred to as ichthyoplankton and typically consider inert

particles. The presences of specialized structures that exist during early life also

supports fishes have larvae.

Jalilah et al. (2011) reported a study of the embryonic and larvae

development of Malaysian Anabas testudineus for samples taken from Marang,

Terengganu. In their experiment, three females and nine matured males

broodstock samples were selected for induce breeding by using intramuscular

injections of Ovaprim hormone (0.5 ml/kg fish) near the caudal peduncle. The

ratio of female to male brood stock was 1:3 by using three breeding containers.

Their results revealed that fertilization occurred between more than 7h(s) to

8h(s) at temperature range between 26°C to 29°C. The samples achieved fully

hatching around 28h(s) at temperature range 25°C-27°C. The female parent

12

released about 4070-43600 eggs with the fertilization rate varied from 55% to

63%, and the percentage of hatching rate vary from 35% to 96%. Their study

concluded that fluctuation in percentage of fertilized and hatching eggs is due to

variations in raining season temperature.

Marimuthu et al. (2009) conducted a study for the fecundity of

Malaysian freshwater fish Climbing perch, (Anabas testudineus, Bloch) with

samples taken near Sungai Petani, Kedah, Malaysia. A total of 70 samples of

matured females were investigated. They reported that the individual fecundity

was 36,804 ± 2,289 for the fishes with total length of 16.13cm±0.249cm and

body weight of 78.596g ± 3.275g. The minimum number of eggs was 3120 for

0.60 g ovary weight and maximum number of eggs 84690 for 16.70 g ovary

weight. Their study revealed that individual freshwater fish of the same length

and body weight give result on variations in egg size.

Amornsakun et al. (2005) reported a study about development in early

life stage of Climbing perch, Anabas testudineus larvae in Thailand. Their

induced spawning was done using chemical injection (Suprefact and Motilium)

to produce fry. The sexually mature fish were cultured in fiber-glass tank (water

volume 300L) with the ratio of male and female brooders 2:1. The 15L glass

aquarium (water volume 10L) containing 7,000-9,000 eggs were used to

determine fertilization rate, hatching out and hatching rate. The average

fertilization rate was 92.67%, hatching out was 20h(s) 30min(s) and average

hatching rate was 87.44% at a range temperature of 27.0ºC-30.5ºC. Sampling of

the newly-hatched larvae was done at 2h(s) intervals, when 20 of them were.


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