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. Fiogbe1, J.-C. Micha2 and C. Van Hove3

1Unite de Recherche sur les Zones Humides, Departement de Zoologie et Genetique, Faculte des Sciences et Techniques, Universited’Abomey-Calavi Benin, Benin; 2Unite de Recherche en Biologie des Organismes, Facultes Universitaires N.D. de la Paix, Namur,Belgium; 3Laboratoire de Botanique, Catholic University of Louvain, Louvain-la-Neuve, Belgium

Summary

An aquatic fern, Azolla microphylla (strain 175 MI, CatholicUniversity 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 withsix different diets in triplicate groups. Diets were formulated

with approximately similar total protein, ranging from 27.25 to27.52%dryweight (dw), gross energy content ranging from 85.1to 96.5 MJ kg)1 dw, and with different levels of dry mealAzolla

(0, 15, 20 30 40, 45% diet dw). All diet levels with incorporatedAzolla meal exhibited weight gain, thus it can be assumed thatAzolla in good combination with local products can be used to

promote fish culture development. The Azolla-free diet and thediet containing 15%Azolla produced the same growth perform-ance. However, the least expensive diet containing 45% Azollaalso exhibited growth and can be used as a complementary diet

for tilapia raised in fertilized ponds.

Introduction

In many developing countries people lack sufficient animalprotein. In Benin, the main protein source is fish; however,

consumption thereof is very low (7 kg year)1) compared to theadult requirement of fish or animal meat per year(30 kg year)1). Fish culture could be a means to increaseanimal 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 from1978 to 1990, tentative fish production in Benin fell due mainly

to the high cost of the feed (350 CFA Franc [FCFA] kg)1 vs600 FCFA kg of fish produced, Fiogbe, 1985). (655.9FCFA 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 aquaticecosystems is often the bioavailability of nitrogen,

(ii) approximately 95% of the cost of formulating an averageproduction diet is related to meeting protein and energyneeds 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 bluealga Anabaena azollae (Van Hove, 1989), and is thus a very

promising supply of nitrogen to aquatic ecosystems. Azolla hasbeen used for centuries as green manure in rice fields and isgiven 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 dwbasis]. Azolla is also able to store phosphorus and potassiumfrom 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 Azollacontains 0.8–6.7% dw crude fat, with 6.1–7.7% and 12.8–

26.4% total fat for polyunsaturated acids (PUFA) omega 3and omega 6. Azolla seems to be rich in some vitamins, notablycarotenes and vitamin A (300–600 ppm dw, Leonard, 1997).

According to these reports on Azolla composition, sixexperimental diets containing different levels of A. microphyllawere formulated for this study to feed the omnivorous–

planktophagous tilapia, Oreochromis niloticus. The dietaryprotein content was calculated based on the protein content oflocal products (Luquet, 1984) and fixed to 27% dw. The dietscost less than 75 FCFA kg)1 (655.9 FCFA ¼ 1 euro;

600 FCFA ¼ 1 U.S.$). As result of this experiment the bestdiet will be recommended for fish culture in rural areas, mainlywetlands, 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 fromcommercial nursery ponds in Songhai Centre, Porto-Novo,Benin, transported to the laboratory and divided among

eighteen 25-L plastic tanks. The experimental diets wereformulated with a calculated energy content ranging from85.1 to 96.5 MJ kg)1 dw and total protein content rangingfrom 27.25 to 27.52% dw. Diets contained different combina-

tions of dry Azolla meal, local marine fish Sardinella auritameal, and other local products (Table 1). These values werecalculated 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 thelaboratory and maintained at the linear phase of their

population growth curve. The quantity of wet Azolla was

J. Appl. Ichthyol. 20 (2004), 517–520� 2004 Blackwell Verlag, BerlinISSN 0175–8659

Received: June 6, 2003Accepted: March 1, 2004

U.S. Copyright Clearance Centre Code Statement: 0175–8659/2004/2006–0517$15.00/0 www.blackwell-synergy.com

estimated weekly, considering that Azolla contain only 5% dry

matter, and dried in a shaded area after harvesting. Five dietscontaining 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 coldwater until a stiff dough resulted. These were then dried andthe 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 tanksand the lower level 12 rearing tanks. City water was stocked ina 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. Theused 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-Ltank installed at a 2-m height from the floor. Temperature and

dissolved oxygen were measured with an oxythermometerWTW Oxi 197/Set, pH was measured using a pH meter WTW330/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 oxygenranged 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 randomlydistributed in the 18 rearing tanks (corresponding to 3 · 6% ofAzolla) 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% offish 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 inorder to adjust the daily feed ration (4% of tank biomassday)1) according to the total biomass in each tank. At the endof the feeding period, all fish were counted and individually

weighed in order to calculate growth performances andsurvival. Feeding duration was 30 days.

Growth parameters were calculated as follows:

Ponderal growth ¼ ðW2 � W1Þdt�1

SGR ¼ 100ðlnW2 � lnW1Þdt�1

FCR ¼ TFSðFB� IBÞ�1

where SGR is the specific growth rate (% day)1), W1,2 are theinitial and final body weights (g), FCR is the feed conversionratio, 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 factorialanalysis 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, specificgrowth 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 tothe six different diets, clearly indicating that the significantdifferences 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% Azollameal for all growth parameters with the

exception of the survival rate. According to Fig. 1 whichshows growth over time, it appears that juvenile fish fed the15% Azolla meal diet exhibited the best growth, followed by

the Azolla-free diet, although this latter diet contained thehighest fishmeal amount and is known to be the best quality

Table 1Composition of experimental diets

Ingredients (%)

Experimental diets

T1 T2 T3 T4 T5 T6

Maize meal 32 25 22 20 15 13Palmseed cake 5 5 5 5 5 5Fishmeal 10 9 7 8 5 6Cottonseed cake 30 23 23 14 14 8Shell 2 2 2 2 2 2Azolla 0 15 20 30 40 45Beer bran flour 20 20 20 20 18 20Salt 1 1 1 1 1 1Gross energy (MJ kg)1) 96.5 93.1 91.4 89.0 86.5 85.1Gross Protein (%) 27.52 27.43 27.30 27.25 27.41 27.52Protein/energy (g MJ)1) 2.85 2.95 2.99 3.06 3.17 3.23

1 MJ ¼ 239 Kcal.

Table 2Growth 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 0.06Final 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 0.16Weight gain (g) 1.27a 0.11 1.57a 0.64 0.67b 0.27 0.53b 0.06 0.53b 0.15 0.58b 0.17Feed conversion ratio 2.62a 0.22 2.53a 0.73 5.03b 2.21 5.45b 0.47 5.54b 1.96 5.18b 1.57Specific 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 0.26Ponderal 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 0.01Survival 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

protein for fish. These observations were confirmed by thespecific growth rate variation and those of the food conversionratio (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 decreasedperformance 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 theminimum (5 mg L)1) required for growth of warmwater fish

(Melard, 1999). Some other parameters known to influence fishsurvival and growth, such as temperature and pH, were

checked weekly and appeared to be within the range requiredfor tilapia, O. niloticus.

In order to maintain inexpensive experimental diets withapproximately similar energy and similar protein contents, thelevels 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 fishmealby dry Azolla meal, but the appetency and growth of theomnivorous tilapia O. niloticus fed with low-cost diets

containing different levels of Azolla. From a biological pointof view, as a first result this study shows that A. microphyllacan 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 Azollalevels 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 theAzolla-free diet and the diet containing 15% Azolla were moreappreciated than the others diets. This is probably due to the

fact that food conversion ratio is very high in groups fed dietscontaining up to 20% Azolla (5.03–5.54) (Table 2). Table 2indicates that the specific growth rate decreases with incor-

poration of up to 15% Azolla in the diet. Similar results wereobserved 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 ofspecific 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 otherhand, the increase of Azolla in the diet can reduce dietdigestibility and growth rate. It is well established thatcarbohydrates from vegetables are generally consumed as

complex molecules, the most common forms being starch andcellulose (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 provideenergy. This last statement explains the significant effect ofAzolla incorporation in diet digestibility in O. niloticus, as

reported by Leonard (1997). Amino acid composition ofAzolla 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 andsemi-essential amino acids for many single-stomach animals

(such as fish and pigs) to total amino acids] observed is similarin Azolla spp. and other aquatic plants of around 55%. As inother aquatic plants, aspartic acid (+ asparagine) and

glutamic acid (+ glutamine) are generally the most concen-trated amino acids in Azolla spp. Azolla spp. and other aquaticplants are generally deficient in sulphured amino acids and

sometimes in lysine. However, Azolla spp. seems to be richerthan aquatic plants in cystine and can therefore be a bettersource for this amino acid. The low growths observed in fishfed diets containing up to 20% Azolla might be due to excesses

or deficiencies of these amino acids. As reported by Cole andVan Lunen (1994), inadequate levels of indispensable aminoacids resulted in depression of food intake and growth.

Deficiencies of one or more amino acids are known to limitprotein synthesis, growth, or both (Cowey, 1992; Cole and VanLunen, 1994). Therefore, for protein synthesis, all amino acid

building blocks must be present. On the other hand, previousstudies reported that the use of vegetable meal, such as lucernemeal or leucaena meal in the fish diet, introduced toxins such

Fig. 1. Weight variation in tilapia Oreochromis niloticus fed dietscontaining different levels of Azolla

Aquatic fern as a main food component for tilapia 519

as saponine and mimosine, which have negative effects onappetency and growth (Jackson et al., 1982; Guillaume et al.,

1999). Although growth was low in fish fed diets containing upto 20% Azolla, the present study indicates that Azolla can beincorporated in tilapia diets in extensive or semi-intensive

systems to reduce food costs significantly. However, forintensive systems, further work is necessary to improve theingestion and digestibility of diets containing high levels ofAzolla. Moreover, mixing Azolla with some agricultural

by-products such as rice bran (Aban, 1989) and the use offermentable by-products such as yeasts or the addition ofpurified enzymes can improve ingestion and digestibility.

Acknowledgements

The authors wish to thank Samuel Ako, Songhai ProjectDirector of Production, who gave us the biochemical compo-sition of local products used in the experimental diets, andBlaise Djogbede for technical assistance. This research was

funded by the General Administration for CooperationDevelopment of Belgium through the University CooperationCIUF-UNB.

References

Aban, S. M., 1989: Growth Performance, Survival, Food Conversionand Biochemical Composition of Nile Tilapia (Oreochromisniloticus) and Zill’s Tilapia (Tilapia zillii) Fed with Azolla (Azollamicrophylla) Feeds in Aquaria. Master of Science in AquacultureThesis, Central Luzon State University, Munoz, Nueva Ecija,Philippines, 68 pp.

Bai, S. C.; Gatling, D. M., 1992: Present and future use of computerlinear programming for fish feed formulations in the UnitedStates. Korean J. Anim. Nutr. Feedstuff 16, 93–104.

Castillo, L. S., 1983: Feeding value of crop residues of food cropsgrown in rice-based farming systems. In: Asian CroppingNetwork, Crop-Livestock Research Workshop, Los Banos, 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 rainbowtrout. Aquaculture 100, 177–189.

Dagnelie, R., 1975: Theorie et Methodes Statistiques, vol. 2. PressesAgronomiques 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 Azollapinnata in practical diets for fingerling and adult Nile tilapia,Oreochromis niloticus (L.). Aquacult. Fish. Manag. 23, 167–173.

Fiogbe, E. D., 1985: Contribution a l�etude de l’alimentation du tilapiaOreochromis niloticus en enclos dans les lagunes du Sud Benin.

Memoire presente en vue de l’obtention du diplome d’ingenieuragronome, Faculte des sciences agronomiques de l’UniversiteNationale du Benin, 126 pp.

Guillaume, J.; Kaushik, S.; Bergot, P.; Metailler, R., 1999: Nutrition etalimentation des poissons et crustaces. 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 TwoSpecies of Tropical Fish (Oreochromis niloticus and Tilapiarendalli). PhD Thesis. Presses Universitaires de Namur, Belgium,276 pp.

Li, Z.; Luo, X.; Xu, T.; Jiang, Y., 1991: Ecological techniques onbroad water body (ETBWB) and effects of Azolla raising and itsapplication. 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 TropicalAgriculture Series 5, Westview Press, Boulder, CO, USA, 230 pp.

Luquet, P., 1984: Alimentation. Rapport de mission d’appui au projetde developpement de la pisciculture. Ministere des Fermes d’Etat,de l’Elevage et de la Peche, Republique Populaire du Benin.Centre Technique Forestier Tropical, Nogent-sur-Marne, France,29 pp.

Melard, C., 1999: Choix des sites, qualite de l’eau et systemes d�elevageen aquaculture. Notes de cours. Centre de formation et deRecherche 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 withAzolla. pp. 347–355. In: Second International Symposium onTilapia in Aquaculture, ICLARM Conference Proceedings 15. R.S. V. Pullin, K. Tonguthai and J. L. Maclean (Eds). Departmentof Fisheries, Bangkok, Thailand, and ICLARM, Manila,Philippines, 623 pp.

Micha, J. -C.; N’Guessan, B.; Van Hove, C., 1989: The aquatic fernAzolla as feed for fish. pp. 677–681. In: Aquaculture, A Biotech-nology in Progress, vol. 2. E. de Pauw, E. Jaspers, H. Ackeforsand N. Wilkins (Eds). European Aquaculture Society, Bredene,Belgium.

Ogino, C., 1980: Requirements of carp and rainbow trout for essentialamino 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 seasonalvariation. 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 ZonesHumides, Departement de Zoologie et Genetique,Faculte des Sciences et Techniques, Universited’Abomey-Calavi Benin B.P. 526 Cotonou, Benin.E-mail: fiogbe@bj.refer.org

520 E. D. Fiogbe, J.-C. Micha and C. Van Hove