Dietary protein requirement for fingerling Channapunctatus (Bloch), based on growth, feed conversion,protein retention and biochemical composition

Seemab Zehra • Mukhtar A. Khan

Received: 4 December 2010 / Accepted: 8 August 2011 / Published online: 31 August 2011� Springer Science+Business Media B.V. 2011

Abstract An 8-week feeding trial was conducted in a flow-through system (1–1.5

L min-1) at 27�C to determine dietary protein requirement for Channa punctatus finger-

lings (4.58 ± 0.29 g) by feeding six isocaloric diets (18.39 kJ g-1, gross energy). Diets

containing graded levels of protein (300, 350, 400, 450, 500 and 550 g kg-1) were fed to

triplicate groups of fish to apparent satiation at 09:00 and 16:00 h. Maximum absolute

weight gain (AWG; 8.11 g fish-1), specific growth rate (SGR; 1.82%) and best feed

conversion ratio (FCR; 1.48) were recorded in fish fed diet containing 450 g kg-1 protein,

whereas protein efficiency ratio (PER; 1.52), protein retention efficiency (PRE; 25%),

energy retention efficiency (ERE; 78%) and RNA/DNA ratio (3.01) were maximum for the

group fed dietary protein at 400 g kg-1. Second-degree polynomial regression analysis of

AWG, SGR and FCR data against varying levels of dietary protein yielded optimum

dietary protein requirement of fingerling between 462.24 and 476.72 g kg-1, whereas the

regression analysis of PER, PRE, ERE and RNA/DNA ratio data showed a lower protein

requirement of 438.28–444.43 g kg-1 of the diet. Considering the PER, PRE, ERE and

RNA/DNA ratio as more reliable indicators, this protein requirement is recommended for

developing quality protein commercial feeds for C. punctatus fingerlings.

Keywords Growth � Protein requirement � RNA/DNA ratio � Channa punctatus

Introduction

Formulation of nutritionally balanced feed is important for successful aquaculture (Schulz

et al. 2007). Feed accounts for about 60% of the operational cost, largely due to the

incorporation of high percentage of protein needed for tissue growth, maintenance and

reproduction. There is an optimum requirement of dietary protein to supply adequate

amino acids for maximizing growth. Increase in dietary protein has often been associated

S. Zehra � M. A. Khan (&)Department of Zoology, Fish Nutrition Research Laboratory, Aligarh Muslim University,Aligarh 202 002, Indiae-mail: khanmukhtar@yahoo.com

123

Aquacult Int (2012) 20:383–395DOI 10.1007/s10499-011-9470-8

with higher growth rate in many species. However, there is a protein level beyond which

further growth is not supported and may even decrease (Alam et al. 2008; Siddiqui and

Khan 2009). If too much protein is supplied in the diet, only part of it is used to make new

protein, and the remainder will be converted into energy, which results in increased feed

cost and increased ammonia nitrogen excretion. Therefore, from both economical and

environmental perspective, it is important that inclusion of the dietary protein should be

optimized (Siddiqui and Khan 2009). The utilization and levels of inclusion of protein in

feeds for carnivorous fish have been addressed by many authors (Alam et al. 2008;

Siddiqui and Khan 2009; Zhang et al. 2010; Kumar et al. 2010; Akpinar et al. 2011).

The RNA/DNA ratio reflects the nutritional condition of the fish (Park et al. 2008). It is

a reliable biochemical indicator of protein synthesis and thus growth (Mustafa 1977;

Bulow 1987; Abidi and Khan 2009). For this reason, in this study, changes in RNA/DNA

ratio in response to varying levels of dietary protein intake were determined.

Channa punctatus, commonly known as murrel, is an important, highly priced

(Rs 180 kg-1) freshwater food fish species. It is found to be distributed throughout the

Southeast Asian countries and is a natural inhabitant of stagnant muddy pond waters,

paddy fields, weedy derelict swamps, beels, canals and reservoirs (Chondor 1999). Its air-

breathing characteristics and general hardiness allow it to be cultured in areas that are not

suitable for the culture of Indian major carp and other carp species. It has great market

demand due to its good taste and high nutritional value. It is recommended in diet during

convalescence and, therefore, is a good candidate for intensive aquaculture (Marimuthu

et al. 2009). Yaakob and Ali (1992) also noted the importance of murrels for hastening the

healing of wounds and internal injuries due to the presence of certain fatty acids such as

prostaglandin and thromboxane. Hence, this species is gaining attention as a cultured

freshwater fish for medicinal purposes in the Asian market. Generally, semimoist feeds

containing 45% dietary protein are used by the farmer for this fish (Abdel-Hameid et al.

2011). The fish are often cultured in grow-out ponds at densities of 40–80 fish/m2 with

annual yields ranging from 7 to 156 tonnes ha-1 (Wee 1982). Although it is very popular

and highly demanded fish in India, the production of this fish is not organized due to the

lack of nutritionally balanced feed. Moreover, a steady decline in the wild population of

the species is also observed mainly due to overexploitation as well as loss and degradation

of natural breeding and feeding grounds. As such, IUCN has listed this species of murrels

in the low-risk near threatened category. Although little information on some aspects of

nutrition and culture of C. punctatus is available (Bhuiyan et al. 2006; Marimuthu et al.

2009; Jindal et al. 2010; Saikia and Das 2010; Abdel-Hameid et al. 2011), no information

is available on dietary protein requirement in relation to growth, RNA/DNA ratio and

protein retention of this fish. The present study was, therefore, aimed at determining the

dietary protein requirement for developing quality protein feeds to maximize growth, feed

conversion and protein retention for C. punctatus fingerlings.

Materials and methods

Preparation of experimental diets

Six isoenergetic (18.39 kJ g-1, gross energy) casein-gelatin-based diets with different

levels of protein (300, 350, 400, 450, 500 and 550 g kg-1 of the diet) were prepared (Table 1).

A combination of corn oil and cod liver oil (5:2) was used as a source for lipid in the test

diets to provide the proper balance of n-3 and n-6 fatty acids (Halver 2002). Vitamin and

384 Aquacult Int (2012) 20:383–395

123

mineral premixes were prepared as per Halver (2002). Experimental diets were prepared

according to the method adopted by Siddiqui and Khan (2009) that has been briefly given

here. For making soft cake, calculated quantities of dry ingredients were thoroughly mixed

and stirred in 30 mL of hot water (80�C) in a steel bowl attached to a Hobart electric mixer

(Hobart, Troy, OH, USA). Gelatin powder was dissolved separately in 20 mL of water

with constant heating and stirring and then transferred to the above mixture. Other dry

ingredients and oil premixes were added to the lukewarm bowl one by one with constant

mixing at 50�C. Carboxymethylcellulose was added last, and the speed of the blender was

gradually increased as the diet started to harden. The final diet with the bread dough

consistency was poured into a Teflon-coated pan and placed in a refrigerator. The prepared

diets were in the form of semimoist cake from which cubes were cut and stored at 20�C in

sealed polythene bags until used.

Experimental design and feeding trial

Fingerlings of C. punctatus were obtained from fish breeding and larval rearing facility of

the Department of Zoology, Aligarh Muslim University (Aligarh, U.P., India). The fin-

gerlings were then transported to the wet laboratory, given a prophylactic dip in KMnO4

solution (1:3,000) before stocking in indoor circular aqua-blue-colored, plastic-lined fish

tanks (1.22 m 9 0.91 m 9 0.91 m; water volume 600 L) for a week. During this period,

the fish were acclimated to a casein-gelatin-based (450 g kg-1 CP) H-440 diet (Halver

2002).

Acclimated fingerlings (4.58 ± 0.29 g) were stocked in triplicate groups in 70-L

circular polyvinyl troughs (water volume 55 L) fitted with a continuous temperature-

Table 1 Ingredients and proximate composition of experimental diets

Ingredients (g kg-1 diet) Dietary protein level (g kg-1 diet)

300 350 400 450 500 550

Casein 300 350 400 450 500 550

Gelatin 75 87.5 100 112.5 125 137.5

Dextrin 457.4 369.6 281.7 193.9 106.1 18.3

Cod liver oil 20 20 20 20 20 20

Corn oil 50 50 50 50 50 50

Mineral mix 40 40 40 40 40 40

Vitamin mix 30 30 30 30 30 30

Carboxymethyl cellulose 27.6 50 50 50 50 50

a-cellulose – 2.9 28.3 53.6 79 104.2

Total 1,000 1,000 1,000 1,000 1,000 1,000

Proximate analyses (g kg-1 of the dry diet)

Calculated crude protein 300 350 400 450 500 550

Analyzed crude protein 304.2 345.2 392.4 443.5 493.6 542.3

Analyzed lipid 69.85 69.91 70.12 69.79 70.14 70.11

Gross energy (kJ g-1)a 18.39 18.39 18.39 18.39 18.39 18.39

Protein/energy ratio (mg kJ-1) 16.31 19.03 21.75 24.47 27.19 29.91

a Calculated on the basis of fuel values 23.07, 20.19, 16.01 and 37.62 kJ g-1 for casein, gelatin, dextrin andfat, respectively, as estimated on Gallenkamp ballistic bomb calorimeter

Aquacult Int (2012) 20:383–395 385

123

controlled water flow-through (1–1.5 L min-1) system at the rate of 15 fish per trough for

each dietary treatment level. Fish were fed test diets in the form of soft cake to apparent

satiation twice daily at 09:00 and 16:00 h. The total weight and number of the fish were

determined every week. Feeding was stopped at the sampling day. The feeding trial lasted

for 8 weeks. The fish were anaesthetized with 0.01% aqueous solution of tricaine methane

sulfonate (MS-222) before sampling. The effectiveness of the MS-222 dose was found to

be similar to be that reported by Mahajan and Agrawal (1979). Fecal matter was siphoned

before every feeding. Water quality parameters including water temperature, dissolved

oxygen, free carbon dioxide, pH, salinity and total alkalinity were monitored daily during

the feeding trial and were recorded following standard methods (APHA 1992). The

average water temperature, dissolved oxygen, free carbon dioxide, pH, salinity and total

alkalinity based on daily measurements were 26.5–27.3�C, 6.8–7.5, 5.8–9.3 mg L-1,

7.2–7.6, 0.21–0.24 ppt and 65.4–81.4 mg L-1, respectively.

Chemical analyses

Proximate composition of the experimental diets and initial and final carcass were deter-

mined using standard methods (AOAC 1995) for dry matter (oven drying at 105 ± 1�C for

22 h.), crude protein (Kjeltec TecatorTM

Technology 2300, Hoegenas, Sweden), crude fat

(solvent extraction with petroleum ether B.P 40–60�C for 2–4 h using Socs Plus, SCS 4,

Pelican equipments, Chennai, India) and ash (oven incineration at 650�C for 2 h using

muffle furnace, S.M. Scientific Instrument (p) ltd. Jindal Company, Delhi, India 2–4 h).

Gross energy was determined on a Gallenkamp Ballistic Bomb Calorimeter-CBB 330

010L (Gallenkamp, Loughbrough, UK). Six subsamples from a pooled sample of 20 fishes

were analyzed for initial body composition. At the end of the experiment, 10 fish from each

replicate of dietary treatments were pooled separately and six subsamples were analyzed

for final body composition.

Determination of RNA and DNA

RNA and DNA were determined by the method of Schneider (1957). Muscle samples

(100 mg mL-1, w/v) were homogenized for 5 min in 5% trichloroacetic acid (TCA) at

90�C and then centrifuged at 5,000 rpm for 20 min. For the estimation of RNA, 2.0 mL of

distilled water and 3.0 mL of orcinol reagent were added in 1.0 mL of supernatant. The

reaction mixture was kept in boiling water bath for 20 min. The greenish-blue color thus

developed was read at 660 nm in a spectrophotometer (Genesis 10-UV, Thermo Spec-

tronic, Madison, USA). For DNA determination, 1.0 mL of distilled water and 4.0 mL of

freshly prepared diphenylamine reagent were added to 1.0 mL of the supernatant. The

reaction mixture was kept on a boiling water bath for 10 min. The blue color developed

was measured at 600 nm. Standard curves for RNA and DNA were drawn using different

concentrations of yeast RNA and calf thymus DNA, respectively. The values were

expressed as lg 100 mg-1 fish muscle tissue on dry basis.

Statistical analyses

At the end of the experiment, the following parameters were determined:

Absolute weight gain (AWG, g fish-1) = Final body weight-Initial body weight

Protein efficiency ratio (PER) = Wet weight gain (g)/Protein fed (g)

386 Aquacult Int (2012) 20:383–395

123

Specific growth rate (SGR, %) = In final body weight-In initial body weight/No.

of days 9 100

Feed conversion ratio (FCR) = Dry feed fed (g)/Wet weight gain (g)

Protein retention efficiency (PRE, %) = Protein gain/Protein intake 9 100

Energy retention efficiency (ERE, %) = Energy gain/Energy intake 9 100

Survival rate (SR, %) = Final number of fish/Initial number of fish 9 100

All growth data were subjected to one-way analysis of variance (Snedecor and Cochran

1968; Sokal and Rohlf 1981). Differences among treatment means were determined by

Duncan’s multiple range test at a P \ 0.05 level of significance (Duncan 1955). All the

growth parameters were subjected to a second-degree polynomial regression

(Y = aX2?bX?c) analysis as described by Zeitoun et al. (1976). Data were analyzed

statistically using Origin (version 6.1; Origin Software, San Clemente, CA) and Matlab

(version 7.1; MATLAB Software, Natick, MA).

Results and discussion

Growth performance

Growth performance of C. punctatus fingerlings in response to varying levels of dietary

protein is depicted in Table 2. The AWG, SGR and FCR improved significantly (P \ 0.05)

as dietary protein level increased from 300 to 450 g kg-1 of the diet. Maximum AWG

(8.11 g fish-1), SGR (1.82%) and best FCR (1.48) were obtained for the group fed dietary

protein at 450 g kg-1. Inclusion of dietary protein above this level (500 g kg-1) did not

produce any additional gain in weight or improvement in FCR. Moreover, a significant fall

in these parameters was recorded in fish fed dietary protein above 500 g kg-1. However, in

contrast to this, PER, PRE and ERE (Table 2) were maximum in the group fed dietary

protein at 400 g kg-1 (Table 2). No further improvement in these parameters was noted for

the groups fed dietary protein at 450 g kg-1. However, PER, PRE and ERE declined

significantly (P \ 0.05) with further increase in dietary protein levels at 500 and

550 g kg-1. The poorest AWG (4.38 g fish-1), SGR (1.21%), PER (1.02), PRE (13%),

ERE (35%) and FCR (3.27) were recorded in fish fed diet containing 300 g kg-1 protein.

Survival was not significantly affected by the varying levels of dietary protein and found to

range between 94 and 96% (Table 2).

Body composition

Data on body composition in response to varying levels of dietary protein are summarized in

Table 2 which indicates that body composition was significantly influenced (P \ 0.05) by the

protein levels in the diet. Body protein content increased significantly (P \ 0.05) with the

increased inclusion of dietary protein up to 400 g kg-1 and remained almost unchanged in

fish fed higher protein levels. Linear (P \ 0.05) increase in body fat was noticed with the

increase in dietary protein, whereas moisture content decreased significantly in contrast to the

body fat content. However, ash content was not significantly affected by the treatment levels.

RNA/DNA ratio

Nucleic acid indices were significantly influenced (P \ 0.05) by the levels of dietary

protein. Muscle DNA concentration was found to decrease significantly with the increase

Aquacult Int (2012) 20:383–395 387

123

Ta

ble

2G

row

th,

feed

con

ver

sio

n,

nu

trie

nt

rete

nti

on

and

bio

chem

ical

com

po

siti

on

of

fin

ger

lin

gC

.p

un

cta

tus

fed

die

tsw

ith

var

yin

gle

vel

so

fp

rote

in

Die

tary

pro

tein

lev

el(g

kg

-1

die

t)

Init

ial

30

03

50

40

04

50

50

05

50

Av

erag

ein

itia

lw

eig

ht

(g)

–4

.52

±0

.05

a4

.53

±0

.03

a4

.58

±0

.06

a4

.58

±0

.02

a4

.49

±0

.04

a4

.51

±0

.03

a

Av

erag

efi

nal

wei

ght

(g)

–8

.90

±0

.07

f1

0.3

7±

0.1

2e

12

.35

±0

.09

b1

2.6

9±

0.1

1a

12

.12

±0

.13

c1

1.5

3±

0.1

4d

Ab

solu

tew

eig

ht

gai

n(g

fish

-1)

–4

.38

±0

.02

f5

.84

±0

.04

e7

.77

±0

.08

b8

.11

±0

.06

a7

.63

±0

.03

c7

.02

±0

.02

d

Sp

ecifi

cg

row

thra

te(%

)–

1.2

1±

0.0

4e

1.4

8±

0.0

6d

1.7

7±

0.0

2b

1.8

2±

0.0

5a

1.7

7±

0.0

3b

1.6

8±

0.0

4c

Fee

dco

nv

ersi

on

rati

o–

3.2

7±

0.0

6a

2.1

2±

0.0

2b

1.6

5±

0.0

3c

1.4

8±

0.0

2e

1.4

9±

0.0

1e

1.5

8±

0.0

2d

Pro

tein

effi

cien

cyra

tio

–1

.02

±0

.02

d1

.35

±0

.03

b1

.52

±0

.02

a1

.50

±0

.02

a1

.34

±0

.01

b1

.20

±0

.01

c

Pro

tein

rete

nti

on

effi

cien

cy(%

)–

13.0

1±

0.2

1f

20

.03

±0

.17

d2

5.1

4±

0.2

7a

24

.11

±0

.19

b2

1.0

4±

0.2

3c

18

.03

±0

.21

e

Ener

gy

rete

nti

on

effi

cien

cy(%

)–

34.9

9±

0.3

5d

59

.03

±0

.41

c7

8.1

5±

0.6

1a

77

.13

±0

.64

a6

6.0

5±

0.5

7b

57

.91

±0

.51

c

Su

rviv

al(%

)–

94

±0

.86

a9

6±

0.9

1a

94

±0

.84

a9

6±

0.8

7a

95

±0

.81

a9

6±

0.9

3a

Mo

istu

re(%

)7

6.7

1±

0.4

17

6.8

4±

0.5

1a

75

.53

±0

.47

b7

4.4

8±

0.4

9c

73

.09

±0

.45

d7

2.1

5±

0.4

4e

71

.26

±0

.46

f

Pro

tein

(%)

12

.53

±0

.18

12

.68

±0

.07

d1

3.7

4±

0.1

7c

15

.17

±0

.27

a1

4.9

1±

0.1

5ab

14

.66

±0

.19

b1

4.5

7±

0.1

8b

Fat

(%)

3.6

8±

0.0

83

.41

±0

.04

e3

.92

±0

.06

d4

.57

±0

.03

c4

.83

±0

.07

c5

.62

±0

.05

b5

.98

±0

.04

a

Ash

(%)

3.2

1±

0.0

43

.19

±0

.02

a3

.23

±0

.06

a3

.21

±0

.02

a3

.25

±0

.02

a3

.24

±0

.01

a3

.22

±0

.03

a

RN

A(l

g1

00

mg

-1

dry

wei

ght

bas

is)

–7

84

±4

.02

d8

56

±4

.16

d9

87

±4

.1a

97

2±

3.1

2a

95

2±

3.8

b9

15

±4

.3c

DN

A(l

g1

00

mg

-1

dry

wei

gh

tb

asis

)–

46

8±

2.1

4a

39

5±

2.6

b3

28

±3

.5d

32

6±

2.6

2d

36

5±

2.8

3c

39

2±

3.5

b

RN

A/D

NA

rati

o–

1.6

8±

0.0

2e

2.1

7±

0.0

3d

3.0

1±

0.0

4a

2.9

8±

0.0

5a

2.6

1±

0.0

2b

2.3

3±

0.0

2c

Mea

nval

ues

of

3re

pli

cate

s±S

EM

;M

ean

val

ues

shar

ing

the

sam

esu

per

scri

pts

inth

esa

me

row

are

insi

gnifi

can

tly

dif

fere

nt

(P[

0.0

5)

388 Aquacult Int (2012) 20:383–395

123

in dietary protein up to 400 g kg-1 and remained almost insignificantly different for the

group fed dietary protein at 450 g kg-1 and then increased in 500 and 550 g kg-1 treat-

ments (Table 2). The muscle RNA concentration and RNA/DNA ratio were found to

increase significantly (P \ 0.05) in fish fed dietary protein up to 400 g kg-1. No signifi-

cant improvement (P [ 0.05) was found in muscle RNA concentration and RNA/DNA

ratio in fish fed 450 g protein kg-1 diet. A significant fall in muscle RNA concentration

and RNA/DNA ratio was recorded for the groups fed at surplus levels of dietary protein

(500–550 g kg-1).

To obtain more precise information on dietary protein requirement for the C. punctatusfingerlings, the growth parameters and RNA/DNA ratio were subjected to second-degree

polynomial regression analysis. Second-degree polynomial regression analysis of AWG

(Y) to dietary protein levels (X) yielded maximum AWG at a dietary protein level of

462.24 g kg-1. The relationship being;

Y ¼ �0:000143X2 þ 0:1322X � 22:55 R2 ¼ 0:970� �

The SGR% data (Y) when regressed against the dietary protein levels (X) yielded the

highest SGR% at a dietary protein level of 466.33 g kg-1. The relationship being;

Y ¼ �0:00002257X2 þ 0:02105X � 3:08486 R2 ¼ 0:983� �

When FCR data (Y) were regressed against dietary protein levels (X), best FCR was

obtained at a dietary protein level of 476.72 g kg-1. The relationship being;

Y ¼ 0:000058X2 � 0:0553X þ 14:54 R2 ¼ 0:973� �

However, second-degree polynomial regression analysis of PER (Fig. 1a), PRE%

(Fig. 1b), ERE% (Fig. 1c) and RNA/DNA ratio (Fig. 1d) yielded maximum PER, PRE%,

ERE% and RNA/DNA ratio to be at 438.46, 438.28, 444.43 and 440.44 g protein kg-1 of

the diet. The equations pertaining to these parameters are shown in the respective figures.

Protein is the most important and expensive item of the feed that should be supplied in

adequate amounts to support good growth with minimal cost. In the present study, weight

gain in C. punctatus fingerlings improved significantly with the increase in dietary protein

from 300 to 450 g kg-1. Inclusion of dietary protein at 500 g kg-1 exhibited no additional

gain in weight. However, significant growth retardation was recorded in fish fed still higher

level of dietary protein in this study. Similar trend of growth depression at surplus levels of

protein intake in the diets has also been observed in various other cultivable finfish species

such as bagrid catfish, Mystus nemurus (Ng et al. 2001); yellow catfish, Pseudobagrusfulvidraco (Kim and Lee 2005); pike perch, Sander lucioperca (Schulz et al. 2007); silver

barb, Puntius gonionotus (Mohanta et al. 2008); stinging catfish, Heteropneustes fossilis(Siddiqui and Khan 2009); African catfish, Clarias gariepinus (Farhat and Khan 2010).

This could be attributed to the reduction in the available energy for growth due to inad-

equate non-protein energy necessary to deaminate and excrete excess absorbed amino

acids (Vergara et al. 1996; Kim et al. 2002). Also, the diets in this study contained

incremental levels of cellulose from 0 to 104.2 g kg-1. The diets with highest level of

protein (550 g kg-1) contained maximum amount of cellulose. Higher cellulose level in

diet may be harmful to the growth of carnivorous fish. Since C. punctatus, the fish under

study, has been reported to harbor endogenous cellulolytic bacteria in its gut (Kar and

Ghosh 2008), the cellulose content of the diet may not be greater problem for the fish fed

surfeit levels of protein. Feeding 500 g kg-1 protein diet did not show any detrimental

effect neither on growth parameters nor on body composition but would be uneconomical

Aquacult Int (2012) 20:383–395 389

123

and hence inclusion of 450 g kg-1 dietary protein for the fingerling is adequate for growth

and more economical.

Energy retention is the amount of energy from feed that is retained for somatic growth (De

Silva and Anderson 1994). Energy retention can be increased either through reduced main-

tenance costs or by retaining consumed energy at levels above maintenance (Lin et al. 1979).

In this study, energy retention efficiency increased with the increase in dietary protein from

300 to 400 g kg-1 but decreased when protein level was further increased to 500–550 g kg-1.

This reduction could be caused by the utilization of part of the retained energy in the

deamination process of excess amino acids (Nuangsaeng and Boonyaratapalin 2001).

Protein efficiency ratio and protein retention efficiency were used as indices of protein

utilization in this study. Weight gain in the present study was found to increase with the

300 350 400 450 500 550

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

aYmax=1.57

Xmax=438.46 g kg-1

Y=-0.000026X2+0.0228X-3.429 (r2=0.974)

Pro

tein

eff

icie

ncy

rat

io (

PE

R)

Dietary protein levels (g kg-1 of the diet)

300 350 400 450 500 550

10

12

14

16

18

20

22

24

26

Pro

tein

ret

enti

on

eff

icie

cy (

PR

E%

)

Dietary protein levels (g kg-1 of the diet)

Xmax

=438.28 g kg-1

Y=-0.000593X2 + 0.5198X - 89.237 (r2=0.963)

Ymax=24.67%

b

Fig. 1 Second-degreepolynomial relationships of PER(a), PRE (b), ERE (c) and RNA/DNA ratio (d) to varying levelsof dietary protein

390 Aquacult Int (2012) 20:383–395

123

increasing levels of dietary protein up to 450 g kg-1, whereas the PER and PRE data did

not follow this trend. Increase in dietary protein improved these parameters up to

400 g kg-1 dietary protein. Decrease in the protein utilization above optimal level of

dietary protein is a well-documented phenomenon (Ng et al. 2001; Yang et al. 2003; Kim

and Lee 2005, 2009; Alam et al. 2008; Siddiqui and Khan 2009). The highest PER and

PRE values observed in the 400 g protein kg-1 group were due to the higher amount of

carbohydrate content in the 400 g kg-1 protein diet compared to the 450, 500 and

550 g kg-1 diets where it decreased with increasing the protein level (Table 1). The

greater the amount of carbohydrate, the lesser will be the probability of deaminating amino

acid and, therefore, the higher protein utilization efficiency and retention. When the dietary

supply of protein exceeds the requirement, large quantity of nitrogenous compounds is

300 350 400 450 500 55030

40

50

60

70

80

En

erg

y re

ten

tio

n e

ffic

ien

cy (

ER

E%

)

Dietary protein levels (g kg-1 of the diet)

Ymax=78.08%

Xmax

=444.43 g kg-1

Y=-0.0020X2+1.7955X-320.91 (R2=0.961)

300 350 400 450 500 550

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

RN

A/D

NA

rat

io

Dietary protein levels (g kg-1 of the diet)

Xmax=440.44 g kg-1

Y=-0.000062X2+0.0551X-9.348 (r2=0.915)

Ymax=2.89

c

d

Fig. 1 continued

Aquacult Int (2012) 20:383–395 391

123

used for gluconeogenesis resulting to an increased catabolism of dietary protein for energy

purposes (Fagbenro and Akegbejo-Samsons 2000). Since the diets were formulated to be

isocaloric, the quantity of dextrin as energy source decreased with the increase in protein in

the diet (Table 1). Diet containing 400 g protein kg-1 and 18.39 kJ g-1 energy with a

protein/energy ratio of 21.75 mg kJ-1 satisfied the protein and energy requirements for

C. punctatus fingerlings and resulting in maximum PER and PRE.

Based on the second-degree polynomial regression analysis of protein efficiency ratio,

protein retention, energy retention and RNA/DNA ratio, the dietary protein requirement for

the fingerlings was found to range between 438.28 and 444.43 g kg-1 of the diet which is

higher than the requirement reported for other carnivorous fish species like Indian catfish,

H. fossilis 400–430 g kg-1 (Siddiqui and Khan 2009), Malaysian catfish, M. nemurus420 g kg-1 (Khan et al. 1993) but lower than the requirement for striped murrel, C. striatus550 g kg-1 (Kumar et al. 2010) and comparable to the requirement reported for bagrid

catfish M. nemurus 440 g kg-1 (Ng et al. 2001) and African catfish, C. gariepinus430–460 g kg-1 (Farhat and Khan 2010). The differences among the dietary protein

requirements may be due to the differences in fish sizes and species (Luo et al. 2004),

experimental conditions (Kim and Lee 2009), diet formulations, methodology and

assessment criteria (Yuan et al. 2010).

Body composition traits mainly the protein and lipid contents are of particular interest in

nutritional studies due to their association with product quality. Therefore, they serve as

important indicators for the requirement of an essential nutrient under question. From a

metabolic point of view, data suggest that feeding dietary protein above optimum level

(400 g kg-1) could not be used for body protein synthesis or tissue building. Body protein

content of the fish tended to increase significantly with the increased inclusion of dietary

protein up to 400 g kg-1 beyond which it remained almost unchanged indicating that

higher levels of dietary protein yielded no particular benefit in improving the carcass

quality of the fish. Additionally, fish fed higher levels of dietary protein (500 and

550 g kg-1) exhibited significantly higher body fat content which has also affected the

carcass quality as increase in body fat is not desirable. The high body fat observed in diets

containing protein above the optimum level is probably caused by the deamination of the

excess protein, thus the remaining carbon skeletons are used to produce fat. Similar trend

was also recorded in yellow puffer, Takifugu obscyrus (Bai et al. 1999), black Sea Bass,

Centropristis striata (Alam et al. 2008), and Indian catfish, H. fossilis (Siddiqui and Khan

2009). From these data on body composition, we further concluded that diet containing

400 g kg-1 protein is optimum for improving the growth performance, carcass quality and

maximizing the protein retention in fingerling C. punctatus.

The highest RNA concentration and RNA/DNA ratio at 400 g kg-1 dietary protein

indicate that the fish were able to synthesize more RNA than other treatments. Mohanta

et al. (2008) reported similar trend for silver barb, P. gonionotus, up to 300 g kg-1 protein.

The decrease in RNA concentration and RNA/DNA ratio in fish fed dietary protein at 500

and 550 g kg-1 points to the fact that there are limits for the amount of dietary protein

intake that fish can convert to its body material (Love 1980). In this study, a significant

reduction in DNA concentration of fish muscles was also noticed with the increase in

dietary protein up to 400 g kg-1. Further increase in dietary protein (450 g kg-1) did not

show significant differences in the DNA concentration. However, further inclusion of

dietary protein at 500 and 550 g kg-1 showed slight increment in DNA concentration.

Since DNA carries the genetic material in each cell and is present in the nucleus in fixed

quantities (Love 1980), it is considered an index of cell numbers contributing to unit

weight of tissue. In fish losing weight, the size of cells decreases and thus number of cells

392 Aquacult Int (2012) 20:383–395

123

contributing to unit weight of tissue increases, enhancing the number of nuclei and con-

tributing to increased DNA content. In a weight-gaining fish, on the other hand, the DNA

content becomes diluted with larger volume of cells per unit weight (Khan and Jafri 1991).

Similar microscopic observations related to increase in cell size were also observed in fish

under study.

Based on the second-degree polynomial regression analysis of PER, PRE, ERE

and RNA/DNA ratio data, it appears that the optimum dietary protein requirement for

C. punctatus fingerlings is 438.28–444.43 g kg-1 with a protein/energy ratio of

23.83–24.17 mg kJ-1. Results of this study would be useful in developing protein-bal-

anced cost-effective commercial feeds for the intensive culture of C. punctatus fingerlings.

Channa punctatus is very popular and highly demanded fish in most of the tropical

countries including India. Information on most of its essential nutrient requirements are not

available which is a bottleneck in developing nutritionally balanced feeds for its intensive

culture. Therefore, further studies are required to generate data on nutrient requirements of

this fish, so that nutritionally balanced feeds could be developed to commercialize the

production of C. punctatus.

Acknowledgments The authors are grateful to the Chairman, Department of Zoology, Aligarh MuslimUniversity, Aligarh, India, for providing necessary laboratory facilities and also to Prof. John E. Halver forsupporting the Fish Nutrition Research Programme at this laboratory. We also gratefully acknowledge thefinancial assistance of University Grant Commissions awarded to one of us (Seemab Zehra) and generousfunding received under DST-FIST Programme, New Delhi, in the priority area of Fish Nutrition.

References

Abdel-Hameid NA, Abidi SF, Khan MA (2011) Dietary vitamin E requirement for maximizing the growth,conversion efficiency, biochemical composition and haematological status of fingerling Channapunctatus. Aquac Res doi:10.1111/j.1365-2109.2011.02819.x

Abidi SF, Khan MA (2009) Dietary arginine requirement of fingerling Indian major carp, Labeo rohita(Hamilton) based on growth, nutrient retention efficiencies, RNA/DNA ratio and body composition.J Appl Ichthyol 25:707–714

Akpinar Z, Sevgili H, Ozgen T, Demir A, Emre Y (2011) Dietary protein requirement of juvenile shi drum,Umbrina cirrosa (L). Aquac Nutr. doi:10.1111/j.1365-2109.2011.02845.x

Alam MS, Watanabe WO, Carroll PM (2008) Dietary protein requirements of Juvenile Black Sea Bass,Centropristis striata. J World Aquac Soc 39:656–663

AOAC (1995) Official methods of analysis of the association of official analytical chemists. In: Cunniff P(ed) Association of official analytical chemists, 16th edn. Arlington

APHA (1992) Standard methods for the examination of water and waste water, 18th edn. American PublicHealth Association, Washington, DC

Bai SC, Wang XJ, Cho ES (1999) Optimum dietary protein level for maximum growth of juvenile yellowpuffers. Fish Sci 65:380–383

Bhuiyan AS, Afroz S, Zaman T (2006) Food and feeding habit of the juvenile and adult snake head, Channapunctatus (Bloch). Life Earth Sci 1:53–54

Bulow FJ (1987) RNA-DNA ratio as indicators of growth in fish: a review. In: Summerfelt RC, Hall GE(eds) The age and growth of fish. Iowa State University Press, Ames, pp 45–64

Chondor SL (1999) Biology of finfish and shellfish. SCSC Publishers, HowrahDe Silva SS, Anderson TA (1994) Fish nutrition in aquaculture. Chapman and Hall, New York, p 319Duncan DB (1955) Multiple range and multiple ‘F’ tests. Biometrics 11:1–42Fagbenro O, Akegbejo-Samsons Y (2000) Optimum protein requirements of diets formulated for economic

growth of Heterotis niloticus (Cuvier 1829). J Fish Technol 2:20–29Farhat, Khan MA (2010) Effects of varying dietary protein levels on growth, feed conversion, nutrient

retention efficiencies and biochemical composition of fry African catfish, Clarias gariepinus (Burc-hell). J Appl Aquac (Accepted)

Halver JE (2002) The vitamins. In: Halver JE, Hardy RW (eds) Fish nutrition, 3rd edn. Academic Press, SanDiego, pp 61–141

Aquacult Int (2012) 20:383–395 393

123

Jindal M, Yadava NK, Jain KL, Gupta RK (2010) Effect of two dietary protein levels on body weight andcomposition in Channa punctatus (Bloch) fingerlings. Turkish J Fish Aquat Sci 10:203–208

Kar N, Ghosh K (2008) Enzyme producing bacteria in the gastrointestinal tracts of Labeo rohita (Hamilton)and Channa punctatus (Bloch). Turkish J Fish Aquat Sci 8:115–120

Khan MA, Jafri AK (1991) Protein and nucleic acid concentrations in muscle of the catfish Clariasbatrachus at different dietary protein levels. Asian Fish Sci 4:75–84

Khan MS, Ang KJ, Ambak MA, Saad CR (1993) Optimum dietary protein requirement of a Malaysianfreshwater catfish, Mystus nemurus. Aquaculture 112:227–235

Kim LO, Lee SM (2005) Effects of dietary protein and lipid levels on growth and body composition ofbagrid catfish, Pseudobagrus fulvidraco. Aquaculture 243:323–329

Kim SS, Lee KJ (2009) Dietary protein requirement of juvenile tiger puffer, (Takifugu rubripes). Aqua-culture 287:219–222

Kim KW, Wang XJ, Bai SC (2002) Optimum dietary protein level for maximum growth of juvenile oliveflounder, Paralichthys olivaceus (Temminck et Schlegel). Aquac Res 33:673–679

Kumar D, Marimuthu K, Haniffa MA, Sethuramalingam TA (2010) Optimum dietary protein requirement ofstriped murrel Channa striatus fry. Malays J Sci 29:52–61

Lin PY, Romsos DR, Vander Tuig JG, Leveille GA (1979) Maintenance energy requirements, energyretention and heat production of young obese (ob/ob) and lean mice fed a high-fat or a high-carbo-hydrate diet1. J Nutr 109:1143–1153

Love RM (1980) The chemical biology of fishes, vol II. Academic Press, LondonLuo Z, Liu YJ, Mai KS, Tian LX, Liu DH, Tan XY (2004) Optimal dietary protein requirement of grouper

Epinephelus coioides juveniles fed isoenergetic diets in floating net cages. Aquac Nutr 10:247–252Mahajan CL, Agrawal NK (1979) Vitamin C deficiency in Channa punctatus Bloch. J Fish Biol 15:613–622Marimuthu K, Arokiaraj AJ, Haniffa MA (2009) Effect of diet quality on seed production of the spotted

snakehead Channa punctatus (Bloch). Int J Sustain Agric 1:6–9Mohanta KN, Mohanty SN, Jena JK, Sahu NP (2008) Protein requirement of silver barb, Puntius gonionotus

fingerlings. Aquac Nutr 14:143–152Mustafa S (1977) Influence of maturation on the concentrations of RNA and DNA in the flesh of the Catfish

Clarias batrachus. Trans Am Fish Soc 106:449–451Ng WK, Soon SC, Hashim R (2001) The dietary protein requirement of a bagrid catfish, Mystus nemurus

(Cuvier and Valenciennes), determined using semi purified diets of varying protein level. Aquac Nutr7:45–51

Nuangsaeng B, Boonyaratapalin M (2001) Protein requirement of juvenile soft-shelled turtle Trionyx sin-ensis Wiegmann. Aquac Res 32:106–111

Park SU, Lim HK, Han HS (2008) Changes in RNA/DNA ratio and growth of slime flounder, Microstomusachne, larvae until metamorphosis. J Appl Ichthyol 24:50–54

Saikia AK, Das DN (2010) Survival and growth of Channa punctatus (Bloch) under controlled condition incemented cistern. Sibcoltejo 5:78–81

Schneider WC (1957) Determination of nucleic acids in tissue by pantose analysis. In: Calowick SP, KaplonNO (eds) Methods of enzymology. Academic press, New York, p 680

Schulz C, Huber M, Ogunji J, Rennert B (2007) Effects of varying dietary protein to lipid ratios on growthperformance and body composition of juvenile pike perch (Sander lucioperca). Aquac Nutr 13:1–8

Siddiqui TQ, Khan MA (2009) Effects of dietary protein levels on growth, feed utilization, protein retentionefficiency and body composition of young Heteropneustes fossilis (Bloch). Fish Physiol Biochem35:479–488

Snedecor GW, Cochran WG (1968) Statistical methods, 6th edn. Iowa State University Press, AmesSokal RR, Rohlf FJ (1981) Biometry. W.H. Freeman and Company, New YorkVergara JM, Fernandez-Palacios H, Robaina L, Jauncey K, Higuera M, Izquierdo M (1996) The effects of

varying dietary protein level on the growth, feed efficiency, protein utilization and body composition ofgilthead sea bream fry. Fish Sci 62:620–623

Wee KL (1982) The biology and culture of snakeheads. In: Muir JF, Roberts RJ (eds) Recent advances inaquaculture. Westview Press, Boulder, pp 180–211

Yaakob WAA, Ali AB (1992) Simple method for backyard production of snake head (Channa striata Bloch)fry. Naga 15:22–23

Yang SD, Lin TS, Liou CH, Peng HK (2003) Influence of dietary protein level on growth performance,carcass composition and liver lipid classes of juvenile Spinibarbus hollandi (Oshima). Aquac Res34:661–666

Yuan YC, Gong SY, Luo Z, Yang HJ, Zhang GB, Chu ZJ (2010) Effects of dietary protein to energy ratioson growth and body composition of juvenile Chinese sucker, Myxocyprinus asiaticus. Aquac Nutr16:205–212

394 Aquacult Int (2012) 20:383–395

123

Zeitoun IH, Ullrey DE, Magee WT, Gill JL, Bergen WG (1976) Quantifying nutrient requirements of fish.J Fish Res Board Can 33:167–172

Zhang J, Zhou F, Wang LL, Shao QJ, Xu A, Xu JZ (2010) Dietary protein requirement of juvenile black seabream, Sparus macrocephalus. J World Aquac Soc 41:151–164

Aquacult Int (2012) 20:383–395 395

123