Biochemical composition of zooplankton community grown in

freshwater earthen ponds: Nutritional implication in

nursery rearing of fish larvae and early juveniles
Gopa Mitra
a,

, P.K. Mukhopadhyay
a
, S. Ayyappan
b
a
Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar-751 002, Orissa, India
b
Krishi Anusandhan Bhawan-II, ICAR, Pusa, New Delhi-110 012, India
Received 8 November 2006; received in revised form 7 August 2007; accepted 8 August 2007
Abstract
This study was conducted to obtain a database describing the nutritional value of freshwater mixed zooplankton that are widely
used for larval and grow-out rearing of freshwater fish. The macro and micronutrient composition of mixed zooplankton samples
collected from 6 fertilized earthen ponds were analysed for protein, lipid, carbohydrate, ash and these ranged from 7379%, 10.79
14.55%, 34.79% and 3.2010.07%, respectively on a dry matter (DM) basis. Amino acid profile showed the presence of all the
ten essential amino acids with low level of methionine. The content of saturated fatty acids (SAFA), mono unsaturated fatty acids
(MUFA) and polyunsaturated fatty acids (PUFA) ranged from 6481%, 715% and 620% of total fatty acids, respectively. The
predominant fatty acids were SAFA (16:0), MUFA (18:1n-9), PUFA viz. linoleic acid (LA 18:2 n-6) and linolenic acid (LNA 18:3
n-3). Among the vitamins, ascorbic acid (1540.01 g/gDM) was less than the requirement of fish especially for larvae, vitamin-A
(13.6163.95 g/g DM) and vitamin-E (218348g/g DM) were more than the requirement of fish. Mineral and trace element
content showed the presence of P, Ca, Fe, Cu, Zn and Mn. Seasonal variation of all biochemical components was evaluated in the
study. Vitamin E had strong co-relation (r
1
=0.72; r
2
=0.88; r
3
=0.83; r
4
=0.86; r
5
=0.36 and r
6
=0.88) with seasonal variation in
lipid content of zooplankton of different ponds and varied inversely with that of rising temperature. Enzyme content from the
mixed zooplankton of different ponds showed availability of protease (6.217.92 g leucine/ mg protein/h), lipase (25.8239.1 g
-naphthol/mg protein/h) and amylase (100226.1 g maltose/mg protein/h), which could be used as an exogenous source of
digestive enzymes for fish and shellfish during ontogenesis. Absence of l-gulonolactone -oxidase activity confirmed the
incapability of these zooplankton to synthesize ascorbic acid (AA) de novo. The average dry weight in zooplankton in different
ponds was 1.24.2 mg/l and different species present in these ponds were Moina (Moina dubia), Daphnia (Daphnia lumholtzi,
Daphnia carinata); Cyclops (Mesocyclops hyalimus, Mesocyclops leuckarti); Diaptomus (Heliodiaptomus viddus, Neodiaptomus
handeli); Rotifer (Brachionus). These results indicate that the biochemical composition and subsequently the nutritional value of
these planktonic organisms are not only genetically determined but also influenced by its maturity stage and type of ingested food.
These data may be helpful for reference purpose and for formulated feed preparation accessing the nutritional potentiality of these
freshwater zooplankton in the nursery rearing of freshwater fish larvae and early juveniles.
2007 Elsevier B.V. All rights reserved.
Keywords: Mixed zooplankton; Protein; Lipid; Vitamins; Minerals; Amino acids; Fatty acids and enzymes
Available online at www.sciencedirect.com
Aquaculture 272 (2007) 346360
www.elsevier.com/locate/aqua-online

Corresponding author. Tel.: +91 674 2465446; fax: +91 674 2465407.
E-mail address: gopamitra@yahoo.com (G. Mitra).
0044-8486/$ - see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2007.08.026
1. Introduction
Zooplankton occupy a central position between the
autotrophs and other heterotrophs maintaining an im-
portant link in the sustainability of the food chain form-
ing one of the most important components of freshwater
aquaculture species (Chakrabarti and Sharma, 1998).
Larvae of all cultivable fish species require live plank-
tonic organisms as first food (Garcia-Ortega et al., 1998).
In semi-intensive or intensive culture conditions, aqua-
culture species derive a substantial part of their dietary
nutrient needs from naturally available zooplankton as
they are a valuable source of protein, amino acids, lipid,
fatty acids, vitamins and enzymes (Millamena et al.,
1990; Munilla-Moran et al., 1990; Pillay, 1990; Evjemo
et al., 2001). Thus the relative contributions of zoo-
plankton in the nutrition of freshwater fish larvae have
immense significance (Jana and Chakrabarti, 1993).
Despite the effort that has been put in the development of
formulated starter feed (Verreth et al., 1987) for larval
fish, live food still remains a better option in terms of
survival and growth compared to formulated diet alone.
Live food seems to provide a good source of exogenous
enzymes, and also helps in chemoreception and visual
stimuli (Kolkovski et al., 1995). However, the nutritional
quality of zooplankton varies considerably and thus
plays a major role in producing quality larvae and juve-
niles (van der Meeren et al., 2001) as well as they would
aid in determining the suitability of the organisms in fish
larvae culture (Kibria et al., 1999).
Although in semi-intensive fish culture the cultured
species draws a significant part of nourishment from zoo-
plankton grown in ponds, however quantitation of its
nutritional contribution to fish growth is limited. However
the dependence on live food as starter feed rather than on
formulatedfeedin fish larvae makes it pertinent to evaluate
the nutritional composition of live food in aquaculture
(Srivastava et al., 2006). Due to escalation in the cost of
Artemia cysts, generally used during larval rearing, use
of pond grown zooplankton is justifiably gaining more
importance in the hatcheries in different regions (Evjemo
et al., 2003; Velu and Munuswamy, 2003). However,
colonization of planktonic organism can be found in si-
milar water bodies of different countries (Welch, 1952).
Studies were thus conducted to determine the proximate
composition, content of amino acids, fatty acids, vitamins
(A, C and E), minerals (P, Ca), trace elements (Fe, Cu, Zn,
Mn) and certain metabolic enzymes of mixed zooplankton
from different freshwater fish ponds. Also their nutritional
contribution are evaluated with the aim to consider the
nutritive potentiality of this zooplankton for nursery
rearing of carp larvae and early juveniles.
2. Materials and methods
2.1. Pond preparation and collection of samples
Zooplankton samples were collected twice every month
from6 culture ponds (area 0.04 ha, mean depth 1.5 m,) of CIFA
farmat Kausalyaganga, Orissa, India (Lat 20 20 N; Long 85
49 E) for one year. The pre-sampling pond preparation was
carried out for eradication of predatory and undesirable fish
according to Jena et al. (1998). The ponds were dried initially
and exposed to sunlight for eradication of predatory and unde-
sirable fish prior to stocking and filling with canal water filtered
through nylon net (40 mesh) for preventing the entry of pre-
datory species. Pond fertilization was carried out according to
Jena et al. (1998) with application of raw cow dung (0.39% N
and 8.0% C) and super phosphate at 3 tonnes/ha and 7 kg/ha,
respectively, as a basal dose with alternating applications every
fortnight at 1 tonnes/ha/month and 20 kg/ha/month, respec-
tively. Water levels in the ponds were maintained at 1.4 to 1.5 m
throughout the period compensating for seepage and evapora-
tion losses.
The samples were collected between 0800 and 0900 h by
filtering 200 l of water using plankton net made of bolting silk
(0.06 mm mesh size) fromfour sites of each pond with a plastic
container (5 l) from the subsurface (at 0.50.6 m depth) water
with least disturbance. By passing the water of the plastic
container through a net (0.064 mm), the plankton density was
increased (any plankton stuck to the side of the net was washed
down by gently splashing water on the outside of the net) and
transferred to a 10 l plastic container (having 9 l water) through
a 1050 m mesh net (to exclude large predatory insects like
notonectids). Then it was thoroughly mixed to get a represen-
tative of a composite sample of each pond. The samples were
then brought to the laboratory for analysis of nutritive compo-
sition. Prior to analysis of nutritive composition, the plankton
was suspended at a density of 150200 numbers/ml in tap
water in 20 l well aerated glass jars. Dead plankton and organic
debris were removed by siphoning with 2 mm diameter pipe.
Before preparation for biochemical analysis, the samples were
checked for zooplankton viability and no organic debris were
present in the samples. After 24 h the zooplankton samples
were harvested by sieving (through mesh #25 bolting silk) the
water from each glass jar and rinsed in distilled water. After
harvesting some portion of the zooplankton samples were dried
at 60 C to constant weight to determine the dry matter content
from which protein, ash and amino acids were analysed. Sub-
samples of harvested zooplankton were packed and immedi-
ately frozen in liquid nitrogen and stored at 80 C until
analysed for lipid, fatty acids, enzymes and vitamins. From all
samples a known volume was immediately fixed in 4% forma-
lin for quantitative estimation of plankton species.
2.2. Nutrient composition
Dry matter (DM), crude protein (CP) and ash content were
determined according to the standard methods (AOAC, 1998).
DM content was determined after drying in an oven at 60 C to
347 G. Mitra et al. / Aquaculture 272 (2007) 346360
constant weight and crude protein (CP) content by the Kjeldahl
method and calculated as nitrogen content multiplied by 6.25.
Lipid was analysed using the method of Bligh and Dyer
(1959). Isolated zooplankton was homogenised with 15 ml
chloroform:methanol (2:1 v/v) containing 0.05% butylated
hydroxy toluene (BHT) as an antioxidant in a 20 ml glass
homogenizer. Lipid extracted was re-dissolved in chloroform/
methanol (2:1, v/v containing 0.05% BHT) at a concentration
of 10 mg/ml and stored under nitrogen atmosphere at 20 C
until fatty acid analysis.
Ash content was determined by incinerating dried samples
in muffle furnace at 550 C for 3 h and then preserved for
mineral estimation. Organic matter (OM) was calculated by
subtracting the total ash value from DM. Total carbohydrates
(TCHO) was determined by subtracting CP and lipid values
from OM.
2.3. Amino acid
For amino acid analysis defatted plankton samples were
dried under vacuum in a HPLC work station (Model: 2690) for
30 min (Waters, USA). After purging with nitrogen the sam-
ples were hydrolysed with 6N HCl at 110 C for 20 h under
vacuum. Hydrolysed samples were dried and redried with a
mixture of ethanol:water:triethylamine (TEA) (2:2:1) at
(6586.13/760) N/m
2
. Phenyl isothiocyanate (PITC) deriva-
tives were prepared by adding PITC reagent (ethanolTEA
WaterPITC:: 7:1:1:1) and mixed well and incubated at room
temperature for 30 min, the samples were then allowed to dry
under vacuum. Diluent was added in each sample, which was
then processed for filtration (45 filter paper) and analysed.
Operating condition was: column temperature: 38 C: column:
pico-tag, absorbance 254 nm, pump pressure 1000 psi.
The chemical score (CS) was calculated based on the
limiting essential amino acid in zooplankton multiplied by 100,
where limiting essential amino acid is that which has the
following lowest ratio of essential aminoacid in plankton:
essential amino acid in fish tissue protein (Hepher, 1988). As in
other animals, arginine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, threonine, tryptophan and valine
were considered the essential amino acids (Tacon and Cowey,
1985). The non-essential amino acids cystine and tyrosine can
only be synthesized by the fish from methionine and phenyla-
lanine, respectively (Tacon and Cowey, 1985). Therefore, the
methionine and phenylalanine requirement of the fish will
partially depend on the cystine and tyrosine content of the diet.
For the calculation of the CS, the values of methionine and
cystine were summed and taken as one essential amino acid in
fish. The same was carried out with phenylalanine and tyrosine.
Tryptophan was not considered in this calculation because it
was not determined.
2.4. Fatty acid
Fatty acid methyl ester (FAME) derivatives of the fatty
acids were prepared by adding 10 ml 20% borontrifluoride
(BF
3
) in methanol to 100 mg lipids and heating for 30 min at
100 C. After cooling, water (same volume as the BF
3
) and
hexane (half the volume of BF
3
) were added, and centrifuged
at 3000 rpm for 5 min. The content of the centrifugation tube
was then transferred into a small separating funnel and allowed
to separate for 510 min. The lower phase was discarded and
the upper phase was recovered in new flasks and FAME in
hexane were dried with sodium sulfate and the sodium sulfate
was removed by filtering through a Pasteur pipette containing
glass wool. The operating conditions of the gas chromatograph
(PYE UNICAM, GC 104) were: flame ionization detector
(FID), stainless steel column packed with 10% diethylene gly-
col succinate polyester (DEGS), column temperature: 195 C
(maintained for 10 min isothermal), injection port temperature:
210 C, detector temperature 210 C, carrier gas (N
2
) with 35
40 ml/min flow rate, recorder chart speed 640 mm/h. The
methyl ester peaks were identified by co-chromatography with
standard fatty acid methyl ester (Sigma Chemical Co, USA)
mixtures by comparing their retention time with the retention
time of known fatty acids and quantified by a Spectraphysic SP
4270 integrator.
2.5. Analysis of vitamins
Ascorbic acid content in sub samples was determined
according to Roe and Kuether (1943) modified by Dabrowski
and Hinterleitner (1989). The plankton were homogenised in
5% trichloroacetic acid (TCA) solution containing 250 mM
(HClO
4
) and 0.08% ethylenediamine tetra acetic acid (EDTA)
using an motor driven glass Teflon homogeniser in ice and
centrifuged at 29,000 g for 30 min at 4 C. Total ascorbic acid
content in tissue homogenate was measured by 2,4 dinitro
phenylhydrazine (DNPH) method by Roe and Kuether (1943)
modified by Dabrowski and Hinterleitner (1989) in which 2, 4
dinitrophenyl hydrazine derivative of ascorbate was measured
spectrophotometrically at 524 nm. Modification of original
method included incubation with dichloroindophenol (DCIP)
shortened to 20 min, incubation temperature was 30 C and
additional blank per sample was included to account for in-
terfering substances according to Dabrowski and Hinterleitner
(1989). For dry weight measurement sub samples of plankton
collected from same batch were oven dried at 60 C for 24 h till
constant weight.
Vitamin A of sub zooplankton samples was analysed
according to the method of Soni et al. (1997). Known amount
of zooplankton was homogenised with chloroform (CHCl
3
).
The filtrate was added with benzene:toluene (2:1v/v) fol-
lowed by acid mixture containing H
2
SO
4
: glacial acetic acid
(1: 4 v/v) which gave blue colour. The blue colour initially
produced immediately changed to pink colour and the solu-
tion remained covered with benzene or toluene, which stabi-
lizes the colour by probably preventing the oxidation of
chromophore. The max was measured at 525 nm against a
reagent blank.
Vitamin E was extracted following the method Quaife and
Dju as described by Oser (1960) and estimated by the method
of Emmerie-Eangel Reaction (ferric chloride-dipyridyl meth-
od). Extracted homogenate was mixed with ferric chloride
348 G. Mitra et al. / Aquaculture 272 (2007) 346360
reagent (0.1 g FeCl
3
dissolved in 50 ml absolute ethanol) and
0.5% dipyridyl in absolute ethanol. The mixture was mixed by
swirling. The mixture was again mixed with absolute ethanol
and shaken vigorously. The max measured at 520 nm against
a reagent blank.
2.6. Mineral analysis
The ash was moistened with a small amount of glass-
distilled water and 5 ml of 6 N hydrochloric acid (AR grade)
was added. Calcium was measured by flame photometry, P
content by spectrophotometry following Fiske and Subbarow
method (Fiske and Subbarow, 1925) and trace elements (Zn,
Cu, Fe and Mn) by atomic absorption spectrophotometry
(AAS).
2.7. Enzyme assays
The total digestive enzymes: protease, lipase and amylase
were measured in the pooled zooplankton homogenates. For
homogenate preparation 100 mg sample was ground in a potter
blender with 0.89% NaCl solution. After homogenisation the
samples were sonified for 30 s and centrifuged at 12,000 g for
10 min at 4 C. During preparation the homogenates were
continuously kept on ice.
Total protease activity was measured in a medium contain-
ing 0.05 M trisHCI buffer (pH 8.0) using bovine serum
albumin (0.5 mg/ml) as substrate. The assay mixture consisted
of 200 l BSA solution, 100 l enzyme solution and 200 l
buffer was incubated at 37 C for 30 min, the reaction was
stopped with addition of cold trichloroacetic acid (10%). The
enzymatically liberated amino acids were assayed according to
the method of Marks and Lajtha (1963). The results are ex-
pressed as g leucine liberated per mg protein in sample per
hour. Protein content of the supernatant solution was deter-
mined by the method of Lowry et al. (1951), using bovine
serum albumin (BSA) as standard.
The assay of amylase activity was based on method of
Bernfeld (1955). The activity is expressed as mg maltose
liberated from starch/g protein/h.
Activity of lipase was measured using the method of
Seligman and Nachlas (1963) using the substrate, 0.2% -
napthyl laurate in acetone: water (1:9 v/v). A unit activity is
defined as g -napthol liberated from -napthyl laurate in
60 min at 37 C. Specific activity is expressed as lipase activity
per mg protein (Lowry et al., 1951).
Activity of l-gulonolactone -oxidase was measured using
the method as described by Mukhopadhyay et al. (1998).
Known quantity of plankton was homogenised in a glass ho-
mogeniser with 2 ml 50 mM phosphate buffer (pH 7.4) con-
taining 1 mM EDTA and 0.2% sodium deoxycholate. The
supernatant was assayed spectrophotometrically for ascorbic
acid by method described above. Protein was assayed in super-
natant by the method of Lowry et al. (1951).
Table 1
Composition of mixed zooplankton collected from different ponds
Pond 1 Pond 2 Pond 3 Pond 4 Pond 5 Pond 6
Proximate composition
Dry matter (DM) 11.812.10
c
11.012.61
c
10.093.18
c
9.592.22
b
7.410.49
a
10.493.11
c
Crude protein (% of DM) 79.911.6
d
78.911.97
c
78.851.65
c
76.142.3
b
76.480.98
b
73.153.87
a
Crude lipid (% of DM) 12.072.98
b
13.701.89
c
12.212.4
b
10.793.6
a
14.550.97
d
13.91.87
c
Total carbohydrate(% of DM) 4.792.56
a
4.111.99
a
4.061.79
a
3.002.1
a
3.092.6
a
3.622.98
a
Ash (% of DM) 3.221.33
a
3.283.68
a
4.883.93
b
10.071.26
e
5.881.98
c
9.330.98
d
Enzymes
Amylase (g maltose/mg protein/h) 241.227.10
e
222.635.61
d
109.394.42
a
105.974.33
a
153.923.12
b
185.444.81
c
Protease (g leucine/mg protein/h) 6.710.21
ab
6.540.21
a
7.570.25
c
6.820.12
b
6.770.19
b
7.610.29
c
Lipase (g naphthol/mg protein/h) 34.000.37
a
35.380.46
ab
37.680.65
c
36.450.57
b
35.650.33
ab
35.162.98
a
Vitamins
Vitamin A (g/gm DM) 21.840.976
b
52.041.15
d
62.311.50
f
15.591.39
a
53.931.46
e
47.371.05
c
Vitamin E (g/gm DM) 230.6318.4
a
267.2215.06
b
258.6611.26
b
228.937.58
a
333.4710.4
d
319.3911. 06
c
Vitamin C (g/gm DM) 15.160.16
a
15.410.34
a
17. 210.88
b
24.014.61
c
39.070.64
d
38.670.74
d
Minerals
Ca (mg/100 gm DM) 1.660.19
b
1.361.16
a
1.740.07
b
2.360.43
c
2.640.17
d
2.770.24
d
P (mg/100 gm DM) 0.860.20
c
0.370.07
b
0.940.09
d
0.320.08
ab
0.330.03
ab
0.240.05
a
Mn (mg/100 gm DM) 0.310.21
d
0.020.0
a
0.210.11
c
0.080.02
b
0.390.03
e
0.340.24
d
Zn (mg/100 gm DM) 1.010.05
b
1.020.07
b
0.060.03
a
2.580.17
d
1.310.21
c
2.640.19
d
Fe (mg/100 gm DM) 0.840.04
d
2.200.32
e
0.320.06
b
0.130.03
a
0.530.03
c
0.160.01
a
Cu (mg/100 gm DM) 0.080.01
b
0.070.07
b
0.160.17
c
0.080.14
b
0.070.02
b
0.050.02
a
Values are given as meanStandard deviation (SD) (n=12). Mean values with different superscripts in a row are significantly (Pb0.05) different
from each other.
349 G. Mitra et al. / Aquaculture 272 (2007) 346360
2.8. Collection of water sample and qualitative analysis of
plankton
Regular fortnightly sampling of water from the above aqua-
tic bodies was done in the morning (08:00 h) and relevant
physico-chemical parameters were analyzed. The phosphate-
phosphorus (PO
4
P) content of the water was estimated by the
stannous chloride method (Clesceri and Greenbreg, 1989), pH
by Elico digital pH meter L 1120, free CO
2
(titrimetric method
using N/44 Na
2
CO
3
), alkalinity (titrimetric method using
0.02NH
2
SO
4
; Clesceri and Greenbreg, 1989), Nitrite-N(NO
2

N) and Nitrate nitrogen (NO

3
N) (spectrophotometric diazo-
tization method; Strickland and Parson, 1984), total ammonia
nitrogen (TAN) (phenolhypochlorite method), and O
2
by the
method described in Stirling (1985). Plankton content and
species identification was done as described in APHA (1989).
Table 2
Amino acid content (% of total protein), chemical score of mixed zooplankton from different ponds and essential amino acid requirements of different
carps
Pond-1 Pond-2 Pond-3 Pond-4 Pond-5 Pond-6 Catla Rohu Mrigal Common carp
Arg 8.130.05
f
7.440.07
d
6.900.05
b
8.010.11
e
5.180.03
a
6.980.03
c
4.8 5.8 5.84 6.2
Lys 10.300.07
a
12.550.04
c
14.110.06
e
13.700.03
d
15.310.04
f
12.440.03
b
6.2 5.7 7.58 7.0
His 1.970.03
d
1.720.05
a
1.880.04
bc
2.060.23
e
1.840.04
b
1.950.03
cd
2.5 2.3 3.02 2.9
Ile 3.740.06
ab
3.920.04
c
3.890.08
c
3.720.03
a
3.990.07
d
3.780.03
a
2.4 3.0 3.69 3.8
Leu 6.930.04
a
7.070.07
c
7.030.05
b
7.020.04
b
7.150.03
d
7.480.03
e
3.7 6.93 7.2
Val 4.500.11
a
4.760.06
c
4.790.05
c
4.620.05
b
5.130.04
d
4.790.04
c
3.6 4.32 4.5
Met 1.980.16
c
1.960.06
c
1.780.09
b
2.060.05
d
1.660.04
a
1.690.05
a
3.6 2.9 1.55 2.7
Phe 3.230.05
a
3.530.08
b
3.520.13
b
3.220.04
a
3.85.0.03
c
3.910.04
d
3.7 4.0 8.07 6.5
Thr 3.780.09
b
4.040.11
c
4.110.06
d
4.170.04
e
3.300.05
a
4.210.05
e
5.0 3.99 4.2
Tyr 5.540.09
d
5.460.11
c
5.620.05
e
5.690.03
f
3.920.06
a
4.900.03
b

Asp 7.320.05
c
6.970.07
b
7.480.04
d
8.170.04
e
6.730.04
a
8.310.03
f

Glu 12.570.05
c
12.440.08
b
12.140.05
a
13.380.03
e
12.770.04
d
12.170.03
a

Ser 3.290.04
b
3.360.07
c
3.460.09
d
4.060.05
e
2.860.04
a
4.220.02
f

Gly 5.830.03
a
6.570.05
f
6.410.13
d
6.500.07
e
6.090.04
c
5.930.03
b

Ala 7.180.03
c
7.360.06
d
7.420.07
e
6.960.03
b
7.810.05
f
6.470.03
a

TAA 86.29 89.15 90.54 93.34 87.59 89.23
TEAA 50.10 52.45 53.63 54.27 51.33 52.13
Chemical score (methionine)
Catla 55.00 54.44 49.44 57.22 46.11 46.9
Rohu 68.27 67.58 61.4 71.03 57.24 58.27
Common carp 73.33 72.59 65.92 76.62 61.48 65.00
TAA total amino acid, TEAA total essential amino acids.
Values are given as meanStandard deviation (SD). Means bearing different superscript in a row differ significantly (Pb0.05).
Table 3
Fatty acid composition of mixed zooplankton from different ponds
Fatty acids Pond-1 Pond-2 Pond-3 Pond-4 Pond-5 Pond-6
14: 0 7.281.59
d
Trace 2.020.87
b
2.291.20
b
0.230.08
a
4.921.08
c
16: 0 63.6 14.95
a
66.4515.6
a
64.1614.8
a
70.2510.39
a
81.23.34
b
70.035.55
a
16: 1 0.73 0.03
c
0.100.0
a
1.010.01
d
0.200.01
b
1.10.05
e
2.10.04
f
18: 0 2.12 0.7
c
2.011.18
c
2.340.89
c
1.150.61
b
0.10.0
a
0.160.09
a
18: 1 6.300.27
a
12.1310.34
a
13.829.85
b
7.146.72
a
8.32.54
a
6.424.15
a
18:2 n-2 13.446.89
a
12.154.40
a
10.564.53
a
16.55.82
b
10.12.19
a
10.12.52
a
18:3 n-3 6.343.03
a
5.252.61
a
4.602.17
a

20:0 Trace Trace 2.510.73
a
2.10.74
a
20:1 4.122.95
Unidentified 0.180.02
a
2.400.16
b
2.00.03
b

SAFA 73.0214.8
ab
68.4617.11
a
68.5215.48
a
76.212.08
ab
81.33.38
b
77.217.12
ab
MUFA 7.036.7
a
12.219.96
a
14.759.97
b
7.346.92
a
8.182.66
a
12.576.25
a
PUFA 19.789.47
b
17.46.94
b
15.166.28
ab
16.55.00
b
10.12.17
a
10.12.51
a
n-3/n-6 0.47 0.43 0.44
SAFA Saturated Fatty Acid, MUFA Monounsaturated fatty acid, PUFA Polyunsaturated fatty acid. Values are given as meanStandard
deviation (SD) (n=12). Mean values with different superscripts in a row are significantly (Pb0.05) different from each other.
350 G. Mitra et al. / Aquaculture 272 (2007) 346360
2.9. Statistical analysis of data
Data are presented as means standard deviation of means.
To correlate vitamin E content in the zooplankton with lipid
content correlation coefficient (R value) analysis between these
two parameters was performed using microsoft excel software.
One-way analysis of variance (ANOVA) with multiple means
tests DMRT (Duncan's Multiple Range test, Duncan, 1955) in
respect of ponds was applied over all the parameters to inves-
tigate the difference existing between the ponds.
3. Results
The protein, lipid, carbohydrate and ash compositions of
zooplankton are given in Table 1 in relative % of dry matter
(DM) values. The relative concentration of zooplankton DM
was on average 9.61% of wet weight through the whole sam-
pling period, varying between 7.4111.81%. Protein content
was relatively higher in the zooplankton of pond 1, 2 and 3
than other ponds and varied from 7379% of DM. The total
lipid content was between 10.7914.55% of DM. The magni-
tude of seasonal variation in lipid content was relatively high
in zooplankton of different ponds and it was inversely related
to environmental temperature. Carbohydrate content varied
between 34.79% and ash content 3.2210.07% of DM, res-
pectively. Increase in organic content surpassed the increase in
inorganic content, which resulted in lower percentages of ash
content (Table 1).
Pond-wise variation of amino acid composition and
chemical score of protein of mixed zooplankton are given in
Table 2. Nine essential amino acids were present in mixed
zooplankton. In most cases slightly over 50% by weight of the
Fig. 1. Monthly variation of SAFA a PUFa content in mixed zooplankton form different ponds.
351 G. Mitra et al. / Aquaculture 272 (2007) 346360
total amino acids were essential amino acids. In general no
seasonal changes were found among individual amino acids
of zooplankton from different ponds but significant differ-
ences (Pb0.05) were present pond wise (Table 2). Chemical
score based on the requirement of different carp species
(Mohanty and Kaushik, 1991; Ravi and Devaraj, 1991; Mur-
thy and Varghese, 1998) revealed that methionine was limit-
ing for rohu (Labeo rohita), catla (Catla catla)and common
carp (Cyprinus carpio) in mixed zooplankton from all the
ponds. The quantity of phenylalanine was also less in respect
of carp requirement. Threonine was deficient for catla in the
amino acids of mixed zooplankton from all the ponds. How-
ever for mrigal (Cirrihinus mrigala) and catla it was deficient
from pond 1 and pond 5.
Fig. 2. Monthly variation of Ca, P and Fe content in mixed zooplankton form different ponds.
352 G. Mitra et al. / Aquaculture 272 (2007) 346360
Selected fatty acids composition of mixed zooplankton is
indicated in Table 3. Nearly all the fatty acids found in mixed
plankton were straight chain molecules with even number of
carbon atoms. The content of total saturated fatty acids (SAFA),
mono unsaturated fatty acids (MUFA) and polyunsaturated
fatty acids (PUFA) ranged from 6481%, 715%, 620% of
total fatty acids. Among the saturated fatty acids myristic acid
(14:0), palmitic (16:0) and stearic acid (18:0) were present in
the plankton of all the ponds, and 16:0 was the dominant fatty
acid. Among the unsaturated, linoleic acid (LA, 18:2 n-6) was
present in the plankton of all the ponds. Whereas linolenic acid
(LNA, 18:3 n-3) was present only in the plankton of pond 1, 2
and 3, MUFAsuch as palmitoleic acid (16:1 n-7) and oleic acid,
(18:1 n-9) were present as 0.10 to 14.1% of total FA. Seasonal
Fig. 3. Monthly variation of Mn, Zn and Cu content in mixed zooplankton form different ponds.
353 G. Mitra et al. / Aquaculture 272 (2007) 346360
variation in FA composition (Fig. 1) of zooplankton was pre-
sent in all the ponds irrespective of plankton composition
(Table 5). The percentage of SAFA increased appreciably
during June to September (water temperature 35 C) compared
with NovemberJanuary (18 C) and MarchApril (23 C)
(Fig. 1). Contrary to this, the percentage of total PUFAs was
greater during NovemberJanuary. The PUFA were at their
lowest levels at 23 C.
The content of ascorbic acid in zooplankton from different
ponds ranged from 15 to 40.01 g/g DM. Although pond wise
variation in the amount of ascorbic acid was present (Table 1),
no seasonal trend (apart from an increase ascorbic acid in
zooplankton of pond 4) was observed. No enzyme activity of l-
gulonolactone -oxidase could be detected in zooplankton.
Among the lipid soluble vitamins, average value of vitamin A
(retinol) was 13.6163.95 g/gm DM while vitamin E (total
tocopherol) averaged 218348 g/g DM. Vitamin A content
of zooplankton from all the ponds differed significantly (Pb
0.05) from each other. The analysis revealed that vitamin E had
a strong correlation (r
1
=0.72, r
2
=0.88, r
3
=0.82, r
4
=0.86,
r
5
=0.36, r
6
=0.88) with the lipid content of zooplankton. The
vitamin E content significantly varied (Pb0.05) pond wise
except for ponds 1, 4 and ponds 2, 3, respectively. Also the
vitamin E content was inversely proportional with that of
rising temperature.
The amylase, protease and lipase activity of mixed zoo-
plankton ranged from 100226.1 g maltose/mg protein/h,
6.217.92 g leucine/mg protein/h, 25.8239.1 g -naphthol/
mg protein/h, respectively (Table 1).
Mineral and trace element composition is presented in
Table 1. Among the minerals P, Ca, and trace elements Fe, Cu,
Zn and Mn content were determined. Monthly variation of
mineral content is depicted in Figs. 2 and 3.
Different water quality variables prevailing in the ponds
during the study period are presented in Table 4. The water
temperature varied between 1835 C with minimum recorded
in DecemberJanuary and maximum in MayJune reflecting
seasonal impact. No distinct trends in other physicochemical
parameters were discernible. Variation in population (%) and
average dry weight of zooplankton in different ponds is presented
Table 5
Variation in population (%) and average dry weight of zooplankton in different ponds
June July Aug. Sept. Oct. Nov. Dec. Jan Feb Mar. Apr May Av. dry wt. mg/L
Pond-1 Cladoceran 71 80 73.9 74.6 74.0 77.3 76.7 70.7 69.2 81.3 74.0 59.1 4.2
Copepod 18.6 10.0 20.8 17.9 20.0 12.2 10.0 10.3 23.2 8.9 12.6 32.6
Rotifer 10.4 10.0 5.2 6.8 6.1 10.5 12.8 18.9 7.4 9.9 13.4 8.6
Pond-2 Cladoceran 92.3 67.9 62.8 59.7 63.6 60.8 69.6 66.4 57.6 68.5 63.6 80.2 4.2
Copepod 4.8 11.9 26.2 25.7 23.3 15.0 21.4 14.2 35.7 21.5 12.5 8.1
Rotifer 2.9 20.2 11.0 14.6 13.1 24.2 9.0 19.4 6.7 10.0 23.9 11.7
Pond-3 Cladoceran 82.7 60.0 73.5 66.9 60.7 60.3 62.1 50.8 51.5 72.0 69.4 69.9 3.7
Copepod 8.0 36.2 14.7 21.9 22.3 27.5 19.4 33.0 30.1 18.0 20.7 13.0
Rotifer 9.3 3.8 11.8 11.2 17.0 12.2 18.5 16.2 18.4 10.0 12.0 7.1
Pond-4 Cladoceran 65.8 79.2 63.2 77.5 77.4 69.8 74.3 71.8 54.9 78.0 71.8 71.0
Copepod 26.1 10.1 22.6 15.5 19.0 18.9 19.7 78.8 27.8 11.7 11.7 24.0 1.8
Rotifer 8.1 10.7 14.2 7.0 3.5 11.3 5.9 9.7 173 10.3 10.3 5.1
Pond 5 Cladocerans 61.0 43.3 63.7 86.6 71.5 49.5 47.8 66.0 77.9 54.0 68.4 78.7 1.2
Copepods 33.9 13.4 6.7 6.7 13.3 42.1 46.79 24.9 10.2 28.2 23.6 18.5
Rotifer 5.1 43.3 29.6 6.7 15.2 8.4 5.3 10.1 11.1 17.8 8.0 2.8
Pond-6 Cladocerans 52.2 81.3 70.5 49.7 53.9 51.7 63.7 40.2 65.0 70.6 65.9 63.9 2.3
Copepods 40.6 8.6 12.6 42.2 32.9 42.9 10.6 31.6 21.5 14.6 27.1 20.1
Rotifer 7.2 10.1 16.9 8.1 13.2 5.4 26.7 28.3 13.5 15.8 7.0 16.0
Table 4
Variations of water quality parameters in different ponds
Pond-1 Pond-2 Pond-3 Pond-4 Pond-5 Pond-6
Temp (C) 1835 18.235.2 18.335.0 18.134.9 1835.1 1835
pH 7.17.3 7.07.3 7.27.3 7.17.3 7.37.4 7.10.4
Free CO
2
(mg/L) 16.2 1.68.0 1.86.0 1.08.0 2.08.0 1.88.0
Dissolved oxygen (mg/L) 3.15.8 2.585.5 2.345.3 2.886.1 3.25.9 3.885.8
Alkalinity (mg CaCO
3
/L) 85101 89103 90100 88100 87100 86100
Total ammonia nitrogen (mg/L) 0.160.35 0.110.40 0.120.29 0.140.33 0.180.40 0.160.28
Nitrite nitrogen (mg/L) 0.080.18 0.060.12 0.030.15 0.020.09 0.020.09 0.010.09
Nitrate nitrogen (mg/L) 0.230.59 0.250.48 0.210.60 0.180.48 0.240.57 0.200.58
Phosphatephosphorus (mg/L) 0.160.19 0.110.23 0.150.20 0.100.22 0.150.24 0.170.30
354 G. Mitra et al. / Aquaculture 272 (2007) 346360
in Table 5. Among the different groups of zooplankton Clado-
cerans were dominant because of their rapid proliferation. The
species present in the ponds were Moina (Moina dubia), Daphnia
(Daphnia lumholtzi, Daphnia carinata); Cyclops (Mesocyclops
hyalimus, Mesocyclops leuckarti); Diaptomus (Heliodiaptomus
viddus, Neodiaptomus handeli); Rotifer (Brachionus), and the
phytoplankton were Chlorella vulgaris, Volvox sp., Cyclotella
glomerata, Navicula, Cryptocyphala, Pediastrum, Oscillatoria,
Melosira, and Anabaena.
4. Discussion
Data on the chemical composition of mixed zoo-
plankton presented above provide basic information on
the nutritive potential of mixed zooplankton. The dry
matter content however was lower than what was re-
ported earlier (Yurkowski and Tabachek, 1979; Wata-
nabe et al., 1983; van der Meeren et al., 2001). This was
because the plankton consisted of cladocerans that
contained less dry matter compared to other zooplank-
ton such as copepods (van der Meeren et al., 2001). This
might limit the required nutrient availability to the fish
larvae, emphasizing the essentiality of formulated feeds.
The protein content in D. carinata and Moina austra-
liensis is 54.34% and 64.80%, respectively (Kibria et al.,
1999) and in Daphnia sp. it is reported to be 49.7070%
(Yurkowski and Tabachek, 1979; Watanabe et al., 1983)
whereas for Moina sp. it varies between 59.00 and
77.85% (Tay et al., 1991). The protein content of natural
zooplankton dominated by Temora longicornis varies
from 31% to 54% (Helland et al., 2003). Present in-
vestigation showed that the protein content of mixed
plankton varied from 7380% DM which is somewhat
higher than reported earlier (Tay et al., 1991) which
could be due to analytical methods used. So mixed
zooplankton might serve as a good source of protein for
Indian major carp larvae and early juveniles as well as
other freshwater fish larvae because larval fish generally
have high demand for dietary protein due to rapid
growth rates and extensive catabolism of amino acids
for production of metabolic energy (Srivastava et al.,
2006). The total protein content of some copepods,
Eurytemore affinis, Centropages hamatus and Aeastia
grani from the Svartatjern lagoon of Norway was
quite stable, around 38% of DM (van der Meeren et al.,
2001). Present study showed the variability of protein
content in zooplankton from pond to pond but not much
variation within a pond. Though the species composi-
tion in the zooplankton population of different ponds
remained the same the relative abundance of different
zooplankton varied remarkably which might have
caused such variation in protein content.
The amino acids composition of mixed zooplankton
from different ponds had a relatively similar essential
and non-essential amino acids composition and the re-
lative amount was higher than previously reported (Yur-
kowski and Tabachek, 1979; Watanabe et al., 1983;
Kibria et al., 1999), which may be due to the mixed
community of zooplankton. Amino acid profile of plank-
ton is generally genetically programmed than diet re-
lated. Different kinds of food did not have any impact on
amino acid compositions of rotifers (Frolov et al., 1991).
Amino acid analysis by Kibria et al. (1999) revealed that
both D. carinata and M. australiensis contained appre-
ciable levels of both essential and non-essential amino
acids for fish however, values of some essential amino
acids in D. carinata and M. australiensis were lower
than those previously cited for Daphnia sp. (Yurkowski
and Tabachek, 1979; Hepher, 1988) and Moina sp.
(Hepher, 1988). Artemia nauplii of different origin had
different amino acid profiles (Watanabe et al., 1983). But
the essential amino acid content of mixed zooplankton in
the present study could meet the general fish require-
ments particularly of carp (Table 2). But methionine was
deficient in the zooplankton of all the ponds, revealed
fromCS (Table 2), which is similar with the other studies
(Yurkowski and Tabachek, 1979; Watanabe et al., 1983;
Srivastava et al., 2006).
Lipid content in mixed zooplankton from 6 ponds
varied from 10.79 to 14.55% DM and was inversely
related to water temperature, which is in agreement with
the findings of Jana and Manna (1993). The lipid con-
tent in freshwater zooplankton is known to have consi-
derable importance (Vijverberg and Frank, 1976) and
might be influenced by seasonal succession of phyto-
plankton species or source of food fed zooplankton
(Proulex and de la Nove, 1985; Kibria et al., 1999).
Watanabe et al. (1983) analysed various zooplankton
with Daphnia containing 13% and Moina 1227%
lipids whereas in D. carinata and Moina australiensis it
ranged from 7.297.73% (Kibria et al., 1999). Our
results also corroborated the above findings and also
could meet the lipid requirement of carp which ranges
from 69% (Mukhopadhyay and Kaushik, 2001). The
percentage of SAFA increased remarkably during May
to July when the water temperature was 35C compared
with SeptemberOctober and MarchApril (23 C).
These differences in the levels of SAFA as the tempe-
rature decreases, were due to the decreased content of
16:0, which decreased by about half as the temperature
decreased from 35 C to 18 C. Contrary to this, the
percentage of total PUFA were greater at 18 C (23%)
compared to 35 C (10%). The PUFA were at their
lowest levels at 23 C. Substantial variation of fatty acid
355 G. Mitra et al. / Aquaculture 272 (2007) 346360
composition in zooplankton along with seasonal varia-
tion of temperature was presumably an adaptation for
maintaining constant membrane fluidity. Water temper-
ature affects the fatty acid composition of algae and light
intensity affects the production of fatty acids (Tatsuzawa
and Takizawa, 1995), for which there was a seasonal
change in fatty acid composition of mixed zooplankton.
Our result showed close similarity with the finding of
Nanton and Castell (1999) who found significantly
higher content of PUFA at 6 C compared with 20 C,
but at intermediate temperature (15 C) it was lowest
where as MUFAwere at their highest levels at 15 C in
both Amonardia and Tisbe, perhaps making up for the
lower levels of PUFA at this temperature. However, the
zooplankton was low in n-3 fatty acids, particularly of
LNA in some ponds and DHA and EPA in all ponds
which might have the similarity in the fatty acid pattern
of these herbivorous zooplankton and its phytoplank-
tonic food (Claus et al., 1979) containing LA and LNA
not EPA and DHA. However, freshwater algae unlike
marine algae generally have 18:3 n-3 and not 20:5 n-3
and 22:6 n-3 as their principal PUFA, in addition to 18:2
n-6 (Bell et al., 1994). It was noted that Daphnia sp. was
low in DHA and Moina sp. in both DHA and EPA. The
fatty acid composition of B. plicatilis closely reflected
the fatty acid composition of the microalgae consumed
as food (Frolov et al., 1991) although biosynthesis rate
of PUFA de novo by B. plicatilis was rather slow
(Lubzens et al., 1985). Despite the different fatty acid
content of microalgae, Scenedesmus abundans, Mo-
noraphidium minitum and Chlorella vulgaris, cultural
B. calyciflorus utilizing these algae was able to produce
its own lipid and fatty acid content to satisfactory levels
both qualitatively and quantitatively (Isik et al., 1999).
But in case of crustaceans fatty acid synthesis is gene-
rally low in Daphnids (de Lange and Arts, 1999). The
MUFA such as palmitoleic acid (16:1 n-7) and oleic acid
(18:1 n-9) in the mixed zooplankton might be syn-
thesised from the corresponding SAFA by the action of
9 desaturase. It has been suggested that harpacticoid
copepods contained sufficient amounts of the C18 to 20
and C20 to 22 elongase, and 6, 5 and putative 4
desaturase enzymes to be capable of this conversion
(Norsker and Stottrup, 1994; Nanton and Castell, 1998).
This desaturating ability a characteristic of the harpacti-
coid class could be an adaptation to a long-chain EFA-
poor benthic environment when compared to the pelagic
calanoid copepods. A calanoid copepod, Paracalanus
parvus also appears to have some limited ability to
elongate and desaturate 18:3 n-3 to the longer chain n-3
HUFA (Nanton and Castell, 1998). Our results showed
that the calanoid copepods as well as other cladocerans
and rotifers present in the mixed zooplankton were in-
capable of the above discussed coversions which could
decrease its EFAvalue for larval fish as well as adults.
Absence of l-gulonolactone -oxidase activity con-
firmed the inability of these zooplankton to synthesize
ascorbic acid (AA) de novo. So the zooplankton must
depend on an exogenous supply. In this study the
presence of ascorbic acid in zooplankton from different
ponds demonstrated that the occurrence and variability
of AA in the organism was related to the feeding status
of the species (Poulet et al., 1989; Hapette and Poulet,
1990; Merchie et al., 1995). In the pond zooplankton
were grown feeding on phytoplankton. Generally the
phytoplankton (different micro algal sp) are rich source
of AA, but show a considerable variability among the
different species and different phase of life cycle (Mer-
chie et al., 1995) as well as environmental condition
such as pH, CO
2
, light intensity, photoperiod, temper-
ature, cells physiological response to inorganic macro-
nutrients such as nitrate, and concentration of trace
metals (Brown and Lavens, 2001; de Castro Araujo and
Garcia, 2005). Studied environmental condition proba-
bly contributed less production of ascorbic acid in the
phytoplankton which was reflected in zooplankton. The
National Research Council (1993) recommends 25
50 mg AA/kg diet as a requirement to secure an optimal
performance of juvenile fish. Dabrowski (1990) sug-
gested that the metabolic rate is the primary factor re-
gulating the AA requirements. Therefore, larval fish,
displaying a relatively faster growth and metabolism
than juveniles and adults might need high dietary AA
levels to sustain optimal growth and physiological con-
dition (Dabrowski, 1990). Six hundred fifty to 750 mg/
kg diet of AA has been recommended for Cirrhinus
mrigala during early growth (Mahajan and Agrawal,
1980). This study clearly indicated the impairment of
the zooplankton to fulfil the vitamin C requirement for
fish larvae and early juveniles. The content of vitamin C
in zooplankton from ponds 1, 2, 3 and 4 was below the
recommended level of NRC and the content of vitamin
C from other ponds was within the recommended level.
So additional dietary source of vitamin C might be
mandatory for healthy growth and development of carp
larvae. Vitamin A content varied between 13.61
63.95 g/gm DM. The requirement of dietary vitamin
A for L. rohita has been recommended as 606.6 g/kg
dry feed (Rangacharyulu et al., 1999) and 121.21
606 g /100 g dry diet for young carp. The values
obtained in the present study were much higher than the
reported values in other zooplankton (Moren et al.,
2004). Less work has been done on the vitamin E re-
quirement of carp larvae. The vitamin Esupplementation
356 G. Mitra et al. / Aquaculture 272 (2007) 346360
at the minimal requirement level as defined by NRC
(1993) is 50 g/kg feed. Assessed data were 228
333 g/g DM which is more than the NRC recom-
mended value. The broodstock diets containing 60 g
ascorbic acid (pure ascorbic acid powder)/g dry feed and
300 g vitamin E/g dry feed are sufficient to ensure
proper reproduction and offspring quality (Mukhopad-
hyay et al., 2003).
Information about mineral and trace element require-
ment in Indian major carp is limited. Relatively few data
exist about the influence of minerals in prey on larval
feeding. Some data exist for the macro mineral and trace
element compositions of Artemia, which is variable
depending on stock (Rabin and Gatesoupe, 2001). In the
present study the concentration of Ca in zooplankton
varied from 1.052.99 mg/100 g, which is less than the
requirement of common carp 0.33.0 g/kg (Steffens,
1989; Kaushik, 2001). The bioavailability of phospho-
rus from zooplankton is not known. The phosphorous
content 0.240.94 mg/100 gm DM of zooplankton was
much less than common carp requirement (600 mg/
100 g) (Kaushik, 2001). The concentration of iron in the
zooplankton of pond 2 was highest among the others.
Iron level in the zooplankton was lower than the level
used in prepared feed and the requirement reported for
common carp (200 mg/kg) (Steffens, 1989). High levels
of dietary iron for fish have recently been questioned
with regard to negative health effects in relation to
pathogen bacteria development (Lie et al., 1997). It
might be prudent to look into ways to maintain the iron
concentration in zooplankton. The zinc concentration of
the zooplankton was found to be 0.022.88 mg/100g
DM. This concentration range is not much less than the
requirement found for fast growing fish (Maage and
Julshamn, 1993) as well as common carp (1530 mg/
kg) (Steffens, 1989; Lim et al., 2000). As absorption of
dissolved zinc (occurs mainly across the gills) is low in
freshwater fish (Kaushik, 2001), this dietary source has
great relevance in freshwater aquaculture. Manganese
content of zooplankton varied from 0.020.49 mg/
100 g, which is also less than the requirement of com-
mon carp (1213 mg/kg) (Steffens, 1989). Dietary cop-
per level of 3 mg/L improved growth in common carp
fry (Ogino and Yang, 1980). The concentration of cop-
per in mixed zooplankton varied from 0.0010.181mg/
100g DM.
The role of zooplankton proteases as activators of
fish zymogen might be of importance because of the
direct contribution of proteolytic activity to the autolytic
processes of food organisms (Dabrowski and Kaushik,
1984). Kurokawa et al. (1998) demonstrated that pro-
teases derived from live food had only a small contri-
bution to the enzymatic activity measured in sea bass
and sardine larva, respectively. The role of zooplankton
amylase and lipase in larval nutrition might also be
significant. The present study revealed that zooplankton
from different ponds were a good source of amylase,
protease and lipase. The contribution of lipolytic en-
zymes to the total lypolytic process was less than 6.5%
of the total enzyme activity of stripped bass (Morone
saxatilis) during ontogenic development (Ozkizilcik
et al., 1996). The role of dietary enzymes in larval
digestion, the characterization and quantitative estima-
tion of digestive enzymes from zooplankton might be
necessary to determine the enzyme input from live food
to fish larvae.
In the present study though the fertilization condi-
tions were same in all the ponds, the phytoplankton
species as well as the zooplankton population varied
greatly among the ponds, which might resulted in the
pond wise variation of chemical composition of zoo-
plankton. Particular sequence of changes in temperature,
photoperiod and light intensity might alter the propor-
tion of the individual constituents of phytoplankton, the
main food of zooplankton. However zooplankton in
ponds has two sources generally: allochthonous popu-
lation from the water used to fill and the autochthonous
population from resting form in the sediment. In the
present study though zooplankton were present in water
source it was the same for all the ponds. Therefore only
autochthonous population from resting form in the sedi-
ment developed and might have added in the variation in
zooplankton population (Milstein et al., 2006). Frolov
et al. (1991) found a positive correlation between lipid
and fatty acid content, of rotifer and content of these
compounds in their food. However, not all biochemical
components (like carbohydrate, amino acid) changed to
the same degree since, in the course of evolution, an
increase in the conservation of some groups of sub-
stances and stability of others had taken place, resulting
in high stability of organisms as a whole. However,
apart from environmental conditions and food, harvest-
ing techniques, and sample preparation are vital factors
that may affect the nutrient composition (Millamena
et al., 1990). The nutritional quality in Artemia also
varied considerably with the variation of the geograph-
ical origin (Leger et al., 1986; Han et al., 2001), to
differences among different batches of cysts from the
same origin, and to the methods of analysis. Among the
water quality variables variation in temperature reflected
seasonal impact. Certain degree of variation in other
water quality variables was registered between different
ponds, and between the different samplings during the
course of investigation. The results of such variations
357 G. Mitra et al. / Aquaculture 272 (2007) 346360
were obvious in field conditions. The inorganic nut-
rients such as NO
2
N, NO
3
N, and PO
4
P values also
showed no particular trend during study period, attri-
butable to the intermittent fertilization practices (Jena
et al., 1998).
5. Conclusion
Freshwater mixed zooplankton contained a high
amount of protein (although deficient in methionine),
vitamin A and vitamin E and low in vitamin C with no
ascorbic acid synthesizing capacity de novo. Although
the lipid content was high, it was low in some essential
fatty acids like LNA, EPA and DHA, which could be
improved through different fatty acid enrichment stra-
tegies according to the requirement of the desired spe-
cies. Appreciable amount of digestive enzymes was
present in mixed zooplankton that could be utilized
during ontogenesis for better nutrient management
through increasing nutrient digestibility of the larvae
for healthy stockable seed production. Substantial dif-
ferences were found in the mineral composition of the
zooplankton studied to the requirement of fish. This
information may have nutritional implication in culture
of fish larvae and raising of juveniles so that provision of
the prepared feed should be to complement what a pond
ecosystem provides rather than to supplement with a
diet of generic nature chosen empirically.
Acknowledgements
The authors are grateful to the Director, Central
Institute of Freshwater Aquaculture, Bhubaneswar for
providing facilities for carrying out the work. Financial
assistance from Indian Council of Agricultural Research
as Senior Research Fellowship to the senior author is
thankfully acknowledged.
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