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

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 www.blackwell-synergy.com

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. 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. 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.
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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: fiogbe@bj.refer.org
520 E. D. Fiogbe´ , J.-C. Micha and C. Van Hove