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(Epinephelus lanceolatus)


Thesis Submitted in Fulfillment of the Requirements for the
Degree of Doctor of Philosophy in the Faculty of Fisheries and Aqua Industry
Universiti Malaysia Terengganu

September 2014


Abstract of thesis presented to the senate of Universiti Malaysia Terengganu
fulfillment of the requirement for the degree of Doctor of Philosophy

OF THE GIANT GROUPER (Epinephelus lanceolatus)

September 2014

Chairperson : Associate Professor Abol Munafi Ambok Bolong, Ph.D.
Member : Associate Professor Yeong Yik Sung, Ph.D.
Safiah Jasmani, Ph.D.
Institute : Faculty of Fisheries and Aqua Industries

The characterization of Vitellogenin (Vtg), a female-specific
glycolipophosphoproteins and its application role as a biomarker for sex
identification in the Giant grouper (Epinephelus lanceolatus) was determined. Vtg is
synthesized in liver of female grouper under stimulation of estrogen and will be
transported into ovary via bloodstream through receptor-mediated endocytosis.
Electrophoresis on plasma sample proof that female was confirmed Vtg positively
presence rather than male. Identification of Vtg by aligning the N-terminal sequence
revealed that the sequence homology for the giant grouper FLELVQLLR matched
the Vtg of other fish species and the de novo peptide sequencing with MALDI-TOF
yields peptide ions with high homology to plaice, flounder, medaka, mummichog,


eelpout and goby. A conceptual translation of the open reading frame resulted in a
1,704 amino acid protein sequence, with the molecular weight of giant grouper Vtg
occurs at 187 kDa.

An experiment was conducted to produce Vtg polyclonal antibody of Giant
grouper. The proposed of this experiment was to develop an immunologic assay for
Giant grouper Vtg in order to establish an accurate method for aiding the sex
identification. A number of techniques allow proteins to be tested including enzyme-
linked immunosorbent assay (ELISA) and Western blot are based on the use of
antibodies specific to Vtg. The results found that the ELISA technique used in this
study was successfully measure Vtg in the immunized rabbit IgG titer. The presence
of Vtg as a biomarker in gender identification was confirmed using Western Blot on
the positive control (females naturally) and negative control (natural male).

The presence of Vtg by induction of the male Giant grouper (as negative control)
with 17-Estradiol (E2) and compared with female (as positive control). Male fish
do not produce Vtg but can be induced by exposure to compounds possessing
estrogenic activity. The anti-Giant grouper Vtg polyclonal antibody developed
recognized the purified Giant grouper Vtg in female and male treated with E2. This
reactivity demonstrates that Vtg polyclonals from immunization were highly


antigenic epitopes. Subsequently, this antibody could be used to measure the Vtg
titer in female blood plasma.

As revealed by the Vtg status and levels in the giant grouper, hormone E2 was
effective in controlling reproductive parameters in females. Greatly increasing the
level of Vtg in female plasma rather than male by implant with E2 hormone was
influenced sex change from unknown sex to the female condition and also to
enhance the early puberty of female Giant Grouper.

The information on Vtg through this experiment could provide the basis of an
immunological assay for sex identification and maturational status of the Giant
grouper. Consequently, the aim of the present study was to establish a simple,
efficient and non-lethal technique by using Vtg as biomarker indicator for rapid sex
identification without killing or seriously injuring the sampled individuals.


Abstrak tesis yang dikemukakan kepada senat Universiti Malaysia Terengganu
sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

KERTANG (Epinephelus lanceolatus)

September 2014

Pengerusi : Profesor Madya Abol Munafi Ambok Bolong, PhD.
Ahli : Profesor Madya Yeong Yik Sung, PhD.
Safiah Jasmani, PhD.
Institut : Fakulti Perikanan dan Akua Industri

Pencirian vitellogenin (Vtg), iaitu glycolipophosphoproteins yang khusus terdapat
pada induk betina ikan kerapu kertang (Epinephelus lanceolatus) telah dikenalpasti
penggunaannya sebagai penanda biologi jantina. Vtg disintesis di dalam hati
daripada ikan betina di bawah rangsangan estrogen dan akan diangkut ke ovari
melalui saluran darah melalui reseptor-pengantara endositosis. Elektroforesis sampel
plasma membuktikan, induk ikan betina disahkan positif kehadiran Vtg dan
bukannya dari induk ikan jantan. Pencirian Vtg dengan menjajarkan urutan N-
terminal menunjukkan bahawa homologi urutan untuk ikan kerapu Kertang
FLELVQLLR berpadanan dengan Vtg spesies ikan yang lain dan de novo peptida


urutan dengan MALDI-TOF menghasilkan ion peptida dengan homologi tinggi
untuk Plaice, Flounder, Medaka, Mummichog, Eelpout dan Goby. Satu terjemahan
konsep rangka bacaan terbuka menyebabkan asid amino urutan sebanyak 1,704
protein, dengan berat molekul kerapu Kertang Vtg, 187 kDa.

Kajian telah dijalankan untuk menghasilkan Vtg polyclonal antibodi ikan kerapu
Kertang. Percubaan untuk membangunkan assay immunologi ikan kerapu Kertang
telah dijalankan bertujuaan untuk pengecaman jantina ikan telah dibuat dengan
menggunakan hormon 17-estradiol (E2). Secara semulajadinya, ikan jantan tidak
menghasilkan Vtg tetapi boleh didorong berbuat demikian dengan cara penanaman
hormon dari jenis sebatian yang mempunyai aktiviti estrogenik. Teknik analisa
protein telah diuji dengan mengunakan Enzyme-Linked Immunosorbent Assay
(ELISA) dan Western blot yang berdasarkan kepada penggunaan antibodi khusus
kepada Vtg. Keputusan mendapati, teknik ELISA yang digunakan dalam kajian ini
telah berjaya mengukur Vtg dalam IgG titer arnab yang diimunisasikan.

Kehadiran Vtg sebagai penanda bio didalam pengecaman jantina telah disahkan
dengan menggunakan kaedah Western Blot terhadap kawalan positif (betina semula
jadi) dan kawalan negatif (jantan semulajadi). Ia mendedahkan bahawa kerapu anti-
Giant Vtg polyclonal antibodi mengiktiraf kehdiran Vtg secara semula jadi ikan
betina dan yang dirawat dengan menggunakan hormon estradiol. Kereaktifan Ini


menunjukkan bahawa polyclonals Vtg dari imunisasi adalah epitopes dan sangat
antigenik. Kemudian, antibodi ini boleh digunakan untuk mengukur titer Vtg dalam
plasma darah ikan betina.

Seperti yang diperkatakan dari status dan paras Vtg dalam ikan kerapu Kertang,
hormon E2 adalah berkesan dalam mengawal parameter pembiakan dalam ikan
betina. Hormon E2 didapati meningkatkan tahap Vtg dalam plasma ikan betina
dengan begitu berkesan. Hormon E2 juga mempengaruhi penukar jantina daripada
status jantina ikan yang tidak diketahui kepada keadaan betina dengan lebih cepat
dan juga untuk mempercepatkan kematangan awal kerapu kertang betina.

Maklumat mengenai Vtg melalui kajian ini dapat menjadi asas bagi kajian
immunologi seterusnya. Adalah bertepatan dengan maksud kajian ini dilaksanakan
kerana ia akan dapat menjadikan Vtg sebagai penanda biologi yang mudah, efisien
dan tidak merosakkan dapat dicapai.



Praise, to Almighty ALLAH, for his blessing to me for finish my work on
preparing this thesis. I would like to express my sincere gratitude to Assoc. Prof Dr.
Abol Munafi Ambok Bolong, the Chairman of the Supervisory Committee for his
invaluable guidance and supervision during my PhD program in the Universiti
Malaysia Terengganu, Malaysia. Without his constant encouragement, this work
would never be completed.

I am proudly indebted to the co-supervisors, Assoc. Prof. Dr. Yeong Yik Sung,
Faculty of Fisheries and Aqua Industries, Universiti Malaysia Terengganu and Dr.
Safiah Jasmani, Institute of Aquaculture Tropical (AKUATROP), Universiti
Malaysia Terengganu for their helpful suggestions, constructive comments, and
meaningful contribution to my study.

I express my gratefulness to the Director General of Department of Fisheries,
Ministry of Agriculture and Agro Industries, and Director of Research, Fisheries
Research Institute (FRI) for permission and encouraging me to pursue the study. I
acknowledge my gratitude to Tn. Haji Hussin Mat Ali, Director of Marine
Aquaculture Research, FRI for giving me the opportunities for studying.


I would like to thanks to staff at Institute of Marin Biotechnology, Universiti
Malaysia Terengganu, especially to Science Officer and staff, Julius and Aishah for
their kind assistance during my analysis work there.

I am gratefully indebted to my friends and staff members of Fisheries Research
Institute Tanjong Demong who helped me during the study. I also thank to Head of
Center, FRI Tg. Demong, Hajjah Nik Haiha Nik Yusoff, and research officers; Dr.
Shaharah Mohd Idris, Haji Nik Razali Nik Lah, Sufian Mustafa, Saiful Anwar Deris
and research assistance Baihaqi Othman for their kind assistance and cooperation in
hatchery, laboratory works and data analysis.

Words are not enough to express my heart feelings to my parents, Om bin Manan
and Embon binti Maidin, who provide me their untiring support since my childhood.
Last but not least, gratitude to my wife, Melor @ Ramlah Abdul Hamid, and my
daughter, Najihah, Najwa, Nasuha and my son, Bazli Hilman, Faris Hakimi and
Ikmal Hakim for their patience, sacrifice, understanding and encouragement during
my study. For my child, lets this success story for motivate you all in future.

All the best Alhamdullilah


I certify that an Examination Committee has met on 16 January 2014 to conduct the
final examination of Ahmad Daud Om on his Doctor of Philosophy thesis entitled
Vitellogenin as a biomarker for sex identification of the Giant grouper (Epinephelus
lanceolatus) in accordance with the regulations approved by the senate of Universiti
Malaysia Terengganu. The committee recommends that the candidate be awarded
the relevant degree. The members of the Examination Committee are as follows:

Profesor Dr. Sakri Ibrahim
Faculty of Fisheries and Aqua-Industry,
University Malaysia Terengganu,
Kuala Terengganu, Terengganu

Dr, Shahreza bin Md. Shariff
Faculty of Fisheries and Aqua-Industry,
University Malaysia Terengganu,
Kuala Terengganu, Terengganu
(Internal Examiner)

Profesor Dr.Sharr Azni bin Harmin,
Faculty of Biotechnology,
University Industry Selangor,
Bestari Jaya, Selangor.

(External Examiner)



I hereby declare that the thesis is based on my original work except for quotations and
citations, which have been duly, acknowledge. I also declare that it has not been
previously or concurrently submitted for any other degree at UMT or other institutions.





1.1 Background of the study 1
1.2 Statement of the problem 3
1.3 Significant of the study 5
1.4 Objectives of the study 7

2.1 Life history of giant grouper 8
2.2 Characteristic of grouper reproduction 12
2.3 Hormonal control in fish production 13


2.4 Biochemical methods for sex identification and gonad
2.5 Vitellogenin as a biomarker indicator 20

3.1 Source of broodstock 24
3.2 Blood sampling 28
3.3 Hormonal induction of vitellogenin synthesis 29
3.4 Electrophoresis of Vitellogenin 30

(Epinephelus lanceolatus)
4.1 Introduction 31
4.2 Materials and Methods 34
4.2.1 Sex status identification 34
4.2.2 Experimental design 36
4.2.3 Induction of Vitellogenin in male Giant grouper 36
4.2.4 In gel digestion procedure 37
4.2.5 MALDI-TOF preparation 37
4.2.6 Identification of vitellogenin 40
4.2.7 Molecular characterization of Giant grouper vitellogenin 40
4.3 Results 43
4.3.1 Sex status of giant grouper 43
4.3.2 Determination of the size and sequence of Giant grouper
4.3.3 Molecular characterization of Giant grouper vitellogenin 53
4.4 Discussion 60


GROUPER (Epinephelus lanceolatus)
5.1 Introduction 66
5.2 Materials and Methods 69
5.2.1 Hormonal induction of vitellogenin synthesis and
5.2.2 Production steps of polyclonal antibody production 69
5.2.3 Purification of Vitellogenin 70
5.2.4 Immunization of Vitellogenin 71
5.2.5 Antibody Purification 73
5.2.6 Quantification of Vitellogenin with Enzyme-linked
Immunosorbent Assay (ELISA)
5.2.7 Validation of vitellogenin with Western immunoblotting 78
5.3 Results 79
5.3.1 Development of polyclonal antibody 79
5.3.2 Validation of vitellogenin 81
5.4 Discussion 83
FEMINIZATION OF GIANT GROUPER (Ephinephelus lanceolatus)
6.1 Introduction 91
6.2 Materials and Methods 90
6.2.1 Preparation and usage of hormone 90
6.2.2 Broodstock selection 92
6.2.3 Sampling procedure 93
6.3 Results 87


6.3.1 Morphological characteristic of genital sex 87
6.3.2 Estradiol effects of vitellogenin profile 89
6.4 Discussion 99





Water parameter in Giant grouper broodstock tank

2 Comparison of MS/MS spectra of Giant grouper vitellogenin
sequences from other species using the Mascot search engine
3 Peptide mass fingerprint (PMF) of the trypsin digestion of male
Giant grouper Vtg induce with E2

4 Distance matrix viewer showing distance and their standard errors
for sequence pairs.

5 Effect of E2 on sex reversal, from unknown status to
female status of Giant grouper after 6 months experiments. (+
indicate positive sex change from unknown to female, - indicate
negative change from unknown to female).

6 Effect of E2 on early maturation of puberty Giant grouper after 5
months experiments. (+ indicate positive sex change from unknown to
female, - indicate negative change from unknown to female).



1 Morphology of the Giant grouper 9
2 Schematics drawing on simplified overview of fish
hormonal control and physiological responses to estrogenic
chemical exposures of Giant grouper.
3 Cement concrete tank (a) 150 ton tank 12 m diameter x 2.0
water depth and (b) 400 ton (10 x 2.0 x 2.5 m) used for
rearing Giant grouper broodstock.
4 Broodstock management of Giant grouper (a) weighing the
broodstock (b) check broodstock sex status (c) feed
5 Preparation of vitamin with using capsule before give to the
6 Collecting blood from the afferent filamentary artery
(AFA) in the gill filament by heparinized needles (21G).
7 The procedure to inject male Giant grouper by E2 for
synthesis of Vtg
8 The procedure of electrophoresis on Giant grouper Vtg. 31
9 Determination of Giant grouper sex status by using cannulation. A
and B. Sampling the broodstock with using catheter, C. Semen, D.
Observation of eggs size using microscope, E. Sample eggs or
semen in Ringers solution
10 Schematic outline of protein identification steps by mass
spectrometric MALDI-TOF) analysis.
11 Presence of vitellogenin in plasma of broodstock detected with
cannulation technique
12 SDS-PAGE result on Vtg band only in female (F1 and F2) and
not in male (M1 and M2), Unknown (UK1 and UK2), and M


13 Vitellogenin band appeared in negative control (male naturally)
and positive control (female naturally) of Giant grouper
14 Denaturing (SDS) polyacrylamide gel of purified plasma
induction with estrogen-treated (E2) of male Giant grouper
plasma band appeared in male Giant grouper after being
induced with estradiol (E2)
15 MALDI-MS mass fingerprinting generated by in gel 1D
electrophoresis tryptic digestion of vitellogenin a) Male giant grouper
after induced with E2, b) natural male giant grouper and c) natural
female giant grouper. (*) Means Vtg peaks at 2292 m/z
16 Significant level 95% confidant Vtg, where P is probability the
observed match is a random event
17 Amino acid sequence (1,704) in single letter code of Giant
grouper vitellogenin peptide showing sequence coverage after
trypsin digest
18 Polygenetic distributions of 14 species of Vtg sequences.
Numbers besides nodes indicate the percent of bootstrap values
for each branch of the tree in the 1,000 bootstrap trials.
19 Schematic representation of the domain in Vtg peptide
sequence of (A) Domain architecture of teleost fish Vtg (Babin,
et al., 2007). Large lipid transfer (LLT) module also referred to
as Vtg_N and LPD-N domain, polyserine track (PT) domain,
and von Willebrand-factor type-D domain (VWD). The
horizontal line indicates the receptor-binding region to VtgR.
Lipovitellin I (Lv-I) and II (Lv-II), phosvitin (Pv), and !-
component (!-C) are the yolk protein generated by the
enzymatic cleavage of Vtg inside the oocyte. Conserved
domains of (B) Epinephelus lanceolatus, (C) Icthyomyzon
unicuspis, (D) !"#$%&'(#)*+ -(.'(/0 anu (E) 1-('"(+
2(#'3#)$4(-*+ Vtg by using NCBI software.
20 Secondary structure of Epinephelus lanceolatus Vtg as
predicted by the Phyre2 software. The green, blue color and the
faint lines symbols represent "-helix, !-sheet and coil
respectively. See secondary structure of other species domain


in Appendix F.
21 The 3-D structure of Vtg in fishes from different species.
Figure A, B, C and D are the 3-D structure of Epinephelus
lanceolatus, Ichthyomyzon unicuspis, Dicentrachus labrax and
Clarias macrocephalus respectively. The location of helix;
blue: helix1; green: helix 2; light green: helix 3; red: helix 4
22 The production step of polyclonal antibody of Giant Grouper 70
23 Project flow and time line of production of vitelllogenin
polyclonal antibody of Giant grouper..
24 The chromatogram of purification using Giant grouper
vitellogenin plasma
25 Vitellogenin ELISA of estrogen-treated Giant grouper plasma
at various dilutions ranging from 1: 125 to 1:2,204,800.
Specific IgG Titre of rabbits immunized with Vtg: a) Primary
immunization vs Pre-Immunized Controls. B) Control vs
Primary Immunization vs Booster 1 vs Booster 2
26 Denaturing (SDS) polyacrylamide gel of purified plasma from
esxtrogen-treated (E2) of male Giant grouper Vtg band
appeared in male Giant grouper after being induced with
estradiol (E2
27 Western blot showing immunoreactivity of polyclonal
antivitellogenin rabbit plasma to estrogen-treated (E2) and
control (c) Giant grouper male plasma.
28 Preparation of pellet, A; ingredient, B; Steel pellet mold, C;
ingredients fixed in the mold, D; pellet ready to be implanted

29 Specification and diagram of the mold pellet. A; Mold overview
from top, B; Cross section overview of the holes, C; Steel plate, D;
Steel bar for fixing in and pulling out of the pellet and implanter.



30 Papila genital morphological structure of Giant grouper; A: Female,
B: Male and C: unknown
30 SDS PAGE result of sex reversal and puberty of Giant grouper
during 5 months sampling.



AFLP Amplified fragment length polymorphism
ACN Acetonitrile
DMY Male sex-determining gene
DNA Deoxyribonucleic acid
FDA US Food and Drug Administration
HPLC High performance liquid chromatography
Enzyme immune assay
Endocrine disruptive chemicals
ELISA Enzyme-linked immunosorbent assays
E2 Hormone estradiol
ESI Eectrospray Ionization
FRI Fisheries Research Institute
FOM Final oocyte maturation
GSI Gonad somatic index
GMP Good management practice
GtH Gonadotropins hormone
GnRH Gonadotropins releasing-hormone
K Potassium
MS-222 Tricane methane sulfonate
MALDI Matrix-assisted laser desorption/ionization
LLT Large Lipid Transfer (LLT)
PT Polyserine track


OD Optical density
PCR Polymerase Chain Reaction
IUCN International Union for Conservation of Nature
T Testosterone
ppm Parts per million
Vtg Vitellogenin
11KT 11-keto-testosterone
VWD Von Willebrand-factor type-D domain
2D-PAGE Two-dimensional polyacrylamide gel



1.1 Background of the study
The culture of high-value species (e.g. groupers, snappers, cobia, pompano and
tuna) is popular in many parts of the world, and continues to develop rapidly in the
Southeast Asian region (Rimmer, 2004). Grouper is an important fish species in
marine, brackishwater and off-shore cage aquaculture. Asia produced approximately
62,088 MT of groupers in 2005, representing 24% of the total world fisheries
production for this species (FAO, 2007). Production increased to 250,000 MT in 2009
(Vi et al., 2009), with major grouper-producing countries including China, Taiwan,
Indonesia and Thailand.

More than 20 species of marine finfish species are cultured in Malaysia, and
half are groupers with major species including the Orange-spotted grouper
(Epinephelus coioides), Brown-marbled grouper (Epinephelus fuscoguttatus), Malabar
grouper (Epinephelus. malabaricus), Dusky-tail grouper (Epinephelus bleekeri),
Humpback grouper (Cromileptes altivelus), Coral trout/Leopard grouper
(Plectropomus leopardus) and Giant grouper (Epinephelus lanceolatus). The pilot
scale marine finfish culture was initiated in Penang (Teng and Chua, 1978) and Setiu
Lagoon of Terengganu in the 1970s (Hussin, 1986) and it has since expanded to more


than 99,749 cages encompasing an area of 1,936,493.82 m
with 2,251 famers in 2011
(DOF, 2011).

The Giant grouper is one of the most popular cultured grouper species in Malaysia
due to its fast growth rate and high market demand. Hong Kong represents one of the
major importing countries of Malaysia, with approximately 2.4 tonnes traded in 2004
(Lau and Parry-Jones, 1999). Giant grouper culture is quite successful in Taiwan but
the production is low and the proportion of traded individuals from wild versus
hatchery production is unknown. The status of Giant grouper culture in Indonesia and
Thailand is unknown but both of these countries are actively studying the breeding
method of this species.

One of the major problems which hinders the development of the Giant grouper
culture worldwide is the lack of seedlings. Though the captive rearing techniques are
quite developed, the breeding method for the Giant grouper has yet to be established,
mainly because the breeding physiology is not fully understood. In addition, sex
identification is difficult when they are hemaphrodites in nature.

Therefore, in order to develop Giant grouper culture technology, the seed
production technology must be developed. The success of seed production activity


depends on the gonad maturity of broodstock, which naturally depends on the age,
size, feed, environment, season and broodstock management. A range of factors
imposed by hatchery practices has been found to influence egg quality such as stress
on broodstock brought about by handling, over-ripening of eggs and hormone
administration in induced spawning.

One of the striking features in teleost fish is the flexibility of the sex identification
with a range of gonochoristic species, where individuals are either males or females, or
species displaying a change in gender during the lifetime. Research works directed to
developing the biotechnology tools to assess gender and maturational status of Giant
grouper are needed.

1.2 Statement of the Problem

The Giant grouper, like other groupers of the family of Serranidae, are protogynous
hermaphrodites (Smith, 1971). Sex change occurs from female to male as the age
increases, depending on the environment of the populations studied (Donaldson,


Sex identification is necessary to maintain the desired sex ratio for breeding
purposes, either by way of induced or natural breeding. Sex identification is
problematic in grouper because they can change sex at certain ages or sizes. Therefore
separation of male, female and transitional fish from each other for breeding purpose is
problematic. Sex identification in cultured species is a prerequisite to broodstock
constitution. Individual identi#cation of the breeder gender is indispensable to
maintain the desired sex ratio to produce the appropriate #ngerling number for
aquaculture production.

In most species, phenotypic sex is more or less easily identified in pubertal male
or female by specific external characteristics. In some cases, intra-ovarian biopsies or
endoscopies or cannulation technique on anaesthetised animals allow gender
identification, even outside the breeding season or in immature fish. However, this
technique has limitation to predict the gender of broodstock. Easy identification is
necessary for the sake of improving the efficiency of aquaculture operations.

Throughout the years, many researchers have been involved in pursuit of
obtaining reliable quantitative values of nutrient in blood plasma. Researchers focused
on developing the biochemical indicators to determine the sex and maturational status
of fish in the absence of the availability of gonad tissues, generating the biological
knowledge necessary to evaluate the impacts of a shift in sex change phenomenon.


Non-invasive techniques based on the level of sexual steroid and /or vitellogenin
(Vtg) have been reported as efficient techniques in sex identification of asian sea bass
(Fazielawanie et al., 2013), tuna (Ceapa et al. 2002; Takemura and Oka, 1998), catfish
(Sundararaj and Nath, 1981), sturgeon (Cuisset et al. 1994) Vtg, a pubertal female
specific glycolipophosphoproteins, is correlated to oocyte development in sexually-
mature fish during the reproductive cycle (Nilsen et al. 2004, Man !anos et al. 1994,
Kishida et al. 1992).

Therefore, a non-invasive sexing technique for Giant grouper using both the Vtg
detection in maturing females by enzyme immune assay (EIA) and the sexual steroid
technique based on 17!-estradiol (E
) plasma levels in mature and immature
individuals of Giant grouper were applied in this study.

1.3 Significance of the Study

Identification of the sex and gonadal maturity of fish is necessary for successful
commercial aquaculture operations. Currently, there are several techniques used for
sex identifications in fish. One example, the amplified fragment length polymorphism
(AFLP) technique, generates large numbers of molecular markers and provides a rapid
method for scanning the genomes of different individuals for sequence variation. The


AFLP technique can be used to quantify genetic variation, and assist in marker-
assisted breeding programmes.

In some cases, sex identification is an integral part of the characterisation of
forensic samples containing genomic DNA. PCR technique (Nagahama, 2005) offers
an efficient and sensitive method for sex identification by amplifying gender-specific
sequences, which results in different size PCR product depending on the gender of the
donor. Genetic sex identification in Japanese Medaka is based on the presence or
absence of the medaka male-sex determining gene, DMY, which is located on the Y

In general, determining the sex and sexual maturity of the protogynous Giant
grouper is difficult, because both males and females are found simultaneously and
there is no definite size or age at which the sex begins. In some cases, biopsies (Alam
and Nakamura, 2008) or endoscopies allowed sex identification; however, most of the
works above were not reliable method for sex identification. An easy identifiable
marker for identification characteristics of maturation in female fish is needed.

Vtg, the hepatically-synthesised precursor to egg-yolk, could be an easily
identifiable marker for the onset of maturation in female fish (Idler et al., 1981: Le
Bail and Breton, 1981) and proposed as indicator in the plasma of maturing female
fish. Immunoassay for Vtg has been developed and validated in Asian sea bass, Lates


calcarifer (Fazielawanie, et al., 2013), tilapia, Oreochromis mossambicus (Kishida and
Specker, 1994) and European seabass, Dicentrachus labrax (Man !anos et al., 1994),
striped bass, Morone saxatilis (Tao et al., 1993; Kishida et al., 1992), channel catfish,
Ictalarus punctatus (Goodwin et al.,1992), white-spotted char, Salvelinus leucomenis,
(Kwon et al., 1990), Sole, Solea vulgaris (Nunes-Rodriguez et al., 1989), It presents a
simple and effective technique to determine the sex and sexual maturity of Giant
grouper which is proposed in this study.

1.4 Objectives of the Study

The aim of the present study is to establish a non-lethal technique for sex
identification of the giant grouper. The specific objective of this research were:

1) To identify and characterize the vitellogenin sequence of the Giant grouper .
2) To develop polyclonal antibody of giant Grouper Vtg.
3) To assess the a viability of Vtg after implant with Estradiol



2.1 Life history of the Giant grouper
The Giant grouper is the largest grouper and represents one of the high
commercial fish species in the Asian markets. There is a gap between demand and
supply due to lack of seed production especially in this region, which includes
Malaysia. The market price of an imported 7.5 cm sized fingerling is about RM30.00
each. A 10-20 kg fish ranges from RM 50-70/kg. Malaysia targets to produce 122,000
metric tons of marine finfish production by 2020, which will comprise 20% grouper
(Department of Fisheries, 2011). Fast growth and high economic value are the
advantages of this species in comparison with other grouper species.

Large specimens are hard to chew with a strong flavour and are therefore not very
much appreciated as food fish. Small specimens (1.5-1.8 kg) are on the other hand,
considered a delicacy and the meat is appreciated worldwide. The meat of this fish is
quite popular around the world. It is one of the most highly valued live food dishes and
retails at over US$ 100/kg for the smaller specimens; larger specimens have a lower
wholesale price per kg (Lau and Parry-Jones 1999, Sadovy and Vincent 2002).
Juvenile Giant Groupers are sometimes found in the pet (aquarium) trade. As a young
fish, this grouper sports beautiful colors and moves gracefully, hence the appeal.


The Giant grouper is also known as the brindle bass, brown spotted cod,
bumblebee grouper, Lance grouper and Queensland grouper in Australia. According to
Heemstra and Randall (1993) this species is found throughout the Indo-Pacific region,
with the exception of the Persian Gulf. It occurs throughout the Indo-Pacific region
from the Red Sea to Algoa Bay (South Africa) and eastward to the Hawaiian and
Pitcairn Islands throughout Micronesia.

The Giant grouper can be distinguished by its closely set, fine and elongated body
with white streaks absent on head. This grouper's stocky shape and brown colouration
give it a nearly potato-like profile (Fig. 1). Watch for it resting motionless on the
bottom or hovering effortlessly in midwater, using only tiny fin movements to
maintain its position. Giant grouper have a big mouth and a rounded tail.

Figure 1.The external morphology of the Giant groper, E. lanceolatus


The fish will change colouration as it matures. Juvenile specimens are black with
irregular yellow patches (Randall et al, 1990). Body is blackish brown to black, molted
with white to light gray; fin dark gray to black with lemon to yellow spot/stripes in
young. However, in large specimen fins without spots/stripes (Khono, et al.1990). As
the fish grows older, the yellow patches will turn into patches that are more ornate and
the body will eventually become green-grey or grayish brown with faint mottling.

Since groupers are long-lived, this specimen could live for decades. The behaviour
of the Giant grouper tends to be solitary and it inhabits lagoon and seaward reefs at a
depth of a few to at least 50 m. Large individuals often have a "home" cave or wreck in
which they frequently stay (Myers, 1991). It is somewhat unusual for a large species in
that large individuals can be found in shallow inshore waters, including rocky areas
caves and wrecks, harbours and estuaries, (Lau and Li, 2000) and down to 100 m;
overall it is more often found in shallow water. It even swims into brackish areas
(Delbelius, 1993). Specimens more than several metres long have been caught from
close to shore and in harbours (Heemstra and Randall, 1993).

Its favorite food is spiny lobsters lives on coral reefs and rocky areas. It is also
known to eat fishes, including small sharks, and juvenile sea turtles (Heemstra and
Randall, 1993). All food is swallowed as whole. The species can grow as large as 2.7


meters long, weighing up to 600 kg (Myers, 1991); there are unconfirmed reports of it
growing much bigger. They are common in shallow waters and feed on a variety of
marine life, including small sharks and juvenile sea turtles.

Twenty species of grouper, a globally important group of 162 coral reef food
fishes, are threatened with extinction unless management or conservation measures are
introduced. At least 12.4% of the worlds 162 grouper species are now listed in
threatened categories (Critically Endangered, Endangered, or Vulnerable), another
14% are Near Threatened, and 30% are considered to be data deficient (Vi et al.,,
2009). The Giant grouper is now listed as vulnerable on the IUCN Red List (Shuk
Man and Ng, 2006). It is not just the live fish trade that threatens the grouper species.
Heavy fishing for these highly-desired food fish also poses a significant threat.

Major threats from over-fishing include targeting of spawning aggregations and
uncontrolled fishing throughout the entire range of the species on multiple life history
phases, from small juveniles to adults. For example, in Southeast Asia, juveniles are
sometimes the major fishery targets, as they are taken at sub-market size and grown-
out in captivity (a practice often referred to as mariculture) until they reach a larger
market size. As for other marine fishes, the most susceptible groupers to these threats
are generally the longest-lived and largest species. In some cases, little is known about
the species biology or impact of fishing on its population, including several species of


considerable economic importance that are traded for the live seafood restaurant trade
in Southeast Asia and are widely sourced in the Indian and Pacific Oceans.
2.2 Characteristic of grouper reproduction

The available reproductive biological information on the Giant grouper is limted;
in particular, information on gonadal development such as the ovarian maturation
process, reproductive cycle and hormone action on gonad is needed. It is necessary to
develop reproductive information to establish seed production in the Giant grouper.

Typically, when the reproductive behaviour of the Giant grouper is discussed, this
species is similar with other groupers which are protogynous hermaphrodites. When
females reach a particular size or range, they can change sex from female-to-male
(Smith, 1971) The average age or body size at which groupers naturally change sex
varies among different species. For example, the honeycomb grouper, Epinephelus
merra, sex change occurs when females reach at least 20 cm in length (Bhandari et al.
2003). However, the average size of males E. Merra are from 23 to 27 cm in total
length. In the dusky grouper (Epinehelus marginatus), the sexual maturity is first
reached when females are 5 years old and 40-50 cm total length, while sex change
occurs when individuals are 9 to 16 years old and 70-90 cm in total length (Harmelin
et al., 1999).


Most groupers are relatively large and require culture periods of several years
before first maturation and spawning is achieved. In the seven-band grouper
(Epinephelus septemfasciatus), a culture period of more than 6 years is needed to reach
maturity while in a small grouper such as the honeycomb grouper (Epinephelus merra)
and red-spotted grouper (Epinephelus akaara), maturity is attained within 2 to 3 years
(Okumura et al. 2002). The female gag grouper (Mycteroperca microlepis) reaches
reproductive maturity between ages three and five years at a minimum length between
450-600 mm (Collins et al.1987; Hood and Schlieder 1992: Collins et al.1998). There
is recent evidence to indicate that the minimum size at maturity for gag is decresing
(McGovern et al. 1998). The male gag have been identified at more than five years
old, and the female have been found not more older than seventeen years old.

2.3 Hormonal control in fish reproduction

Most fishes when reared in captivity, do not exhibit normal reproduction.
Normally, female fail final oocyte maturation (FOM) and male exhibit little or even
lack of milt and reproduce just by chance in environmental conditions. Breeding in
captivity may be approached by managing the proper environmental condition suitable
for the species. Artificial environments lack natural spawning stimuli and are not able
to reduce appropriate endogenous response from the fish.


Reproduction in teleost fishes is synchronised by complex interactions of biotic
and abiotic factors. It is controlled by the internal and external environment cues such
as water temperature and photoperiod (day/length), and social behaviour interactions.
Successful reproduction requires adequate nutrients for gamete production, integration
of environmental cues with hypothalamus-pituitary gland-gonad neuroendocrine (Kah
et al. 1993) cascade that brings about growth and maturation of gamete, integration of
cues for expression of appropriate courting, mating, gamete release, and subsequent
parental behaviour.

The primary transduction centre in the teleost brain is the hypothalamus, which is
releases gonadotropins releasing-hormone (GnRH) as primary effects on the pituitary
gland (Yu et al. 1991). Among reproductive hormones, GtH is the most important
hormone in the regulation of gonadal development and maturation. GtH is synthesised
in the pituitary gland and induces the synthesis of sex steroids in the gland.

In female fish, GtH induces the secretion of 17!-estradiol (E2), which in turn
stimulates the hepatic cell to synthesise the egg yolk protein precursor vitellogenin
(Vtg), a phosphorlipoglycoprotein that can circulate at extremely high levels in the
blood (Matsubara,, 1994). Vtg were transported to the ovaries and incorporated
into developing oocytes. In the cellular level (liver cell response), estrogen (E2) is
transported in plasma via the sex-hormone-binding globulin (SHBG). Simultaneously,


it will effect binding to the estrogen receptor (ER) in the liver, and at the same time
will trigger production of mRNA, followed by translation to Vtg protein.

Figure 2: Schematics drawing on simplified overview of fish hormonal control and
physiological responses to estrogenic chemical exposures of the Giant

External or internal stimuli integrated in the hypothalamus activate release of
gonadotropin hormones (GtHs) from the pituitary, triggering maturation / steroid
hormone production in gonads. Estrogen-induced vitellogenin production in liver. Vtg
is then transported to the ovaries and incorporated into developing oocytes (Fig.2).

Vtg, is a synonyms term for the gene and the expressed protein (from latin vitellus
= yolk and gener = to produce). The protein molecule is classified as a
glycolipoprotein; having properties of sugar, fat and protein. It belongs cursor protein


to a family of several lipid transport proteins. Vtg is an egg yolk precursor expressed in
the females of nearly all oviparous species including fish, amphibians, reptiles, birds,
most invertebrates, and the platypus. Vtg is the precursor of the lipoprotein and
phosphoproteins that make up most of the protein content of yolk. In the presence of
estrogenic endocrine disruptive chemicals (EDCs), male fish can express the Vtg gene
in a dose dependent manner. Vtg gene expression in male fish can be used as a
molecular marker of exposure to estrogenic EDCs. Exogenous estrogen can induce
synthesis of Vtg in males and juveniles, making this protein a useful indicator of
environmental contaminants (Sumpter and Jobling, 1995).

Both males and females have the gene for Vtg. These genes are all present in the
liver, a target organ for E2. Vtg, the main nutrient source for developing embryos.
Thus, Vtgs were critical to reproduction, is normally present in females undergoing
oogenesis (Mommensen and Walsh, 1988). In males, the gene for Vtg is normally
suppressed, but it can be turned on by exposure to estrogen or estrogen mimics. Vtg
expression in male fish has, therefore, become an accepted assay for measuring
exposure to estrogenic chemicals (Jobling et al., 1995; Sumpter and Jobling, 1995).
Vtgs are typically only produced by maturing females and are low or undetectable in
the plasma of immature animals, males, and non-reproducing females (Copeland and
Thomas 1988, Mommsen and Walsh 1988; Tyler and Sumpter 1990; Hara et al. 1993).
In plasma, Vtg binds Ca
, Mg
, and K+ to provide minerals to developing fish. The


primary degradation products of Vtg were also shown to have a role in regulating the
oocyte hydration and buoyancy of teleost eggs (Matsubara et al. 1999).

The levels of Vtg in females vary, depending on season. In largemouth bass
(Cline, 2002) levels are generally low in summer and early fall. Soon after estradiol
starts to increase, vitellogenin starts to be synthesised; plasma levels normally reach
their maximal levels by late February or early March, just prior to spawning.

2.4 Biochemical methods for sex identification and gonad differentiation

Information on sexual demographics of a fishs status, such as sex ratio and age-or
size-at-maturity is needed to accurately assess the reproductive potential of a
population. The standard method for fishes is to perform histological analysis of
gonads collected during fishery census. Some of the standard test and end point and
more recently identified biomarkers are gonad somatic index (GSI); histopathology,
general blood chemistry and molecular measures of endocrine balance.

GSI changes can be indicative of reproductive success. Significant variation in
gonadal size can be measured throughout a reproductive cycle for many species and
can be an indicator of reproductive maturity. This parameter is not specific to a
particular mechanism of action, and may reflect a variety of factors like seasonal cycle.
The GSI parameter provides a guide to comparative reproductive status. However, this


technique is difficult to perform and fish must be sacrifice, and if the organisms are
rare or endangered or expensive like the Giant grouper, it is not suitable to be used.
Because of these reasons, researcher should work on establishing non-gonadal markers
for sex and maturity.

With that purpose, several researchers believed that Vtg could be an easily
identifiable marker for onset of maturation in female fish (Idler et al., 1981; Nunez-
Rodriguez, 1989; Kwon, 1990). Vtg can be easily collected from the plasma of
maturing female and also easily purified for further analysis. It is also quite antigenic,
making it ideal for the development of immunoassays for detection. Immunoassay for
Vtg have been developed and validated for several species of fish including Asian sea
bass, Lates calcarifer (Fazielawanie, et al., 2013), African catfish, Clarias gariepinus
(Manohara et al., 2005), sole, Solea vulgaris (Nunez-Rodriguez et al. 1989), channel
catfish, Ictalurus punctatus (Goodwin et al, 1992), tilapia, Oreochromis mossambicus
(Kishida and Specker, 1994), California halibut, Paralichthys California (Palumbo et.
al., 2007), and European seabass, Dicentrachus labrax (Mananos et al.1994).

Despite that technique, gonadal steroids have been used to evaluate the sex
identification. Typically, these are the reproductive steroid hormones in part because
there are commercial kits available for these assays. In female fish, the important
steroid hormones are estradiol (E2) and testosterone (T) and in males, the important
hormones are these as well as 11-keto-testosterone (11KT). The concentration of these


steroids hormones in plasma varies according to the natural reproductive cycle of the
fish. Chu-Koo, et al, (2009), had established used levels of 11KT to distinguish
females from males in Paiche or Pirarucu (Araipama gigas). A useful measure of the
ratio of the hormones also can be used for sex determined. Females are expected to
have an E2/T ratio greater than 1, and males are expected to have a ratio less than 1.
Gross et al. (1995) used E2/T ratio to distinguish between male and female hatchling
loggerhead sea turtles (Caretta caretta).

Genetic identification of sex has been used to distinguish gonad identification.
Analysis of chromosomes (X and Y) by using PCR could be an efficient and sensitive
method for sex identification by amplifying gender-specific sequences, which results
in different size PCR products depending on the gender of donor. Latest technology
uses ion-pair reversed-phase high-performance liquid chromatography (IPRP HPLC)-
DNA chromatography could identify sex at the molecular level (Devlin and
Nagahama, 2002). Clifton and Rodriguez (1997) identified a sex-linked marker that
could be used to identify the sex of chinook salmon (Oncorhynchus tshawytscha).

All the above techniques for sex identification showed promise, but may have
limited applicability to some species. However, identification on sex determination by
using biochemical markers such as Vtg is more rapid and practical to use. Therefore, in
this thesis attempts were made to discover the usefulness of Vtg as biomarker indicator
on sex identification of the Giant grouper.


1.5 Vitellogenin as a biomarker indicator

The FDA (US Food and Drug Administration) defines a biomarker as, A
characteristic that is objectively measured and evaluated as an indicator of normal
biological processes, pathogenic processes, or pharmacologic responses to a
therapeutic intervention.

Therefore, vitellin is the generic name given to the protein constituents of the yolk
granules. The term vitellogenin was first coined in Pan et al. (1969), who referred to
Vtg as the female specific protein found in the blood of mature fish. Vitellogenesis is
defined as the estradiol-induced hepatic synthesis of Vtg, its secretion and transport in
blood to the ovary, and its uptake into maturing oocytes (Mommsen and Walsh, 1988,
Tyler and Sumpter, 1996).

Vtg are large, multi-subunit proteins generally ranging in molecular weight from
180 to 600 kDa in fishes, and circulate as multimers (typically dimmers or tetramers)
in native form (Byrne et al. 1989; Specker and Sullivan, 1994). Molecular weights of
Vtg were different among species. Analysed on molecular mass of Vtg rainbow trout
(Oncorhynus mykiss) by SDS-PAGE, obtained values in the range of 170-180 kDa
(Watts et al., 2003). Meanwhile, in Gag grouper (Mycteroperca microlepis), Vtg
molecular weight was 183 kDa (Heppell et al., 2006).


There are several methods to measure Vtg plasma level. Over the past two
decades, wide spectrums of analytical methods have been developed to meet this
purpose. The first indirect estimates of Vtg were performed calorimetrically by
determining the alkaline-labile phosphorus content of fish plasma (Wallace and Jared,
1968). Other techniques have also been used, such as immunoagglutinations (Le Bail
and Breton, 1981), densititometry following electrophorisis (Van Bohemen et al.,
1981), immunoblotting (e.g. Western blot) (Denslow, 1999), radio immunodiffusion
(Hara and Hirai, 1978) and by liver tissue slice immunoassays (Schmieder et al, 2000).

Beside the above techniques, the new proteomics technologies of protein
repertoires of developing oocytes have been discovered. Proteomics is a new analytical
field aimed at simultaneously characterising entire protein repertoires of a biological
system. Proteomics is emerging as one of the most powerful new tools for research in
developmental biology similarity to other fields. It involves accurate measurements of
the masses of protein or their derived proteolytic peptides and is followed by vacuum-
phase fragmentation and mass fingerprinting of the resulting fragments. Using this
technique we can characterize the giant grouper, Vtg.

Proteomic technologies based on one or two-dimensional polyacrylamide gel
electrophoresis (2D-PAGE), multidimensional high-performance liquid
chromatography (HPLC) and mass spectrometry were employed for following
molecular change. Since peptide and protein (Vtg) is nonvatile molecules, their


introduction into the gas phases of the mass spectrometers is achieved by interfacing
liquid chromatography through electrospray ionisation (ESI) or dried-down peptide
samples through matrix-assisted laser desorption ionization (MALDI) (Aebersold and
Mann, 2003). Cohen and Banoub, (2011) have used MALDI-TOF to analysts Vtg of
Atlantic salmon (Gadus morhua) and rainbow trout (Salmo gairdnerii).

Despite the widespread use of antibodies in immune assays, polyclonal antibodies
with high specificities can be useful alternatives in sex identification studies (Azarm,
et al., 1988). Polyclonal antibodies can be used for develop polyclonal reagents
directed against fish immunoglobulin (Ig) are easily and economically produced and
have been used extensively to monitor teleost antibody responses to antigenic
stimulation by immunisation. From a fish reproduction perspective, monitoring of
antibody production, together with evaluation of prospective Vtg level in bloodstream,
is integral to successful development of sex identification detection kit. The
measurement of specific antibody allows immunogenicity of specific antigens to be
assessed and the establishment of a correlation between the adaptive humoral immune

Estrogens are generally responsible for development of the reproductive tract and
for female secondary sexual characteristic. There are different physiological forms of
estrogen, the most potent and abundant of which is 17!-estradiol (E2). The ovarian
follicles mainly produce estrogen forms. They secreted from the ovary and released


into the bloodstream bound to albumin or to sex hormone-binding globulins
(Goodman, 1996). The mechanism of estrogen action begins when estrogen enters the
cell via passive diffusion and binds to the intracellular estrogen receptor, which resides
in the nucleus. Binding of the steroids to the receptor forces the heat shock protein to
dissociate and allows the receptor to bind to specific nucleotide sequences called
hormone response elements (Goodman, 1996). It is needed to clarify the role of E2 in
Vtg production of the Giant grouper.

All these consideration led to clarify and this would allow us to obtain an overall
view of using Vtg as biomarker indicator in sex identifications of Giant Grouper. It
might help in our understanding of complex interactions in reproduction.




3.1 Giant grouper Broodstock
Giant grouper broodstock (males and females were kept together) were reared in
150,000 litres and 400,000 litres capacity cement tanks with stocking densities of 20 -
25 fish per tank (2-4 kg/ton), at the Fisheries Research Institute (FRI) Tanjong
Demong, Besut, Terengganu. (Fig.3 and Appendix A).

Broodstock management was based on standard operational protocol (SOP) of
Fisheries Research Institute (FRI) Tanjong Demong Hatchery Operation (2004) (Fig. 4
and Appendix B). For the purpose of good management practice (GMP) of broodstock
maintenance, the shape (round), depth (2 metres) and tank size (150 ton) were
designed to more easily handle the broodstocks. Water changes were done 100% each
alternate day and water-quality management also followed the SOP of FRI Tanjong
Demong Hatchery Operation (2004).

Water-quality measurement was taken once a week by using a YSI 5000 device,
which is monitored on dissolved oxygen, pH, salinity, ammonium and temperature
(Table 1). Broodstock were fed at one-day intervals with Longtail tuna (Thunnus sp.)


at feeding rate of 1% of body weight. The supplementation feed used capsule vitamin
E (200 I.U./ pieces) and capsule fish oil (300 mg/pieces) was given to the fish once a
week (Fig. 5). To safeguard broodstock from parasites, the symptom of fish diseases
were checked once a week by taking mucus sample and analysing it at FRI Tg.
Demong fish diseases laboratory. Once a month for a 3-day duration, prophylactics
treatments of 25ppm formalin were used as a precautionary task of broodstock

Table 1. Water parameter in Giant grouper broodstock tank
Parameter Optimum range Optimal range observed
Dissolve Oxygen
4.0-8.00 ppm 5.0-6.0 ppm
pH 7.5-8.5 6.8-8.8
Salinity 30-33 ppt 30 ppt
Temperature 28-30
C 28-30
Ammonium <1 ppm 0 ppm


Figure 3: Cement concrete (a) 150 ton tank 12 m diameter x 2.0 meter uepth anu (b)
4uu ton (1u x 16 x 2.Sm) useu foi ieaiing uiant gioupei biooustock


Figuie 4: Biooustock management of uiant gioupei; (a) Weighing the biooustock
(b) Checking biooustock sex status (c) Feeu foi biooustock.

Figure 5: Preparation of vitamin with using capsule before give to the broodstock.


3.2 Blood collection

Prior to blood collection, fish were anaesthetised by immersion in a 50mg/l
solution of tricane methane sulphonate (MS-222, Sigma-Aldrich). Bloods were
collected from the afferent filamentary artery (AFA) (Fig. 6) in the gill filament by
heparinised needles (21G) into 1 ml plastic syringes and transferred into cryovials
containing heparin (32.9 IU) and aprotinin (C 0.132 TIU, 50$l/2.5ml of blood). In the
normal condition, blood was taken from peduncle, but due to the thickness of Giant
grouper peduncle and the short of needles, this technique cannot be done. Samples
were allowed to clot at 4
C overnight and serum was separated by centrifugation at
15,000 rpm for 15 min at 4
C and stored at -30
C until use.

Figure 6: Collecting blood from the afferent filamentary artery (AFA) in the gill
filament by heparinised needles (21G).


3.3 Hormonal induction of vitellogenin synthesis
The starting material for Vtg is vitellogenic male plasma with estradiol-injected.
Vitellogenin synthesis was induced in three male Giant grouper (15.0 2.57kg) by
single injection of Estradiol-17! (E2) (Syndel Asia Sdn. Bhd.). The fish was injected
intraperitoneally with a peanut oil solution and ethanol, with 17- Estradiol dissolved
in peanut oil (2 mg E2/kg of body weight, diluted in ethanol: peanut oil, 1:9) (Kishida,
et al., 1992). After 3, 5, and 7 days, fish were anaesthetised and plasma was collected
(Fig.7) as mentioned in previous method. Blood sample from hormonal induction were
pooled and use for characterization (chapter 4) and antigen production (chapter 5).

Figure 7: The procedure to inject male Giant grouper by E2 for synthesis of Vtg.


3.4 Electrophoresis of Vitellogenin
Vtg fractions were determined by SDS-polyacrylamide gel electrophoresis (SDS-
PAGE) analysis (Laemmli, 1970) in 6.5% gel (Figure 8). Five microlitres of thawed
plasma with concentration of 15.0 $g/ml protein (ratio 2:1) from both experimental
and control fish were loaded onto a 6.5% polyacrylamide SDS-PAGE, diluted in 0.125
M Tris base, 6.5% SDS, 20% glycerol, 10% mercaptoethanol (sample buffer) and
boiled for 5 min at 95
C. Electrophoresis was conducted using 6.5% gels in 50 mM
Trisbase, 192 mM Glycine, 0.1% SDS, pH 8.3, in polyacrylamide at a constant
voltage of 120 V for the first 15 minutes, and 150 V for the following 45 minutes.
Refer to appendix C for electrophoresis protocol. PageRuler Protein Ladder as
standard was used for molecular weight identification. The gels were fixed and stained
with 0.025% Coomassie Brilliant Blue. The band corresponding to the induced Vtg
was easily recognised by comparison of the protein band from both control (positive-
nature female and negative-nature male) fish.


Figure 8: The procedure of electrophoresis on Giant grouper Vtg.


(Epinephelus lanceolatus)


Development of gonad can be observed both at the morphological level by visual
inspection of their size, colour and transparency and at molecular level by following
changes in the protein and mRNA patterns accompanying their maturation.
Vitellogenesis is the principal event responsible for the enormous growth of oocytes
in many teleost, and this is when most nutritive products are taken up and stored for
future use by the developing embryo. The factors involved such as amino acids,
energy, lipid and calcium, are derived from plasma during vitellogenesis, most of
them originating from the uptake of large complex molecule, called vitellogenin

One and two-dimensional gel electrophoresis (2-DE) and multidimensional
protein identification technology combined with tandem mass spectrometry
(MS/MS) are among the most powerful tools available for comparative proteomic
studies. With these methodologies, facilities detection was important for the
identification and characterisation of proteins participating in different cellular


process, such as the oocytes maturation (Coonrod et al., 2002). De novo N-terminal
sequencing of proteins is important, because the key information about the
proteolytic processing such as the nature of modification or site of degradation might
not be available otherwise.

Molecular characterization of Vtg gene is important because it indirectly leads to
the understanding of the role-play in the molecular basis of gonad development in
terms of their structure and function. Basically, each gene has its own molecular
characteristic that is specific to their action. This includes the Vtg genes, which has
certain features that are fundamental and responsible for its actions. The Vtg gene
sequence acts as an indicator to the fish reproduction, which can adapt to the
environment factor or can influence the gonad development. Study on the molecular
levels could permit the understanding of gonad development, gene regulation,
structural-function relationships, evolution and adaptation to environment.

Vtg, plays a role in maintaining the fertilization ability of the oocyte and allows
subsequent normal development. Quantification of Vtg in blood plasma is useful for
different purposes. The reproductive status and degree of sexual maturation of fishes
can be assessed according to the levels of Vtg in the plasma. Vtg functions as a
nutritional source for the developing embryo, rather than as an important functional
protein (Denslow et al. 1999). Therefore, Vtg is an ideal biomarker for female sex


identification. Males normally do not synthesize Vtg; however, they will be induced
to synthesize. The expression of this protein can also be induced in males under the
effects of estrogenic compounds. Relying on this observation, some studies have used
Vtg as a biomarker of environmental endocrine disruption in many species (Heppell et
al., 1995; and Folmar et al., 1996).

Therefore, the purpose of this study was to obtain fundamental knowledge on
biochemical, immunological and molecular biological characteristic of Giant grouper
Vtg for eventual use in sex identification and as a maturation indicator of this species
as well as for reproductive studies in developing aquaculture.

4.2.1 Sex status identification

All broodstock (71 pieces) samples sex status was determined by using
cannulation methods (Appendix A). A cannulation tube was used to cannulate fish
broodstock to check egg and sperm maturation. The outer diameter of the eggs was
measured making sure that the egg size was slightly smaller than the inner diameter
of the cannulation tube. For that purpose, the cannulation tube used was 1.0 mm
diameter tube plastic catheter. Sample gonad was sucked by mouth out and kept in
Ringers solution before being observed with microscope. At female stage, oocyte


will be found in the plastic tube catheter and at male stage the white cream of sperm
easily appeared when abdomen (gonad) was pushed slowly (Fig. 9). However, at
unknown stage, body liquid or some blood were sample out from the gonad. The
sex status of broodstock (71 pieces), male, female and unknown stage was
confirmed with the presence of Vtg in the plasma by using SDS-PAGE method
(method described in chapter 3).

Figure 9: Determination of Giant grouper sex status by using cannulation.
A and B. Sampling the broodstock with using catheter
C. Semen, D. Observation of eggs size using microscope
E. Sample eggs or semen in Ringers solution


4.2.2 Experimental design

Giant grouper broodstock was chose from three categories; which is males (35.0
4.5 kg; body weight, 115 7.5 cm; total length) as negative control and females (20.0
3.15 kg; body weight, 95 3.12 cm; total length) as positive control (sample number
from each categories was 3 pieces). The fish was reared in indoor concrete tank 10-
ton, and broodstock management was based on Standard Operational Protocol (SOP)
of FRI Tg. Demong Hatchery Operation (2004), which is described in previous

4.2.3 Induction of vitellogenin in male Giant grouper
The method for induction of vitellogenin synthesis with using male giant grouper
was describe in chapter 3. Injection on intramuscular by 2 mg E2/kg body weight on
the initial day. Blood samples were collected accordingly on days 0,1,3,5 and 10 after
injection and process as describe in chapter 3. To determine the Vtg band of every
categories sample, the plasma Vtg was electrophoresed (SDS-PAGE) as described in
chapter 3.


4.2.4 In-gel digestion procedure
Protein bands from SDS-PAGE gels (preparation was describe in chapter 3) of male
giant grouper after induced with E2, natural male giant grouper (negative control) and
natural female giant grouper (positive control) were washed four times with Mili-Q
water, and then, each interesting band was diced into approximately 1-mm square (Fig.
10A) using surgical blades and placed into 2-ml plastic vials for enzymatic digestion
and dried in a SpeedVac. Unstained gel from one corner of gel slab served as a blank.

Briefly, the protein of interest (approximately 1-mm square) was transferred to a
microfuge tube and washed with 50 $l of 50 mM ammonium bicarbonate, 50 % (v/v)
acetonitrile (ACN) was added and incubated at 37
C for 20 min on a rotating mixer.
Samples were destained with 50% (v/v) methanol and 5% (v/v) acetic acid over a
period of 24 h (Fig. 10B).These processes are critical for removing excess SDS and
salts in the gel pieces, which interfere in most MALDI and ESI analyses.

This process was repeated until all stain had been removed. The plugs were then
washed in 100 mM ammonium bicarbonate and subsequently discarded.
Iodoacetamide (IA) (150 $l, 55 mM) was added to each plug and incubated at 37
After 20 min, the supernatant was discarded; iodoacetamide (500 $l, 100 mM) was
added to each tube and incubated in the dark for 60 min. The gel was dehydrated using


50 $l of 100% ACN, and incubation at 37
C was resumed for 15 min. The supernatant
was removed from the dehydrated plug, which was allowed to air dry (Fig. 10C). Once
dry, the gel was rehydrated in 50 mM ammonium bicarbonate (25 $l) containing
trypsin (6ng/l trypsin in 50 mM ammonium bicarbonate) vortexed briefly, spun down
at 200 rpm for 1 min, and allowed to rest for 5 min; then 50 mM ammonium
bicarbonate (50 $l) was added to each tube, and digestion was allowed to continue
overnight at 37
C; the reaction was heated by the addition of 10 $l of formic acid.

Figure 10: Schematic outline of protein identification steps by mass spectrometric
MALDI-TOF) analysis. A) Cut protein spot B) Protein digestion C) Spot
onto MALDI chip D) Peptide purification E) MALDI_TOF analysis
F) Peptide fragment fingerprint.


4.2.5 MALDI-TOF preparation
MALDI sample preparation with the mixed matrix/analysed solution droplet was
air-dried on the sample plate (Fig. 10D). With this method, inhomogeneous sample
distribution and sweet spots can be expected. For peptide and protein samples, each
compound was prepared to 1 nmol/$l as a stock concentration then diluted in 50%
ACN to desired concentration for analysis. Matrix of ACHCA (alpha-cyano-cinnamic
acid) was dissolved in 50% ACN to final compound of 50 nmol/$l. Each compound
and matrix was mixed at equal volume and deposited onto a 0.7 $l sample plate area.
The mixture was allowed to air dry or vacuum dry prior to analysis.

Samples to be analysed for nominal molecular weight were exchanged into low-
salt (<20mM) buffers, then spotted onto the MALDI target with an equivolume
amount of MALDI matrix (Sinapinic acid in 50% ACN/0/1% TFA) and allowed to air
dry. Spectra were acquired for 2500 shots with an accelerating voltage of 25,000 V.
Calibration with external standards resulted in typical mass accuracies of 0.1%.
(Calibration mix of Mass Standard Kit ABSCIEX TOF/TOF). Analysis on Vtg
molecular weight was performed with 4800 Plus MALDI TOF/TOF mass
spectrophotometer (Applied Biosystems/MDS SCIEX USA (Fig. 10E). Fractions of
tryptic peptides were desalted using Zip-Tips (Millipore). Peptides were eluted from
the Zip-Tips (Millipore) with 0.1%TFA-acetonitrile ACN (50:50). Samples were


mixed in a 1:1 ratio with a saturated solution of "-cyano-4-hydroxycinnamic acid in
ACN/ water/ trifluoroacetic acid (50:49:1, v/v/v).

4.2.6 Identification of vitellogenin

The relative molecular mass of the Vtg and its derived peptide mass fingerprints
were searched for in the corresponding organism database or the Swiss-Prot, in-house
database by using the MASCOT search engine with a probability-based scoring
algorithm (Fig. 10F) (http: and free-access internet search
engine tools.

The interpretation of the MS/MS spectra was performed with BioAnalyst, the
sequencing algorithm provided with the GPS Explorer Software from ABSCIEX
(AppliedBiosystem, Foster City, CA, USA). The production spectra obtained from the
MALDI-MS/MS of the tryptic in-gel digestion of Giant grouper Vtg were analysed
using a computer-based sequencing algorithm called Lutefisk.


4.2.7. Molecular Phylogenetic Analysis of Giant grouper Vtg

The Vtg nucleotide sequence from Giant grouper was characterized using
bioinformatics software. A homology search of the deduced amino acid sequence of
the obtained Vtg DNAs was carried out using the National Center for
Biotechnology Information website ( The Vtg Giant
grouper sequence was compared from 13 species such as lamprey (Ichthyomyzon
unicuspis, GenBank; AAA49S27.1), sailfin molly (Poecilia latipinna, GenBank;
ACV65040.1), rohu (Catla catla; GenBank; ABPu4uS4.2), tuna (Thunnus thynnus;
GenBank;), catfish (Clarias macrocephalus; GenBank; ABW96S64.1), carp (Cyprinus
carpio; GenBank; AuZ8u88u.1), european seabass (Dicentrarchus labrax; GenBank;
AFA2667u.1), mummichog (Fundulus heteroclitus; GenBank; AAB171S2.1), mangiove
iivulus (Kryptolebias marmoratus; GenBank; AAQ16635.1 ), striped bass (Morone
sexatillis; GenBank; ABZS7172.1), rainbow trout (Oncorhynchus mykiss; GenBank;
CAA6S421.1), zebra fish (Danio rerio; GenBank; NP 001157843.1), and Japanese eel
(Anguilla japonica; GenBank; AAv48826.1 ) (Appendix D).

The deduced amino acid sequences were aligned using the ClustalW (Thompson
et al., 1994) program hosted by the DNA Data Bank of Japan
( and subjected to ClustalW analysis to


construct a phylogenetic tree using the bootstrapped neighbor-joining method
(Saitou and Nei, 1987). The sequence obtained was exported to FASTA format in
notepad and then, was edited using Bioedit software to remove the unwanted and
vector sequences to identify the location of the insert sequence. Multiple alignments
from 14 fish peptide sequences of Vtg were conducted using eBiox 5.2.2 program and
it were used in the phylogenetic analysis. The phylogenetic analysis was carried out
using Molecular Evolutionary Genetic Analysis MEGA version 5.2.2 (Tamura et al.,
2011) with Maximum Likelihood and Neighbor Joining algorithms in order to estimate
the phylogeny.

In order to verify the Vtg gene sequences obtained and elucidate structure-function
relationship in Vtg, the conserved and essential domains and residues in Vtg and other
members of the gene family such as Lipovitellin I (Lv-I) and II (Lv-II), phosvitin (Pv),
polyserine track (PT), von Willebrand-factor type-D domain (VWD) were determined
by using DELTA BLAST ( The molecular
characterization of primary structure Giant grouper Vtg gene such as protein domain,
families and functional sites were determined by comparing the sequence to other fish
(Marchler-Bauer and Stephen, 2004).

Furthermore, in this study the prediction of 3-D structure of Giant grouper with
others fishes from different orders were also viewed using Protein Homology / analogy


Recognition Engine v 2.0 Phyre2 server (Kelley and
Sternberg, 2009).
4.3.1 Sex status of giant grouper
The sex status of unknown sex was very unreprodocible in the sampling (Fig. 11).
Group 1; (Body weight 35- 54 kg; and Total length 98-136 mm, n-sampled, 50) shows
there is 50% unknown sex status, followed by the male 18% and female 32%.
However, group 2 (Body weight 14-21 kg, total length 92-110mm and n-sampled, 18)
show the sex status from the early puberty broodstock, which was 100% unknown and
no sample, was detected either female or male.

Analysis on 6.5% SDS-PAGE gel showed that the samples containing the Giant
grouper Vtg were successfully expressed at about between 130 KDa to 290 kDa. It was
confirmed with MALDI-TOF. From analysis SDS-PAGE, the sex status from every
tail Giant grouper was confirmed (Fig. 12). The female showed Vtg band but male and
unknown status was absent. All the individual broodstock were checked and shown in
the appendix A list. Once again, Vtg band clearly shown in fig. 13 appears only in
neutrally female and not in male.


Figure 11: Presence of vitellogenin in plasma of broodstock detected with cannulation

Figure 12: SDS-PAGE result on Vtg band only in female (F1 and F2) and not in
male (M1 and M2), Unknown (UK1 and UK2), and M (Marker).
Female Nale 0nknown



Size SS-SS kg Size 14-21 kg


Figure 13: Vitellogenin band appeared in negative control (male naturally) and
positive control (female naturally) of Giant grouper.

4.3.2 Determination the size and sequence of Giant grouper Vitellogenin
The Vtg was synthesised from liver after hormone E2 induction at day 3 and
continued to day 10 (Fig. 14). The fraction that contained Vtg were pooled, dialysed,
and lyophilised as antiplasma by using dry-freezer technique and was kept in frozen
C prior use.

Control and E2-induced plasma samples were analysed in parallel by SDS-
PAGE. After staining with Coomassie Blue, the Vtg band was easily distinguished by


comparing the intensity of the bands between both samples. This is a routine analytical
technique, which permits the perusal of qualitative and semi-quantitative results,
obtained in protein biochemistry experiments.

Figure 14: Denaturing (SDS) polyacrylamide gel of purified plasma induction with
estrogen-treated (E2) of male Giant grouper plasma band appeared in
male Giant grouper after being induced with estradiol (E2).

Naturally female Giant grouper was confirmed by the appearance of Vtg if
compared with naturally male Giant grouper. The bands corresponding to Vtg were
excised and subjected to in-gel digestion with trypsin and the results were analyzed by
MALDI-MS. The MALDI-MS spectrum obtained for Giant grouper Vtg is shown in


Figure 15a, b and c. The experimental protocol used in this study generated good
quality spectra enabling easy identification of Vtg. Vitellogenin MS spectra from
naturally female (Fig.15a) were not similar in relative intensity of the spectra in
natural male. However, the MS spectra of natural male (Fig. 15b) did not appeared in
mass fingerprinting and it is revealed that naturally male Giant grouper does not
synthesize Vtg as long as no bands were also detected in naturally male (Fig. 15a). It
was confirmed that MS spectra of Vtg, were quite reproducible in terms of detection
of abundant ions at 2292 m/z.


Figure 15: MALDI-MS mass fingerprinting generated by in gel 1D electrophoresis tryptic
digestion of vitellogenin a) Male giant grouper after induced with E2, b) natural
male giant grouper and c) natural female giant grouper. (*)Vtg peaks at 2292 m/z.

The major peaks from this spectrum were used for protein identification using the
PMF search in MASCOT. Database references were referred to Lafleur et al., (1995).
The proteins from database showed a statistically significant hit from the list of peaks


(Fig. 16), which is 34, and indicated extensive homology (p < 0.05). By matching the
peptide masses generated following the trypsin digestion with those available in the
database, Table 1 shows the most matching peptide of Giant grouper Vtg. It was
confirmed that molecular weight of Giant grouper Vtg was 187,805 Da and the
peptide sequence was FLELVQLLR, and matched only with sequence homology with
reference sequence. The top and bottom spectra correspond to a m/z ranges of 804 to

Figure 16: Significant level of 95 % confidant Vtg, where P is probability
the observed match is a random event.


Table 2: Peptide mass fingerprint (PMF) of the trypsin digestion of male Giant
grouper Vtg induce with E2
Sequence Position Match

835.52 834.47 1.05 K.ALHPELR.M 543-549 N.M
898.58 897.58 1.01 K.IAARLNIK.E 880-887 N.M
939.61 938.61 1.00 K.VVENPLLR.E 423-430 N.M
1089.60 1088.59 1.01 K.NPALSESTDR.I 692-701 N.M
1106.62 1105.53 1.09 K.ISWGEQCRK.Y 1310-
1120.64 1119.53 1.11 K.CQEETKNLR.G 205-213 N.M
1130.76 1129.69 1.07 K.FLELVQLLR.I # 331-339 M
1464.86 1463.79 1.07 R.TKQSLEFLEIEK.E 256-267 N.M
1770.00 1769.15 0.85 R.ITPLLPTKVLVIPIR.R 926-941 N.M
1793.02 1791.75 1.27 R.RNSSSSSSSSSSSESR.S 1079-
1918.05 1916.92 1.13 K.NSISFAHSWILPAESCR.D 1601-
2231.20 2229.97 1.23 K.NKMPSSCYQVAAQDCTDELK.F 1461-
2253.33 2252.17 1.16 R.SPATIHPDVAAACSAAMKILGTK.L 595-617 N.M
2274.32 2272.96 1.36 R.TSSASSLASFFSDSSSSSSSSSSDR.R 1185-
2486.60 2485.36 1.24 K.ALLLSGINFHYAKPVLAAEMRR.I 768-789 N.M
3097.85 3096.43 1.42 R.NSMLDSSELLPMEEEDVEPIPEYKFR.R 952-977 N.M

# Only this sequence were matched with ions score 41. N.M, indicate not matched with
sequence homology and M, indicate matched with sequence homology. Nominal mass of Vtg
was 187,805 Da and calculated pI value was 8.93.


A conceptual translation of the open reading frame resulted in a 1,704 amino acid
protein sequence (Fig. 17). A signal peptide was predicted by aligning the Giant
grouper sequence with the N-terminal sequence of several other piscine Vtg. It shows
that FLELVQLLR was the sequence homology for Giant grouper Vtg and the only
single sequence matched with references in database. Meanwhile, de novo peptide
sequencing with MALDI-TO of Giant grouper Vtg yields peptide ions showed high
homology to 6 sequences from several other fish species, including Plaice, Flounder,
Medaka, Mummichog, Eelpout and Goby (Table 3).

Figure 17: Amino acid sequence (1,704) in single letter code of Giant grouper
vitellogenin peptide showing sequence coverage after trypsin digest.


Table 3. Comparison of MS/MS spectra of Giant grouper vitellogenin to vitellogenin
sequences from other species using the Mascot search engine

Matching sequences







(Pleuronectus plactessa)



E. Flounder
(Platichthys felsus)
(Oryzias latipes)
(Fundulus heteroclitus)
Y. goby
(Acanthogobies flavimanus)
(Zoarces viviparous)
Rainbow trout
(Oncorhynchus mykiss)
F. minnow
(Pimephales promelas)
J. Sillago, smelt
(Sillago japonica)
(Corogonus lavaretus)
Giant Grouper
(Epinephelus lanceolatus)
(in this experiments)




4.3.3 Molecular characteristic of Giant grouper Vitellogenin

Figure 18: Polygenetic distributions of 14 species of Vtg sequences. Numbers besides
n nodes indicate the percent of bootstrap values for each branch of the tree in
t the 1,000 bootstrap trials.

Result of phylogenetic analysis using Maximum Likelihood (ML) and Neighbor
Joining (NJ) methods showed tree analysis generated two separated tree topology
(Figure 18). In general, showed that Epinephelus lanceolatus Vtg is evolutionary
more related to Poecilia latipinna and Kryptolebias marmoratus. It is noted that the
distribution of Vtg phylogenetic was significantly different, between freshwater
species (1('(++"*+ (*'(&*+0 1(&-( #(&-(0 !(%"3 '$'"3 (%5 1-('"(+ 2(#'3#)$4(-*+) as one


gioup of vtg compaie to seawatei anu euryhaline species (64"%$4)$-*+ -(%#$3-(&*+0
73$#"-"( -(&"4"%%(0 8'94&3."(+ 2('23'(&(0 :3'3%$ +$/(&"-"+0 ;)*%%*+ &)9%%*+0 <*%5*-*+
)$&$'3#"&&*+0 !"#$%&'(#)*+ -(.'(/ anu =%>*"--( ?(43%"#() foi anothei gioup. The
constructed a phylogenetic tree that places closely related sequences under the same
interior node and whose branch lengths closely reproduce the observed distances between

Table 4: Distance matrix viewer showing distance and their standard errors for
sequence pairs.

The results from evolutionary distance estimations are displayed in the distance
matrix explorer (Table 4). Results describe the accuracy of pair wise alignment by


Clustal under the specific simulation conditions and alignment parameters.
Estimation of evolutionary distances between vtg sequences is important for
constructing phylogenetic trees (figure 18), dating species divergences and
understanding the mechanism of evolution of protein. Vtg sequence of giant grouper
(Epinephelus lanceolatus) was closed (0.015) with Poecilia latipinna (Genbank:
ACV65040.1) and very far from Icthyomyzon unicuspis (Genbank: AAA49S27.1
(1.041). Estimating the number of nucleotide or amino acid substitutions needed to
compute evolutionary distances is one of the most important subjects in molecular
evolutionary genetics and comparative genomics. Estimation of evolutionary
distance of Giant grouper Vtg with alignment of 13 other fish homologous sequence,
revealed that Giant grouper Vtg was belongs to the marine fishes species rather than
freshwater species group.

Babin et. al., 2007, was proposed the domain architecture and conserved
sequence of teleost fish Vtg (Figure 19A). Based on the analysis, Epinephelus
lanceolatus shows four main domains (Vitellogenin-N, DUF1943, DUF1944 and
VMD) (Fig 19B), similarly found in Dicentrachus labrax but different compare to
Clarias macrochepalus, Catla catla, and Danio rerio (see in Appendix E) where
VMD domains, was absent. This indicate characteristic of Vtg domain for
freshwater species is control by present of VMD in Vtg. Analysis on domain of Vtg
with PROSITE (http// clarified the amino acid sequence
for Giant grouper was from sequence number 22 till number 660 (Figure 20).


Figure 19: Schematic representation of the domain in Vtg peptide sequence, (A)
Domain architecture of teleost fish Vtg (Babin, et al., 2007). Large lipid
transfer (LLT) module also referred to as Vtg_N and LPD-N domain,
polyserine track (PT) domain, and von Willebrand-factor type-D domain
(VWD). The horizontal line indicates the receptor-binding region to
VtgR. Lipovitellin I (Lv-I) and II (Lv-II), phosvitin (Pv), and !-
component (!-C) are the yolk protein generated by the enzymatic
cleavage of Vtg inside the oocyte. Conserved domains of (B)
Epinephelus lanceolatus, (C) Icthyomyzon unicuspis, (D) !"#$%&'(#)*+
-(.'(/0 anu (E) 1-('"(+ 2(#'3#)$4(-*+ Vtg by using NCBI software. See
more schematic representation of other species domain in Appendix E.

!!" !"

!" !!-!! !-!
!"# !


The predicted secondary structures of Giant grouper Vtg are in figure 20 (and
Appendix F). It was clearly seen that "-helix was predominantly present in the
Giant grouper Vtg sequence and helix can be grouped into four major groups,
which are located in domain region. Analysis indicated that the "-helix, !-sheet and
the coil structure configurations have 39.96%, 25.54% and 34.48% respectively. As
it can see in figure 21, the 4-helics can recognize in the different color of domain

Figure 20: Secondary structure of Epinephelus lanceolatus Vtg as predicted by the
Phyre2 software. The green, blue color and the faint lines symbols
represent "-helix, !-sheet and coil respectively. See secondary structure
of other species domain in Appendix F.


Analysis of the 3-D structure found that E. lanceolatus Vtg gene shows this
protein has the typical 4!-helices bundle protein that runs in anti-parallel (Figure
21A). Based on the color, its can categorized in 4 helix structure, which is Blue,
Red, Light green and Green respectively. In the present study, the main structure of
Vtg gene in Giant grouper from different species was similar at the 4-helic region
(Figure 21 B-F). However, the difference can be seen in Helix-1 (blue) and Helix-4
(red) where the structure was totally different in Lamprey (Icthyomyzon unicuspis)
but similar in Catfish (Clarias macrocephalus), Japanese Eel (Anguilla japonica)
and European Seabass (!"#$%&'(#)*+ labrax). However, Zebra fish (Danio rerio) 3-
D vtg structure was different with giant grouper in the position of reddish color



Figure 21: The 3-D structure of Vtg in fishes from different species. Figure A,
B, C, D, E, and F are the 3-D structure of Epinephelus lanceolatus,
Ichthyomyzon unicuspis, Dicentrachus labrax, Clarias
macrocephalus, Anguilla japonica and Danio rerio respectively.
The location of helix; blue: helix1; green: helix 2; light green: helix
3; red: helix 4.

The studies demonstrate that administration of estradiol-17 (E2) induces the
synthesis of Vtg in male Giant grouper within 3 to 10 days. Vtg was present naturally
only in mature females, but its can produce with induction technique from male itself
as proof in this experiments. E2 was used for the induction of Vtg synthesis in male,
because its known to be the most potent estrogen for the induction of Vtg synthesis in
fish (van Bohemen et al., 1981).


Vtg, a phospholipoglycoprotein, plays a role in maintaining the fertilization
ability of the oocyte and allows subsequent normal development. Vtg functions as a
nutritional source for the developing embryo, rather than as an important functional
protein (Denslow et al. 1999). Vtg can be defined as a set of amino acids arranged in a
specific sequence to yield a defined activity and has specific characteristics to have a
degree of homology or sequence similarity with other proteins. It can be identified and
characterised through its peptide mass fingerprint (PMF) (Henzel et al., 1993), in the
case of organisms with fully sequenced genome, or by analysis of the fragmentation
spectra of such peptide (PFF, peptide fragment fingerprinting or even de novo
sequencing) obtained through tandem MS (Chapovetsky et al., 2007).

Proteomic studies are now becoming powerful tools for the discovery of new
proteins involved in the developmental processes. To date, several proteomic studies
have been conducted to quantify the reproductive status and degree of sexual
maturation of the fishes (Cohen and Banoub, 2011). Proteomic approaches are also
feasible ways to identify species-specific (Lpez, et al., 2002), and organ-specific (Sun
et al., 2003) peptides in some species.

One and two-dimensional gel electrophoresis (2-DE) (reviewed in Gorg et al.,
2005) and multidimensional protein identification technology combined with tandem
mass spectrometry (MS/MS) (reviewed in Aebersold and Mann, 2003) are among the


most powerful tools available for comparative proteomic studies. Matrix-assisted laser
desorption/ionisation mass systems (MALDI-MS) is a soft ionisation technique in
mass spectrometry, allowing the analysis of biomolecules (such as proteins and
peptides), which tend to be fragile and fragment when ionised by more conventional
ionization methods. The ionization is triggered by laser beam. A matrix is used to
protect the biomolecules from being destroyed by direct laser beam and to facilitate
vaporization and ionization.

The molecular mass values of the trypsinised peptides obtained by MALDI-MS
are then used to identify the predicated proteins using Web-based search engines such
as MASCOT. The present study demonstrates that molecular mass of Giant grouper
Vtg was 187 kDa and is close to 184 kDa for brook trout (Salvelinus fontinalis;
Schafhauser-Smith and Benfey, 2002); 180 kDa for Atlantic halibut (Hippoglossus
hippoglossus; Norberg, 1995), 180 kDa for sea bass (Dicentrarchus labrax L;
Man !anos et al., 1994), 180 kDa for seabream (Sparus aurata; Mosconi et al., 1998),
170 kDa for barfin flounder (Verasper mosri; Matsubara et al., 1999) and 118.80 kDa
in Asian sea bass; (Lates calcarifer, Fazielawanie et al., 2013).

One approach is to create the so-called sequence ladder of an isolated peptide
that can be read following MALDI-MS analysis. These sequence ladders can be
obtained either chemically or enzymatically. Chemical ladder sequencing is mainly


based upon Edman degradation. Through MALDI-MS, the sequences of peptides
could be obtained with mass difference between consecutive ions in the obtained
spectrum corresponding to masses of amino acids and thus lead to peptide sequencing.
Sequence ladder with MALDI-MS technique is rapid compared to conventional
Edman sequencing method, because a reaction yield is not required and the one-step
readout is very fast (less than 1 minute). An Edman degradation rate of 10
residues/hour has been achieved by the use of an automated instrument (Wang,,1994).

Relevant studies on reproductive status and degree of sexual maturation of the fish
have correlated with vitellogenin characteristic and can be assessed through proteomic
studies. Vtg was produced at specific tissue (liver) as a yolk protein precursor
undergoes processing into smaller molecules and subsequent accumulates in
developing oocytes as vitellin. It is essential to attain complete knowledge of the
structure of the Vtg molecule itself.

Peptide sequencing is essential for understanding antibody structure, function and
diversification by the immune system. Antibody variable and hypervariable regions
were initially identified by sequence comparisons. Similarly, sequencing greatly
contributed to the understanding of how additional mechanisms further introduce
diversity to fine-tune the interaction with an antigen.


The information from identification of Vtg (such as molecular mass and
sequencing) could be useful during preparation of Vtg antibody production. Antibodies
production is generated by in vivo or in vitro approaches, their identification relies
mainly on screening of hybridoma supernatants or bacterially expressed antibody
fragments. The molecular approach can be done on Giant grouper to understand the
molecular respond towards fish growth and determine the individual of Giant grouper
that has potential to increasing the Vtg production for increase eggs quality.

Application of Vtg gene in aquaculture is promising in many aspects especially in
molecular approach. This includes in the production of GMOs to improve the fish
performance and gene regulation study to produced the high-quality eggs and
determination of SNPs that can be used as genetic marker. Nutritional genomics is an
area of science to studying how genes influence response of genetically to feed.
Knowledge of these interactions and variations can be applied in the field of
nutrigenetics to improved maturation diet for broodstock.

The results of the present study facilitate a better understanding on Vtg as a
biomarker in sex identification of the Giant grouper (Epinephelus lanceolatus). This
information can be used for improving the production of Giant grouper for broodstock


(Epinephelus lanceolatus))


The term "antibody production" has both general and specific meanings. In the
broad sense, it refers to the entire process of creating a usable specific antibody,
including steps of antigen preparation, immunization, collection, screening,
purification, and labeling for direct use in a particular method. In the more restricted
sense, antibody production refers to the steps leading up to antibody generation but
does not include various forms of purifying and labeling the antibody for particular
uses. Antibody production involves preparation of antigen samples and their safe
injection into laboratory, so as to evoke high expression levels of antigen-specific
antibodies in the serum, which can then be recovered from the animal. Polyclonal
antibodies are heterogeneous and bind to several different antigen epitopes. While
decreasing the specificity and potentially increasing non-specific reactions, the
polyclonal antibody is also more likely to successfully bind to the specific antigen in a
variety of different test conditions.

Polyclonal antibodies can be used for develop polyclonal reagents directed
against fish immunoglobulin (Ig) are easily and economically produced and have been
used extensively to monitor teleost antibody responses to antigenic stimulation by


immunisation. From a fish reproduction perspective, monitoring of antibody
production, together with evaluation of prospective Vtg level in bloodstream, is
integral to successful development of sex identification detection kit.

In aquaculture management, understanding and controlling reproduction is principal
to the efficient propagation of organisms due to differential growth rates of the sexes
and a need for synchronous and reliable maturation. Part of this understanding
involves knowledge of the strategies and mechanisms involved in reproduction and sex
identification to better facilitate investigation into the organism points of impact.
Direct observation in sex identification of broodstocks is not always possible due to
water turbidity, and this visual method is not compatible with an efficient broodstocks
management. Specifically, it does not allow the constitution of male and female mating
pairs before the breeding period in order to optimise fry production in this valuable
species. By using immunological technique, this could be realize with develop a
simple and rapid detection kit.

Vtg has been detected in plasma by a number of different methods, including
radioimmunoassay, enzyme-linked immunosorbent assays (ELISA) and western
blotting methods (Campbell and Idler, 1980; Man !anos et al., 1994; Nunez-Rodriguez
et al., 1989; Specker and Anderson, 1994). Among these methods, ELISA is the most
favourable tool because it has no radioactivity, is easy to set up, and has high


sensitivity. ELISA, can quickly determine antigen or antibody levels in plasma or other
fluids. The standard ELISA measures antibody levels, using an antigen adhered to a
surface (the immunosorbent). If antibody is present in the test fluid, it will bind firmly
to the antigen. Prior to using these ELISA methods, one must validate that the antibody
is specific for Vtg.

It is proposed to develop an immunologic assay for Giant grouper Vtg in order to
establish an accurate method for aiding the identification of spawning status, egg
quality and egg viability. This antiplasma would be used to measure the Vtg titer in
female plasma. With these measures it will be possible to assess the reproductive
maturity of the female and the health and viability of her eggs and potential developing
embryos. In general, antibodies recognise Vtg cross-reactive epitopes between related
species (Nilsen et al., 1998).

The purpose of this experiment was to acquire information on the Vtg in the sample
plasma from induction with Estradiol (E2), natural female and male Giant grouper as
basic information to develop Vtg as biomarker indicator in sex identification of Giant



5.2.1 Hormonal induction of vitellogenin synthesis and Electrophorisis
The method for vitellogenin antigen preparation and SDS-PAGE was discussed in
chapter 3.

5.2.2. Production steps of polyclonal antibody production
The summaries of production step for Giant grouper polyclonal antibody are in
figure 22. Induction of Vtg and antigen preparation was described in chapter 3.
Followed by purification antigen, immunization of Vtg and antibody purification of
polyclonal antibody. During immunization process, ELISA and Western Blot, was
used for quantified and verified the development of the polyclonal antibody.


Figure 22. The production step of polyclonal antibody of Giant Grouper
5.2.3 Purification of Vitellogenin
Vitellogenin was purified using a modification of the method developed by
Palumbo et. al. 2007. Plasma from estrogen-treated males (induction) was pooled
and used as source of vitellogenin. The plasma was fractionated on a 6B sepharose
separation column (2.5 x 20 cm column).

The equilibration buffer was 20 mM Tris-HCL pH 8.0, 1 mM monothioglycerol,
pH 7.5 and gradient buffer was 500 mM NaCl in equilibration buffer. Prior, plasma
was diluted 1:1 in equilibration buffer and loaded onto the column. The column was


washed with 30 ml of equilibration buffer to remove proteins. To know the exactly
Vtg band was extracted, SDS-Page methods was done. Three ml fractions were
collected throughout the procedure, and then protein levels were determined
separately for each fraction utilising a Coomassie protein assay (Appendix G).

The fractions found to contain Vtg were pooled and dialyzed against DEAE
equilibration buffer and lyophilised using dry freezer technique and was kept in
frozen at -20
C prior use.

5.2.4 Immunization of Vitellogenin

Polyclonal antibodies against Giant grouper Vtg were generated in two New
Zealand white rabbits (size 2-3 kg). The rabbit was purchased from Innobiologics,
Sdn. Bhd, Nilai, Negeri Sembilan and was keep in ambient temperature and
acclimatize for 2 weeks before experiments started. Time line of antibody production
was 72 days starting from primary immunization (Figure 23). The rabbits were
immunised with 0.5 mg of purified Vtg in Freunds complete adjuvant
subcutaneously. Booster immunizations were given at 30-day intervals with purified
Vtg dissolved in Freunds incomplete adjuvant. One week after primary
immunization, the booster 1 was performed. While, 3 weeks after that, booster II


immunization was made and finally Booster III was done for production bleed or
about 6 weeks from beginning. Bleeding (20 ml) was performed prior to the initial
and 10 days after each subsequent immunization.

The ELISA was used to test quality of antibody to determine if there are any
cross-reacting antibodies to other plasma proteins. The antibody was tested starting
from first bleed immunization, followed with sample after booster I, II and III and
finally from last production bleed. Blood was removed from the ear vein and was
then permitted to clot for 4 hour at 37
C, and then the anti-plasma was extracted by
centrifugation at 2500x g for 10 min. Centrifugation was repeated and the plasma
was stored at -20


Figure 23. Project flow and time line of production of vitelllogenin polyclonal
antibody of Giant grouper.

5.2.5 Antibody Purification

Protein Affinity chromatography is used for the purification of polyclonal antibodies.
This is mainly due to their advantages i.e. high specificity and reversible binding of
antibodies. The protein A resin has a binding capacity of ~25mg of antibodies per 1
ml of resin. The column used for the purification purpose is Tricon
10/10 (Column
height 12 cm & Diameter 10mm, from GE Life Sciences) and packed with
Protein A resin Sepharose 4 Fast Flow (GE Life Sciences) at a bed height of 10cm.
The total column volume after packing is ~7.85ml.


For the purification process, phosphate buffers and Glycine-HCl buffers are used
for the binding and elution of polyclonal antibodies respectively. Prior to
chromatography run, the plasma sample (from terminal bleeding of rabbits) was
diluted in phosphate buffer to achieve the binding condition. The prepared sample was
injected into the column, followed by post-load wash of binding buffer and eventually
elution using Glycine-HCl buffer. Due to acidic condition of elution, the collected
elate was neutralized immediately with Tris-HCl buffer. Analysis steps including
column equilibration, sample loading, post-load wash, elution and re-equilibration of
column are performed by AKTA Explorer System 100 (GE Life Sciences). Due to
high titer of total IgG in the plasma sample, two cycles of chromatography runs were
performed. Once the run was completed, the column was washed with the application
buffer to equilibrate for 15 minutes. The fractions were covered and stored in 4

Figure 24 illustrates the complete chromatogram of each cycle and their respective
elution profile. From the figures, it can be observed that the chromatography was run
in continuous mode using different block functions to perform the purification process
including sample injection (indicated by dotted-pink lines), flow through (unbound
impurities), elution, regeneration and post operation (cleaning, flushing and re
equilibration of column). Overall, the elution peak reached the maximum absorbance


of ~3000mAu and there were no other significant peaks noticeable neither regeneration
or post operation process.

Figure 24. The chromatogram of purification using Giant grouper vitellogenin

The efficiency of any chromatography cant be determined without performing
definite analysis of targeted product. In this protein Affinity chromatography, the
concentration of antibodies in the initial plasma sample was analyzed by Enzyme-
linked Immunosorbant Assay (ELISA). Subsequently, the purified samples were
analysed by UV @ 280 nm for the estimation of antibodies concentration. Only the
final product, i.e. sterile filtrate after formulation with respective buffer (1xPBS with
Pol y05 (02 ) 07022013:10_UV1_280nm Pol y05 (02 ) 07022013:10_Cond Pol y05 (02 ) 07022013:10_Conc
Pol y05 (02 ) 07022013:10_pH Pol y05 (02 ) 07022013:10_Fracti ons Pol y05 (02 ) 07022013:10_Inj ect
Pol y05 (02 ) 07022013:10_Logbook
180.0 200.0 220.0 240.0 ml
buffer_0.1M Glycine-HCl, pH 3.00
buffer_0.1M Glycine-HCl, pH 2.8
Buffer_50mM NaOH+ 1M NaCl
Waste F5 Waste


0.05% sodium azide as preservative), has extensive analysis including total IgG and
Indirect ELISA.

5.2.6 Quantification of Vitellogenin with Enzyme-linked Immunosorbent Assay

Quantify polyclonal antibody production was able with assay Vtg by binding it
indirectly to a solid support, the surface of a microtiter well, and then detecting how
much is bound. This work is applicable to the detection and semi-quantitative
estimation of specific rabbit IgG antibodies. An easily-purified standard needs to be
characterized so that its concentration (in $M or mg per ml) can be easily estimated by
using its OD 405 nm. Quantification of Vtg is based on absorbance that was present in
sample with determined through the use of a standard curve.

Two $l samples (plasma and purified Vtg) were diluted with 1998 $l phosphate-
buffered saline (PBS, 136 mM NaCl, 1.5mM KH
, 8.2 mM Na
, 2.7 mM
KCl, pH 7.2) to obtain a 1:1000 dilution by using a single-channel micropipette. Of
this, 1 ml was added to another 2 ml of PBS (1:3,000) and 1 ml of this dilution was
added to another 2 ml of PBS (1:9000). One hundred $l of the 1:9000 dilutions were


added to individual wells of a polystyrene microtiter plate, and 100$l of PBS was
added to the first row as a blank (minimum 2 replicates).
Incubation took place for 1.5 hour at room temperature on an orbital shaker at 180
rpm. The plate was then washed four times with PBS. PBS blotto (5g nonfat dry milk
and 100 ml PBS) was used to block for 30 minutes at room temperature on the shaker.
The plate was incubated overnight at 2-8
C with primary antibody at a dilution of 1 $
of antibody specific for Vtg (Rabbit #s, 2
immune) to 9 $l of distilled water and 3 $l
of this to 30 ml of blotto (1:100,000 dilution). The plate was washed 3 times with PBS
and incubated for 2 hour on the shaker at room temperature with goat anti-rabbit
immunoglobulin conjugated to horseradish diluted 1:10,000 in PBS blotto. The plate
was sealed and incubated at room temperature for 1 hour on a microplate shaker, at
180 rpm.

The plate was washed 5 times with 300 $l washing buffer. Finally, blot and
vigorously bang out residual liquid over tissue paper. Followed with 100$l substrate
solution in each well. Once again, the plate was seal and incubated in the dark for 1
hour and do not shake. The reaction was stopped with 50 $l 2M H
. The
absorbance was then read by QC-Reader-02 microplate reader at 405 nm, with
reference wavelength at 492 nm, and read again 5 min later by using the microplate
reader. Standard curves were run for each experiment.


5.2.7 Validation of Vitellogenin with Western Immunoblotting
Protein, separated on a gel using electrophoresis, was immediately transferred to a
polyvinyl diflouride (PVDF) membrane. The gel was equilibrated in transfer buffer
(25 mM Tris, pH 8.3, 192mM glycine, 20% v/v methanol) for 15 min while the
membrane was equilibrated in 100% methanol for 5 min then placed in the transfer
buffer with the gel for the remaining 10 min. The proteins were transferred to the
PVDF membrane under a constant voltage (100 V) electrical field for 1 hour. After the
transfer, the PVDF membrane was blocked overnight at 4
C in 5% (W/V) non-fat dry
milk in nanopure water (blotto; Bio-Rad cat. No 170-6404). The membrane was
incubated for 1 hour with primary antibody at a dilution of 1 $l of antibody specific
for vitellogenin (taken from Rabbit 2, 2
immune) to 9 $l of distilled water and 3 $l
of this to 30 ml of blocking buffer in TBS-T 0.1% tween-20 (1:100,000 dilution).

The membrane was washed two times in Tris-Tween for 10 min each and then
once in Tris-saline for 5 min. The membrane was incubated for 1 hour with a goat
anti-rabbit immunoglobulin conjugated to horseradish peroxidase (Bio-Rad) diluted
1:1000 in blocking buffer in TBS-T 0.1% tween-20. The PVDF membrane was
washed again in Tris-Tween (2 x 10 min) and Tris-saline (1 x 5 min). A sigma Fast
3.3 diaminobenzidine tetra hydrochloride (DAB) kit was used to develop the
membrane (contains DAB buffered and H
) and locate the reactive proteins. After
developing, the membrane was placed in distilled water to stop the reaction.


5.3.1 Development of polyclonal antibody
The polyclonal antiplasma against Vtg was used to develop an enzyme-linked
immunosorbent assay (ELISA) for Vtg. Rabbit #2 produced higher absorbance with
the experimental samples showing that it had a higher titer than rabbit #1.Therefore,
rabbit #2 was used in all future ELISA protocols (data not shown). Figure 26 shows
the results of a Vtg ELISA using the polyclonal antibody and various dilutions of
estrogen-treated Giant grouper and control (1:125 to 1:2,048,000).

There was a difference in absorbance OD between two-time line of production of
Vtg polyclonal antibody of the Giant grouper. The first 2 weeks, after primary
immunization (Fig 25a), the greatest difference in absorbance range from the 1:250
dilutions to the 1:8,000 dilutions. The optimal antibody dilution for the assay was
determined to be 1:8,000 and was chosen as the standard dilution for the ELISA
protocol. The primary antibody was used at a dilution of 1:8,000. However, after
booster 1 and 2 (Fig. 25b), there is no increased absorbance OD in titer of Booster 2,
compared with Booster 1, absorbance OD in Booster 1 and 2 positive was higher at
1:32,000. Primary immunization at 1:512,000 and 1:2,048,000 were omitted,
absorbance OD was already negative at 1:128,000 and therefore had no impact on


Figure 25: Vitellogenin ELISA of estrogen-treated Giant grouper plasma at various
dilutions ranging from 1: 125 to 1:2,204,800. Specific IgG Titre of rabbits
immunised with Vtg: a) Primary immunization vs Pre-Immunised Controls. b)
Control vs Primary Immunization vs Booster 1 vs Booster

1:12S 1:Suu 1:2uuu 1:8uuu 1:S2uuu


-)%./ 012.31$'
Pie Immunization
Post Immunization
Negative contiol
1:8,uuu 1:S2,uuu 1:128,uuu 1:S12,uuu 1:2,u48,uuu


-)%./ 012.31$'
Pie Immunization
Post Immunization
Boostei 1
Boostei 2
Negative contiol


From UV @ 280nm, the concentration of anti-vitellogenin antibodies in pool elute
of two cycles is 4.8 mg/ml in the total volume of 40ml with the step recovery of~84%.
After formulation, the product will be delivered at the same concentration of ~5mg/ml
but in aliquots of 10ml per tube.

5.3.2 Validation of vitellogenin
In order to evaluate the anti-Vtg polyclonal, positive (estrogen-treated) and
negative (untreated) Giant grouper plasma samples were analyzed using western blot
to determine if the antiplasma was able to bind to the Vtg. Fig. 26 shows the SDS-
PAGE revealed that the samples of estrogen-treated and female naturally contained
Vtg band, while the untreated samples, male naturally did not show any Vtg band.
Specificity of the antibodies was confirmed by Western blot, as shown in Fig. 27
Western blot recognized the protein indicating specific binding of the Vtg antiplasma
and showed reaction clearly happened in estrogen-treated and natural female but not in
natural male as similar as in SDS-PAGE results.


Figure. 26: Denaturing (SDS) polyacrylamide gel of purified plasma from
esxtrogen-treated (E2) of male Giant grouper Vtg band appeared in
male Giant grouper after being induced with estradiol (E2)

Figure 27: Western blot showing immunoreactivity of polyclonal antivitellogenin
rabbit plasma to estrogen-treated (E2) and control (c) Giant grouper
male plasma





Several methods have been developed for the purification of Vtg, ultracentrifugation
(Redshaw and Follett, 1971), precipitation with dimethylformamide (Ansari et. al,
1971), selective precipitation with Mg
-EDTA and chromatography (Wiley et al.,
1979). In this study, Giant grouper Vtg was purified from plasma of E2 treated using a
double chromatography method. Similar procedures have been successfully used for
the purification of Vtg in Anguilla japonica (Hara et al., 1980) and Anguilla Anguilla
(Burzawa-Gerard and Dumus-Vidal, 1991).

The production of polyclonal antibodies remains a common research procedure
involving vertebrate animal use. Rabbits are the most commonly used laboratory
animals for the production of polyclonal antibodies. The rabbit offers many advantages
over other species. The adequate body size of the rabbit and the ready accessibility of
the marginal ear vein and central auricular artery afford a technically easy procedure
for the collection of large blood samples. Polyclonal antibody production is essential in
research activity and rabbits continue to serve as one of the primary species used in
polyclonal antibody production.

Immunoblotting (Western blotting) is a technique, which provides information not
only on antibody-antigen binding, as do ELISA and RIA procedures, but also on the


electrophoretic migration and numbers of electrophoretically distinct antigens in a
sample. Thus, the specificity of antibodies can be productively examined in purified
and complex mixtures by this method. There are a number of techniques was
developed for the measurement of Vtg in the blood of fish. Plasma calcium and
protein-bound phosphate have been used as indirect indices of Vtg (Elliott et al., 1979;
Bjornsoon and Haux, 1985). Immunoassays have been also developed, including radial
immunoagglutination (Le Bail and Breton, 1981), rocket immunoelectrophoresis
(Maitre et al., 1985 and radioimmuoassays (Tyler and Sumpter, 1990). The ELISA
was employed in this study and success to measure Vtg in specific IgG titer of rabbits

Other reports on the measurement of Vtg by ELISA include the whitespotted char
Salvelinus leucommaenis (Kwon et al., 1990), sole Sole vulgaris (Rodriguez et al.,
1989), brown trout Salmo trutta (Maisse et. al., 1991) and channel catfish Ictalurus
punctatus (Pacoli et al., 1990).

There are several types of ELISA that can be performed, including a direct assay
(plasma containing Vtg is absorbed directly to the microtiter plate), a competition
assay (a known amount of control Vtg adsorbed to the plate competed with Vtg) in
plasma samples for antibodies, and a sandwich assay (antibodies are adsorbed to the
plate and they bind Vtg in plasma). The idea behind this assay is to quantify Vtg by


binding it directly to a solid support, the surface of a microtiter well, and then
detecting how much is bound with specific antibody to Vtg.

A potential disadvantage of Vtg ELISA is that, depending on the particular anti-
plasma or antibody employed, it can be relatively species-specific. The immunological
and structural features of Vtg can vary considerably in fishes, even among species in
the same family (Campbell and Idler, 1980; Benfey et al., 1989; Lee et al. 1992). This
diversity can require development of a new Vtg assay for each species or genera of
interest, which is time-consuming and costly, but still feasible for researchers working
on a single or limited number of species.

The Western blot is a variation of enzyme-immune assays. Antigen is
electrophoresed through an acrylamide gel and the proteins are separated according to
their size. It is blotted, to allow the transfer of proteins to a nitrocellulose matrix that is
processed to visualise the reaction. The western blot can be used to detect antibodies
directed against Vtg.

Western blotting is used extensively in research to determine the presence of
specific proteins, to quantify their expression levels, and to determine whether they
have undergone genetic or post-translational modifications. This method categorically


identifies proteins of interest based on two distinguishing features: molecular mass and
antibody-binding specificity.

Western blotting provides information on the identity, size and quantity of proteins.
This information is useful for many applications such as disease diagnosis, agriculture,
and biomedical research. Western blot was confirmed Vtg and its could qualified by
polyclonal antibody and validated back with positive control (natural female) and
negative control (natural male). It showed that the anti-Giant grouper Vtg polyclonal
antibody recognised the purified Giant grouper Vtg in naturally female and treated
estradiol male. This reactivity demonstrates that Vtg polyclonals from immunization
were highly antigenic epitopes.
Maltis and Roy (2009), demonstrated shorthead redhorse and copper redhorse Vtg
was successfully detected by using Western blot technique. They used carp anti-Vtg
polyclonal antibody to recognise the purified redhorse Vtg. Antibodies developed
against carp Vtg have demonstrated good cross-reactivity with Vtg of cyprinids also
report by Tyler et al., 1990.


From the discussion above, its can be revealed that Vtg is clearly capable to be
used as a biomarker indicator in sex identification of the Giant grouper. These results
indicate that Vtg may be of potential for in the quantified analysis as a non-lethal test
for female maturity in these economically important fishes.


(Ephinephelus lanceolatus)


Sex-control techniques are becoming an important tool to enhance stocks for
aquaculture production. The benefits of sex control include controlling early
maturation, production of larger fish, and control of fish quality in relation to the
marketing season and the culture of the sex with the highest commercial value.

Hormonal manipulation to produce female populations of fish has been
implemented with using direct method of feminisation, which introduces estrogens to
fish with undifferentiated gonads. This alters the normal path of differentiation so the
undifferentiated gonadal tissue will develop into ovaries, resulting in a phenotypic
female stock (Devlin and Nagahama, 2002). Estradiol has been found in higher levels
in females and is believed to be the major sex steroid responsible for ovarian
development (Pandian and Sheela, 1995). Estradiol was reported to successfully
induce the synthesis of many fish species (Utaraband and Bunlipatanon 1996;
Mendoza et al. 2011).

Estradiol would be expected to play a central role in the determination of egg mass
and quality. This hormone stimulates the fish liver to produce the yolk precursors


vitellogenin and very-low density lipoprotein (VLDL), the primary sources of yolk
protein and lipid, respectively (Wallace, 1985). The production of these yolk
precursors occurs not only in females forming eggs, but also in males and immature
females treated with estradiol (Wallace, 1985).

Typically, reproductive behaviour of the Giant grouper is similar with other
groupers, which are protogynous hermaphrodites. When females reach a particular size
or range, they can change sex from female-to-male after exceeding a certain age and
body size. On the other hand, getting early maturity for breeding purposes is necessary
in order for producers to obtain mature spawners. Early puberty is a major problem in
many farmed-fish species due to negative effects on growth performance, flesh
composition, external appearance, behaviour, health, welfare and survival, as well as
possible genetic impact on wild populations. Late puberty can also be a problem for
broodstock management in some species, while some species completely fail to enter
puberty under farming conditions. Knowledge of reproductive physiology including
vitellogenesis is very important in managing fish broodstock for reproduction in most
farmed animals including fish.

Consequently, the purpose of this experiment is to test the performance of using
estradiol-17! (E2) for sex-change reversal in unknown status to female and early
puberty Giant grouper for getting early candidate spawner to be used in breeding


programmes. At the same time to prove a viability to produce Vtg through
implantation technique. The information on gonadal development, such as the ovarian
maturation process, reproductive cycle and hormone action on gonad, is necessary to
establish seed production in the Giant grouper.


6.2.1 Preparation and usage of hormone

The estradiol-17! (E2), Cholesterol and cocoa butter (Fig. 28A) were purchased
from Syndel Asia Sdn. Bhd. Initially; E2 dosage per treatment was based on
preliminary trial. Then, the amount of E2 per fish was calculated based on the weight
of fish. Every single of pellet was for 30 mg of weight and the amount of the
ingredients were about 15 mg of E2 plus 15 mg of cholesterol and at little drop of
melted cocoa butter.

For example; 30 kg of weight of broodstock: 30 x 500 g = 15,000 $g or 15 mg of
E2 was used, and this can consist of a single pellet for every implantation of pellet
made. To make a solid pellet, molds from steel were used (Fig. 28B and Fig. 29).
Prior, E2 was dissolved well with a little drop of ethanol and mixed with cholesterol
and cocoa butter. After stirring well, the ingredients were placed in the holes of mold
(Fig. 29C) and pushed little by little with a hammer on steel bars until the hole was


fixed. To get pellet, after 2 hours placement in refrigerator (5
C) the backside of the
steel was pushed with hammer and steel bar (Fig. 28D) to collect the pellet before
storage in the container prior to using.

Figure 28: Preparation of pellet, A; ingredient, B; Steel pellet mold, C; ingredients
fixed in the mold, D; pellet ready to be implanted


Figure 29: Specification and diagram of the mold pellet. A; Mold overview from
top, B; Cross section overview of the holes, C; Steel plate, D; Steel bar
for fixing in and pulling out of the pellet and implanter.

6.2.2 Broodstock selection

The experiments were carried out for six months between December 2010 and May
2011 in the facilities of FRI, Tanjong Demong, using 8-year-old (for sex-reversal
experiment) and 4-year-old (for early puberty experiment). Second experiment was
done in six months between June and November 2011. All experimental fish were
individually marked with passive integrated transponder (PIT). Fish were maintained
during the experiment in 150ton concrete round tanks supplied with seawater.



Two different range size of broodstock was chosen for the experimental purpose. For
sex-reversal experiment, three of Giant groupers ranging from 31.0 kg - 53.5 kg were
used and for early puberty (5 pieces) experiment range was between 14.2 kg- 21.5 kg.
Initially, for both experiments, the fish were checked for gender status using
cannulation technique and validated with SDS-PAGE technique later in laboratory. The
fish had been taken from an unknown status, before beginning the experiment.

Three treatment groups were assigned randomly to the tanks three fish per treatment.
For the experiment of E2 on sex reversal, the treatment was, 0 $g/kg, 50 $g/kg and 500
$g/kg. While, for effect of E2 on early puberty treatment was 0 $g/kg, 500 $g/kg and
1,000 $g/kg. The trial was conducted for 6 months experiments.

6.2.3 Sampling procedure

Before handling, fish were anaesthetised in a bath with MS-222 at 50 ppm. Implant
E2 pellet to the fish in both experiments was made by using TROVAN ID-100A
Microtransponder (Syndel Asia, Malaysia) to the peritoneal body of fish. During the
sampling, cannulation technique was detected egg development from initial to final
experiment and confirmed with SDS-PAGE methods.


6.3.1 Morphological characteristic sex genital of Giant grouper

On occasions, the estimation of age and sex differentiate is more difficult to
distinguish. The lack of biological data on sex status of the Giant grouper was difficult
to predict. However, in general there is little guide as can see in the morphological
structure during maturity. Figure 30 shows differentiate sex on genital structure of the
mature broodstock of the Giant grouper. In general, morphological structure of genital
female showed a u-shaped curve on genital structure and red colour rather than
unknown and male. Meanwhile, male were easily differentiated with white
colour/cream (sperm) when gonad was gently pushed by hand and unknown stage,
were showed different than the above mentioned.


Figure 30: Papila genital morphological structure of Giant grouper; A: Female, B:
Male and C: unknown

Six months data on sex reversal is shown in Table 5 66% of broodstock implanted
with E2 was confirmed the change sex from unknown to female in high concentration
(500g/kg) treatment rather than 33% broodstock change in low concentration
(50g/kg). There was no sex reversal to female in control treatments (0g/kg). This
showed that E2 significantly can induced sex reversal from unknown to female status
in the Giant grouper. The concentration of E2 (500g/kg) also improved results to
produce 100% puberty of Giant grouper (Table 6) when all broodstock changed to
female as early as 2 months after being implanted with E2.



6.3.2 Effect estradiol on vitellogenin profile
All broodstock 100%, change to female after 5 months after being implanted
with E2. However, using high concentration (500g/kg) only 33% change to female
and this only happened after 6 months experiments. From the above results it would
suggest that E2 be used for induced sex change of Giant grouper at a dosage of
500g/kg, and higher or lower than of this concentration will give slow impact for
sex change.

Results from SDS-PAGE analysis on Vtg plasma on sex reversal (Fig. 32)
showed 500$g/kg was confirmed developed after 4 months compared to control
treatment (0$g/kg). This result confirmed the analysis of gonad development by
sampling with cannulation technique (Appendix A).

Figure 31: SDS PAGE result of sex reversal and puberty of Giant grouper during
5 months sampling.


Table 5: Effect of E2 on sex reversal, from unknown status to female status of
Giant grouper after 6 months experiments. (+ indicate positive sex
change from unknown to female , - indicate negative change from
unknown to female).


Table 6: Effect of E2 on early maturation of puberty in Giant grouper after 6
months experiments. (+ indicate positive sex change from unknown to
female , - indicate negative change from unknown to female).



Sex change is an interesting phenomenon and common among certain groups of
teleost fishes. Two social factors are involved as the primary regulators of sex change:
inhibition by males and stimulation from other females (Robertson, 1972 and Shapiro,
1979). Without sufficient social stimulation, fish capability of sex reversal might not
initiate sex change (Carlisle et al., 2000). Size advantage (Warner et al., 1975)
contributes to the identification of which animal changes sex, but behavioural
interactions are also critical determinants (Munday, 2002).

In general, identification of sex and sexual maturity of the Giant grouper is
difficult, because both males and females are found simultaneously and there is no
definite size or age at which the sex reversal begins. In some cases, biopsies (Alam and
Nakamura, 2008) or endoscopies technique allowed sex identification; however, most
of the procedures were depended on a reliable method. An easily identifiable marker
for the onset of maturation in female fish has to be discovered.

Steroid treatments have been used successfully in several species in order to
control sex in fish culture. The ability of sex steroids to regulate sex differentiation has
been successfully exploited in fish farming for the manipulation of the onset of puberty
(Munakata and Kobayashi, 2010) or the induction of the change in both gonochoristic
and hermaphroditic fishes (Pandian and Shella, 1995; Piferrer, 2001).


The present study demonstrates that the E2 stimulated reproductive in females of
the Giant grouper. This is the first confirmation of the sex-reversal and early puberty
of the Giant grouper by implant with E2. It revealed that this fishs reproductive status
can be manipulated successfully using E2. The results from both experiments revealed
that exposure of Giant grouper to a dosage 500 $g/kg was effective for sex reversal
and stimulated early maturation within 2-5 months.

The onset of puberty and first maturity has been shown to be strongly influenced by
E2, which stimulates vitellogenesis pubertal Giant grouper and may be involved in the
onset of puberty via positive feedback on the GnRH system. GnRH has its primary
effects on the pituitary gland where it stimulates the release of gonadotropins (GtH)
(Yu et al.1991). In females, GtH induces estradiol-17! (E
), which in turn stimulates
the hepatic cell to synthesise the egg-yolk protein-precursor vitellogenin (Vtg), a
phospholipoglycoprotein that can circulate at extremely high levels in the blood. Vtgs
were transported to the ovaries and incorporated into developing oocytes. In the
cellular level (liver cell response), estrogen (E
) is transported in plasma via the sex
hormone-binding globulin (SHBG). Simultaneously, it will effect the binding to the
estrogen receptor (ER) in the liver, and at the same time will trigger production of
mRNA, followed by translation to Vtg protein.


The Giant grouper are protogynous hermaphrodites, which is mature as female
and later change to male. This kind of sexuality raises several problems for
broodstocks management, concerning the prediction of natural sex inversion (age and
/or size) and artificial sex control. The development of sex-change technology is
instrumental for improving the efficiency of an aquaculture operation. Alternatively,
the ability to change the gender of sexually-mature fish is extremely useful for
overcoming shortage of female broodstock (Hassin et al., 1999).

Throughout the years, many researchers have been developing indicators in pursuit
of obtaining reliable quantitative values of nutrient in blood plasma. Researchers were
focussed on developing biochemical indicators to determine the sex and maturational
status of fish in the absence of the availability of gonad tissues, generating the
biological knowledge necessary to evaluate the impacts of a shift in sex change
phenomenon. In this regard, the first clues indicating a relation between the sexual
maturation and the appearance of changes in the plasma profiles were obtained by
experiments which demonstrated that changes in the blood component were reflected
by the administration of estrogens (MacDonald and Riddle, 1945).

Therefore, non-invasive sexing techniques for the Giant grouper using both the
vitellogenin detection in maturing females by enzyme immune assay (EIA) and the
sexual steroid technique based on 17!-estradiol (E2) plasma levels in mature and
immature individuals of the Giant grouper, were suggested in this study.



The only positive way to determine the sex of sexually monomorphic species
(males and females look similar) is by examination of the vent. This practice is
commonly referred to as venting. The vent is the opening between the anus and the
anal fin where the fish will excrete either eggs or sperm. The vent is also referred to as
the genital papilla. For females, the term ovipositor (egg tube) also refers to the
vent. In the present study, the Giant grouper showed u-shaped patterns of vent when
female characteristic was observed. However, the u-shaped of genital papilla was
clearly absent in males and fish with unknown gender.

Sex identification is difficult and almost impossible in juveniles. Sex identification
is only possible when they begin to pair. In some cases, some morphological data
could help to determine fish gender (e.g. freshwater fishes), such as both male and
female have a rounded dorsal fin, until they begin to mature and a difference can be
detected. However, this condition is not visible in this species. Another sign is that
males usually have thicker lips to aid in fights to protect the females, and they will be
more aggressive during spawning. Sexual dimorphism is a phenotypic difference
between males and females of the same species, meaning that there are obvious
differences between the male and female of the species. Conversely, the morphological


data (Appendix A) of the Giant grouper showed size of female is not bigger than male,
as seen in the animal kingdom other species.

The ability to identify sex and maturational status of broodstock is essential for
fishery biologists. Routine methods used to evaluate maturity of females include
histological examination of the gonads after dissection or biopsy, manual expression of
ovulated eggs or semen from matured fish, and evaluation of external characteristic for
sexually dimorphic species. This method is limited because histological technique is
labour-intensive, expensive and slow to generate sufficient usable data. To overcome
these complications, rapid and sensitive tests were developed to detect reproductive
hormones or protein in the blood of fish.

Foi biooustock management puiposeu, inteiim time was consumeu to get eaily
pubeity. Eaily matuiity is uesiieu in gioupei faiming foi the puipose of biooustock
management. Eaily female pubeity is a significant economic pioblem in faiming of the
uiant gioupei. It is theiefoie necessaiy to uevelop stiategies to fastei pubeity. These
stiategies shoulu meet both, consumei acceptance anu the economic inteiests of
aquacultuie. 0n the othei hanu, uiant gioupei sex behavioui coulu be change sex
phenomena fiom female to male aftei ceitain size oi age. Thus, theie is a neeu to claiify
the sex iuentification mechanism in gioupei so that sex iuentification easy can be
manipulateu foi commeicial benefit. It is necessaiy to uevelop smait anu iapiu kit sex


uetection foi that kinu of ieason. Theiefoie, with new technology such as biosensoi
technology, iapiu kit foi vtg uetection coulu be easy to iealize in the neai futuie.

The cannulation technique was performed in this experiment (as discussed in
Chapter 6), which is the usual method for sex identification in marine hatchery
operation for grouper. Cannulation is a cheap and easy method to observe the vent.
However, there is a requirement to initiate a rapid, simple and easy method without
stressing the fish when sex identification is carried out.

Biochemical properties of fishs blood had potential concern to examine and need to
be investigated sufficiently. Information about the existence, status of possible
sickness in organism can be rapidly obtained by with use of hematological and
biochemical parameters, but for sex identification purpose is rare and need to clarify.
Therefore, identification and characterization of the giant grouper plasma Vtg was
determined in this study. It is revealed that the occurrence Vtg could be used as a way
to distinguish sex of the Giant grouper. Instead, matrix-assisted laser
desorption/ionization-Time of Flight (MALDI-TOF) was used for Vtg identification.
Identification of Vtg by aligning the N-terminal sequence revealed that the sequence
homology for the Giant grouper FLELVQLLR matched the Vtg of other fish species
and the de novo peptide sequencing with MALDI-TOF yields peptide ions with high


homology. The indication to relate protein sequencing of Vtg as biomarker indicator in
sex identification of the Giant grouper was related with this study.

It is demonstrated that only matured female has detectable level of plasma Vtg and
this is absent in male, indicating its potential as a biomarker for sex identification in
the giant grouper. On an applied note, males normally do not synthesize Vtg except
when they are exposed to contamination or hormone such as estrogen (Denslow et al.,

Greatly increasing the level of Vtg in female by hormone E2 implants influence sex
change in the giant grouper. E2 implants could induce those with unknown sex to
female and at the same time boost them to early puberty. It is revealed that E2 can
influence the reproductive status of Giant grouper successfully and it confirmed that,
E2 were definitely precursor the production of Vtg at certain level.

A number of techniques allow proteins to be tested including western blot, enzyme-
linked immunosorbent assay (ELISA) or mass spectrometry, which were, discussed in
chapter 4 and 5. Immunoassay procedure, (chapter 5) enzyme-linked immunosorbent
assay (ELISA) are based on the use of antibodies specific to Vtg. However, the


specifics of the antibodies sometimes can lead to high degree of species specificity
preventing the use of assay across species. ELISA has been the preferred method for
immunoassay detection due to its relative simplicity, use of non-radioactive reagents,
amenability to automation and good sensitivity, generally in the ng/ml range. In this
study, the polyclonal antiplasma developed for use in ELISA detection was specific
against Vtg of estrogen-treated and natural giant grouper female. On the contrary, from
SDS-Page results, Vtg was not detected in natural male. This indicates the possibility
of using the developed antibody to produce a rapid detection kit because ELISA is
relatively simple and inexpensive to perform.

On an applied note, results from present study could also be useful in the
development of a Giant grouper maturation rapid kit. As one example, detection of
maturation by Immunochromatokit is established on Vtg analysis correlated with
ovarian development in the Giant grouper could develop in future. The test device
consists of a membrane strip and plastic case (e.g. pregnancy rapid test for human).
The concept for detecting Vtg-matured fish is by using monoclonal and polyclonal
Giant grouper antibody. Antibody-conjugated reacts with IgG antibody to produced a
visible-coloured band.


Since it has been observed that plasma protein occurs in skin mucus (Smith and
Ramos, 1976), it seemed logical that sex-specific proteins or their catabolic products
may be present in this medium. Vtg has been detected in surface mucus of several fish
induced with estrogenic chemical (Kishida et al. 1992) including copper redhorse
(Maltis and Roy, 2009). In this context, it is possible to detect Vtg of the giant grouper
via surface mucus. This technique if successful, offers a decided advantage of sex
identification in the giant grouper.

For recommendation, further study on Vtg of Giant grouper to identifying the role
of Vtg in maturation and other study related such as gene expression could be use vtg
in relating to enhance egg quality of this species. The science of nutritional genomics
should increase understanding of maturation diet interactions. Dietary factors and
related metabolic interactions have direct and indirect nutrient influence on specific
gene regulations and expression. Other studies have demonstrated nutritional
regulation of gene expression in lipogenesis, and suppression of fatty acid synthase
transcription by PUFA (Walker and Blackburn, 2004). Fabiola et al., (2011) was
reported that the Vtg of the white-leg shrimp (Litopenaeus vannamei) has been found
to reflect the degree of female shrimp ovarian development, and to be useful predictor
of ovarian development. The levels of Vtg in hemolymph could be proposed in a
definitive manner as a predictor or indicator of gonad maturation stages. Vtg content in
hemolymph was correlated with the number and diameter of oocytes in gonads. In


simple electrophoresis methods, proteins are separated based on molecular weight, and
the relative intensity of the protein band at the molecular weight corresponding to Vtg
is assessed either qualitatively or quantitatively with proteomic analysis as discussed in
this thesis.

From the discussion above, it is revealed that Vtg could be used as a biomarker
indicator in sex identification of Giant grouper. Development of a new non-invasive
technique for sex identification could help to manage this species successfully and
provide the appropriate technology for improving the efficiency of aquaculture


The results of the present study facilitate a better understanding on vitellogenin as
biomarker indicator in sex identification of Giant grouper (Epinephelus lanceolatus).
This information can be used for improving production of Giant grouper and for
broodstock management.

Overall, this study has achieved the objectives to use Vtg as a biomarker indicator in
sex identification purposed. This may contribute to solving the sex identification
problems of Giant grouper due to sex reversal phenomenon in social behaviour life.
The identification and characterization of Vtg was rapid with the MALDI-TOF
technique, can be applied in gene regulation or expression study where the fish culture
in different condition to find the best culture condition. Information on molecular
characteristic can be used in gene expression study for understanding the molecular
respond towards fish growth in aquaculture for propagation of fish.

Estradiol-17! (E
) significantly affected sex change of Giant grouper at dosage of
500g/kg. This may contribute to solving sex ratio of Giant grouper when its prepared
for breeding programmes. Antibody production of Giant grouper was produced
successfully by immunization of rabbit and could be used for ELISA and Western Blot
analyses for quantity and quality of Vtg in other samples.


For recommendation, future study on vtg as a biomarker in maturation rapid kit
could be developed after polyclonal and monoclonal Giant grouper is successfully


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Data on Body weight (kg), Total length (cm), Vtg presence and sex status of Giant
grouper used in this experiment.

















SDS-PAGE (Laemmli) Buffer System
Stock Solution and Buffers.
1. Acrylamide/Bis (30%T, 2.67%C)
2. 10% (w/v) SDS
3. 1.5 M Tris-HCl, pH 8.8
4. 0.5 Metris-HCl, pH6.6
5. Sample Buffer (SDS Reducing Buffer)
6. 10x Electrode (Running Buffer, pH 8.3
7. 10% (w/v) APS

Gel Formulations (10ml)
1. Prepare the monomer solution by mixing all reagents except the TEMED and 10%
Percent Gel DDI H2O (ml) 30% Degassed
Gel Buffer *
10% w/v SDS






* Resolving Gel Buffer 1.5 M Tris-HCl. pH 8.8

* Stacking Gel Buffer 0.5 M Tris-HCl, pH 6.8

2. Immediately prior to pouring the gel, add:
For 10 ml monomer solution:
Resolving gel : 50 $l 10%APS and 5 $l TEMED
Stacking gel : 50 $l 10% APS and 10 $l TEMED
Swirl gently to initiate polymerisation.



List of Vtg sequence of various fish species from NCBI data with accession number

1) Anguilla japonica AAv48826.1




2) Carassius auratus AuZ8u88u.1



3) Catla catla ABPu4uS4.2



4) Clarias macrocephalus ABW96S64.1


5) Danio rerio NP 001157843.1



6) Dicentrachus labrax AFA2667u.1



7) Fundulus heteroclitus AAB171S2.1



8) Ichthyomyzon unicuspis AAA49S27.1



9) Krytolebias marmoratus AAQ16635.1



10) Morone sexatilis ABZS7172.1



11) Oncorhynchus mykiss CAA6S421.1



12) Poecillia latipinna ACV65040.1



13) Thunnus thynnus ACX32463.1




Schematic representation of the domain in Vtg peptide sequence of A) Anguilla japonica
B) Poecilia latipinna, C) Kryptolebias marmoratus, D) Morone sexatilis, E) Thunnus
thynnus, F) Fundulus heteroclitus, G) Oncorhynchus mykiss, H) Catla catla, I) Carassius
auratus, J) Danio rerio.















Secondary structure of A) Danio rerio, B) Anguilla japonica, C) Icthyomyzon
unicuspis Vtg as predicted by the Phyre2 software. The green, blue color and the faint
lines symbols represent "-helix, !-sheet and coil respectively.







Determination of Protein Concentration

The protein concentration was determined by Coomasive (Bradford) Protein Assay Kit
(Thermo Scientific, USA) following the manufacturers protocol. Firstly, the diluted
Albumin (BSA) Standards were prepared. The contents of one Albumin Standards
(BSA) ampule (2 mg/mL) was diluted into several cleans vials, preferably using the
same diluents as the samples. The dilution scheme for standard test tube protocol
procedure (working range 20- 2,000g/mL) was prepared according to the Table 1.

Table 1. Preparation of Diluted Albumin (BSA) Standards
Vial Volume of Diluent (l) Volume and Source of BSA (l) Final BSA

300 of stock
375 of stock
325 of Stock
175 of vial B dilution
325 of vial C dilution
325 of vial E dilution
325 of vial E dilution
100 of vial G dilution

Next, the BCA Working Reagent (WR) was prepared. Generally, 2.0mL of the WR is
required for each sample in the test-tube procedure. This WR was prepared by mixing
50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1, reagent A: B). The
volume WR required in the analysis was calculated according to this formula.


(Standards + Unknowns) x (replicates) x volume of WR per sample) = total volume WR

After mixing the WR, the test tube procedure (sample to WR ratio = 1:20) was
prepared. A total of 0.1 ml of each standard and unknown sample replicate were
pipetted into an appropriate labeled test tube. After that, 2.0 ml of WR was added to
each tube and were mixed well. Then, the tubes were covered and incubated at 37
for 30 minutes and after that, the tubes were cooled at room temperature. With the
spectrophotometer set to 562 nm, the instrument was zero on a cuvette filled only with
water. Subsequently, the absorbance measurement of the blank standard replicates was
subtracted from the 562 nm absorbance measurements of the blank standard replicates
was subtracted from the 562 nm absorbance measurement of all other individual
standards and unknown samples replicates.

After obtaining the absorbance measurement, a standard curve was organized by
plotting the average blank-corrected 562 nm measurement for each BSA standards
versus its concentration in m/ml .This standard curve was use as a template to determine
the protein concentration in g/ml . This standard curve was use as a template to determine the
protein concentration of each sample. Microsoft Excel was used in order to plot the standard in
Column A and concentration data in Column B were enter. Then, both column were highlight
and from the Insert menu Chart and XY (Scatter) was selected. Next, click on the resulting
graph, and Add Trend line was selected from the Chart menu and Polynomial was choose. To


display the equation on the Chart, the Display Equation on Chart from the Options tab was
selected. The resulting equation was used to determine protein concentration (y) of an
unknown sample by inserting the sample absorbance value (x). Once the concentration of
samples has been known, the final volume of protein samples that need to be loaded into SDS-
PAGE gel were calculated so that they have similar final concentration.



Mr. Ahmad Daud bin Om was born in Teluk Intan, Perak on 18
October 1966. He
had his early education in 1973 in Sekolah Kebangsaan Selabak and continued his
education in elementary and secondary school in Sekolah Menengah Seri Perak. He
entered Universiti Pertanian Malaysia, in 1984 for a 3-year Diploma of Agriculture and
from 1987 he entered Bachelor program in Fisheries Science and completed his B.Sc.
degree in 1991. For higher education, he entered Monbushokagaku Scholarship program
in 1998 and graduated in Master Sc. of Agriculture (Fish nutrition) at Hiroshima
University, Japan in 2001.

After obtaining his Bachelors degree, he was offered to a position as hatchery
supervisor for 15 months in a private farm at Pasir Gudang, Johor before he joined
Department of Fisheries as Fisheries Officer in 1992. In 1995 he was appointed as
research officer at the Marine Finfish Research Center, Besut, Terengganu. Besides that
he attended several short course at Seafdec Iloilo, Philippines (Fish Nutrition in 1997),
Gondol Research Institute of Mariculture (GRIM) (Grouper Hatchery Production
Training Course), Bali, Indonesia in 2006 and Mariculture Technology at Fujian Institute
of Oceanography, Xiamen, China in 2008.


Ahmad Daud O, Safiah J., Nosrihah I., Yeong Y.S., and Abol-Munafi A.B., 2013.
Application MALDI-TOF on protein identification of vitellogenin in Giant grouper
(Ephinephelus lanceolatus). Fish Physiology Biochem (2013) 39:12771286.
Ahmad Daud O, Safiah J., Yeong Y.S., and Abol-Munafi A.B., 2013. Vitellogenin as
profile and its potential role as a biomarker for sex identification of the Giant grouper
(Epinephelus lanceolatus). Submitted to Fish Physiology Biochem.
Ahmad Daud O, Safiah J., Yeong Y.S., and Abol-Munafi A.B., 2013. Feminization of
Giant grouper (Epinephelus lanceolatus) by using estradiol. Submitted to Malaysia
Fisheries Journal.


Ahmad Daud Om, Safiah Jasmani, Yeong Yik Sing and Abol Munafi Abol Bolong,
2011. Vitellogenin as biomarker indicator in sex determination of Giant grouper.
(Epinephelus lanceolatus). Presented at 1
International Fisheries Symposium (IFS)
2011 in Kuala Terengganu. Terengganu. 3-5 October 2011.
Ahmad Daud Om, Safiah Jasmani, Yeong Yik Sung and Abol Munafi Ambok Bolong,
2013. Feminization of Giant grouper (Epinephelus lanceolatus) by using Estradiol.
Presented at International Seminar on Marine Science and Aquaculture (ISOMSA)
2013, in Kota Kinabalu, Sabah. 23-25 March 2013.
Ahmad Daud Om, Safiah Jasmani, Azizah Mohd Taib, Yeong Yik Sung and Abol
Munafi Ambok Bolong, 2013. Polyclonal antibody production of Giant Grouper.
(Epinephelus lanceolatus). Presented at 3
International Fisheries Symposium (IFS)
2013 in Pattaya, Thailand. 28-31 November 2013.