Aquacult Int (2012) 20:383–395 DOI 10.1007/s10499-011-9470-8
Dietary protein requirement for fingerling Channa punctatus (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 Ó 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 fingerlings (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, India e-mail:
[email protected]
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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 airbreathing 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
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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
-1
Calculated on the basis of fuel values 23.07, 20.19, 16.01 and 37.62 kJ g fat, respectively, as estimated on Gallenkamp ballistic bomb calorimeter
for casein, gelatin, dextrin and
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 fingerlings 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-
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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 determined using standard methods (AOAC 1995) for dry matter (oven drying at 105 ± 1°C for TM 22 h.), crude protein (Kjeltec Tecator 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 Spectronic, 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)
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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
123
123
–
–
–
–
76.71 ± 0.41
12.53 ± 0.18
3.68 ± 0.08
3.21 ± 0.04
Protein efficiency ratio
Protein retention efficiency (%)
Energy retention efficiency (%)
Survival (%)
Moisture (%)
Protein (%)
Fat (%)
Ash (%)
-1
dry weight basis)
–
–
b
b
d
c
a
b
c
d
a
d
f
d
a
a
d
e
a
1.68 ± 0.02
e
468 ± 2.14
a
784 ± 4.02
d
3.19 ± 0.02
3.41 ± 0.04
12.68 ± 0.07
76.84 ± 0.51
94 ± 0.86
34.99 ± 0.35
13.01 ± 0.21
1.02 ± 0.02
3.27 ± 0.06
d
2.17 ± 0.03
395 ± 2.6
b
856 ± 4.16
d
3.23 ± 0.06
3.92 ± 0.06
13.74 ± 0.17
75.53 ± 0.47
96 ± 0.91
59.03 ± 0.41
20.03 ± 0.17
1.35 ± 0.03
2.12 ± 0.02
1.48 ± 0.06
a
1.21 ± 0.04
d
5.84 ± 0.04
e
4.38 ± 0.02
e
f
10.37 ± 0.12
e
8.90 ± 0.07
4.53 ± 0.03a
f
350
4.52 ± 0.05a
300
a
3.01 ± 0.04
328 ± 3.5
d
987 ± 4.1
a
3.21 ± 0.02
a
4.57 ± 0.03
c
15.17 ± 0.27
a
74.48 ± 0.49
c
94 ± 0.84
a
78.15 ± 0.61
a
25.14 ± 0.27
a
1.52 ± 0.02
a
1.65 ± 0.03
c
1.77 ± 0.02
b
7.77 ± 0.08
b
12.35 ± 0.09
b
4.58 ± 0.06a
400
2.98 ± 0.05
a
326 ± 2.62
d
972 ± 3.12
a
3.25 ± 0.02
a
4.83 ± 0.07
c
14.91 ± 0.15
ab
73.09 ± 0.45
d
96 ± 0.87
a
77.13 ± 0.64
a
24.11 ± 0.19
b
1.50 ± 0.02
a
1.48 ± 0.02
e
1.82 ± 0.05
a
8.11 ± 0.06
a
12.69 ± 0.11
a
4.58 ± 0.02a
450
Mean values of 3 replicates±SEM; Mean values sharing the same superscripts in the same row are insignificantly different (P [ 0.05)
RNA/DNA ratio
DNA (lg 100 mg
RNA (lg 100 mg
–
–
Feed conversion ratio
dry weight basis)
–
Specific growth rate (%)
-1
–
Absolute weight gain(g fish )
–
Average final weight (g)
-1
–
Average initial weight (g)
Initial
Dietary protein level (g kg-1 diet)
c
2.61 ± 0.02
b
365 ± 2.83
952 ± 3.8
b
3.24 ± 0.01
a
5.62 ± 0.05
b
14.66 ± 0.19
b
72.15 ± 0.44
e
95 ± 0.81
a
66.05 ± 0.57
b
21.04 ± 0.23
c
1.34 ± 0.01
b
1.49 ± 0.01
e
1.77 ± 0.03
b
7.63 ± 0.03
c
12.12 ± 0.13
c
4.49 ± 0.04a
500
Table 2 Growth, feed conversion, nutrient retention and biochemical composition of fingerling C. punctatus fed diets with varying levels of protein
2.33 ± 0.02c
392 ± 3.5b
915 ± 4.3c
3.22 ± 0.03a
5.98 ± 0.04a
14.57 ± 0.18b
71.26 ± 0.46f
96 ± 0.93a
57.91 ± 0.51c
18.03 ± 0.21e
1.20 ± 0.01c
1.58 ± 0.02d
1.68 ± 0.04c
7.02 ± 0.02d
11.53 ± 0.14d
4.51 ± 0.03a
550
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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 treatments (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 significant 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. punctatus fingerlings, 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 0:000143X 2 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 0:00002257X 2 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 0:000058X 2 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, Pseudobagrus fulvidraco (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 inadequate 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
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390
Y=-0.000026X2+0.0228X-3.429 (r2=0.974)
a
Ymax=1.57
1.6
Protein efficiency ratio (PER)
Fig. 1 Second-degree polynomial relationships of PER (a), PRE (b), ERE (c) and RNA/ DNA ratio (d) to varying levels of dietary protein
Aquacult Int (2012) 20:383–395
1.5 1.4 1.3 1.2 1.1 1.0 0.9 Xmax=438.46 g kg-1
0.8 300
350
400
450
500
550
Dietary protein levels (g kg-1 of the diet)
Protein retention efficiecy (PRE%)
b
Y=-0.000593X2 + 0.5198X - 89.237 (r2=0.963)
26 24 Ymax=24.67%
22 20 18 16 14 12
Xmax=438.28 g kg-1
10 300
350
400
450
500
550
Dietary protein levels (g kg-1 of the diet)
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 maintenance 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
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c Energy retention efficiency (ERE%)
Fig. 1 continued
391
Ymax=78.08%
80
70
60
50
40 Xmax=444.43 g kg-1
30 300
350
400
450
500
550
Dietary protein levels (g kg-1 of the diet)
d
3.2
Y=-0.000062X2+0.0551X-9.348 (r2=0.915)
3.0
RNA/DNA ratio
2.8
Ymax=2.89
2.6 2.4 2.2 2.0 1.8 Xmax=440.44 g kg-1
1.6 300
350
400
450
500
550
Dietary protein levels (g kg-1 of the diet)
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
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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. nemurus 420 g kg-1 (Khan et al. 1993) but lower than the requirement for striped murrel, C. striatus 550 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. gariepinus 430–460 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
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contributing to unit weight of tissue increases, enhancing the number of nuclei and contributing 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-balanced 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 Muslim University, Aligarh, India, for providing necessary laboratory facilities and also to Prof. John E. Halver for supporting the Fish Nutrition Research Programme at this laboratory. We also gratefully acknowledge the financial assistance of University Grant Commissions awarded to one of us (Seemab Zehra) and generous funding received under DST-FIST Programme, New Delhi, in the priority area of Fish Nutrition.
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