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Use of a natural aquatic fern, Azolla microphylla, as a main component in food for
the omnivorous–phytoplanktonophagous ti...
estimated weekly, considering that Azolla contain only 5% dry
matter, and dried in a shaded area after harvesting. Five di...
protein for fish. These observations were confirmed by the
specific growth rate variation and those of the food conversion
as saponine and mimosine, which have negative effects on
appetency and growth (Jackson et al., 1982; Guillaume et al.,
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Use of a natural aquatic fern, Azolla microphylla, as a main component in food for the omnivorous–phytoplanktonophagous tilapia, Oreochromis niloticus L.


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Use of a natural aquatic fern, Azolla microphylla, as a main component in food for the omnivorous–phytoplanktonophagous tilapia, Oreochromis niloticus L.

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Use of a natural aquatic fern, Azolla microphylla, as a main component in food for the omnivorous–phytoplanktonophagous tilapia, Oreochromis niloticus L.

  1. 1. Use of a natural aquatic fern, Azolla microphylla, as a main component in food for the omnivorous–phytoplanktonophagous tilapia, Oreochromis niloticus L. By E. D. Fiogbe´ 1 , J.-C. Micha2 and C. Van Hove3 1 Unite´ de Recherche sur les Zones Humides, De´partement de Zoologie et Ge´ne´tique, Faculte´ des Sciences et Techniques, Universite´ d’Abomey-Calavi Be´nin, Be´nin; 2 Unite´ de Recherche en Biologie des Organismes, Faculte´s Universitaires N.D. de la Paix, Namur, Belgium; 3 Laboratoire de Botanique, Catholic University of Louvain, Louvain-la-Neuve, Belgium Summary An aquatic fern, Azolla microphylla (strain 175 MI, Catholic University of Louvain, Belgium), a natural source of protein, was used in this study to produce low-cost feeds for the omnivorous–phytoplanktonophagous tilapia, Oreochromis nil- oticus L. Fish were grown in a recirculating system and fed with six different diets in triplicate groups. Diets were formulated with approximately similar total protein, ranging from 27.25 to 27.52% dry weight (dw), gross energy content ranging from 85.1 to 96.5 MJ kg)1 dw, and with different levels of dry meal Azolla (0, 15, 20 30 40, 45% diet dw). All diet levels with incorporated Azolla meal exhibited weight gain, thus it can be assumed that Azolla in good combination with local products can be used to promote fish culture development. The Azolla-free diet and the diet containing 15% Azolla produced the same growth perform- ance. However, the least expensive diet containing 45% Azolla also exhibited growth and can be used as a complementary diet for tilapia raised in fertilized ponds. Introduction In many developing countries people lack sufficient animal protein. In Benin, the main protein source is fish; however, consumption thereof is very low (7 kg year)1 ) compared to the adult requirement of fish or animal meat per year (30 kg year)1 ). Fish culture could be a means to increase animal protein consumption not only in Benin but also in most of the developing countries that lack sufficient animal protein. However, in a project financed by the European Union from 1978 to 1990, tentative fish production in Benin fell due mainly to the high cost of the feed (350 CFA Franc [FCFA] kg)1 vs 600 FCFA kg of fish produced, Fiogbe´ , 1985). (655.9 FCFA ¼ 1 euro; 600 FCFA ¼ 1 U.S.$). Indeed, fish meal, vitamin premix and mineral premix used in the feed formu- lation were imported and increased the formulated feed costs. Considering the reports of Micha (1990) and Bai and Gatling (1992) assuming respectively that: (i) the first limiting factor for productivity of tropical aquatic ecosystems is often the bioavailability of nitrogen, (ii) approximately 95% of the cost of formulating an average production diet is related to meeting protein and energy needs of the fish, an attempt is made here to use the natural aquatic fern Azolla microphylla (strain 175 MI, Catholic University of Louvain, Belgium) as a main component in food for the omnivorous– phytoplanktonophagous tilapia, Oreochromis niloticus L. Azolla is an aquatic fern able to fix unlinked nitrogen (N2) directly from the atmosphere because of its endosymbiotic blue alga Anabaena azollae (Van Hove, 1989), and is thus a very promising supply of nitrogen to aquatic ecosystems. Azolla has been used for centuries as green manure in rice fields and is given as a food supplement to poultry, pigs and cattle in China and Vietnam (Lumpkin and Plucknett, 1982; Van Hove, 1989). However, reports on the use of Azolla for fish are rare. According to Lumpkin and Plucknett (1982) and Van Hove (1989), Azolla under good conditions presents a high produc- tivity and high protein content [generally 20–30%, on a dw basis]. Azolla is also able to store phosphorus and potassium from water (Leonard, 1997). Azolla is also rich in Fe (1000– 8600 dw), Cu (3–210 ppm dw) and Mn (120–2700 ppm dw) (Leonard, 1997). Paoletti et al. (1987) found that Azolla contains 0.8–6.7% dw crude fat, with 6.1–7.7% and 12.8– 26.4% total fat for polyunsaturated acids (PUFA) omega 3 and omega 6. Azolla seems to be rich in some vitamins, notably carotenes and vitamin A (300–600 ppm dw, Leonard, 1997). According to these reports on Azolla composition, six experimental diets containing different levels of A. microphylla were formulated for this study to feed the omnivorous– planktophagous tilapia, Oreochromis niloticus. The dietary protein content was calculated based on the protein content of local products (Luquet, 1984) and fixed to 27% dw. The diets cost less than 75 FCFA kg)1 (655.9 FCFA ¼ 1 euro; 600 FCFA ¼ 1 U.S.$). As result of this experiment the best diet will be recommended for fish culture in rural areas, mainly wetlands, to reduce the fishing effort in aquatic ecosystems. Material and methods Experimental fish and diets O. niloticus juveniles weighing 1.62–1.75 g were obtained from commercial nursery ponds in Songhai Centre, Porto-Novo, Benin, transported to the laboratory and divided among eighteen 25-L plastic tanks. The experimental diets were formulated with a calculated energy content ranging from 85.1 to 96.5 MJ kg)1 dw and total protein content ranging from 27.25 to 27.52% dw. Diets contained different combina- tions of dry Azolla meal, local marine fish Sardinella aurita meal, and other local products (Table 1). These values were calculated based on the results of analyses of local Beninese products as performed by Luquet (1984). A. microphylla (strain 175 MI) were cultivated in ponds 7 km from the laboratory and maintained at the linear phase of their population growth curve. The quantity of wet Azolla was J. Appl. Ichthyol. 20 (2004), 517–520 Ó 2004 Blackwell Verlag, Berlin ISSN 0175–8659 Received: June 6, 2003 Accepted: March 1, 2004 U.S. Copyright Clearance Centre Code Statement: 0175–8659/2004/2006–0517$15.00/0
  2. 2. estimated weekly, considering that Azolla contain only 5% dry matter, and dried in a shaded area after harvesting. Five diets containing different levels of dry Azolla meal and one Azolla- free diet (control) were prepared by thoroughly mixing the dry pulverized ingredients (particle size <63 lm) and adding cold water until a stiff dough resulted. These were then dried and the blends ground in a mortar. Experiment design and feeding The experiment was conducted in a recirculating system with two rearing tanks levels. The upper level contained six tanks and the lower level 12 rearing tanks. City water was stocked in a 300-L tank and used during 1 week for rearing fish in eighteen 25-L tanks at a constant flow rate of 0.5 L min)1 . The used water coming from the rearing tanks flowed, respectively, through a 150-L tank for decantation, a 150-L tank for biological filtration and a 150-L tank containing a pump (Nautilus 3000 OASIS) which was able to fill the initial 300-L tank installed at a 2-m height from the floor. Temperature and dissolved oxygen were measured with an oxythermometer WTW Oxi 197/Set, pH was measured using a pH meter WTW 330/set0. Nitrites and ammoniac were analyzed by the colo- rimeter method based on sulfanilamide and 1-naphtylamine. Temperatures were between 26.4 and 28.9°C, dissolved oxygen ranged from 5.30 to 7.47 mg L)1 , pH between 5.92 and 8.20, and nitrites and ammoniac were <0.01 mg L)1 . Before the start of the experiment, fish were randomly distributed in the 18 rearing tanks (corresponding to 3 · 6% of Azolla) at a density of 25 fish tank)1 (mean initial den- sity ¼ 1650 g m)3 ) and fed a mixture of the different experi- mental diets (Table 1) for 1 week. Thereafter, each group (three tanks) received treatment (one diet) at a ration of 4% of fish biomass per tank. The daily ration was calculated on a dw basis and distributed six times daily (8.00, 10.00, 12.00, 14.00, 16.00 and 18.00 hours). Survival was determined daily by removing dead fish from each rearing tank; weight gain was recorded each week in order to adjust the daily feed ration (4% of tank biomass day)1 ) according to the total biomass in each tank. At the end of the feeding period, all fish were counted and individually weighed in order to calculate growth performances and survival. Feeding duration was 30 days. Growth parameters were calculated as follows: Ponderal growth ¼ ðW2 À W1ÞdtÀ1 SGR ¼ 100ðlnW2 À lnW1ÞdtÀ1 FCR ¼ TFSðFB À IBÞÀ1 where SGR is the specific growth rate (% day)1 ), W1,2 are the initial and final body weights (g), FCR is the feed conversion ratio, and IB and FB are the initial and final biomass (g), dt)1 is the experiment duration. Analysis of data Values of the different parameters were subjected to factorial analysis of variance using one-way ANOVAANOVA (Dagnelie, 1975). Treatment effects were considered significant at P < 0.05. Results The dietary Azolla meal level had a significant effect (P < 0.05) on the final body weight, weight gain, specific growth rate and ponderal growth (Table 2). Statistical analysis of the results (Table 2) showed no significant difference (P < 0.05) in the initial body weight of the fish submitted to the six different diets, clearly indicating that the significant differences observed for the final body weight and the other growth parameters were effects of the experimental diets. However, for the Azolla-free diet and the diet containing 15% Azolla, no significant differences were observed for final body weight, weight gain, food conversion ratio or ponderal growth (Table 2). The same observations were made for diets con- taining 30–45% Azolla meal for all growth parameters with the exception of the survival rate. According to Fig. 1 which shows growth over time, it appears that juvenile fish fed the 15% Azolla meal diet exhibited the best growth, followed by the Azolla-free diet, although this latter diet contained the highest fishmeal amount and is known to be the best quality Table 1 Composition of experimental diets Ingredients (%) Experimental diets T1 T2 T3 T4 T5 T6 Maize meal 32 25 22 20 15 13 Palmseed cake 5 5 5 5 5 5 Fishmeal 10 9 7 8 5 6 Cottonseed cake 30 23 23 14 14 8 Shell 2 2 2 2 2 2 Azolla 0 15 20 30 40 45 Beer bran flour 20 20 20 20 18 20 Salt 1 1 1 1 1 1 Gross energy (MJ kg)1 ) 96.5 93.1 91.4 89.0 86.5 85.1 Gross Protein (%) 27.52 27.43 27.30 27.25 27.41 27.52 Protein/energy (g MJ)1 ) 2.85 2.95 2.99 3.06 3.17 3.23 1 MJ ¼ 239 Kcal. Table 2 Growth performances of juvenile tilapia Oreochromis niloticus fed diets containing different levels of Azolla Growth parameters 0% Azolla 15% Azolla 20% Azolla 30% Azolla 40% Azolla 45% Azolla Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Initial body weight (g) 1.72a 0.06 1.67a 0.07 1.67a 0.04 1.64a 0.02 1.64a 0.07 1.70a 0.06 Final body weight (g) 3.00a 0.06 3.23a 0.59 2.34b 0.27 2.17b 0.04 2.18b 0.09 2.28b 0.16 Weight gain (g) 1.27a 0.11 1.57a 0.64 0.67b 0.27 0.53b 0.06 0.53b 0.15 0.58b 0.17 Feed conversion ratio 2.62a 0.22 2.53a 0.73 5.03b 2.21 5.45b 0.47 5.54b 1.96 5.18b 1.57 Specific growth rate (% day)1 ) 1.84a 0.16 2.18b 0.70 1.10c 0.39 0.93c 0.10 0.94c 0.25 0.98c 0.26 Ponderal growth (g day)1 ) 0.04a 0.00 0.05a 0.02 0.02b 0.01 0.02b 0.00 0.02b 0.00 0.02b 0.01 Survival rate (%) 77.33a 12.22 56.00b 28.84 66.67c 8.33 68.00c 12.00 52.00b 24.98 61.33c 8.33 Data on the same line followed by a, b and c is significantly (P < 0.05) different. 518 E. D. Fiogbe´ , J.-C. Micha and C. Van Hove
  3. 3. protein for fish. These observations were confirmed by the specific growth rate variation and those of the food conversion ratio (Table 2). However, the best survival rate was observed (Table 2) with fish fed the Azolla-free diet (77.33%) followed by the fish groups fed 30% Azolla (68.00%), 20% Azolla (66.67%) and 45% Azolla (61.33%). Discussion In aquaculture, ammonia and nitrite are toxic to fish at relatively low levels (10 mg L)1 ) and can cause decreased performance at levels between 1 and 10 mg L)1 (Spotte, 1979). The highest values obtained in this study for nitrite and ammonia were 0.0066 and 0.0008304 mg L)1 , respectively, assuming that the experiment was done under good conditions. Dissolved oxygen (5.3–8.15 mg L)1 ) was higher than the minimum (5 mg L)1 ) required for growth of warmwater fish (Me´ lard, 1999). Some other parameters known to influence fish survival and growth, such as temperature and pH, were checked weekly and appeared to be within the range required for tilapia, O. niloticus. In order to maintain inexpensive experimental diets with approximately similar energy and similar protein contents, the levels of fishmeal were kept low and the increase of dietary Azolla did not follow the decrease of dietary fishmeal. Thus, what we were testing here was not the replacement of fishmeal by dry Azolla meal, but the appetency and growth of the omnivorous tilapia O. niloticus fed with low-cost diets containing different levels of Azolla. From a biological point of view, as a first result this study shows that A. microphylla can be incorporated at a high level (45%) in O. niloticus diets without a decreasing effect on the weight gain (Table 2; Fig. 1). The significant differences among the dietary Azolla levels for the growth parameters might be due to different appetency between experimental diets. Indeed, at the begin- ning of the trials, we observed during the first week that the Azolla-free diet and the diet containing 15% Azolla were more appreciated than the others diets. This is probably due to the fact that food conversion ratio is very high in groups fed diets containing up to 20% Azolla (5.03–5.54) (Table 2). Table 2 indicates that the specific growth rate decreases with incor- poration of up to 15% Azolla in the diet. Similar results were observed by Micha et al. (1988, 1989) and El-Sayed (1992). These authors noted that Azolla incorporation in the diet of O. niloticus decreased the specific growth rate. Such decrease of specific growth is often related to the decrease in food intake. As reported by Ogino (1980) in common carp and trout, under-feeding decreased the growth rate of fish. On the other hand, the increase of Azolla in the diet can reduce diet digestibility and growth rate. It is well established that carbohydrates from vegetables are generally consumed as complex molecules, the most common forms being starch and cellulose (De Silva and Anderson, 1995). In general, however, cellulose is not digested by fish. Starch is broken down to produce glucose, which again is further degraded to provide energy. This last statement explains the significant effect of Azolla incorporation in diet digestibility in O. niloticus, as reported by Leonard (1997). Amino acid composition of Azolla spp. and other aquatic plants is quite variable (Li et al., 1991). Proportions of amino acids in Azolla spp. total protein (N · 6.25) show values from 45.3 to 87.3%, comparable to those found in other aquatic plants of 52.2–77.7% (Castillo, 1983; Li et al., 1991). Furthermore, the ratio [essential and semi-essential amino acids for many single-stomach animals (such as fish and pigs) to total amino acids] observed is similar in Azolla spp. and other aquatic plants of around 55%. As in other aquatic plants, aspartic acid (+ asparagine) and glutamic acid (+ glutamine) are generally the most concen- trated amino acids in Azolla spp. Azolla spp. and other aquatic plants are generally deficient in sulphured amino acids and sometimes in lysine. However, Azolla spp. seems to be richer than aquatic plants in cystine and can therefore be a better source for this amino acid. The low growths observed in fish fed diets containing up to 20% Azolla might be due to excesses or deficiencies of these amino acids. As reported by Cole and Van Lunen (1994), inadequate levels of indispensable amino acids resulted in depression of food intake and growth. Deficiencies of one or more amino acids are known to limit protein synthesis, growth, or both (Cowey, 1992; Cole and Van Lunen, 1994). Therefore, for protein synthesis, all amino acid building blocks must be present. On the other hand, previous studies reported that the use of vegetable meal, such as lucerne meal or leucaena meal in the fish diet, introduced toxins such Fig. 1. Weight variation in tilapia Oreochromis niloticus fed diets containing different levels of Azolla Aquatic fern as a main food component for tilapia 519
  4. 4. as saponine and mimosine, which have negative effects on appetency and growth (Jackson et al., 1982; Guillaume et al., 1999). Although growth was low in fish fed diets containing up to 20% Azolla, the present study indicates that Azolla can be incorporated in tilapia diets in extensive or semi-intensive systems to reduce food costs significantly. However, for intensive systems, further work is necessary to improve the ingestion and digestibility of diets containing high levels of Azolla. Moreover, mixing Azolla with some agricultural by-products such as rice bran (Aban, 1989) and the use of fermentable by-products such as yeasts or the addition of purified enzymes can improve ingestion and digestibility. Acknowledgements The authors wish to thank Samuel Ako, Songhai Project Director of Production, who gave us the biochemical compo- sition of local products used in the experimental diets, and Blaise Djogbede for technical assistance. This research was funded by the General Administration for Cooperation Development of Belgium through the University Cooperation CIUF-UNB. References Aban, S. M., 1989: Growth Performance, Survival, Food Conversion and Biochemical Composition of Nile Tilapia (Oreochromis niloticus) and Zill’s Tilapia (Tilapia zillii) Fed with Azolla (Azolla microphylla) Feeds in Aquaria. Master of Science in Aquaculture Thesis, Central Luzon State University, Munoz, Nueva Ecija, Philippines, 68 pp. Bai, S. C.; Gatling, D. M., 1992: Present and future use of computer linear programming for fish feed formulations in the United States. Korean J. Anim. Nutr. Feedstuff 16, 93–104. Castillo, L. S., 1983: Feeding value of crop residues of food crops grown in rice-based farming systems. In: Asian Cropping Network, Crop-Livestock Research Workshop, Los Ban˜ os, Phil- ippines, 25–28 April 1983, 23 pp. Cole, D. J. A.; Van Lunen, T. A., 1994: Ideal amino acid patterns. In: Amino Acids in Farm Animal Nutrition. J. P. I. D’Mello (Ed.). The Scottish Agricultural College, Edinburgh, UK, pp. 99–112. Cowey, C. B., 1992: Nutrition: estimation requirements of rainbow trout. Aquaculture 100, 177–189. Dagnelie, R., 1975: The´ orie et Me´ thodes Statistiques, vol. 2. Presses Agronomiques de Gembloux, Belgium, 463 pp. De Silva, S. S.; Anderson, T. A., 1995: Fish Nutrition in Aquaculture. Chapman & Hall Aquaculture, London, UK, 319 pp. El-Sayed, A.-F. M., 1992: Effect of substituting fish meal with Azolla pinnata in practical diets for fingerling and adult Nile tilapia, Oreochromis niloticus (L.). Aquacult. Fish. Manag. 23, 167–173. Fiogbe´ , E. D., 1985: Contribution a` lÕe´ tude de l’alimentation du tilapia Oreochromis niloticus en enclos dans les lagunes du Sud Be´ nin. Me´ moire pre´ sente´ en vue de l’obtention du diploˆ me d’inge´ nieur agronome, Faculte´ des sciences agronomiques de l’Universite´ Nationale du Be´ nin, 126 pp. Guillaume, J.; Kaushik, S.; Bergot, P.; Metailler, R., 1999: Nutrition et alimentation des poissons et crustace´ s. INRA Editions Ifremer, 489 pp. Jackson, A. J.; Capper, B. S.; Matty, A. J., 1982: In: Fish Nutrition, 3rd edn. Copyright 2002. John Halver (Ed.). Elsevier Science, USA , Aquaculture 27, 97–109. Leonard, V., 1997: Use of Aquatic Fern (Azolla filiculoides) in Two Species of Tropical Fish (Oreochromis niloticus and Tilapia rendalli). PhD Thesis. Presses Universitaires de Namur, Belgium, 276 pp. Li, Z.; Luo, X.; Xu, T.; Jiang, Y., 1991: Ecological techniques on broad water body (ETBWB) and effects of Azolla raising and its application. Chin. J. App. Ecol. 2, 113–120. Lumpkin, T. A.; Plucknett, D. L., 1982: Azolla as a Green Manure: Use and Management in Crop production. Westview Tropical Agriculture Series 5, Westview Press, Boulder, CO, USA, 230 pp. Luquet, P., 1984: Alimentation. Rapport de mission d’appui au projet de de´ veloppement de la pisciculture. Ministe` re des Fermes d’Etat, de l’Elevage et de la Peˆ che, Re´ publique Populaire du Be´ nin. Centre Technique Forestier Tropical, Nogent-sur-Marne, France, 29 pp. Me´ lard, C., 1999: Choix des sites, qualite´ de l’eau et syste` mes dÕe´ levage en aquaculture. Notes de cours. Centre de formation et de Recherche en Aquaculture (CEFRA), 80 pp. Micha, J. -C., 1990: Ecologie dulcicole. Diffusion Universitaire Ciaco, Louvain-la-Neuve, Belgium, 262 pp. Micha, J. -C.; Antoine, T.; Wery, P.; Van Hove, C., 1988: Growth, ingestion capacity, comparative appetency and biochemical com- position of Oreochromis niloticus and Tilapia rendalli fed with Azolla. pp. 347–355. In: Second International Symposium on Tilapia in Aquaculture, ICLARM Conference Proceedings 15. R. S. V. Pullin, K. Tonguthai and J. L. Maclean (Eds). Department of Fisheries, Bangkok, Thailand, and ICLARM, Manila, Philippines, 623 pp. Micha, J. -C.; N’Guessan, B.; Van Hove, C., 1989: The aquatic fern Azolla as feed for fish. pp. 677–681. In: Aquaculture, A Biotech- nology in Progress, vol. 2. E. de Pauw, E. Jaspers, H. Ackefors and N. Wilkins (Eds). European Aquaculture Society, Bredene, Belgium. Ogino, C., 1980: Requirements of carp and rainbow trout for essential amino acids. Bull. Jpn. Soc. Sci. Fish. 46, 171–174. Paoletti, C.; Bocci, F.; Lercker, G.; Capella, P.; Materassi, R., 1987: Lipid composition of Azolla caroliniana biomass and its seasonal variation. Phytochemistry 26, 1045–1047. Spotte, S., 1979: Seawater Aquariums. John Wiley & Sons, New York, NY, USA. Van Hove, C., 1989: Azolla and its Multiple Uses, Emphasis on Africa. FAO, Rome, 53 pp. Author’s address: E. D. Fiogbe´ , Unite´ de Recherche sur les Zones Humides, De´ partement de Zoologie et Ge´ ne´ tique, Faculte´ des Sciences et Techniques, Universite´ d’Abomey-Calavi Be´ nin B.P. 526 Cotonou, Be´ nin. E-mail: 520 E. D. Fiogbe´ , J.-C. Micha and C. Van Hove