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 Aquaculture Nutrition
2009 15; 627–637
..........................................................................................
1 1
1 2
Central Institute of Freshwater Aquaculture, Bhubaneswar, Orissa, India;
Maharashtra, India
Five iso-nitrogenous (300 g protein kg)1 diet) and iso-lipidic
(80 g kg)1 diet) semi-purified experimental diets with variable
energy levels of 10.5 (D-1), 12.5 (D-2), 14.6 (D-3), 16.7 (D-4)
and 18.8 (D-5) MJ kg)1 diets were fed to Puntius gonionotus
fingerlings (average weight 1.79 ± 0.02 g) in triplicate groups
(15 healthy fishes per replicate) for a period of 90 days to assess
the optimum dietary energy level and protein-to-energy ratio
(P/E). Fifteen flow-through cement tanks of 100 L capacity
with a flow rate of 0.5 L min)1 were used for rearing the fish.
Maximum specific growth rate, protein efficiency ratio, protein
productive value, RNA : DNA ratio, whole body protein
content, digestive enzyme activity and minimum feed conver-
sion ratio was found in fish-fed diet D-3 with 14.6 MJ kg)1
energy level. There were no improvements in all these
parameters with the further rise in dietary energy level. Hence,
it may be concluded that the optimum dietary gross energy
level for maximum growth and nutrient utilization of silver
barb is 14.6 MJ kg)1 diet with a resultant P/E ratio of 20.2 g
protein MJ)1 diet, when the dietary protein and lipid are
maintained at optimum requirement levels of 300 and
80 g kg)1 diet, respectively, for this species.
WORDS: diet, energy, growth, nutrient, protein, Puntius
KEY WORDS:
gonionotus, silver barb
Received 24 April 2008, accepted 29 August 2008
Correspondence: Kedar Nath Mohanta, ICAR Research Complex for Goa,
Ela, Old Goa, Goa, 403 402, Goa, India. E-mail: knmohanta@yahoo.com
Successful fish culture depends up on the provision of diets
containing adequate levels of energy and appropriate balance
..............................................................................................
 2008 The Authors
Journal compilation  2008 Blackwell Publishing Ltd
doi: 10.1111/j.1365-2095.2008.00632.x
1 2
Central Institute of Fisheries Education, Mumbai,
of nutrients to permit the most efficient growth and to
maintain the health of the animal under given circumstances
(Cho & Bureau 1995). Dietary energy level is also critical
because protein in the feed is utilized as an energy source
when feed deficient in energy is fed to fish; whereas when feed
excessive in energy is fed, feed consumption decreased and
result in growth reduction because of lack of other necessary
nutrients for normal growth (Lovell 1989; Hemre et al. 2002;
Ali et al. 2008). As protein constitutes the single most
expensive item in fish diets, it is imperative to incorporate
only the amount necessary for normal maintenance and
growth and the use of protein as dietary energy source is
undesirable (Watanabe 2002). Any excess dietary protein is
considered as biologically and economically wasteful (Cho &
Kaushik 1990). Incorporation of appropriate levels of non-
protein energy sources in the diet determines the efficiency of
protein utilization and hence the growth of fish (Wilson &
Halver 1986). Carbohydrate and lipid are the major non-
protein sources in fish diet. Compared with lipid, carbohy-
drate is much less expensive, available abundantly and a
ready source of energy. Carbohydrate also improves the
pelleting quality of the diet due their reasonably good bind-
ing properties. Therefore, it is suggested that the carbohy-
drate may be added in excess of the required amounts that
can be efficiently utilized for energy by fish (Krogdahl et al.
2005). Again, use of high level of lipid as dietary energy
source may create problem in pelleting and keeping quality of
feed in addition to adversely affecting the fish whole body
composition (NRC 1993; Erfanullah & Jafri 1998).
Not only the assessment of dietary protein (P) and energy
(E) are necessary but also understanding the relationship
between these two requirements has become increasingly
important. A proper balance between dietary P and E is
necessary to maintain high growth rate and good food con-
version efficiency, improve protein utilization, minimize
excessive accumulation of lipid and glycogen in somatic tis-
sue and liver, decrease undesirable nitrogen waste output and
improve the quality of fish farm effluents (Ellis & Reigh 1991;
Tibbetts et al. 2005). In contrast, improper dietary protein,
energy levels and/or their ratio lead to an increase of fish
production cost and deterioration of water quality resulting
from waste feed and fish excreta; thus, they are important in
formulating commercial feed (Lee et al. 2000). It has been
shown that at low protein-to-energy (P/E) ratios, the use
dietary protein for growth and maintenance of body protein
is maximized, while at higher P/E ratios, more protein is used
for energy or stored as fat (Ali et al. 2008).
Silver barb, Puntius gonionotus, is an important carp
cultured in several Asian countries like Indonesia, Thai-
land, Vietnam and Bangladesh. It is used for biological
control of aquatic weeds as it is very efficient in checking
the weed menace in freshwater aquaculture system
(Jhingran 1997). The fish attains marketable size of 300 g
in 4–5 months and even grows up to 1 kg in a year (Shetty
et al. 1989). Although silver barb is cultured extensively in
many of the Asian countries, the knowledge of the nutri-
tional requirement of this fish is very limited. Using
casein-gelatin-dextrin based semi-purified diets, Mohanta
et al. (2007a,b, 2008a,b) reported that the optimum dietary
protein, lipid and carbohydrate levels of silver barb are
300, 80 and 260 g kg)1 diet, respectively. For formulation
of cost-effective, nutritionally balanced practical diets for
pond culture of this species, the determination of optimum
dietary energy levels and P/E ratio is prerequisite.
According to Hemre et al. (2002), warm water and
omnivorous fishes can utilize higher dietary carbohydrate
than cold water and carnivorous fishes. Silver barb being
an omnivorous fish, the use of carbohydrate as a dietary
energy source is very important. Therefore, in the present
study, an attempt was made to elucidate the effect of dif-
ferent energy levels (derived from carbohydrate) and P/E
ratio on growth, nutrient retention and digestibility, muscle
nucleic acid content and digestive enzyme activity of silver
barb fingerlings. The experiment results may be useful in
developing cost-effective practical diets for pond produc-
tion of silver barb.
The experiment was conducted at the nutritional yard facil-
ities of the Central Institute of Freshwater Aquaculture,
Bhubaneswar, India. Fifteen indoor flow-through cement
..............................................................................................
Journal compilation  2008 Blackwell Publishing Ltd Aquaculture Nutrition 15; 627–637
tanks of 100 L capacity each (60 · 60 · 42 cm3) were used
for rearing the fish. The flow rate of water was maintained at
0.5 L min)1. The natural light cycle was 12 h light/12 h
darkness (12 L : 12 D) for the experiment. Seasoned ground
water was used for rearing of the fish.
Six hundred fingerlings of P. gonionotus (average weight;
1.22 ± 0.01 g) were procured from Central Institute of
Freshwater Aquaculture Farm (Bhubaneswar, India). After
15 days of acclimatization, a group of 15 fishes (average
weight; 1.79 ± 0.02 g) were stocked randomly into triplicate
tanks for each dietary treatment following a completely ran-
domized design. All groups of fish were fed ad libitum to a
level close to apparent satiation. Fishes were batch-weighed at
every fortnight interval to know the growth and health status
of the fish. The experiment was run for a period of 90 days.
Before commencement of the feeding trial, 100 fishes were
randomly killed with an over dose of MS 222 solution (60 mg
tricaine methane sulfate per liter), and the samples were ta-
ken in triplicates for determining the initial whole body
composition (AOAC 1990).
At the end of experiment, fishes were batch-weighed to
know the final weight and then pooled from each tank for
chemical analysis. The four fishes from each replicate (n = 12
for each treatment) were used for determination of whole
body composition, three fishes per replicate (n = 9 for each
treatment) used for muscle DNA and RNA analysis, three
fishes per replicate (n = 9 for each treatment) for gut enzyme
assay and five fishes per replicate (n = 15 for each treatment)
used for hepatosomatic index (HSI) and viserosomatic index
(VSI) determination (Mohanta et al. 2007b, 2008a,b).
Five iso-nitrogenous (300 g protein kg)1 diet) and iso-lipidic
(80 g kg)1 diet) semi-purified experimental diets with vari-
able energy levels of 10.5 (D-1), 12.5 (D-2), 14.6 (D-3), 16.7
(D-4) and 18.8 (D-5) MJ kg)1 diets were prepared. While
the dietary protein and lipid levels were kept constant as per
the nutrient requirement of this species (Mohanta 2005,
Mohanta et al. 2008a,b), the carbohydrate levels were var-
ied to obtain the desired energy levels in the diets. Casein
(vitamin-free) and gelatin were used as protein source. The
dextrin was used as a source of carbohydrate and a-cellu-
lose as filler. Equal proportions of sunflower and cod liver
oils were used as dietary lipid. The diets were fortified with
prepared vitamins (slightly modified Lovell et al. 1984) and
minerals (Ogino 1977). Carboxymethyl cellulose was used as
binder. The P/E ratios of the experimental diets were varied
between 15.7 and 28.4 g protein MJ)1 diet (Table 1).
 2008 The Authors
 Table 1 Composition of experimental diets with varying levels of energy

D-1 (10.5 D-2 (12.5 D-3 (14.6 D-4 (16.7 D-5 (18.8
Ingredients MJ kg)1 diet) MJ kg)1 diet) MJ kg)1 diet) MJ kg)1 diet) MJ kg)1 diet)

Ingredient compositions (g kg)1 dry matter basis)

Casein1 268 268 268 268 268
Gelatin1 67 67 67 67 67
Dextrin1 12.5 137.5 263 387.5 512
Carboxymethyl cellulose1 20 20 20 20 20
Mineral mix2 40 40 40 40 40
Vitamin mix3 10 10 10 10 10
Sunflower oil 40 40 40 40 40
Cod liver oil 40 40 40 40 40
a-Cellulose1 502.5 377.5 252 127.5 3
Proximate composition (g kg)1 dry matter basis)
Crude protein 301 301 300 302 300
Ether extract 81 81 80 80 81
Ash 78 76 78 75 76
Gross energy (MJ kg)1 diet) 10.6 12.6 14.8 16.9 19.1
P/E ratio (g protein MJ)1 diet) 28.4 23.9 20.2 17.8 15.7
1
The source is Hi-Media Laboratories, Mumbai, India.
2
Vitamin mixture: vitamin A (3000 IU), vitamin D3 (15 000 IU), menadione sodium bisulphate (10 mg), choline chloride (2000 mg), niacin
(50 mg), riboflavin (20 mg), pyridoxine (10 mg), thiamine mononitrate (10 mg), pantothenic acid (40 mg), folic acid (5 mg), vitamin B12
(0.02 mg), biotin (1 mg), inositol (400 mg), a-tocopherol acetate (50 mg) and vitamin C (200 mg) (Modified Lovell et al. 1984).
3
Mineral mixture: NaCl (1.0 g), MgSO4, 7H2O (15.0 g), NaH2PO4, 2H2O (25.0 g), KH2PO4 (32.0 g), Ca (H2PO4)2, H2O (20.0 g), Fe-citrate (2.5 g),
Ca-lactate (3.5 g) and 4 trace element mixture (1.0 g) (Ogino 1977).
4
Trace element mixture: ZnSO4, H2O (35.3 g), MnSO4, 4H2O (16.2 g), CuSO4, 5H2O (3.1 g), CoCl2, 6H2O (0.1 g), KIO3 (0.3 g) and cellulose
(45.0 g).

For preparation of experimental diets, the required quan-
tities of various dietary ingredients except gelatin were
weighed and put in five different aluminum trays. A desired
quantity of water was heated in a 1-L beaker to 80 C by using
an electric heater and the required amount of gelatin was
dissolved into it with slow stirring. The dextrin, vitamin and
mineral mixture thoroughly mixed earlier with oil were added
to it and blended in a mixer followed by the addition of
remaining dietary ingredients excepting carboxymethyl cellu-
lose, which was added at the last to make dough. The dough
was steam-cooked for 5 min in a pressure cooker and passed
through a hand pelletizer with a 2 mm diameter to prepare the
pellets. The pellets were dried at 60 C and stored in a refrig-
erator at 4 C until use (Sau et al. 2004). The fresh feeds were
prepared at every 15 days using the same lot of ingredients that
were purchased at one time from the market to ensure almost
constant quality of nutrients in the respective diets that were
prepared at different times during the experiment (Mohanta
et al. 2007a, 2008a,b). We presumed that by preparing the
fresh feed at 15 days interval and storing these at 4 C, there
was a very little chance of mold or bacterial contamination.
Apparent nutrient digestibility coefficients of the diets were
determined by indirect method using 10 g chromic oxide kg)1
..............................................................................................
of diet (Gomes et al. 1995) in the expense of a-cellulose
during the last 30 days of the experiment. The unconsumed
feed and feces were removed 1 h after first two feeding of the
day at 1000 and 1300 h and then the freshly voided feces were
collected after 1 h (Mohanta et al. 2007b, 2008a,b). The
feces of initial 5 days were discarded and next 25 days sam-
ples were collected following the immediate pipetting method
outlined by Spyridakis et al. (1989). Pooled fecal samples of
each treatment were dried at 55 C and stored in refrigerator
at )20 C for subsequent analysis (Mohanta et al. 2007b,
2008a,b).
Proximate analysis. The proximate composition of experi-
mental diets, fecal samples and whole body was analyzed in
triplicates (AOAC 1990). Dry matter was estimated by oven
drying the samples at 105 C till a constant weight and crude
protein percent was calculated by estimating nitrogen content
by micro-Kjeldahl method and multiplying with a factor
6.25. Ether extract was determined by solvent extraction with
petroleum ether, boiling point 40–60 C, for 10–12 h. Total
ash content was determined by incinerating the sample at
650 C for 6 h and crude fiber by acid digestion (1.25%)
followed by alkali digestion (1.25%). Gross energy in diets,
fecal samples and fish body was calculated by using Bomb

 2008 The Authors

Journal compilation  2008 Blackwell Publishing Ltd Aquaculture Nutrition 15; 627–637
Calorimeter (Parr, model 1341, Parr Instrument Company,
Moline, IL, USA). The chromic oxide (Cr2O3) content of the
feed and fecal samples was determined as described in
Furukawa & Tsukahara (1966) by using atomic absorption
spectrophotometer.
DNA and RNA analysis. The muscle DNA analysis was
conducted according to Ceriotti (1955) and the RNA by
Burton (1956) and the modified methods of Jayaram (1981).
For DNA and RNA study, muscle sample from each treat-
ment was collected 12 h after last feeding and stored at
)20 C (Mitra & Mukhopadhyay 2002; Mohanta et al.
2008a,b).
To extract and quantify the DNA, a tissue homogenate
with buffered saline (0.14 M buffered with 0.02 M sodium-
citrate, pH 7.4) was centrifuged at 5000 g for 15 min in a
refrigerated centrifuge. Sediment was suspended in 2 M
NaCl, kept in refrigerator overnight and centrifuged at
10 000 g for 15 min in a refrigerated centrifuge. Supernatant
was discarded and two volumes of ethanol were slowly added
with continual stirring. The DNA pellet was removed after
washing with 75.0% ethanol and dissolved in Tris-EDTA. It
was heated with 10.0% trichloroacetic acid (TCA). Concen-
trated H2SO4 was added, and then it was heated in a boiling
water bath for 10 min and cooled. Deoxyribose of purine
nucleotides is converted to highly reactive a-hydroxylevu-
linaldehyde, which reacts with diphenylamine to give a blue
color, and extinction was noted at 595 nm with a test and
standards (Calf thymus DNA, Sigma Chemical Company,
Mumbai, India) against distilled water as blank.
To extract and quantify the RNA, the tissue homogenate
was mixed with concentrated and water saturated phenol,
which disrupts hydrogen bonding in the macromolecules
causing denaturation of protein. After stirring mechanically
for 30 min at room temperature, the mixture was centrifuged
at 3000 g for 15 min in a refrigerated centrifuge. The upper
layer was removed and again centrifuged at 10 000 g for
5 min. Potassium acetate (20.0%, pH 5.0) was added to the
supernatant to a final concentration of 2.0%, and RNA was
precipitated by adding two volumes of ethanol. After cooling
on ice for 1 h, precipitate was collected by centrifuging at
2000 g for 5 min. RNA was collected after washing with
ethanol and water (3 : 1), ethanol and finally ethyl ether. The
amount of RNA was quantified with orcinol reagent (0.1%
ferric chloride in concentrated HCl with 6.0% orcinol),
where orcinol reacts with furfural (formed with the reaction
of HCl and purine base of RNA) in the presence of FeCl3 to
give a green color and the optical density of which was
measured at 665 nm against an orcinol blank. A calibration
..............................................................................................
Journal compilation  2008 Blackwell Publishing Ltd Aquaculture Nutrition 15; 627–637
curve, relating to the optical density to be of RNA, was
prepared using purified yeast RNA as the standard. Nucleic
acid concentration was expressed in lg mg)1 fish tissue.
Digestive enzyme assay. The protease activity was measured
following the methods of Moore & Stein (1948) using bovine
serum albumin (BSA) as substrate and the specific activity of
protease was expressed as lg leucine liberated mg)1 tissue
protein h)1 at 37 C. The lipase activity was measured using
a-napthol liberated mg)1 tissue protein h)1 at 37 C follow-
ing the method developed by Seligman & Nachlas (1963).
The a-amylase activity was quantitatively determined using
soluble starch as substrate (Bernfeld 1955) and expressed as
mg maltose liberated mg tissue protein)1 h)1 at 37 C. The
tissue protein content was determined following Lowry et al.
(1951).
Water analyses. Water quality parameters of the experi-
mental tanks were analyzed at every 15 days (APHA 1989)
and recorded as follows: temperature, 26.5–28.4 C; pH, 7.4–
7.8; dissolved oxygen, 7.3–7.6 mg L)1; total alkalinity,
107.56–113.48 mg CaCO3 L)1; total hardness, 103.45–
109.53 mg CaCO3 L)1; ammonia nitrogen (NH3–N), 0.06–
0.09 mg L)1; nitrate nitrogen (NO3)1–N) 12.2–17.6 mg L)1;
nitrite nitrogen (NO2)1–N), 0.07–0.10 mg L)1 and phosphate
(P2O5), 0.03–0.05 mg L)1. These values were found to be in
the normal range of carp rearing (Renukaradhya & Varghese
1986).
The different nutritional indices were determined as follows:
Specific growth rate (SGR)
ln Final weight  ln Initial weight
¼  100
Experimental duration (days)
Feed conversion ratio (FCR)
Total feed intake (dry weight)
¼
Total live weight gain
Total live weight gain
Protein efficiency ratio (PER) ¼
Total protein intake
Nutrients (protein) and energy productive values (PPV and
Nutrient or energy gain in body
EPV) ¼
Nutrient or energy intake
Liver weight
Hepatosomatic index (HSI) ¼  100
Body weight
 2008 The Authors
 diet gross energy, respectively. But, there was no significant
Viscerosomatic index (VSI)
difference (P > 0.05) found in these parameters between 14.6
Weight of the whole digestive tract
¼  100 (D-3) and 16.7 (D-4) MJ kg)1 diet gross energy. The values
Body weight
of PER are in increasing trend up to 14.6 MJ kg)1 diet gross
energy level and thereafter, it showed a decreasing trend. No
Apparent digestibility coefficient (ADC) of nutrient in the significant difference (P > 0.05) in HSI was observed up to
diet (% ADC of nutrient) ¼ 100 14.6 MJ kg)1 diet gross energy level and then it was
 
% of Cr2 O3 in diet % of nutrient in faeces increased significantly (P < 0.05) reaching maximum at
 100 
% of Cr2 O3 in faeces % nutrient in diet 18.8 MJ kg)1 diet gross energy level (D-5). However, VSI
values were not varied significantly (P > 0.05) up to
where chromic oxide (Cr2O3) is used as indicator and nutrient 16.7 MJ kg)1 dietary gross energy levels and it was signifi-
is protein, lipid or energy (Sullivan & Reigh 1995). cantly higher (P < 0.05) at 18.8 MJ kg)1 diet gross energy
levels than the other dietary energy levels.
The PPV was significantly higher (P < 0.05) in fish-fed
D-3 (14.6 MJ kg)1 dietary gross energy) than the values
The difference among the treatments was tested by one-way obtained in other diets (Table 3). However, the EPV was
ANOVA and comparison between the treatments was carried decreased with increase in dietary energy levels and the
out by DuncansÕ multiple range test at P < 0.05. The sta- significantly higher (P < 0.05) EPV was recorded in fish-fed
tistical package used for the analysis of data was PC-SAS diet D-1 than the other diet fed groups.
program for Windows, release v6.12 (SAS Institute Inc., The maximum apparent nutrient digestibility (ADCProtein,
Cary, NC, USA, 1996). ADCLipid & ADCEnergy) values were obtained in diet D-3,
which were significantly higher (P < 0.05) than the values
found in diets D-1, D-2 and D-5 but on par (P > 0.05) with
the values observed in D-4 (Table 3).

The data on growth and nutrient utilization of fish-fed var-

ious energy diets are presented in Table 2. The fish-fed diet
D-3 (14.6 MJ kg)1 diet gross energy) had registered
significantly higher (P < 0.05) weight gain and SGR and the
lowest (P < 0.05) FCR when compared with fish-fed diets
D-1, D-2 and D-5 containing 10.4, 12.5 and 18.8 MJ kg)1
The digestive enzyme activities (protease, lipase and amylase)
were significantly higher (P < 0.05) in fish-fed D-3 than
those of D-1, D-2, and D-5 (Table 3). But all the three
enzyme activities did not vary significantly (P > 0.05) in fish-
fed diets D-3 and D-4.

Table 2 Growth and diet utilization of P. gonionotus fed diets with varying levels of energy

Experimental diets

D-1 D-2 D-3 D-4 D-5

Nutritional indices (10.5 MJ kg)1) (12.5 MJ kg)1) (14.6 MJ kg)1) (16.7 MJ kg)1) (18.8 MJ kg)1)

Initial weight (g) 1.86 ± 0.1a 1.76 ± 0.08a 1.80 ± 0.04a 1.77 ± 0.07a 1.78 ± 0.03a
Final weight (g) 12.69 ± 0.38d 13.58 ± 0.49cd 16.21 ± 0.52a 15.49 ± 0.44ab 14.56 ± 0.24bc
Weight gain (g) 10.83 ± 0.25d 11.82 ± 0.42cd 14.41 ± 0.51a 13.72 ± 0.40ab 12.78 ± 0.22bc
DFI (mg diet fish)1 day)1) 203 ± 9.0a 221 ± 8.8a 239 ± 10.9a 237 ± 8.8a 225 ± 3.7a
SGR (% day)1) 2.13 ± 0.03c 2.27 ± 0.01b 2.44 ± 0.01a 2.41 ± 0.03a 2.34 ± 0.02b
FCR 1.69 ± 0.04a 1.68 ± 0.01a 1.49 ± 0.02c 1.55 ± 0.02bc 1.58 ± 0.02b
PER 1.96 ± 0.04c 1.97 ± 0.01c 2.22 ± 0.03a 2.13 ± 0.03ab 2.09 ± 0.03b
HSI 1.74 ± 0.03c 1.79 ± 0.01c 1.84 ± 0.01c 2.16 ± 0.03b 3.04 ± 0.06a
VSI 8.40 ± 0.03b 8.44 ± 0.04b 8.47 ± 0.01b 8.52 ± 0.03b 10.11 ± 0.07a

Mean values with same superscripts in each row are not significant (P > 0.05). Values are means of three replicates in each experimental
diet ± SE.
DFI, daily feed intake; SGR, specific growth rate; FCR, food conversion ratio; PER, protein efficiency ratio; HSI, hepatosomatic index; VSI,
viscerosomatic index.
..............................................................................................

 2008 The Authors

Journal compilation  2008 Blackwell Publishing Ltd Aquaculture Nutrition 15; 627–637
Table 3 Nutrient retention, digestibility and enzyme activity of P. gonionotus fed diets with varying levels of energy

Experimental diets

D-1 D-2 D-3 D-4 D-5

Parameters (10.5 MJ kg)1) (12.5 MJ kg)1) (14.6 MJ kg)1) (16.7 MJ kg)1) (18.8 MJ kg)1)

Nutrient productive values

PPV 0.29 ± 0.006c 0.29 ± 0.001c 0.32 ± 00.005a 0.31 ± 0.004b 0.30 ± 0.004bc
a
EPV 0.34 ± 0.007 0.30 ± 0.001b 0.30 ± 0.004b 0.25 ± 0.003c 0.23 ± 0.003d
Apparent nutrient digestibility coefficients (%)
ADCProtein 94.19 ± 0.09d 94.36 ± 0.05cd 94.79 ± 0.05a 94.65 ± 0.06ab 94.53 ± 0.03bc
ADCLipid 94.22 ± 0.09d 94.28 ± 0.05cd 94.74 ± 0.05a 94.57 ± 0.11ab 94.43 ± 0.07bc
d
ADCEnergy 91.63 ± 0.13 91.87 ± 0.08cd 92.39 ± 0.07a 92.22 ± 0.08ab 92.09 ± 0.04bc
Enzyme activity
1
Protease 23.11 ± 0.04d 23.30 ± 0.03cd 23.91 ± 0.16a 23.66 ± 0.12ab 23.43 ± 0.05bc
2
Lipase 29.84 ± 0.07d 29.96 ± 0.06cd 30.43 ± 0.09a 30.29 ± 0.07ab 30.10 ± 0.07bc
3 d
Amylase 11.97 ± 0.05 12.19 ± 0.11cd 12.63 ± 0.06a 12.48 ± 0.11ab 12.29 ± 0.09bc

Mean values with same superscripts in each row are not significant (P > 0.05). Values are means of three replicates in each experimental
diet ± SE.
PPV, protein productive values; EPV, energy productive values; ADCProtein, apparent digestibility coefficient of the diet for protein; ADCLipid,
apparent digestibility coefficient of the diet for lipid; ADCEnergy, apparent digestibility coefficient of the diet for energy.
1
Protease activity: lg of leucine liberated mg)1 tissue protein h)1 at 37 C.
2
Lipase activity: lg of a-napthol reduced mg)1 tissue protein h)1 at 37 C.
3
Amylase activity: mg of maltose liberated mg)1 tissue protein h)1 at 37 C.

The data on RNA and DNA concentrations,

RNA : DNA ratio in fish-fed different dietary energy levels
are presented in Table 4. While there was no variation
(P > 0.05) in DNA concentrations in all dietary treatment
groups, there was a significant variation (P < 0.05) in
muscle RNA concentrations with highest value obtained in
D-3 group which was significantly higher (P < 0.05) than
D-1, D-2 and D-5 but did not vary (P > 0.05) with D-4.
Similarly, the RNA : DNA ratio was found to be highest in
D-3 group, which was significantly higher (P < 0.05) than
those of D-1, D-2 and D-5 groups and on par (P > 0.05) with
D-4 group.
The whole body compositions of the fish-fed various gross
energy diets are presented in Table 4. The initial dry matter
content was significantly lower (P < 0.05) than the all diet
fed groups. However, there was a significant increase
(P < 0.05) in whole body dry matter contents with increase
in dietary gross energy. The whole body protein was signifi-
cantly higher (P < 0.05) in initial group than the diet fed
groups. Among the dietary treatment groups, maximum
whole body protein was found in D-1 group and it was
marginally decreased (P < 0.05) as the gross energy levels in

Table 4 Whole body composition (g kg)1 wet weight) and RNA and DNA concentrations (lg mg)1 muscle tissue) of P. gonionotus fed diets
with varying levels of energy

Final

D-1 D-2 D-3 D-4 D-5

Parameters Initial (10.5 MJ kg)1) (12.5 MJ kg)1) (14.6 MJ kg)1) (16.7 MJ kg)1) (18.8 MJ kg)1)

Whole body composition

Dry matter 231.5 ± 2.0e 250.2 ± 1.3d 253.2 ± 0.9cd 257.7 ± 1.0bc 260.7 ± 1.5ab 262.4 ± 1.8a
Crude protein 151.0 ± 1.4a 147.4 ± 0.9b 146.5 ± 1.0b 145.8 ± 0.8b 144.5 ± 1.1bc 142.3 ± 0.9c
Ether extract 40.0 ± 0.3f 62.8 ± 0.7e 65.5 ± 1.0d 69.1 ± 0.5c 72.8 ± 0.9b 76.5 ± 0.7a
Total ash 38.0 ± 0.7a 36.0 ± 0.5b 34.0 ± 0.6c 32.0 ± 0.3d 29.0 ± 0.5e 27.0 ± 0.3f
Gross energy (MJ kg)1) 21.6 ± 0.21e 25.2 ± 0.37d 25.8 ± 0.25cd 26.5 ± 0.21bc 27.2 ± 0.29ab 27.8 ± 0.33a
Nucleic acid concentrations
Muscle RNA 2.88 ± 0.02d 2.99 ± 0.02cd 3.24 ± 0.04a 3.17 ± 0.05ab 3.10 ± 0.04bc
Muscle DNA 0.27 ± 0.003a 0.27 ± 0.000a 0.27 ± 0.003a 0.27 ± 0.003a 0.27 ± 0.003a
RNA : DNA ratio 10.8 ± 0.21d 11.1 ± 0.06cd 11.9 ± 0.03a 11.6 ± 0.24ab 11.3 ± 0.04bc

Mean values with same superscripts in each row are not significant (P > 0.05). Values are means of three samples ± SE.
..............................................................................................

 2008 The Authors

Journal compilation  2008 Blackwell Publishing Ltd Aquaculture Nutrition 15; 627–637
 the diets increased. The whole body ether extract was mini-
mum in the initial group and it significantly increased
(P < 0.05) with increase in dietary energy levels. However,
the whole body ash contents significantly decreased
(P < 0.05) with increase in dietary energy levels. The whole
body gross energy level of fish was increased with increase in
dietary energy level and it was significantly higher (P < 0.05)
in higher dietary energy levels (D-4 and D-5) than lower
levels (D-1 and D-2) including initial group.
Energy intake is a basic nutritional requirement because
maintenance of life processes takes priority over growth and
other functions. Thus, energy concentration should be the
first nutritional consideration in diet formulation for the fish
(Kim et al. 2004). An optimal balance of energy and protein
components in the diet is important for increasing the
growth, survival and production. If the diet is deficient in
non-protein energy, protein will be used for basal metabolism
and voluntary activity rather than protein synthesis and
growth (Cho & Bureau 1995). Protein utilization can be
improved by replacing protein with lipid or carbohydrate;
however, excess energy is undesirable because it may reduce
feed consumption (Lovell 1979), produce fatty fish (Page &
Andrews 1973) and may inhibit optimum utilization of other
essential dietary components like protein and amino acids
(Prather & Lovell 1973; Winfree & Stickney 1984). There-
fore, a proper balance between protein and non-protein
energy is required to supply calories and amino acids for
rapid growth and efficient feed utilization and to maintain
fish flesh that is high in protein and low in fat (Kaushik &
Medale 1994).
Casein and gelatin are widely used for basic nutrient
requirement study in fish. As casein is deficient in arginine,
the gelatin, a binding agent, is added to make up the amino
acid deficiencies in formulating the test diets (Halver 1972;
Santiago & Lovell 1988; Mohanty et al. 1992). Blending of
casein and gelatin in appropriate proportions improve die-
tary efficiencies by maintaining the desired levels of amino
acids in fish diets (Murai et al. 1984; Mohanty et al. 1992).
Mohanty et al. (1992) and Sau et al. (2004) reported that the
optimum casein and gelatin ratio to meet the amino acid
requirements of tropical carps is 3.5 to 4.0. In our study, the
casein and gelatin ratio was maintained at 4 : 1 and the
amino acid compositions of the experimental diets were
calculated (Halver & Hardy 2002). The calculated amino acid
compositions of the experimental diets were almost matched
with the amino acid requirements of common carp, Cyprinus
..............................................................................................
 2008 The Authors
Journal compilation  2008 Blackwell Publishing Ltd Aquaculture Nutrition 15; 627–637
carpio (Nose 1979) and catla, Catla catla (Ravi & Devaraj
1991). As there is no literature available on amino acid
requirement of silver barb, the amino acid requirement of
common carp and catla could be considered as standard
requirement of this species.
In the present study, no fish mortality was found in any of
the diet fed groups. The experiment results indicated that fish
reared in medium dietary energy levels of 14.6–16.7 MJ kg)1
diet (D-3 and D-4) outperformed the other groups of lower
(10.4 and 12.5 MJ kg)1 diet in D-1 and D-2, respectively)
and higher (18.8 MJ kg)1 diet in D-5) energy levels in terms
of growth, nutrient utilization, retention and digestibility,
digestive enzymatic activities, RNA contents and RNA : DNA
ratio.
The weight gain was in the increasing trend up to
14.6 MJ kg)1 diet energy level (D-3) and then declined with
further rise in dietary energy level. Decrease in weight gain
with increase in energy levels beyond the optimum require-
ment level was also found in flounder Paralichthys olivaceous
(Lee et al. 2000). The maximum weight gain observed in fish-
fed D-3 is attributed to proper balance between protein and
energy (P/E ratio), higher rate of protein synthesis as
reflected by increased RNA : DNA ratio and high nutrient
retention and digestibility with maximum enzyme (protease,
lipase and amylase) activities.
It is reported that when the diet is deficient of non-protein
energy, the protein is used for energy rather than the growth
(Cho & Bureau 1995; Kim et al. 2004). This may be the
reason for the poor performance of low-energy (10.4 and
12.5 MJ kg)1) diets (D-1 and D-2), which are deficient of
non-protein energy because of the low dextrin content in
these two diets compared with other diets. Therefore, the
dietary protein might have used as energy purpose adversely
affecting the growth when the fish were fed with low-energy
diets. Similarly, no improvement in weight gain was observed
beyond the optimum energy level of 14.6 MJ kg)1 as the
excess dietary energy beyond the requirement level had re-
sulted whole body lipid gain rather than growth of fish,
which is in agreement with Kaushik & Medale (1994), sug-
gesting lipogenesis in fish-fed high-energy diet. Several au-
thors also reported that high-energy diet increases whole
body lipid deposition without improving the growth perfor-
mance of fish (Page & Andrews 1973; Marais & Kissil 1979;
Peres & Oliva-Teles 1999). Ellis & Reigh (1991) and Lee et al.
(2000) found decrease in weight gain when fish-fed high-
energy diets.
The best FCR was found in the fish fed at 14.6 MJ kg)1
energy diet (D-3) because of relatively more weight gain in
fish in relation to feed intake (feed intake was independent of
dietary energy in the present study). However, the FCR
improved up to the optimum energy requirement level of
14.6 MJ kg)1 diet, which either leveled off (D-4) or showed a
trend of decline (D-5) with further rise in dietary energy. This
observation compares favorably with the similar pattern of
FCR obtained by Hassan et al. (1995) in mrigal (Cirrhinus
mrigala).
In the present experiment, PER improved with increasing
dietary energy levels and was significantly higher (P < 0.05)
in the medium energy levels of 14.6 and 16.7 MJ kg)1 diet,
beyond which there was no further improvement. This im-
plies that the dietary protein is not being efficiently utilized
beyond a particular dietary energy level for silver barb.
However, Cowey et al. (1975) reported the progressive in-
crease of PER with increase in dietary energy levels for plaice
(Pleuronectes platessa). Similarly, Buhler & Halver (1961)
also found increasing PER values in chinook salmon
(Oncorhynchus tshawytscha) fed increasing levels of carbo-
hydrate energy. Daniels & Robinson (1986) reported
decreased growth, PER and feed intake with increase in
dietary energy in red drum (Sciaenops ocellatus). Ellis &
Reigh (1991) revealed that the growth depression occurred in
red drum when higher dietary energy was supplied to the fish
which was due to appetite loss before sufficient protein was
consumed to support maximum growth. In addition to
reduced weight gain, they observed decreased feed efficiency
and nutrient retention in fish-fed high-energy diets than the
low and medium energy diets. In the present experiment,
although appetite loss was not found among different dietary
energy fed groups as seen from the feed intake data, yet,
growth was not satisfactory because of presence of high levels
of indigestible carbohydrate in low-energy diets (502.5 and
377.5 g kg)1 a-cellulose in D-1 and D-2, respectively) and
digestible carbohydrate in high-energy diet (512.0 g kg)1
dextrin in D-5), which the fish might not have been able to
digest properly as evident from low nutrient digestibility
(ADCs, Table 3). But, from the growth and nutrient digest-
ibility (ADCs) of fish, it is clearly evident that the fish can
digest digestible carbohydrate very well up to 380.0 g kg)1
diet (D-4) and can improve the weight gain when carbo-
hydrate (dextrin) is included up to 380.0 g kg)1 diet by
sparing protein for growth. Degani & Viola (1987) observed
linear increase in weight with increase in dietary carbohy-
drate energy in eel Anguilla anguilla. In the contrary, Hemre
et al. (1989) showed no improvement in weight gain with
increase in carbohydrate energy in the diet for cod Gadus
morhua. Inclusion of high carbohydrate energy in rainbow
trout feed had negative impact on nutrient digestibility
(Singh & Nose 1967; Bergot & Breque 1983; Hillestad et al.
..............................................................................................
Journal compilation  2008 Blackwell Publishing Ltd Aquaculture Nutrition 15; 627–637
2001), on growth (Hilton & Atkinson 1982; Beamish &
Medland 1986) and on FCR (Kim & Kaushik 1992). The
present finding is in agreement with these workers. Ellis &
Reigh (1991) reported that high-energy diet (beyond the
optimum requirement level) had significantly lower weight
gain, feed efficiency and net protein retention in red drum,
which corroborate the present findings in silver barb.
There is a linear increase in HSI with increase in dietary
energy and the maximum HSI value found at highest level of
dietary energy fed to silver barb. Daniels & Robinson (1986)
and Helland & Grisdale-Helland (1998) observed higher HSI
at higher levels of dietary carbohydrate energy in red drum
and Atlantic salmon (Salmo salar), respectively. Hepatoso-
matic index is positively related to dietary dextrin level as
reported in several studies (Daniels & Robinson 1986; Yang
et al. 2002, 2003). As the high carbohydrate energy diet (D-5)
in the present experiment contains 512.0 g kg)1 dextrin, this
may be the reason for increased HSI value in fish-fed high-
energy diet. We presume that the high level of digestible
carbohydrate (dextrin) in high-energy diet (D-5) might have
lead to fat deposition in fish by the process of lipogenesis as
reported by earlier workers (Lin et al. 1977; Likimani &
Wilson 1982) after meeting the energy requirement. The VSI
also followed the same trend as HSI with highest value
obtained at the highest level of dietary energy.
The maximum protein retention (PPV) in silver barb fed
diet D-3 than the other diet fed groups may be due to pro-
portionately more weight gain of fish with respect to protein
in take. The concentration of DNA in normal somatic cells is
constant for a given species. Therefore, a change in the
amount of DNA in a given quantity of tissue is thought to
represent a change in cell size and number. An increase in
DNA would represent an increase in cell number and
decrease in cell size (Barrows et al. 1988). In our study, we
also have found constant DNA content in fish muscle tissue
irrespective of dietary energy levels. DNA is a prerequisite
for RNA synthesis, which in turn is a requirement for protein
synthesis (Mitra & Mukhopadhyay 2002; Mohanta et al.
2008b). An increase in the rate of protein synthesis requires a
concomitant increase in the total quantity of RNA in the cell
(Barrows et al. 1988; Mohanta et al. 2008b). RNA : DNA
ratio is considered as an effective indicator of protein syn-
thesis and hence, the growth of fish (Nandeesha et al. 2000;
Mohanta et al. 2008a; b). Although we have not measured
the protein synthesis, the higher protein retention and growth
of fish-fed diet D-3 could be attributed to maximum
RNA : DNA ratio found in this diet fed group and that
might have resulted more protein synthesis. Carter et al.
(1993) reported that a positive correlation exist between
 2008 The Authors
 RNA content and growth of fish. In the present study, also
higher muscle RNA content complement more growth of
fish-fed diet D-3.
The whole body composition of fish showed a significant
lower (P < 0.05) protein content in treatment groups than
the initial. But, the whole body lipid was significantly
(P < 0.05) higher in treatment groups compared with the
initial group. While the comparison was made among the
treatment groups, there was a decrease in whole body protein
content with increase in dietary energy levels. However, the
whole body lipid content was found to be significantly
increased (P < 0.05) with rise in dietary energy. Similar
results were observed by Zeitler et al. (1984) in common carp
(Cyprinus carpio). Papaparaskeva-Papoutsoglou & Alexis
(1986) suggested that the body lipid content increased with
dietary carbohydrate level could be taken as indication of
lipid synthesized from carbohydrate when dietary lipid was
fixed at constant level in grey mullet Mugil capito. In the
present study, as the dietary lipid level was maintained at
optimum requirement level of 80 g kg)1 diet (Mohanta et al.
2008b) in all diets, therefore, the increase in whole body lipid
(P < 0.05) with increase in dietary energy may be due to
proportionate increase of carbohydrate (dextrin) levels in the
diets. The correlation of whole body compositions showed
that the whole body protein and lipid are also inversely
correlated (r = )0.90, P < 0.01). These observations are
consistent with the observations of earlier workers (Garling
& Wilson 1976; Ellis & Reigh 1991; Shearer 1994; Tibaldi
et al. 1996).
The optimum dietary P/E ratio (g protein MJ)1) has been
reported for several fish are channel catfish Ictalurus punct-
atus, 20.81 (Garling & Wilson 1976); grass carp, 23.92–29.66
(Xianghua 1988); Indian major carp Labeo rohita, 22.68 (Das
et al. 1991); red tilapia Oreochromis species, 26.55 (Santiago
& Laron 1991); Nile tilapia Oreochromis niloticus, 18.96–
26.31 (El-Sayed & Teshima 1992; Ali et al. 2008); sunshine
bass Morone chrysops · M. saxatilis, 23.68 (Webster et al.
1995); African catfish Clarias gariepinus, 20.5–34.69 (Henken
et al. 1986; Ali & Jauncey 2005); Asian seabass Lates calca-
rifer (Catacutan & Coloso 1995); mangrove red snapper
Lutjanus argentimaculatus, 23.3 (Catacutan et al. 2001); olive
flounder Paralichthys olivaceus, 27.5 (Kim et al. 2004); black
catfish Rhamdia quelen, 23.6 (Salhi et al. 2004), and white sea
bream Diplodus sargus, 20.0 (Sa et al. 2006). The wide vari-
ations in dietary P/E ratio as mentioned above indicated that
it depends on type of fish (tropical, temperate, fresh, brackish
and marine) and their feeding habits (herbivorous, carnivo-
rous and omnivorous), rearing conditions (static, flow-
trough and recirculatory), environmental parameters (mainly
..............................................................................................
 2008 The Authors
Journal compilation  2008 Blackwell Publishing Ltd Aquaculture Nutrition 15; 627–637
temperature and photoperiod) and diet composition includ-
ing the digestibility of feed ingredients. From the present
study, it may be concluded that the optimum dietary gross
energy level for maximum growth and nutrient utilization of
silver barb is 14.6 MJ kg)1 diet with a resultant P/E ratio of
20.2 g protein MJ)1 diet, when the dietary protein and lipid
are maintained at optimum requirement levels of 300 and
80 g kg)1 diet, respectively, for this species. The experiment
results on optimum energy level and P/E ratio obtained in the
present study may be useful in developing cost-effective,
nutritionally balanced practical diets for pond culture of
silver barb.
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