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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.
v
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
vi
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
vii
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
viii
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
ix
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
x
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.
xi
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
xii
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
xiii
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.
xiv
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.
4
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.
6
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
7
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
8
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
10
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.