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IGF as a Growth Rate Indicator in Oreochromis niloticus 10RCR3

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IGF as a Growth Rate Indicator in Oreochromis niloticus

Reproduction Control Research 3 (10RCR3)/Experiment/Philippines

Collaborating Institution
Central Luzon State University, Philippines
     Remedios B. Bolivar

Florida International University
     Christopher L. Brown

Objectives
1) Overall: Test the sensitivity and accuracy of the growth mediator, Insulin-like Growth Factor (IGF-1) as an indicator of growth rate in Oreochromis niloticus.

2) Clone the IGF-1 gene in Oreochromis niloticus.

3) Develop an RNAse protection assay for IGF-1 in O. niloticus.

4) Adapt and test available methods for determining RNA and DNA content and RNA/DNA ratios in O. niloticus tissues.

5) Use the methods specified in objectives 4 and 5, above, to test the responsiveness of cellular indicators of growth in young O. niloticus, exposed to nutritional variables.

Significance
Faculty development has been a high priority at Central Luzon State University, and a steadily increasing percentage of faculty at CLSU hold the Ph.D. The Freshwater Aquaculture Center is the highest-ranking academic unit in this regard within CLSU (R. Undan, CLSU President, pers. comm.). Many of the FAC faculty have earned their Ph.D. from US and Canadian universities, and a concerted effort is being made to advance the higher educations of additional faculty. Clearly, the establishment of a high standard of education has contributed to the increasing academic strength of the FAC, and the desire to continue to develop faculty research capabilities is a goal of the CLSU administration.

In order to maintain the necessary numbers of faculty needed to sustain research programs and curriculum offerings, it is usually the case that one faculty member is allowed a Leave of Absence to pursue doctoral studies at a time. Emmanuel Vera Cruz is considered to be one an outstanding young faculty member at CLSU, and he has applied for admission to the Doctoral Program at Florida International University in the Department of Biological Science. He graduated summa cum laude, and is eager to pursue a rigorous doctoral program in fish physiology and genetics. Initiating that program is one goal of this educational development activity.

The proposed activity will include a program of doctoral research designed to add a potentially valuable dimension to the ongoing studies at CLSU on the subject of feeds and fertilizers. Specifically, we have found in large-scale farm trials that either delayed feeding or feeding a sub-satiation-level diet benefit are realized in the cost savings that occurs as a result of the reduction in the amount of processed feed that must be purchased by farmers. Our analysis, based on trials at seven participating commercial farms in the Philippines, showed that delayed feeding could result in a lowering of feed costs by approximately 37% without a significant loss of crop value at harvest (Brown et al., 2000).

One factor limiting the number of experiments that can be carried out on the subject of diet optimization is the extended length of time that is required to see results. We presently stock juvenile fish and sample throughout a full grow-out period, which in the Philippines, typically takes about five months. The research goal in the planned doctoral program will be to evaluate and validate short-term physiological indicators of growth. Both RNA/DNA ratios and the quantification of circulating Insulin-like Growth Factor (IGF-1) will be used for this purpose.

Quantified Anticipated Benefits
The proposed research will provide highly specific cellular and molecular tools that can be used for rapid evaluation of diet and other culture parameters. With the capability to monitor the proximal regulatory mechanisms involved in growth, it will be possible to determine optimal grow-out conditions without having to wait for a complete growth cycle to be completed. These methods should be capable of providing a clear indication of growth stimulation within a few days of exposure to experimental conditions.

We have found already that it is possible to reduce grow-out costs by providing reduced rations, and other means of improving grow-out efficiency are very likely. Presently, the effectiveness of locally produced feeds applied judiciously to reduce farmers' bills must be evaluated in large-scale farm trials. It should be possible to sort through and evaluate the various feeding and environmental variables much more efficiently with the help of short-term indicators of growth, such as the circulating level of IGF-1 or the RNA/DNA ratio. Conceivably, this approach would allow the effectiveness of a dietary or environmental factor or combination of factors to be determined in a matter of days, rather than months.

The scientific benefits and impacts of an educational development activity must ultimately be viewed in terms of the long-term institutional and humanitarian effects. Solid progress toward the Ph.D. will be one such quantifiable benefit. At present the Freshwater Aquaculture Center does not have anyone with the expertise to lead a project in DNA technology, although the interest is certainly there among students and faculty. The proposed project will involve the cloning of a portion of the tilapia genome (the IGF-1 gene) and its detection using standard molecular methods. These techniques will be propagated at the host country institution, allowing CLSU to be a participant rather than an observer in the development of new applications of biotechnology. Documented mastery and specific accomplishments in the use of these techniques will be other quantifiable benefit (number of abstracts produced, successful cloning, etc.).

This approach is in keeping with the spirit of the recent PD/A CRSP white-paper policy document on biotechnology and genetics (Brown and Bart, 2000), which advocated for the development of non-invasive applications of DNA technology. The use of a regulatory compound as a marker or indicator of growth is non-invasive; that is, it does not involve reconstruction or other potentially controversial manipulation of the genome. This is, nevertheless, a potentially valuable technology. The cost of developing a new strain of fish, for example, can be dramatically reduced if the rapidly growing individuals can be identified within a few days using DNA markers, rather than a few months by the conventional feed and weigh approach.

Research Design
Location of Work: Mr. Vera Cruz will travel from CLSU in Nueva Ecija to carry out his doctoral studies at the Biscayne Bay campus of Florida International University, North Miami Beach, Florida. He will be enrolled as a full-time doctoral student, using the typical configuration of doctoral candidates of 50% time in classroom study activities and 50% time as a Research Assistant. Research Assistantships are the usual arrangement for graduate student support.

A very well-equipped laboratory is presently available for these studies, and a new marine lab building is in the late planning stages, and will probably be available before the doctoral program is completed.

Methods: Insulin-like Growth Factor, IGF-I is the hormone accepted as directly responsible for promoting growth in fish, as in other vertebrates. The actions of growth hormone and other anabolic agents are transduced through IGF-1, which is responsible for direct stimulation of growth in muscle and other tissues. For this reason, the detection of IGF-1 represents one of the best conceivable indications of growth at the proximal end of the process.

Not surprisingly, circulating IGF-I levels correlate well with growth rates in animals (Jones and Clemmons, 1995; Beckman et al., 1998). The relationship of IGF-I levels and growth rate is more consistent than that of growth hormone (GH) with growth rate. Growth hormone levels can become dissociated with growth rate under some conditions in which the correlation of IGF-I with growth persists (Duan et al., 1997). For these reasons, the detection of IGF-1 is gaining wide acceptance as one of the most accurate and direct short-term indicators of growth rate available (Fruchtman et al., 1997).

Our colleagues at North Carolina State University have been working with the cloning of IGF genes in the striped bass, and have agreed to provide technical and collaborative assistance as necessary for this project. A letter confirming collaboration from Dr. R. Borski is attached. The detection of IGF-I in Nile tilapia will require the cloning of the IGF-1 gene in O. niloticus, and the adaptation of established methods based on a highly sensitive RNAse protection assay previously developed in rats (Borski et al., 1996; Borski et al., 2000). Primers are available which should make this a straightforward task.

A second approach will also be used in the assessment of cellular indicators of growth rate. Because cellular DNA content remains at baseline as RNA concentrations rise with the rate of cellular protein synthesis, the ratio of RNA to DNA concentrations has often been used as an index of protein synthesis, which reflects changes in growth rate. RNA/DNA data support this association (Bulow, 1987), although the sensitivity of RNA/DNA ratios as an indicator is limited.

Our plan is to develop the tools needed to test and apply both short-term indicators of growth rate in O. niloticus. This approach will dramatically reduce the long and costly production trials required for growth selection inherent to traditional breeding programs. Using the data already obtained in our continuing farm trials, and the data expected to be obtained in the course of Work Plan 10, we will test the levels of these biomarkers in laboratory-reared fish on similar diets as compared with the growth results obtained in the farm trials. This will serve either to validate or refute the efficacy of these tools as predictors of growth rates. The use of both RNA/DNA ratio and heterologous IGF-1 taken in combination may have a more accurate predictive value than either indicator used alone (R. Borski, pers. comm.).

One limitation on the proposed program of study is the use of a genetically selected strain of tilapia in the farm trials (the GIFT Foundation fish) that are unavailable in Florida. Ideally, the correlative studies seeking relationships of growth data in farm trials and biomarkers for growth in laboratory tests should use identical fish. Recognizing this shortcoming, we still believe that the proposed work will be valid, since it will be carried out in the same species. Eventually it is anticipated that the cellular methods we adapt to O. niloticus in the course of this project will be applied in situ (in the Philippines) and elsewhere.

Regional Integration
The enormity of the value of Nile tilapia worldwide provides strong incentive to test and validate cellular tools that can be applied in its selective breeding and domestication. The regional integration of this technology is a long-term prospect, which will be completed after Mr. Vera Cruz has completed his studies and returned to his teaching and research position at CLSU. His technical knowledge and experience will continue to grow, as will his influence as he serves as mentor for students throughout his chosen career as a scholar and educator.

We will post and regularly update research progress on the project website, described in more detail under the Farm Trials investigation. Interest in and frequency of downloads of research products (abstracts, manuscripts, etc.) derived in the course of this educational development activity will be monitored.

Schedule
Year 1

TABLE 1

Year 2

TABLE 1

Note: the typical duration of a doctoral program exceeds the length of time available in the Tenth Work Plan, which is less than two years. We are committed to seeking additional sources of funding, assistantships, etc. during the portion of the program of study that occurs after the completion of this activity in order to see this degree program through to completion.

Literature Cited
Beckman, B.R., D.A. Larsen, S. Moriyama, B. Lee-Pawlak, and W.W. Dickhoff, 1998. Insulin-like growth factor-I and environmental modulation of growth during smoltification of spring chinook salmon (Oncorhynchus tshawytscha). Gen. Comp. Endocrinol., 109:325­335.

Borski R.J., W. Tsai, R. Demott-Friberg, and A.L. Barkan, 1996. Regulation of somatic growth and the somatotropic axis by gonadal steroids: Primary effect on insulin-like growth factor I gene expression and secretion. Endocrinology, 137:3253­3259.

Borski, R.J., W. Tsai, R. DeMott-Friberg, and A. Barkan, 2000. Induction of GHmRNA by pulsatile GHRH in rats is pattern-specific. Amer. J. Physiol. (in press)

Brown, C.L. and A. Bart, 2000 (unpublished). Biotechnology, Genetics, and Disease issues. A white-paper document, prepared for the Pond Dynamics/Aquaculture CRSP.

Bulow, F.J., 1987. RNA-DNA ratios indicators of growth in fish: a review. In: Summersfelt, R.C., and G.E. Hall (Editors), The Age and Growth of Fish. The Iowa St. Univ. Press, Ames, Iowa. pp. 45­64.

Duan, C., 1997. The insulin-like growth factor system and its biological actions in fish. Amer. Zool., 37:491­503.

Fruchtman, S., D.C. McVey, and R.J. Borski, 1997. Insulin-Like Growth Factor-I regulation of prolactin and growth hormone secretion in hybrid striped bass. Amer. Zool., 37:180A.

Jones, J.I. and D.R. Clemmons, 1995 Insulin-like growth factors and their binding proteins: Biological actions. Endo. Rev., 16:3­34.

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