MATERIALS AND METHODS

This work was conducted from December 2003 to March 2004 using common carp seed obtained from the fish farm of the Department of Fisheries, Karnataka.

Diets: Four fish meal free, low protein (17.73-18.91%) test diets (T₀, T₁, T₂ and T₃) were formulated (Varghese et al., 1976) incorporating high levels of maize (Table 1). Dietary ingredients (groundnut cake, rice bran and maize) procured locally were dried, pulverized and sieved to obtain uniform particles (400 mm size). Fish (sardine) oil was incorporated at 3, 6 and 9% levels in diets T₁, T₂ and T₃, respectively. Diet T₀ without oil supplementation served as the control. Oil incorporation to the diets was done by adding requisite amount of oil to 250 mL of water containing a few drops of Tween-80 (Polysorbate-80, Himedia Laboratories, Mumbai, India), mixing thoroughly with the help of a glass rod and using the suspension along with additional 550 mL of water per kg of ingredient(1:0.8 ratio). The diets were prepared following the method described by Jayaram and Shetty (1981) to obtain 3 mm diameter pellets that were dried soon after pelleting in a thermostatic oven at a temperature of 40°C and packed in heavy duty plastic bags.

Experimental set up: The experiment was carried out over a period of 120 days in 12 outdoor cement tanks of 18 m² each, with a 15 cm thick soil base. The tanks were cleaned and dried, limed at 400 kg ha^-1 (0.72 kg tank^-1) and initially fertilized with poultry manure at 2000 kg ha^-1; subsequent fertilization was done at 5% of the initial dose of poultry manure every 15 days. Ground water was used to fill the tanks, maintaining a depth of 90±5 cm throughout the experimental period. Common carp fingerlings (av. wt. 2.21-2.24 g) were stocked at a density of 1 per m² (18/tank). The four diets were fed to triplicate group of fish once daily in the morning at 5% body weight as per Varghese et al. (1976), using trays suspended into the tanks 50 cm below the water surface. The fish were sampled at 15-day intervals for measuring growth. The quantity of feed given was adjusted after each fish sampling, on the basis of new weight of the fish. On termination of the experiment, the surviving fish were weighed, based on which the following parameters were calculated.

Table 1:	Ingredient proportion and proximate composition of diets and ingredients
Values are as Mean±SD, *Supplevite-M (Sarabhai Company Ltd., India)

Water quality: Water quality parameters viz. temperature, pH, dissolved oxygen, free carbon dioxide and total alkalinity were determined every 15 days, by collecting samples between 07.00 and 08.30 h. Water temperature was recorded using a thermometer while pH was measured with a digital pH meter (LI-120, ELICO, India). Dissolved oxygen, free carbon dioxide and total alkalinity were determined following standard procedures (APHA, 1992). Plankton samples were also collected on fish sampling days, using a net made of No. 30 bolting silk cloth having 60 μm mesh size, by filtering 100 L of water from different locations in each experimental tank. Quantitative estimation of plankton was done by the direct census method using a Sedgewick Rafter Cell having 100 equal squares (Jhingran et al., 1969). The planktonic organisms were identified up to the generic level. Wet and dry weights of plankton were also determined.

Biochemical composition: Proximate com position of ingredients, diets and fish carcass was analyzed in triplicate. Three fish from each treatment were randomly selected for carcass analysis. Protein was determined by Kjeltec (Tecator-1002), Lipid by Soxtec (Tecator-1043) and fibre by Fibretec (Tecater-1017) systems. Ash was analyzed by incineration (AOAC, 1995) and NFE was computed by the difference method (Hastings, 1976). The energy content of the feed ingredients and diets was calculated using values of 22.6 kJ g^-1 for protein, 38.9 for lipid and 17.2 kJ g^-1 for carbohydrate as NFE (Mayes, 1990).

Enzyme assay: The activity of digestive enzymes viz. amylase, protease and lipase in the hepatopancreas and intestine of the experimental fish was analyzed on termination of the experiment by the methods of Bernfeld (1955), Kunitz (1946) and Bier (1962), respectively, using six fish from each treatment. Enzyme activity is expressed in μm of product liberated per min per mg of tissue protein.

Statistical analysis: Comparison among different dietary treatments was done by one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test at p<0.05 (Duncan, 1955; Snedecor and Cochran, 1968).

RESULTS

The water quality parameters monitored ranged as follows: water temperature 17.4-23.5°C, pH 7.45-8.36, dissolved oxygen 6.30-8.83 mg L^-1, free carbon dioxide 0-2.4 mg L^-1 and total alkalinity (CaCO₃) 55.84-89.12 mg L^-1.

Among the feed ingredients, groundnut cake had the highest crude protein and fat (39.62 and 8.02%), rice bran the highest crude fibre (24.07%) and maize the highest NFE (74.44%) (Table 2). The control diet (T₀) had the highest protein and NFE (18.91and 47.40%) values while they were the lowest in diet T₃ (17.73 and 40.44%). In contrast, fat content was the least in diet T₀ (5.48%) and highest in diet T₃ (13.28%) (Table 1).

The phytoplankton in the different treatments belonged mainly to Cyanophyceae (Microcystis sp.; Spirulina sp.; Oscillatoria sp.), Chlorophyceae (Eudorina sp.; Scenedesmus sp.; Spyrogyra sp.; Volvox sp.; Ulothrix sp. and Pondorina sp.) and Bacillariophyceae (Melocera sp.; Navicula sp.; Synedra sp. and Fragilaria sp.). The important zooplankton encountered belonged to the groups Rotifera (Brachionus sp.; Asplanchna sp. and Filina sp.), Copepoda (Cyclops sp. and Diaptomus sp.), Cladocera (Moina sp.) and larval forms (crustaceans). Some small protozoans were found occasionally. Table 3 summarizes the number of phytoplankton and zooplankton encountered in the water samples from different treatments. Plankton density in the different treatments ranged from 190 No. L^-1 (T₂, Day 0) to 8292 No. L^-1 (T₂, Day 75) during the experimental period.

Table 2:	Proximate composition (%) of ingredients
Values are as Mean±SD

It was generally higher in the first half of the experiment, reaching peak values on the 75th day; thereafter, a decline was noticed (Table 3).

The mean final weight of common carp under T₂ (64.42 g) and T₃ (74.72 g) treatments was significantly (p<0.05) higher compared to that of the control (57.63 g). Specific growth rate (%) and FCR were the best (p<0.05) under T₃ treatment. FCR improved (p<0.05) with all levels of oil supplementation.

Table 3:	Mean plankton density (No. L-1) in different treatments
Values with same superscript in each column are not significantly different (p>0.05)

Table 4:	Growth parameters and carcass composition of common carp in different treatments
Values in parentheses indicate standard error, Values with same superscript in each row are not significantly different (p>0.05)

Table 5:	Digestive enzyme activity in common carp from different treatments
1Hepatopancreatic, Values in parentheses indicate standard error, Enzyme activity is expressed in μ moles of product liberated per minute per mg of tissue protein at 28°C. Values with same superscript in each column are not significantly different (p>0.05)

PER values were only marginally different (p>0.05) among treatments. The survival of fish ranged from 96.26 to 98.14% (Table 4).

Carcass protein, fat and ash content were affected by dietary oil supplementation (p<0.05). Fat increased with increasing dietary lipid level, the highest being 4.58% in T₂ and T₃ treatments. Fish from T₂ treatment had the highest carcass protein content of 15.51%. Fish under the different treatments had significantly (p<0.05) lower ash than that of the control (Table 4).

Protease activity in the treated fish showed a decrease in the hepatopancreas as well as intestine (p<0.05). Amylase activity was high but did not differ among treatments (p>0.05). Increased lipase activity was observed in the hepatopancreas with increasing level of dietary oil supplementation (Table 5).

DISCUSSION

Sardine oil supplementation of the diets led to an increase in energy and a decrease in protein content and P/E ratio (Table 1). An optimal P:E ratio is considered a crucial factor for protein use efficiency. Nonetheless, growth of fish receiving oil supplemented diets increased significantly over that of the control. This could be related to optimization of the available dietary protein for somatic growth while the lipid adequately meets the energy needs (Caballero et al., 1999; Satpathy et al., 2003). In an earlier study conducted under identical conditions using diets containing 10% fish meal (24% protein) and supplemented with sardine oil, common carp harvest weight ranged from 53.26 g on control diet with no oil addition to 68.78 g on diet with 6% supplemented oil (Manjappa et al., 2002). In contrast, in the present study, the best growth (74.72 g) was obtained with the 9% oil supplemented diet containing no fish meal (17.73% protein) as against the mean weight of 57.63 g of the control fish. This performance reflects the ability of common carp to grow well on a diet with no fish meal, when cultured in the fertilized system. The protein requirement of carps varies between 25-35%, depending upon age (Hossain et al., 1997). Since the diets employed in the present study had low protein (17.73 to 18.91%), it may be presumed that natural food has contributed to fish growth. The plankton number showed an increase till the 75th day of the experiment and then declined in all the treatments. Regular fertilization facilitated plankton multiplication while higher grazing with greater fish biomass and increase in energy demand as the experiment progressed would have led to the decrease noticed. Lovell (1975) observed that natural food plays a key role in the determination of dietary protein requirements of fish under pond conditions. Common carp being an omnivore must have been able to utilize some protein from detritus also, apart from plankton. When mirror carp was grown with both natural food and a high protein supplemental feed, fish growth and specific growth rate were positively correlated with the density of natural food (Lam and Shephard, 1988). Schroeder (1983) stated that the dietary protein level and also percent protein energy level in the diet of tilapia could be reduced in the presence of natural food as it can correct the deficient nutrient in the diet. Hepher (1988) reported 18-35% protein, 7-10% lipid and 27-48% ash content (dry matter basis) for planktonic algae in ponds which indicates the nutritive value of the natural food.

Higher growth of fish fed the oil supplemented diets in the present study in comparison with that of the control indicates protein sparing by lipid. Increasing the energy density of diets has been suggested as a strategy to spare protein and limit ammonia production for several fish species, including common carp (Steffens, 1996). If the growth of common carp obtained in this study is compared with that reported by Manjappa et al. (2002), 3% additional sardine oil supported growth surpassing that by 10% dietary fish meal. Gangadhara et al. (1997) found that the growth of rohu (Labeo rohita) fingerlings fed a diet containing 25% protein and 9% fat was comparable with those fed 30% protein and 6% fat, indicating that growth induced by 3% dietary oil is comparable to that produced by 5% dietary protein. Kheir and Saad (2003) who determined the suitable lipid level which spares dietary protein for maximum growth in three fish species viz. Oreochromis niloticus, Sarotherodon galilaeus and Hypophthalmichthys molitrix, found that a low protein level of 20% with a high lipid level of 6% produced nearly equal growth and FCR similar to that of high protein (30%) and low lipid (2%). They concluded that fish are able to store considerable quantities of lipid in their tissues for utilization as energy source in order to improve growth and feed utilization. Skalli et al. (2004) observed that increasing dietary lipid from 10.6 or 13.5 to 19.7% allowed the protein level to be decreased from 53 or 57 to 50% without affecting the SGR and FCR, suggesting that energy from lipid spares protein in fingerling Dentex dentex. Protein sparing by lipid has also been demonstrated in other fish species such as Rhamdia quelen (Meyer and Fracalossi, 2004), Epinephelus coioides (Luo et al., 2005) and Sander leucioperca (Schulz et al., 2008). More than one-third of dietary protein could be spared by replacing protein with lipid in the diet of L. rohita (Satpathy et al., 2003). The beneficial effects of increased dietary lipid are associated with low protein diets rather with high protein diets (Dias et al., 1998). Hu et al. (2007) reported maximum growth of yellowfin seabream at a dietary lipid level of 13.63%, a level close to that of 9% oil supplemented diet used in the present study (13.28%).

FCR of diets improved with oil supplementation which indicates that oil addition improved diet utilization. Similar observations have been made by Satpathy et al. (2003) in L. rohita and Cho et al. (2005) in Schophthalmus maximus and El-Marakby (2006) in Oreochromis niloticus. Addition of fish oil did not affect fish survival; it was 98.14% in all the treatments as against 96.26% of the control. Dietary oil supplementation enhanced carcass lipid content as has been reported in C. carpio fingerlings by Abbass (2007) and other species viz. Pseudoplatystoma coruscans (Martino et al., 2002), L. rohita (Satpathy et al., 2003), S. maximus (Cho et al., 2005), E. coioides (Luo et al., 2005), Sparus latus (Hu et al., 2007), E. malabaricus (Tuan and Williams, 2007), S. leucioperca (Schulz et al., 2008), E. bruneus (Yoshii et al., 2010) and also the protein content, Tor khudree (Bazaz and Keshavanath, 1993), L. rohita (Gangadhara et al., 1997) and S. maximus (Cho et al., 2005).

High amylase activity recorded in fish from different treatments, including control, in this study could be attributed to the high maize content of the diets. Warm water herbivorous or omnivorous fish utilize high levels of carbohydrate and the efficiency of dietary carbohydrate utilization is better in common carp (Wilson, 1994). Jafri et al. (1995) opined that carps utilize carbohydrate preferentially over fat due to high amylolytic activity. Increased lipase activity was observed in the hepatopancreas with the increasing level of dietary oil addition. Higher lipase activity has been reported in T. khudree, L. rohita and Dicentrarchus labrax fed oil supplemented diets (Bazaz and Keshavanath, 1993; Gangadhara et al., 1997; Peres and Oliva-Teles, 1999). Protease activity of the treated fish showed a decrease in the intestine as well as hepatopancreas. This could be because of the decreased dietary protein content following oil addition. Nonetheless, treated fish had higher carcass protein, reflecting protein synthesis and increased tissue production (Fafioye et al., 2005).

CONCLUSIONS

It is concluded that common carp grown in the fertilized system can perform well on a low protein fish meal free diet when the energy content of the diet is enhanced through oil supplementation. Further, sardine oil spares protein for growth and common carp is capable of effectively utilizing high amount of dietary maize.

ACKNOWLEDGMENT

We thank the Dean, College of Fisheries, Mangalore for providing facilities.