Abstract: The effect of C. dactylon incorporated into diet formulations on the growth and body composition of Indian major carp, Catla (Catla catla) were investigated in a 45 days feeding trial. Different concentration (0, 0.05, 0.5 and 5% with the total fish feed) of C. dactylon ethanolic extract was mixed with feed ingredient. The incorporation of C. dactylon mixed diet improved the feed conversion ratio and body composition in C. catla. The elevated levels of amylase and protease activity were found in hepatopancreas at 30th days of feeding trial in 0.5 and 5% level of mixed diet groups. Non-specific immune parameter of serum antiprotease activity was higher in 5% level of experimental group. The results indicate that 5% inclusion of C. dactylon mixed diet improves the growth performance, feed efficiency, body composition, digestive enzyme and antiprotease activity in C. catla. In general, this study demonstrated the benefits of incorporating C. dactylon into fish feeds.

INTRODUCTION

India is a biodiversity nation and it has a rich background in medicinal herbs, most of which have been used to treat human and animal diseases. Aquaculture is a fast developing industry in India. Fish farming and aquaculture industries take part in contributing fish protein to large Asian population (Ravenhalt, 1982). Fish is a good source of protein and also has essential amino acids with minerals like zinc, magnesium, sodium, etc. (Barlas, 1986). Development of aquaculture is mainly depended on availability of compatible and suitable diet. For the formation of fish diet, Feed Conversion Ratio (FCR) and Specific Growth Rate (SGR) are good tools to compute the acceptability and suitability of artificial diet. Normally, balanced fish feeds contain fishmeal, deoiled cakes and rice bran.

The research mainly focuses on alternative sources of feed ingredients, the main reasons being escalating cost and uncertainty of constant supply and improves the disease resistant capability through prophylactic treatment of normal feed ingredients. Already different plant materials have been studied widely for their nutritive values (Shetty and Nandesha, 1990; Ray and Das, 1992; Harish and Gajaria, 1995; Nandesha et al., 2001).

Many of medicinal herbs and their chemical components are used as an immunostimulants which are used in artificial diet preparation, aquaculture research and their practices. Many of the herbal plants have the ability to inhibit the microbial pathogens and activate the immunity (Immanuel et al., 2004; Chansue et al., 2000; Dügenci et al., 2003). Immunostimulation is an alternative effective method against vaccination. It may be achieved through only feed supplement.

Several ayurvedic medicinal plants are acting as a powerful immunomodulators. Now-a-days, supplemental treatment is popular for preventing the diseases in aquatic animals. Moreover, they are cheaper, safer and biocompatible. Respiratory burst activity of phagocytic cells and plasma lysozyme activity have been significantly increased in common carp (Cyprinus carpio) and large yellow croaker (Pseudosciena crocea) after feeding with Astragalus membranaceus and Angelica sinensis mixed diet (Jian and Wu, 2003, 2004).

The present study was conducted to evaluate the effects of Cynodon dactylon L. mixed artificial diet on growth, survival, body composition, digestive enzyme activity and antiprotease activity of experimental fish Catla catla.

MATERIALS AND METHODS

Preparation of ethanolic extract of Cynodon dactylon L.: C. dactylon L. was collected from Madurai, Tamil Nadu, India and then transferred to PRIST University, Thanjavur, Tamil Nadu, India. It was taxonomically identified and authenticated by Rev Dr. S. John Britto SJ, Director, The Rapinat Herbarium and Centre for molecular systematics, St. Joseph College (Autonomous), Thiruchirapalli, Tamilnadu, India. The voucher specimen was deposited at the Rapinat herbarium and the voucher number is RHCD BP18. Fresh plant of C. dactylon L. was cleaned and shade dried. The dried plants were pulverized by an electrical blender and passed through 20 μ mesh sieve. A powdered plant (550 gm) was extracted successfully with 1:2 w/v in 70% ethanol by using soxhlet apparatus. The extraction was carried out in 24 h at room temperature with mild shaking (Chopra et al., 1992). The extract was filtered and concentrated at 45°C using rotary vacuum evaporator under reduced pressure. The yield of extract was 12.2% w/w in terms of dried starting material. The extract was stored in tight containers in desiccators.

Experimental diet preparation: Artificial balanced diet was prepared by using fish meal, fish grower, wheat flour, cod liver oil (Universal medicare Pvt. Ltd.), vitamin premix (Vetsfarma Ltd.) as feed ingredients which contain 20% carbohydrate; 41% protein; 15% lipid and 9% ash (Table 1). Diet groups were designed to provide with different concentrations of 0.05, 0.5 and 5% ethanolic extract of C. dactylon mixed with the normal diet before pelletization.

All dietary ingredients were mixed thoroughly, moistened, cold-pelleted with a pelletizer and dried at 40°C for 24 h. Diets were stored at -20°C. The dried pellets were hand crumbled into small pieces and stored in airtight PVP containers.

Proximate composition analysis: Proximate composition of feed ingredients were analysed by the following method of AOAC (2003). Moisture was determined by oven drying method. Crude protein and fat content were analysed by Kjeldahl and Soxhlet apparatus (Tempo, Bombay, India). Total ash content was determined by burning the sample at 500°C for 10 h in a muffle furnace.

Experimental animal and their maintenance: Similar age groups of Indian major carp, Catla catla was obtained from Golden fish farm Karandhai, Thanjavur, Tamil Nadu, India. Average weight of fishes used for the experiment was 88.05±4.75 g. All fishes were maintained in Fiber Reinforced Plastic (FRP) tanks.

The water was replaced once in 2 days to maintain the water quality. Bore well water was used to rear the fish with 2 h aeration daily. Dissolved oxygen, dissolved carbon dioxide, pH and total alkalinity of water were monitored at weekly intervals (APHA, 1985). Fishes were kept at the ambient, uncontrolled temperature of 28±2°C under the natural photoperiod. Fishes were acclimated for 15 days and fed ad libitum with balanced fish diet prepared in the laboratory.

Table 1:	Composition of diet ingredients used for experiments

Feeding trial: The feeding trial was conducted over a period of 45 days. The test diets were fed with apparent sanitation twice a day at 09.00 and 18.00 h during 45 days of experimental period. Experimental fishes were fed with supplemented diet at the rate of 2% of their body weight per day.

Fishes were randomly divided into 4 groups, namely, Diet Group I (DG1), Diet Group II (DG2), Diet Group III (DG3) and Diet Group IV (DG4) and they were fed with 0, 0.05, 0.5 and 5% plant extract mixed diet, respectively. The initial body weights of each fishes were recorded. Fishes from each tank were weighed randomly at 10 days of interval. The experiment was conducted in triplicate to determine average readings. The morphological growth parameters were calculated as follows:

(1)

Where:

(2)

(3)

On termination of the experiments all the surviving fishes were harvested, weighed individually and a portion of the dermal muscle was dissected for proximate analysis of protein, carbohydrate, moisture, ash and fat content of the muscle as per the methods of AOAC (2003).

Digestive enzyme activity: Hepatopancreas of fishes were collected every 10 days of experimental period for measure the digestive enzyme activity. Fishes were taken at morning 9-11 a.m. for the removal of hepatopancreas which were kept in frozen condition at -70°C until further use. Approximately 1.0 g of hepatopancreas were homogenized in chilled 10 mM Tris-HCl buffer at pH 7.5 and enzyme extracts were obtained after centrifugation at 10,000 g for 30 min at 4°C. The supernatant of each sample was assayed in triplicate. Total soluble protein was measured by the method of Bradford (1976) using bovine serum albumin as a standard. Amylase activity was assayed by the Bernfeld (1955) method using soluble starch as the substrate and react with 3, 5-dinitrosalicylic acid. Total protease activity was assayed by the method of Anson (1938) with slight modification. Casein used as the substrate and react with Folin reagent. The absorbance of enzyme activities was measured by using a Techcomp UV-2310 spectrophotometer at 630 nm.

Immunization of fish: Another set of experimental diet groups were fed with the supplemental diet at the rate of 2% of the body weight for 30 days. On 5th day, 500 μL of 20% suspension of RaRBC in phosphate buffered saline were injected intraperitoneally to the fishes using 1 mL tuberculin syringe fitted with 28 G needle.

Collection of blood samples: After immunization, blood was collected at 7th, 14th, 21st and 28th day of experimental period. For bleeding each fishes were individually caught using a dip net and were bled from common cardinal vein using 1 mL tuberculin syringe fitted with 24 gauge needle (Mickael et al., 1994).

In order to sample the blood for serum separation, 200 μL of blood was drawn and whole bleeding procedure was completed within 1 min. The blood was collected in serological tubes and allowed to clot at room temperature. The clot was then spun down at 400 g for 10 min. The serum collected by aspiration was stored in sterile eppendorf tubes at -20°C for further use.

Assay of serum anti-trypsin activity: Serum anti-protease activity was performed by incubating 10 μL of serum with 20 μg of trypsin dissolved in 100 μL of Tris-HCl (50 mM, pH 8.2). In serum blank, 100 μL of Tris-HCl was added to 10 μL of serum, instead of trypsin in Tris-HCl and in the positive control, no serum was added to trypsin.

All tubes were filled with 200 μL of Tris-HCl and incubated for 1 h at room temperature. After the incubation, 2 mL of 0.1 mM substrate BAPNA (Na-benzoyl-DL-arginine-p-nitroanilide HCl, Sigma chemicals) was dissolved in Tris-HCl (containing 20 mM calcium chloride) which was added to all tubes and again incubated for further 15 min.

At the end of incubation, the reaction was stopped by adding 500 μL of 30% acetic acid. The optical density was measured at 410 nm by using UV-Visible spectrophotometer (Techcomp UV-2310). The percentage of trypsin inhibition was calculated as described by Rao and Chakrabarti (2004):

Where:

Statistical analyses to compare the mean differences among each diet groups for all the parameters was computed by student t-test at p<0.05 level. All treatments were assayed in triplicate.

RESULTS

Preparations of diet for the experiments were shown in Table 1 and the percentage proximate composition of the feed ingredients used in the trial (DG1-DG4) is shown in Table 2. The nutritional profile of C. dactylon mixed diet is almost equal to the control feed ingredients. Lipid level of the control and experimental diets do not differ largely but crude protein content in the DG4 diet increased marginally. Ash content in the DG1 diet was lower than DG4, probably due to reduction of percentage of C. dactylon extract mixed in the content of diet.

Table 2:	Proximate composition of the experimental diets (%)
Values represent means of duplicate samples DG1-DG4 are represented diet group I-IV, respectively

Table 3:	Feed conversion and morphological parameters in C. catla after feeding with C. dactylon supplemented feeds for 45 days
Values with different superscripts are significantly different (p<0.05). DG1-DG4 are represented diet group I-IV, respectively; IBW-Initial Body Weight (g), FBW-Final Body Weight (g), NWG-Net Weight Gain (g), ADG-Average Daily Growth (g), SGR-Specific Growth Rate, FCR-Feed Conversion Ratio and SR-Survival Ratio

Table 4:	Body composition of C. catla fed with different experimental diets (%)
Values with different superscripts are significantly different (p<0.05). DG1-DG4 are represented diet group I-IV, respectively

Fig. 1:	Specific activity of enzymes in digestive gland of C. catla fed with different experimental diets. Statistical differences (p<0.05) among groups are indicated by different letters. No significant differences appear among the groups marked with the same letter. DG1-DG4 are represented diet group I-IV, respectively

Various feed conversion parameters were studied and shown in Table 3. When compared to other diet groups, a significant difference (p<0.05) was observed in final weight of DG3 and DG4. However, there were no significant difference found among DG1 and DG2. Net and average weight gains are higher in DG3 and DG4 than other groups. Specific growth rate linearly increased in the C. dactylon mixed diet groups.

Even though, no significant difference (p<0.05) was found among the four groups, noticeable Survival Rate of fish (SR 100%) in all experimental groups indicated that the culture condition and composition diets provided to them were acceptable. According to the results of proximate composition of body moisture and ash, significant (p<0.05) differences were not found among the experimental groups (Table 4). Increased level of protein and lipid were found in all C. dactylon incorporated diet groups than DG1 while slight variations observed in the moisture and ash contents. However, crude protein and lipid content were found to be significantly (p<0.05) different among the experimental groups. Specific enzyme activities of amylase and protease activity were recorded at every 10 days of interval in C. dactylon mixed diet treated C. catla (Fig. 1).

Fig. 2:

Fish were immunized with rabbit RBC. Control groups were fed with normal diet and the experimental groups were fed with experimental diet. Total 4 weeks prior to immunization till the end of the experiment. Serum was collected from individual fishes for 4 weeks after immunization. The percentage of trypsin inhibition for each individual serum was determined. The values represented were the mean±SE of 4 fishes (p<0.05). DG1-DG4 are represented diet group I-IV, respectively

Specific activities of amylase and protease activity were significantly (p<0.05) higher in all experimental diet group than the control diet group throughout the experimental period. In DG4, both amylase and protease activity were significantly (p<0.05) higher at 30 days of feeding than DG1-DG3.

Whereas at the end of 40 days of experimental period, all groups of enzyme activity were reduced. Even though, experimental group of DG2-DG4 were slightly higher than DG1. The trend of dominating specific amylase and protease activity in C. catla fed C. dactylon mixed feed was continued until the end of the 40 days. The level of antiprotease activity in serum of test groups exhibited more than the control group throughout the study period (Fig. 2).

Antiprotease activity of DG1-DG4 were significantly (p<0.05) higher during all experimental days. From first and second c ollection (7th and 14th day, respectively) of blood, the serum antiprotease activity was slightly higher than third and fourth collection (21st and 28th day, respectively).

DG3 and DG4 maintained their antiprotease activity in almost all experimental days. Present study revealed that the experimental diet of C. dactylon play a vital role in enhancement of serum antiprotease activity.

DISCUSSION

The results of the present study clearly show that dietary C. dactylon extract supplementation enhances the growth of C. catla. Kono et al. (1987) experimented that the feeding of supplemented diet containing 10% chitin, chitosan or cellulose had not affected the growth of red sea bream, Japanese eel and yellow tail. Dietary chitosan and levamisole supplementation enhances the growth of common carp (Gopalakannan and Arul, 2006). On the contrary, depressed growth in tilapia after feeding with chitin and chitosan at 2, 5 and 10% level were observed by Shiau and Yu (1999).

In the results, net weight, average weight and specific growth rate were increased in the C. dactylon mixed diet groups. Similarly, Sandbrook and Hepher (1978) proved that the better growth of fish fed on algae enriched diets than conventional diets. Possibly, the presence of herbs C. dactylon, FCR value was also increased significantly (p<0.05%) in C. catla between the control and experimental diets. Likewise, increased FCR value found in Sarathrodon niloticus tilapia fingerlings fed with Caldophora glomerata incorporated experimental diet which may due to the higher lysine content in the alga (Appler and Jauncey, 1983).

The high FCR in fish fed with DG3 and DG4 diets could also activate the absorption of diet ingredient in C. catla by fibre rich herbs. Fibre fraction defines extent and rate of feed digestibility (Rubanza et al., 2005). SGR and FCR were significantly increased in DG3 and DG4 of experimental diet groups and attained 100% of SR in all experimental diet groups which proved that the composition of diet is suitable for this experiment and the fish. The significant increase in SGR, FCR and 100% survival rate in the experimental groups can be attributed to the presence of higher essential amino acids in C. dactylon (Stewart, 1973). Significant responses were found in survival, growth, body composition and digestive enzyme activity of white shrimp Litopenaeus vannamei by treating medicinal herbs and Bacillus (Yu et al., 2009). High body condition scores and average daily growth observed in supplemented steers could also be due to high dietary protein and energy intake (Rubanza et al., 2005).

In the study, the diet composition of crude protein and lipid levels in all experimental diet groups were relatively similar, there is no significant variation found in the diet group. In the proximate value of crude protein and lipid composition were higher in DG3 and DG4 than other diet groups. Likewise, direct relationship between the amount of Spirogyra incorporated diet and muscle protein and fat contents in C. catla were demonstrated by Harish et al. (2004). Dietary lipids in aquafeeds are an important source of energy and essential fatty acids (Sargent et al., 2002). Inclusion of 5% Ulva meal at low and high lipid levels significantly improves the growth performance, feed efficiency, nutrient utilization and body composition of Nile tilapia (Ergun et al., 2009). Optimum lipid levels results in improved growth rates, feed conversion ratios, nutrient utilization and reduced nitrogen excretion (Martins et al., 2007).

C. dactylon mixed diet treated C. catla were exhibited the increased level of specific activity of amylase and protease. Administration of MH and/or Bacillus bacteria to shrimps resulted in an increase in the specific activity of amylase and protease in the shrimps’ digestive gland (Yu et al., 2009). The results were also found that the enhancement in growth parameters and specific activities of digestive enzymes of fish. The noticed changes in digestive enzymes may have led to enhanced digestion and increased absorption of feed which in turn contributed to the progression in growth of fish.

Medicinal herbs contained potent bioactive substances which may influence digestive processes by enhancing or impairing enzyme activity and improving or diminishing digestibility of nutrients (Lin et al., 2006). Principally, α1 protease inhibitor and α2 macroglobulin play a role in restricting the ability of bacteria to invade and grow in fish by acting against proteases from pathogenic organisms (Ellis, 2001). The present study revealed that the experimental diet containing C. dactylon has to develop the non specific immunity in C. catla. C. dactylon play a vital role in enhancement of serum antiprotease activity in all experimental days.

This is in agreement with the observation of Rao and Chakrabarti (2004) that the feeding of Catla catla with Achyranthes aspera (0.5%) mixed diet for 4 weeks enhanced the level of serum antiprotease which might provide the resistance against the bacterial pathogens. Experimental result of Tafalla et al. (1999) plasma bactericidal activity and total protein concentrations were considerably increased by oral administration of oxytetracycline to turbot Scophthalmus maximus. Magnadottir (2006) stated that the antiprotease activity was high in serum by immunization or infection.

Similar result was also found in aqueous extract of Eclipta alba increased the serum antiprotease activity in Oreochromis mossambicus after 2 or 3 weeks of feeding (Christybapita et al., 2007). Similarly, the antiprotease activity enhanced in rainbow trout by experimental diet containing taxanthin, a carotenoidfrom natural source (Carophyll pink) supplemented for 4 months (Thomson et al., 1995). When fishes were fed with C. dactylon mixed diet, the protease inhibitor levels were enhanced in C. catla. Thus, the results revealed that the fish can defend more strongly against invading pathogens.

CONCLUSION

The results of the present study clearly demonstrate that C. dactylon, a natural weed can serve as an alternative replacement to the costly feed ingredients like deoiled groundnut cake, jowar powder and reduce the additional expenditure in disease management for the vaccine or immunomodulator/immunostimulator in the feed for C. catla. However, complete investigation is required in long-term feeding trials to assess the potential of C. dactylon and optimum dietary inclusion levels.

ACKNOWLEDGEMENT

The researchers are grateful to PRIST University for awarding the research fellowship.