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

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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 leader Name NRIC : : AHMAD DAUD BIN OM 661018-08-5187 D. No. Project title Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) C. Fax.Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) PROJECT IDENTIFICATION A. : : E. Project number 06-03-06-SF0021 B. No. Key words Giant Grouper Maturation Determination Kit Vitellogenin ELISA Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 1 of 16 . Institution Name Address : : Institut Penyelidikan Perikanan Pengarah Institut Penyelidikan Perikanan (IPP) Batu Maung 6262231 6262210 Tel.

Biological Sciences F1080100 .Biochemistry F1080104 .Fisheries S2020301 . Type of research Applied research C.Animal Production And Animal Primary Products S2020300 . Research Cluster and SEO Categories being addressed by the Project Research Cluster SEO Category SEO Group SEO Area S&T CORE S2020000 .Agricultural Sciences F1090900 . Fields of research Primary Field of Research FOR Category FOR Group FOR Area F1090000 .Immunology Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 2 of 16 .Fish reproduction Secondary Field of Research (if applicable) FOR Category FOR Group FOR Area F1080000 .Aquaculture F1090909 .Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) OBJECTIVES OF THE PROJECT A.Aquaculture product D. 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.

the seed production technology must be prepared. feed. Research works directed to the developing the biotechnology tools to assess gender and maturation status of giant grouper is needed to be developed. One of the striking features in teleost fish is the plasticity of the sex determination with a range of gonochoristc species. environment. A range of factors imposed by hatchery practices has been found to influence egg quality such as stress on broodstock brought about by handling. The success of seed production activity depends on the gonad maturity of broodstock. Literature review summary Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 3 of 16 . over ripening of eggs and hormone administration in induced spawning. where individuals are either males or females. or species displaying a change in gander during the lifetime. season and broodstock management.Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) RESEARCH BACKGROUND B. which is naturally depending on the age. Research background of the project Project status New Project summary In order to develop giant grouper culture technology. size.

Currently. and this visual method is not compatible with an efficient broodstock management. 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. sequencing and amino acid profile. depending on the natural environment of the populations studied (Lasserre. which is difficult particularly concerning the prediction of natural sex inversion (age and/or size) and the artificial control of sex. generating the biological knowledge necessary to evaluate the impacts that a shift in sex change phenomenon. Therefore. phenotypic sex is more or less easily identified in pubertal male or female by specific external characters. Individual identification of the breeder gender is necessary to maintain the desired sex ratio to produce the appropriate fingerling number for aquaculture production. Prelimanary works has discover molecular information on proteomic basic data of vitellogenin such as molecular weight. Which is changing sex from female in early life to male at older age. Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 4 of 16 . 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. 1971). Generally. there are kit determination develop on used of immunoassay concept for detecting WSSV virus in shrimp. In some cases. trout and salmon (Pottinger et al. The point in time at which sex-change occurs is can be interpreted on many different levels (e. except may be some morphological characters observed only when fish have initiated courtship behavior. 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. histological) were effects on broodstocks management of this species. In most species. Throughout the years. 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. this technique has limitation to predict the gander of broodstock. 1998). no reliable method for gender determination has been described in giant grouper. 1945). equilibrated or determinate sex ratios are required when mature male and female pairs are necessary for induced or natural breeding. which is using protein in shrimp tissue. This prelimanary works were important for further study on development of maturation kit.g. 1994). This characteristic were useful as biomarker indicator or as sign on sex determination . In this regard.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. It is similar concept with the purpose experiment on development maturation kit are suggest. behavioral. Direct observation of broodstock is not always possible due to water turbidity. However. From an academic perspective. 2005) and sturgeon (Cuisset et al. 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. In fact. many researchers have been developed in pursuit of obtaining reliable quantitative values of nutrient in blood plasma. Despite that. 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. in development of pregnancy test kit also used the same principle of sandwich immunoassay. morphological. Specifically. intra-ovarian biopsies or endoscopies or cannulation technique on anesthetized animals allowed gender identification. even outside the breeding season or even in immature fish. 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. The shrimp virus detection kit was based on the principle of sandwich immunoassay. Sex determination in cultured species is a constraint to broodstock constitution. 1972). sexual transition is a useful process in which to investigate the mechanisms controlling sex determination and differentiation.

Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 5 of 16 .

Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) 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 Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 6 of 16 .

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 Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 7 of 16 .

By using infomation on early results. better than 50 µg/kg (33%) and control. SDS-PAGE analysis revealed that 500 µg/kg of Estradiol-17ß resulted in 66% sexreversal success. E. Source of Funding Kementerian Pertanian . Description how it is relevant to the proposed project In previous result. Other collaborator that were involved in the project F. 0 µg/kg (0%). It is can be evident in serum. Research Team Programme head (if applicable) Project leader Name Institution : : Ahmad Daud bin Om IPP : C. It showed that Vtg can be use as non-invasive techniques based on the use of sexual steroid for sex determination of this species. Description of the project From the previous result showed. Three treatments were tested using 17ß-estradiol at different dosage (500 µg/kg. Its could be quantified by using SDS PAGE on vitellogenin (Vtg). vitellogenin could be a good indicator to determining the reproductive status and degree of sexual maturation.Escience Fund Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 8 of 16 . steroid treatments (Estradiol) could be used successfully to control sex in grouper culture. Appearance of Vtg is correlated to oocyte development in sexually mature fish during the reproductive cycle. kit determination by implemeted immunoassay concept. These results supported the possibility of using Vtg as biomarkers indicators for reproductive status in giant grouper. we can develop a rapid. 50 µg/kg and 0 µg/kg). 31-53 kg) in a 6months experiment. D.Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) SUMMARY OF RELEVANT PAST RESEARCH PROJECT A. Project title Study on induced spawning of Giant Grouper (Epinephelus lanceolatus) B. showed vitellogenin (Vtg) in serum were clearly found in female and not male or immature spawner. The hormone was implanted in the captive body of the Giant Grouper (size.

blood will collect every 2 days intervals and centrifuge for 15 min at 15. 0. The 23 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. Purification of vitellogenin. The best way to quantify the reaction is to use a chemi-illuminescent method. Specialised Equipment NA Facility N.07 M NaCl. Within 10 days.5. Several are now on the market and they all work well.A Infrastructure Description New Description Existing Description B. Test kit validation and optimization Develop. 4. pH 7. Localization will accomplished by using an indirect immunofluoresceance method in both frozen and paraffin-embedded tissues. 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. Test quality of antibody by ELISA and Western blot to determine if there are any cross-reacting volume antibodies to other serum proteins. the technique present in this study represents a way to detect tiny amounts of Vitellogenin in the liver of fish.700X g to separate the serum and store at -80oC. 5. 0.Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) RESEARCH APPROACH A. than the rabbits will kill and the blood will allow to clot overnight at 4oC.025 M Tris-HCl. detection of yolk protein in eggs served as a positif control. depending on the source of the primary antibody being used. The fraction will concentrate by ultra filtration and analyzed further using size exclusion chromatography and polyacrylamide gel electrophoresis.000 rpm at 4oC to obtain plasma. 2. 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). By using antibody produce in above step. Estradiol-stimulation of giant grouper to produced vitellogenin. Immunohistochemistry analysis on localize of Vitellogenin Vitellogenin in liver of estrogen-induced male and female giant grouper is visualized by immunohistochemical technique. Preparation of polyclonal and monoclonal antibodies against vitellogenin Purified vitellogenin (400 µg/ml) (>98% purity) will prepare above step are use in antibodies production. 3. Hormone estradiol mix with corn oil (1:9) will inject to male giant grouper. devoleped with DAB and hydrogen peroxide . Vitellogenin detection by western Blot (Validation of antibody preparation) Western Blot will give information on the degree of degradation of the sample. 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 Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 9 of 16 . Vitellogenin in the plasma will be purified by ion-exchange using a DEAE-agarose (Bio-RAD) column equilibrated with 0. When the antibody titter is judged to be sufficiently high. Finally. Research methodology 1. 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. Take test bleeds from the rabbit after each month to test for reactivity to vitellogenin by ELISA or Western blot. The blood will centrifuge at 12. 6.1% aprotinin.

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. Risks of the project Factor : Determination on polyclonal and monoclonal of antibody could be little bit problem due to time consume and experience Medium .Optimization and validation could be more longer to succes Medium . Medium . Time schedule Starting date Completion date Duration : : : 01/06/2012 01/05/2014 24 Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 10 of 16 . 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.Reagent. standard chemical and consultancy fee to third party could be subject to approval of the grant Technical risk : Timing risk Budget risk : : E.Determination on vitellogenin antibody could be technically risk due to experiece.

Outputs Expected from the project Research New/improved material Quantity &nbsp. 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. Infrastuctural contributions of the project New equipment Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 11 of 16 . Economic contributions of the project Sales of manufactured product/ device/ equipment Royalties from licensing Revenue from consultancies Time savings C.Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) BENEFITS OF THE PROJECT A.

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.Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) RESEARCH COLLABORATION A.00 Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 12 of 16 .00 Man-Month 75.00 280.00 Man-Month 50. 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.00 Man-Month 300.00 300.Institut Penyelidikan Perikanan UMT . Institutions involved in the project Organisations Involved IPP .Universiti Malaysia Terengganu B.

000 0 0 0 0 0 70. accessories (V 35000) NA Total direct expenses 0 100.Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) 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.000 0 0 0 0 30.000 15.000 5.000 5.000 0 0 0 0 0 0 0 0 10.500 0 0 232. Reagent. glove. standard etc Minor modifications & repairs (V 28000) NA Special services (V 29000) Development Kit validation and optimization NA Special equiment.500 0 0 0 0 0 0 0 0 0 10.000 2.000 0 0 0 0 15.000 0 0 15.000 0 132.500 25.000 100.000 50. needle etc) Chemical.000 2.000 0 45.000 50.500 10.000 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 Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 13 of 16 .000 5.

150 75.000 0 0 0 0 0 100. 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. Total project cost Year 1 (RM) 2012 161.000 0 17.000 65.000 0 0 130.Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) PROJECT COST A.500 Year 3 (RM) 2014 0 Year 4 (RM) 2015 0 Total (RM) 368.500 0 0 232.150 Year 2 (RM) 2013 75.150 Year 2 (RM) 2013 207.150 B.000 45.500 0 0 0 0 0 65.500 0 0 17.500 C.000 Year 3 (RM) 2014 Year 4 (RM) 2015 Total (RM) 136.150 61.000 132.000 0 0 70.650 Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 14 of 16 . Salaried Personnel costs Staff Category Year 1 (RM) 2012 Salaried personnel (V111000) Total salaried 61.000 Year 2 (RM) 2013 5.000 0 0 0 0 0 25.000 Year 3 (RM) 2014 0 Year 4 (RM) 2015 0 Total (RM) 15.000 136.

500 132. by participating research organisation Organisation Year 1 (RM) Year 2 (RM) Year 3 (RM) Year4 (RM) 2012 Institut Penyelidikan Perikanan Total ScienceFund 100. Disbursement schedule for ScienceFund.00 % 100 % B.000 100.500 Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 15 of 16 .500 136. Funding Sources Funding Sources Science Fund Internal Fund Other Sources Total Funding RM 232.150 0 368.000 2013 132.Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Summary of Project Funding A.500 232.93 % 0.07 % 36.650 % of Total Funding 63.500 2014 0 0 2015 0 0 Total (RM) 232.

Contractual obligations under this project none B. Ownership of intellectual property rights IPP .Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) CONTRACTUAL MATTERS A.Institut Penyelidikan Perikanan Determination vitellogenin level and development of maturation broodstock kit in giant grouper (Epinephelus lanceolatus) (06-03-06-SF0021) Page 16 of 16 .

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