Rapid Kit Development

C O N F I D E N T I A L
Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021)
Prepared by
Name Institution Date Submitted Date Printed Produced by
: AHMAD DAUD BIN OM : Institut Penyelidikan Perikanan : 02 May 2012 : 12 December 2012 : Ministry of Science, Technology and Innovation
PROJECT IDENTIFICATION
A. Project number 06-03-06-SF0021
B.
Project title
Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus)
C.
Project leader Name NRIC : : AHMAD DAUD BIN OM 661018-08-5187
D.
Institution Name Address : : Institut Penyelidikan Perikanan Pengarah Institut Penyelidikan Perikanan (IPP) Batu Maung 6262231 6262210
Tel. No. Fax. No.
: :
E.
Key words Giant Grouper Maturation Determination Kit Vitellogenin ELISA
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OBJECTIVES OF THE PROJECT

A. Specific objective (i) to determine the vitellogenin level for fish maturation (ii) to develop a rapid and simple maturation broodstock determination kit of Giant Grouper
B.
Type of research Applied research
C.
Research Cluster and SEO Categories being addressed by the Project Research Cluster SEO Category SEO Group SEO Area S&T CORE S2020000 - Animal Production And Animal Primary Products S2020300 - Fisheries S2020301 - Aquaculture product
D.
Fields of research Primary Field of Research FOR Category FOR Group FOR Area F1090000 - Agricultural Sciences F1090900 - Aquaculture F1090909 - Fish reproduction
Secondary Field of Research (if applicable) FOR Category FOR Group FOR Area F1080000 - Biological Sciences F1080100 - Biochemistry F1080104 - Immunology
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RESEARCH BACKGROUND
B. Research background of the project Project status New
Project summary In order to develop giant grouper culture technology, the seed production technology must be prepared. The success of seed production activity depends on the gonad maturity of broodstock, which is naturally depending 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 plasticity of the sex determination with a range of gonochoristc species, where individuals are either males or females, or species displaying a change in gander during the lifetime. Research works directed to the developing the biotechnology tools to assess gender and maturation status of giant grouper is needed to be developed. Literature review summary
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Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) The giant grouper which is similar to other grouper behavior are well-known protogynous hermaphrodite teleost of the family of Serranidae (Smith, 1971). Which is changing sex from female in early life to male at older age, depending on the natural environment of the populations studied (Lasserre, 1972). The point in time at which sex-change occurs is can be interpreted on many different levels (e.g. behavioral, morphological, histological) were effects on broodstocks management of this species, which is difficult particularly concerning the prediction of natural sex inversion (age and/or size) and the artificial control of sex. Sex determination in cultured species is a constraint to broodstock constitution. Individual identification of the breeder gender is necessary to maintain the desired sex ratio to produce the appropriate fingerling number for aquaculture production. Generally, equilibrated or determinate sex ratios are required when mature male and female pairs are necessary for induced or natural breeding. From an academic perspective, sexual transition is a useful process in which to investigate the mechanisms controlling sex determination and differentiation. In most species, phenotypic sex is more or less easily identified in pubertal male or female by specific external characters. In some cases, intra-ovarian biopsies or endoscopies or cannulation technique on anesthetized animals allowed gender identification, even outside the breeding season or even in immature fish. However, this technique has limitation to predict the gander of broodstock. It is necessary to develop the instrument /device or methods which can easily to define the sex change technology for sake of improving the efficiency of aquaculture operations. Currently, no reliable method for gender determination has been described in giant grouper, except may be some morphological characters observed only when fish have initiated courtship behavior. Direct observation of broodstock is not always possible due to water turbidity, and this visual method is not compatible with an efficient broodstock management. Specifically, it does not allow the constitution of male and female mating pairs before the breeding period in order to optimize fry production in this valuable species. Throughout the years, many researchers have been developed in pursuit of obtaining reliable quantitative values of nutrient in blood plasma. Researchers were focus 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 that 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 serum profiles were obtained by experiments which is demonstrated that changes in the blood component were reflect by the administration of estrogens (MacDonald and Riddle, 1945). Non-invasive techniques based on the use of sexual steroid and /or vitellogenin (Vtg) have been reported as efficient techniques in tuna (Takemura and Oka, 1998), trout and salmon (Pottinger et al. 2005) and sturgeon (Cuisset et al. 1994). 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 (E2) plasma levels in mature and immature individuals of giant grouper were suggest to apply in this study. Despite that, there are kit determination develop on used of immunoassay concept for detecting WSSV virus in shrimp. The shrimp virus detection kit was based on the principle of sandwich immunoassay, which is using protein in shrimp tissue. It is similar concept with the purpose experiment on development maturation kit are suggest. In fact, in development of pregnancy test kit also used the same principle of sandwich immunoassay. Related research Escience fund entitled of study of Induced spawning of giant grouper (Ephinephelus lanceolatus) IPP/AM/15 (05-05-18-SF1007) (2009-2011) indirectly provide infomation on vitellogenin of giant grouper was identically detected only in female broodstock. This characteristic were useful as biomarker indicator or as sign on sex determination . Prelimanary works has discover molecular information on proteomic basic data of vitellogenin such as molecular weight, sequencing and amino acid profile. This prelimanary works were important for further study on development of maturation kit.
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PROJECT SCHEDULE
2012 Activities Chemical / equipment procurement 2013 2014
J F MAM J J A S ON D J F MAM J J A S ON D J F MAM J J A S ON D
Induction of Vtg by using Estradiol in giant grouper Purification of vitellogenin
Development of polyclonal and monoclonal antibodies against vitellogenin Vitellogenin detection by Western Blot
Immunohistochemistry analysis on localize of vitellogenin Test kit validation and optimization
Compilation & Analysis of data & report writing 2012 Milestone Completion of Induction of vitellogenin 2013 2014
J F MAM J J A S ON D J F MAM J J A S ON D J F MAM J J A S ON D
Completion of purified vitellogenin
Completion of development of polyclonal antibodies Completion of vitellogenin detection by Western Blot Completion of vitellogenin locallize
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Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Completion on test kit validation and optimization Project Completion
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SUMMARY OF RELEVANT PAST RESEARCH PROJECT

A. Project title Study on induced spawning of Giant Grouper (Epinephelus lanceolatus)
B.
Research Team Programme head (if applicable) Project leader Name Institution : : Ahmad Daud bin Om IPP :
C.
Description of the project From the previous result showed, steroid treatments (Estradiol) could be used successfully to control sex in grouper culture. Its could be quantified by using SDS PAGE on vitellogenin (Vtg). It is can be evident in serum, vitellogenin could be a good indicator to determining the reproductive status and degree of sexual maturation. Three treatments were tested using 17-estradiol at different dosage (500 g/kg; 50 g/kg and 0 g/kg). The hormone was implanted in the captive body of the Giant Grouper (size; 31-53 kg) in a 6months experiment. SDS-PAGE analysis revealed that 500 g/kg of Estradiol-17 resulted in 66% sexreversal success, better than 50 g/kg (33%) and control, 0 g/kg (0%). These results supported the possibility of using Vtg as biomarkers indicators for reproductive status in giant grouper.
D.
Description how it is relevant to the proposed project In previous result, showed vitellogenin (Vtg) in serum were clearly found in female and not male or immature spawner. It showed that Vtg can be use as non-invasive techniques based on the use of sexual steroid for sex determination of this species. Appearance of Vtg is correlated to oocyte development in sexually mature fish during the reproductive cycle. By using infomation on early results, we can develop a rapid, kit determination by implemeted immunoassay concept.
E.
Other collaborator that were involved in the project
F.
Source of Funding Kementerian Pertanian - Escience Fund
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RESEARCH APPROACH
A. Research methodology 1. Estradiol-stimulation of giant grouper to produced vitellogenin. Hormone estradiol mix with corn oil (1:9) will inject to male giant grouper. Within 10 days, blood will collect every 2 days intervals and centrifuge for 15 min at 15,000 rpm at 4oC to obtain plasma. 2. Purification of vitellogenin. Vitellogenin in the plasma will be purified by ion-exchange using a DEAE-agarose (Bio-RAD) column equilibrated with 0.025 M Tris-HCl, pH 7.5, 0.07 M NaCl, 0.1% aprotinin. A plasma sample from an estradioltreated giant grouper (from above step) will dilute with starting buffer and applied to 27 cm column and the column washed with 80 ml of starting buffer. The fraction will concentrate by ultra filtration and analyzed further using size exclusion chromatography and polyacrylamide gel electrophoresis. 3. Preparation of polyclonal and monoclonal antibodies against vitellogenin Purified vitellogenin (400 g/ml) (>98% purity) will prepare above step are use in antibodies production. The 23 New Zealand white rabbits will inject with 50g of purified vitellogenin in two incomplete Freunds adjuvant at 2 week intervals for 2 months or until the rabbits respond with an appropriate titter. Take test bleeds from the rabbit after each month to test for reactivity to vitellogenin by ELISA or Western blot. When the antibody titter is judged to be sufficiently high, than the rabbits will kill and the blood will allow to clot overnight at 4oC. The blood will centrifuge at 12,700X g to separate the serum and store at -80oC. Test quality of antibody by ELISA and Western blot to determine if there are any cross-reacting volume antibodies to other serum proteins. 4. Vitellogenin detection by western Blot (Validation of antibody preparation) Western Blot will give information on the degree of degradation of the sample. The best way to quantify the reaction is to use a chemi-illuminescent method. Several are now on the market and they all work well. Electroblot of sampel on PVDF membrane by using anti-Vtg antibody (polyclonal) to be test and secondary antibody-linked to alkaline phosphate (antirabit or antimouse), depending on the source of the primary antibody being used. Finally, devoleped with DAB and hydrogen peroxide . 5. Immunohistochemistry analysis on localize of Vitellogenin Vitellogenin in liver of estrogen-induced male and female giant grouper is visualized by immunohistochemical technique; detection of yolk protein in eggs served as a positif control. Localization will accomplished by using an indirect immunofluoresceance method in both frozen and paraffin-embedded tissues. By using antibody produce in above step, the technique present in this study represents a way to detect tiny amounts of Vitellogenin in the liver of fish. 6. Test kit validation and optimization Develop, test and assess the reliability of immune-based tools (using polyclonal and/or monoclonal antibodies) as diagnostic tools for detecting maturating of giant grouper in the laboratory and in field.
Specialised Equipment NA Facility N.A Infrastructure
Description New Description Existing Description
B.
Project activities Activities Chemical / equipment procurement Purification of vitellogenin Vitellogenin detection by Western Blot Immunohistochemistry analysis on localize of vitellogenin From Date 01/06/2012 01/09/2012 01/05/2013 01/05/2013 To Date 01/09/2012 01/11/2012 01/07/2013 01/08/2013
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Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Test kit validation and optimization Compilation & Analysis of data & report writing Induction of Vtg by using Estradiol in giant grouper Development of polyclonal and monoclonal antibodies against vitellogenin 01/06/2013 01/04/2014 01/07/2012 01/11/2012 01/04/2014 01/05/2014 01/09/2012 01/04/2013
C.
Key milestones Milestones Completion of Induction of vitellogenin Completion of purified vitellogenin Completion of development of polyclonal antibodies Completion of vitellogenin detection by Western Blot Completion of vitellogenin locallize Completion on test kit validation and optimization Project Completion Date 01/09/2012 01/11/2012 01/04/2013 01/07/2013 01/09/2013 01/04/2014 01/05/2014
D.
Risks of the project Factor : Determination on polyclonal and monoclonal of antibody could be little bit problem due to time consume and experience Medium - Determination on vitellogenin antibody could be technically risk due to experiece. Medium - Optimization and validation could be more longer to succes Medium - Reagent, standard chemical and consultancy fee to third party could be subject to approval of the grant
Technical risk
Timing risk Budget risk
: :
E.
Time schedule Starting date Completion date Duration : : : 01/06/2012 01/05/2014 24
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BENEFITS OF THE PROJECT

A. Outputs Expected from the project Research New/improved material Quantity Details\Remark Determination kit of Giant Grouper maturation which is applicable for hatcery operator
Human capital and expert development Quantity Doctorate degree 1 Specialisation Area (specific area) Fish reproductive
B.
Economic contributions of the project Sales of manufactured product/ device/ equipment Royalties from licensing Revenue from consultancies Time savings
C.
Infrastuctural contributions of the project New equipment
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RESEARCH COLLABORATION
A. Institutions involved in the project Organisations Involved IPP - Institut Penyelidikan Perikanan UMT - Universiti Malaysia Terengganu B. Industries involved in the project Industry Laboratory work Role Optimization and validation kit Other Role Immunohistochemistry analysis on localize of vitel Validation and optimize kit
C.
Project Team Project Leader AHMAD DAUD BIN OM Researchers Safiah Jasmani Yeong Yik Sung Kua Beng Chu Support Staff Type RA Contract Staff Type Contract Staff Organisation Institut Penyelidikan Perikanan Organisation Universiti Malaysia Terengganu Universiti Malaysia Terengganu Institut Penyelidikan Perikanan Number 2 Number 1 Man-Month 280.00 Man-Month 300.00 300.00 280.00 Man-Month 75.00 Man-Month 50.00
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DIRECT EXPENSES ESTIMATION WORKSHEET

Expense Categories and Items Temporary and contract personnel (V 11000) CONTRACT STAFFS Travel & transportation (V 21000) International Conferences Local seminar Workshop NA Rental (V 24000) NA Research materials & supplies (V 26000) Disposble item (Vials, glove, needle etc) Chemical, Reagent, standard etc Minor modifications & repairs (V 28000) NA Special services (V 29000) Development Kit validation and optimization NA Special equiment, accessories (V 35000) NA Total direct expenses 0 100,000 0 132,500 0 0 232,500 25,000 0 45,000 0 0 0 0 0 70,000 0 0 0 0 15,000 50,000 15,000 50,000 0 0 0 0 30,000 100,000 0 0 0 0 0 0 0 0 10,000 5,000 2,500 0 0 0 0 0 0 0 0 0 10,000 5,000 2,500 10,000 5,000 0 0 15,000 Year 1 Year 2 Year 3 Year 4 Total
SPECIAL EQUIPMENT AND ACCESSORIES Item Justification Similar Equipment available N Justification to purchase Estimation Cost
NA
NA
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PROJECT COST
A. Salaried Personnel costs Staff Category Year 1 (RM) 2012 Salaried personnel (V111000) Total salaried 61,150 Year 2 (RM) 2013 75,000 Year 3 (RM) 2014 Year 4 (RM) 2015 Total (RM) 136,150
61,150
75,000
136,150
B.
Direct Project Expenses Expense Category Year 1 (RM) 2012 Temporary and contract personnel (V11000) Travel and transportation (V21000) Rentals (V24000) Research materials and supplies (V26000) Minor modifications and repairs (V28000) Special services (V29000) Special equipment and accessories (V35000) Total direct 10,000 Year 2 (RM) 2013 5,000 Year 3 (RM) 2014 0 Year 4 (RM) 2015 0 Total (RM) 15,000
17,500
17,500
65,000
65,000
130,000
25,000
45,000
70,000
100,000
132,500
232,500
C.
Total project cost Year 1 (RM) 2012 161,150 Year 2 (RM) 2013 207,500 Year 3 (RM) 2014 0 Year 4 (RM) 2015 0 Total (RM) 368,650
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Summary of Project Funding

A. Funding Sources Funding Sources Science Fund Internal Fund Other Sources Total Funding RM 232,500 136,150 0 368,650 % of Total Funding 63.07 % 36.93 % 0.00 % 100 %
B.
Disbursement schedule for ScienceFund, by participating research organisation Organisation Year 1 (RM) Year 2 (RM) Year 3 (RM) Year4 (RM) 2012 Institut Penyelidikan Perikanan Total ScienceFund 100,000 100,000 2013 132,500 132,500 2014 0 0 2015 0 0 Total (RM) 232,500 232,500
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CONTRACTUAL MATTERS
A. Contractual obligations under this project none
B.
Ownership of intellectual property rights IPP - Institut Penyelidikan Perikanan
Page 16 of 16

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C O N F I D E N T I A L

Name Institution Date Submitted Date Printed Produced by

Project leader Name NRIC : : AHMAD DAUD BIN OM 661018-08-5187

Tel. No. Fax. No.

Key words Giant Grouper Maturation Determination Kit Vitellogenin ELISA

OBJECTIVES OF THE PROJECT

Type of research Applied research

J F MAM J J A S ON D J F MAM J J A S ON D J F MAM J J A S ON D

Induction of Vtg by using Estradiol in giant grouper Purification of vitellogenin

Immunohistochemistry analysis on localize of vitellogenin Test kit validation and optimization

J F MAM J J A S ON D J F MAM J J A S ON D J F MAM J J A S ON D

Completion of purified vitellogenin

SUMMARY OF RELEVANT PAST RESEARCH PROJECT

Other collaborator that were involved in the project

Source of Funding Kementerian Pertanian - Escience Fund

Specialised Equipment NA Facility N.A Infrastructure

Description New Description Existing Description

Timing risk Budget risk

Time schedule Starting date Completion date Duration : : : 01/06/2012 01/05/2014 24

BENEFITS OF THE PROJECT

Infrastuctural contributions of the project New equipment

DIRECT EXPENSES ESTIMATION WORKSHEET

Summary of Project Funding

Ownership of intellectual property rights IPP - Institut Penyelidikan Perikanan

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