Increasing productivity and efficiency of Nile tilapia production using plant saponins and introduction of its culture in areas under the jurisdiction of the Palestinian Authority (PA)
Final Report Abstract
One of the most striking features of the results of this project was the discrepancy between the in vivo and in vitro work. The in vitro work demonstrated clearly that the plant extracts, especially QS80 and TS90 were potent inhibitors of aromatase, and as such should cause fairly comprehensive masculinisation of tilapia, if properly applied, just as MT does. In vivo, the situation was far less clear and in only one of the experiments was there any evidence that the same plant extracts caused any appreciable masculinisation. Furthermore, evidence from other scientists' trials that these plant extracts could enhance growth in tilapia was not confirmed except in one trial where fairly high doses (2000 ppm) of a plant saponin containing 25% sapogenins produced an increase of less than 10% above the control. Manipulation of the dosage level and the time of application of saponin supplements did not result in any improvements. At present, therefore it seems that despite much careful experimental work, feeding saponins as a supplement added directly to the fishes' feed is pointless. However, observations made by the Israeli group indicate that it may be worth looking at the method of application of these supplements. Their results show that TS90 saponins are absorbed through the gut lining of tilapia, but only in small amounts and there is good evidence that the saponins are metabolised (altered) to some extent once they are absorbed. This very low level of uptake may be connected with the fact that it has proved very difficult to solubilise many of the TD90 saponins especially those from 'cluster 3' that are believed to be the most potent aromatase inhibitors. It may therefore be worthwhile to resume large scale in vivo trials in the future if ways can be found of administering saponins more efficiently. Such methods might include coating saponins in lipids or protein to aid absorption, investigating better ways of solubilising saponins as an aid to absorption either in the gut or across the gill membrane, or directly injecting saponins either into the blood stream or else targeting specific organs such as the liver. Any such innovations would involve careful work in the laboratory before proceeding to large scale in vivo trials. Although saponins do not seem to have any useful effects when added directly to the feed in classic feeding trials, there is still the question of their effects at a molecular level in vitro. There is very good evidence that one or more compounds extracted from fenugreek have a profound effect on the enzyme aromatase and that this in turn can have profound effects on reproduction and growth. The nature of the compound(s) and the mechanism(s) by which they alter aromatase activity are topics that are well worth pursuing and may result in fundamental discoveries in molecular biology and reproductive physiology. The compound(s) themselves or derivatives may enable real control of the sex and/or reproduction of tilapia and thus provide a safer and commercially viable alternative to MT. It is now possible to produce a partially refined extract from fenugreek, known as cluster 3, that inhibits aromatase 0.01 to 0.1 times as strongly as MT. There is good reason to believe that it will be possible to further refine this extract so that it will be as potent if not more so than MT because there are at least 10 separate compounds in cluster 3, most of which appear to be sapogenlns, and it is entirely likely that only one or two of these are responsible for the inhibitionof aromatase. Using advanced analytical techniques such as LC-MS MRM it may be possible to identify these compounds and determine their structures and the mechanism of their interactions with aromatase. This in turn may give clues as to whether the molecules can be modified to make them even more potent. Any candidates for enhanced aromatase inhibitors / growth promoters derived from fenugreek or other plants would first have to be comprehensively screened for pharmacological and environmental safety. Even then the problem would still remain of how to produce the candidate molecule(s) in sufficient quantities for commercial exploitation. Attempts In this direction in the present project have met with mixed success. The hydrolysis technique is a cheap and effective way of increasing the yield of "cluster 3" material, and the technique would be easy to scale up to the 10, 100 or 1000 gram level, but the product is still impure. Far less successful were the attempts to purify the "cluster 3" material itself using preparative HPLC. If future research at a "micro" level does produce any promising candidate molecules, maybe some radically different technique will have to be tried for their purification. In the mean time, saponins continue to be useful tools in investigations of fundamental questions concerning transport of materials across biological membranes, control of gene transcription and the regulation of growth and reproduction. Saponins may prove to have as much potential for the microbiologist as they have for the commercial fish farmer. The work reported in the Palestine section of the report has resulted in equipment and facilities for future work on aquaculture in Palestine. Although at present there seems little point in pursuing trials involving the direct supplementation of tilapia feed with saponins there is sure to be other worthwhile work that Palestinian researchers will need to do on other aspects of aquaculture. It is hoped that their efforts will provide information for farmers and extension workers who want to set up aquaculture enterprises in Palestine to provide a badly-needed source of good quality protein for the local population.
Publications
- Assays for the validation of substances for sex reversal of tilapia fry. The Annual conference for the memory of Dan Poper, Eilat, Israel. 2005
Matan Golan & Berta Levavi-Sivan
- Assays for the validation of substances for sex reversal of tilapia fry. Annual Conference of the Israeli Zoological Society, 2006
Matan Golan & Berta Levavi-Sivan
- (2007). Expression and Characterization of Biologically Active Recombinant Tilapia FSH: Immunohistochemistry, Stimulation by GnRH and Effect on Steroid Secretion. Biology of Reproduction 76(4): 692-700
Aizen, J., Kasuto, H., Golan, M., Zakay, H. and Levavi-Sivan, B.
- 8th International Syposium on Reproductive Phvsiologv of Fish fISRPF). San Malo. France. 2007. 17-α-methyltestosterone exerts its masculinizing activity by acting as an androgen rather than as an aromatase inhibitor
Matan Golan & Berta Levavi-Sivan
- 2008. Fish recombinant gonadotropins. CYBIUM, International Journal of Ichthyology, 32,17-21
Levavi-Sivan, B., Golan, M., Aizen. J., &. Elizur, A.
- 2008. Quillaja saponins Inhibit tilapia aromatase activity in vitro. CYBIUM, International Journal of Ichthyology. 32,80-82
Golan, M., Avitan, A., Qutob, M., Dweik, H., Abu-Lafi, S., Focken, U., Francis, G., Becker, K., Kerem Z. &. Levavi-Sivan.B.
- Treatment with saponins from Trigonella foenum-graecum and Quillaja saponaria influences sex ratio in Nile tilapia (Oreochromis niloticus) larvae. (2010) Eighth International Symposium on Tilapia in Aquaculture (ISTA) (Cairo)
Stadtlander T, Focken U, Levavi-Sivan B, Dweik H, Qutob M, Abu-Lafi S, Kerem Z and Becker K
- Effects of saponin fractions from Trigonella foenum-graecum and Balanites aegyptiaca on gene expression of GH, IGF-1 and their respective receptors, grovrth, nutrient utilization, body composition, oxygen consumption and plasma IGF-1 in Nile tilapia, Oreochromis niloticus (L.). (2011) Ninth International Symposium on Tilapia in Aquaculture (ISTA) (Shanghai)
