Maturation Rapid Kit Proposal

Prepared by

CONFIDENTIAL

Name

Institution

Date Submitted

Date Printed

Produced by

:

:

:

:

:

AHMAD DAUD BIN OM

Institut Penyelidikan Perikanan

12 December 2012

Ministry of Science, Technology and Innovation

Determination vitellogenin level and development of maturation broodstock

kit in giant grouper (Epinephelus lanceolatus)

(06-03-06-SF0021)

02 May 2012

Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021)


Page 1 of 16

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

Tel. No.

Fax. No.

:

:

:

:

Institut Penyelidikan Perikanan

Pengarah Institut Penyelidikan Perikanan (IPP) Batu Maung

6262231

6262210

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

S&T COREResearch Cluster

SEO Category S2020000 - Animal Production And Animal Primary Products

SEO Group S2020300 - Fisheries

SEO Area 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

- FOR Category

- FOR Group

- FOR Area

(if applicable)

F1080000 - Biological Sciences

F1080100 - Biochemistry

F1080104 - Immunology



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RESEARCH BACKGROUND

Research background of the project

Project status

B.

Literature review summary

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.



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Related research

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 ß-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.

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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Page 6 of 16

J

2014

J

2014

J

2013

J

2013

PROJECT SCHEDULE

J F M A M JJ A S O N D F M A M JJ A S O N D F M A M JJ A S O N D

2012

Immunohistochemistry analysis on localize of vitellogenin

Vitellogenin detection by Western Blot

Compilation & Analysis of data & report writing

Test kit validation and optimization

Induction of Vtg by using Estradiol in giant grouper

Chemical / equipment procurement

Development of polyclonal and monoclonal antibodies against vitellogenin

Purification of vitellogenin

J F M A M JJ A S O N D F M A M JJ A S O N D F M A M JJ A S O N D

Completion of vitellogenin locallize

Completion of vitellogenin detection by Western Blot

Completion of Induction of vitellogenin

Completion of purified vitellogenin

Completion of development of polyclonal antibodies

2012

Activities

Milestone



Page 7 of 16

Project Completion

Completion on test kit validation and optimization



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SUMMARY OF RELEVANT PAST RESEARCH PROJECT

A. Project title

B. Research Team

Programme head

Project leader

:(if applicable)

Name :

Institution :

C. Description of the project

D. Description how it is relevant to the proposed project

E. Other collaborator that were involved in the project

F. Source of Funding

Study on induced spawning of Giant Grouper (Epinephelus lanceolatus)

Ahmad Daud bin Om

IPP

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 17ß-estradiol at different dosage (500 µg/kg; 50 µg/kg and 0 µg/kg). The hormone was implanted in the captive body of the Giant Grouper (size; 31-53 kg) in a 6-months experiment. SDS-PAGE analysis revealed that 500 µg/kg of Estradiol-17ß resulted in 66% sex-reversal success, better than 50 µg/kg (33%) and control, 0 µg/kg (0%). These results supported the possibility of using Vtg as biomarkers indicators for reproductive status in giant grouper.

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.

Kementerian Pertanian - Escience Fund



Page 9 of 16

RESEARCH APPROACH

A. Research methodology

B. Project activities

Activities From Date To Date

Purification of vitellogenin 01/09/2012 01/11/2012

Chemical / equipment procurement 01/06/2012 01/09/2012

Immunohistochemistry analysis on localize of vitellogenin 01/05/2013 01/08/2013

Vitellogenin detection by Western Blot 01/05/2013 01/07/2013

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 estradiol-treated 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 µg/ml) (>98% purity) will prepare above step are use in antibodies production. The 2-3 New Zealand white rabbits will inject with 50µg 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 Description

NA New

Facility Description

N.A Existing

Infrastructure Description



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C. Key milestones

Milestones Date

D. Risks of the project

Determination on polyclonal and monoclonal of antibody could be little bit problem due to time consume and experience

Factor :

Technical risk :

Timing risk :

Budget risk :

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

E. Time schedule

Starting date :

Completion date :

Duration :

01/06/2012

24

Induction of Vtg by using Estradiol in giant grouper 01/07/2012 01/09/2012

Development of polyclonal and monoclonal antibodies against vitellogenin 01/11/2012 01/04/2013

Test kit validation and optimization 01/06/2013 01/04/2014

Compilation & Analysis of data & report writing 01/04/2014 01/05/2014

Completion of vitellogenin locallize 01/09/2013

Completion on test kit validation and optimization 01/04/2014

Project Completion 01/05/2014

Completion of vitellogenin detection by Western Blot 01/07/2013

Completion of Induction of vitellogenin 01/09/2012

Completion of purified vitellogenin 01/11/2012

Completion of development of polyclonal antibodies 01/04/2013

01/05/2014



Page 11 of 16

BENEFITS OF THE PROJECT

A. Outputs Expected from the project

Research Quantity Details\Remark

New/improved material Determination kit of Giant Grouper maturation which is applicable for hatcery operator

Human capital and expert development

Quantity Specialisation Area (specific area)

1 Fish reproductiveDoctorate degree

B. Economic contributions of the project

Revenue from consultancies

Time savings

Sales of manufactured product/ device/ equipment

Royalties from licensing

C. Infrastuctural contributions of the project

New equipment



Page 12 of 16

RESEARCH COLLABORATION

A. Institutions involved in the project

Organisations Involved Role

UMT - Universiti Malaysia Terengganu

Validation and optimize kit

IPP - Institut Penyelidikan Perikanan Immunohistochemistry analysis on localize of vitel

B. Industries involved in the project

Industry Role

Laboratory work Optimization and validation kit

C. Project Team

Project Leader Organisation Man-Month

AHMAD DAUD BIN OM Institut Penyelidikan Perikanan 280.00

Kua Beng Chu Institut Penyelidikan Perikanan 280.00

Yeong Yik Sung Universiti Malaysia Terengganu 300.00

Safiah Jasmani Universiti Malaysia Terengganu 300.00

Researchers Organisation Man-Month

Support Staff Type Number Man-Month

RA 2 75.00

Contract Staff Type Number Man-Month

Contract Staff 1 50.00

Other



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DIRECT EXPENSES ESTIMATION WORKSHEET

Expense Categories and Items Year 1 Year 2 Year 3 Year 4 Total

Temporary and contract personnel (V 11000)

Travel & transportation (V 21000)

Rental (V 24000)

Research materials & supplies (V 26000)

Minor modifications & repairs (V 28000)

Special services (V 29000)

Special equiment, accessories (V 35000)

Total direct expenses

CONTRACT STAFFS 10,000 5,000 0 0 15,000

Workshop 0 2,500 0 0 2,500

NA 0 0 0 0

International Conferences 0 10,000 0 0 10,000

Local seminar 0 5,000 0 0 5,000

NA 0 0 0 0

Chemical, Reagent, standard etc 50,000 50,000 0 0 100,000

Disposble item (Vials, glove, needle etc) 15,000 15,000 0 0 30,000

NA 0 0 0 0

NA 0 0 0 0

Development Kit validation and optimization 25,000 45,000 0 0 70,000

NA 0 0 0 0

100,000 132,500 232,500

NA NA N

Item Justification Similar Equipment available

Justification to purchase Estimation Cost

SPECIAL EQUIPMENT AND ACCESSORIES



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PROJECT COST

A. Salaried Personnel costs

B. Direct Project Expenses

Expense Category

2012 2013 2014 2015

Total (RM)

Year 1 (RM) Year 2 (RM) Year 3 (RM) Year 4 (RM)

00000Minor modifications and repairs (V28000)

70,0000045,00025,000Special services (V29000)

00000Special equipment and accessories (V35000)

130,0000065,00065,000Research materials and supplies (V26000)

15,000005,00010,000Temporary and contract personnel (V11000)

17,5000017,5000Travel and transportation (V21000)

00000Rentals (V24000)

Staff Category

2012 2013 2014 2015

Total (RM)

Year 1 (RM) Year 2 (RM) Year 3 (RM) Year 4 (RM)

Salaried personnel (V111000)

61,150 75,000 136,150

Total salaried 61,150 75,000 136,150

Total direct 100,000 132,500 0 0 232,500

C. Total project cost

2012 2013 2014 2015

Year 1 (RM) Year 2 (RM) Year 3 (RM) Year 4 (RM) Total (RM)

161,150 207,500 0 0 368,650



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Summary of Project Funding

A. Funding Sources

Funding Sources RM % of Total Funding

Science Fund 232,500

Internal Fund 136,150

0

368,650

Other Sources

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) Total (RM)

2012 2013 2014 2015

Institut Penyelidikan Perikanan 100,000 132,500 0 0 232,500

Total ScienceFund 100,000 132,500 0 0 232,500



Page 16 of 16

CONTRACTUAL MATTERS

A. Contractual obligations under this project

B. Ownership of intellectual property rights

none

IPP Institut Penyelidikan Perikanan-

Maturation Rapid Kit Proposal

Documents

Transcript of Maturation Rapid Kit Proposal