Early Larval Development and Identification of Infectious Microbes and Antibiotic Resistance in Hybrid Climbing Perch, Anabas Sp

Early Larval Development and Identification of Infectious Microbes and Antibiotic Resistance in Hybrid Climbing Perch, Anabas sp.

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

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

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:

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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.

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

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 35 Hybrid Anabas Species 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 39 Anabas species 3.5.1 Sampling and Isolation of Bacteria 40 3.5.2 Tests to Identify Type of Bacteria on Infected Hybrid Anabas 41 species 3.6 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

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 58 Anabas species 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

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.

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

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.

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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).

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 2

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. 3

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. 4

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.

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 6

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.

Dorsal fin

Pelvic fin Soft rays

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

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).

Dorsal fin

Pelvic fin Soft rays

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,

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

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 10

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.