1 Introduction

The focus in aquaculture diet development is largely on cost-effectiveness, since feed cost typically account for between 40 and 60% of the production cost in semi-intensive and intensive aquaculture systems (De Silva and Hasan, 2007). The two dietary options available for reducing production cost are the use of cheaper diets or diets that hasten growth, thereby reducing the period of culture. Feed additives that promote faster growth fall under the latter category. In aquaculture, the most utilized growth-promoting agents include probiotics, prebiotics, hormones, antibiotics, ionospheres and some salts (Faramarzi et al., 2011). Use of natural materials or plant-based growth promoters as feed additives to enhance feed utilization efficiency and productive performance is acceptable as potential alternatives to antibiotics in diets, because of the increased public awareness and ban on the use of antibiotics as growth promoters in aquaculture diets. Probiotics is a new concept in aquaculture (Li and Gatlin III, 2004) where the addition of microorganisms in diets has a positive effect on growth caused by the best use of carbohydrates, protein, and energy. Probiotics stimulate appetite and improve nutrition by the production of vitamins, detoxification of compounds in the diet, and by breakdown of indigestible components (Irianto and Austin, 2002). A number of commercially available probiotic feed additives have been shown to improve feed utilization and growth performance in fish and shellfish (Wang et al., 2005; Pooramini et al., 2009; Tewary and Patra, 2011; Seenivasan et al., 2012; Hossain et al., 2013; Saini et al., 2014, Mona et al., 2015; Gumus et al., 2016; Mohammadi et al., 2016).

The use of probiotics in fish farming has increased considerably as they have received greater attention in recent years (Li and Gatlin III, 2003; Yanbo and Zirong, 2006; El-Dakar et al., 2007; Abdel-Tawwab, 2012; Khalil et al., 2012; Heidarieh et al., 2013; Munir et al., 2016). Probiotics improve feed conversion efficiency and live weight gains (Saenz de Rodriguez et al., 2009). They may serve to keep the host intestinal microbial balance and improve growth performance of the respective culture species (Ezema, 2013). The benefits of yeast and bacterial probiotics upon the gastrointestinal microbial ecosystem in monogastric animals were reported by Chaucheyras-Durand and Durand (2010). Probiotics in aquaculture have several modes of action: competitive exclusion of pathogenic bacteria through the production of inhibitory compounds, improvement of water quality, enhancement of immune response of host species, enhancement of nutrition of host species through the production of supplemental digestive enzymes and direct uptake of dissolved organic material mediated by the bacteria (Garriques and Arevalo, 1995; Moriarty, 1997; Vine et al., 2004; Carnevali et al., 2006; Yang et al., 2009).

This investigation was undertaken to determine the effect of graded levels of dietary G-Pro on growth, body composition and digestive enzyme activity in common carp, Cyprinus carpio.

2 Materials and Methods

2.1 Feed preparation

Pelleted diets were prepared using locally procured ingredients viz. fishmeal, groundnut oil cake, rice bran and tapioca flour. All the ingredients were sieved using ISI standard mesh No.1. G-Pro, containing yeast (Saccharomyces cerevisiae) and B-complex vitamins, was obtained from Vetcare Division of Tetragon Chemie Ltd. Bangalore. Five isonitrogenous and isocaloric diets (T₀, T₁, T₂, T₃ and T₄) were formulated incorporating 0, 1, 2, 3 and 4 g G-Pro/kg diet respectively (Table 1). Each diet was prepared separately according to Jayaram and Shetty (1981) to obtain pellets of 2 mm diameter. G-Pro was added to the cooled dough before pelletizing. The diets were dried in a thermostatic oven at a temperature of 40 ºC and stored at room temperature in air-tight containers until use.

2.2 Experimental set up

The growth experiment of 140-day duration was carried out in triplicate in 15 outdoor cement tanks of 25 m³ (5x5x1 m) each, without a soil base. Ground water was used to fill the tanks, maintaining a height of 90+5 cm over the experimental period. Common carp fry procured from B.R. Project Government Fish Farm, Karnataka were reared in the Fish Farm of College of Fisheries, Mangalore and acclimated to the experimental pond conditions. Fry of av. wt. 0.8+0.03 g were stocked at a density of 50 per tank. They were fed with the formulated diets every day at 5% of body weight in the morning and evening in 2 equal parts. Feeding was done using plastic trays of 25x20x5 cm kept suspended in the tanks at a depth of about 50 cm. Fish were sampled every fortnight to measure body weight and length. The quantity of feed given was re-adjusted after each sampling, based on the weight recorded.

2.3 Water analyses

Water samples from the experimental tanks were collected fortnightly between 09.00 and 10.00 hr for measuring temperature, dissolved oxygen, pH, free carbon dioxide and total alkalinity. Water temperature was recorded using a digital 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 liters of water from different locations of each experimental tank. Dry weight of plankton was determined by drying the filtrate in a hot-air oven at 80 ºC, till a constant weight was obtained.

2.4 Body indices

Hepatosomatic index (HSI) and Viscerosomatic index (VSI) of fishes was computed on termination of the experiment using the following formulae.

HSI (%) = Weight of liver (g)/Weight of fish (g) X 100

VSI (%) = Weight of viscera (g)/Weight of fish (g) X 100

2.5 Muscle DNA and RNA

Muscle DNA was determined by the diphenylamine method of Giles and Myres (1965), while RNA was estimated as described by Ceriotti (1955).

2.6 Proximate composition

Proximate composition of the feed ingredients (Table 2), diets and fish carcass was analyzed. Crude protein, lipid, crude fiber and ash were determined according to AOAC (1995). NFE was obtained by the ‘Difference method’ (Hastings, 1976). The energy content was obtained by multiplying protein, fat and carbohydrate (NFE) content by factors of 5 (Smith, 1976), 9 and 4 (Hastings, 1975) respectively and expressed in kJ/g.

2.7 Digestive enzyme activity

The activity of digestive enzymes viz. protease, amylase and lipase in the pancreas and intestinal segments of the experimental fish was analyzed on termination of the experiment by the methods of Kunitz (1947), Bernfeld (1955) and Bier (1962) respectively. Six fish from each treatment were used to collect the tissues for enzyme assay.

2.8 Performance indices and statistical analysis

Performance indices viz. specific growth rate (SGR), feed conversion efficiency (FCE) and net production were calculated as follows.

Specific growth rate (SGR % day-¹) = [(ln final weight - ln initial weight/Experimental duration in days)] × 100.

Feed conversion efficiency (FCE) = Fish weight gain (g)/Feed consumed (g) x100.

Net production (g) = Gain in body weight (g) x Total number of fish harvested.

Comparison among treatments for various parameters 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).

3 Results

The water quality parameters monitored over the experimental period ranged as follows. Temperature: 28 to 29.5 ºC, pH: 7.4 to 8.4, dissolved oxygen: 5.0 to 8.8 ppm, free carbon dioxide: nil to 1.4 ppm and total alkalinity 42 to 70 ppm. The average plankton wet and dry weight values varied from 4.84 to 17.09 mg and 1.33 to 5.69 mg/100 L. On termination of the experiment, the average fish weight recorded was 67.82 g (2 g G-Pro), 61.82 g (1 g G-Pro), 51.79 g (4 g G-Pro) 51.57 g (3 g G-Pro) and 49.46 g (Control) (Table 3). Weight gain in 2 g treatment was significantly (P<0.05) higher from that of the rest of the treatments and the control, while that of 1 g treatment was significantly higher than that of 3 and 4 g treatments as well as the control. The difference in growth of 3 g and 4 g G-Pro treated fish and the control was not significant (P>0.05). The highest specific growth rate and RNA: DNA ratio was obtained in fish from 2 g treatment; they were the lowest in the control. HSI was highest in the control (0.59) and lowest in 3 g treatment (0.38). VSI was higher in the treated fish; the highest and lowest values (18.18, 9.62) were recorded in 2 g treatment and the control respectively. Survival of fish was maximum at 86.0% under 2 g treatment as against the minimum 76.0% in the control. Net fish production was the highest in 2 g treatment. The highest feed digestibility (dry matter 81.49%, protein 87.68%, fat 91.94%) was recorded under 2 g treatment, whereas it was the lowest in the control (79.02%, 84.01% and 87.23% respectively). FCE was the best under 2 g treatment (27.88%); it was the least under the control (25.26%).

Carcass protein content was the highest in 2 g treatment (19.43%) as against the lowest of the control (18.22%). Lipid content was significantly higher and similar in fish from 1 g and 2 g treatments (2.43%) compared to the control (1.95%). Ash level was the lowest in 2 g G-Pro treated fish and the highest in the control (Table 3). Pancreatic amylase activity was the highest in 4 g G-Pro treated fish (1.04 units); in contrast in the 1^st intestinal segment, its activity was highest in the control (2.36 units). In the 3^rd segment, highest amylase activity was recorded in 2 g G-Pro treated fish (1.23 units), while in the 2^nd segment activity was higher in all the treated fish (P<0.05). Compared to amylase and lipase activities, protease activity was higher in all the fish. Among the treated fish, higher doses induced higher protease activity in the pancreas. However, in the intestinal segments, the activity was higher with the lower doses. Lipase activity was higher in the pancreas of 1 g and 2 g G-Pro treated fish. Intestinal segments of control fish showed better lipase activity (Table 4).

4 Discussion

The water quality parameters monitored were suitable for fish growth (Boyd, 1982), with no drastic variation between treatments. Temperature ranged from 28 to 29.5ºC. Bhatnagar et al. (2004) suggested that a temperature range of 28-32ºC is ideal for tropical major carps, whereas Santhosh and Singh (2007) observed that the suitable water temperature for carp culture is between 24 and 30 ºC. pH between 7 to 8.5 is considered suitable for biological productivity. In general, an aquaculture pond should have a pH range between 6.5 and 9.0 (Wurts and Durborow, 1992; Bhatnagar et al., 2004). pH varied from 7.0 to 8.4 in the present study. The dissolved oxygen content was optimum in all the treatments (5.0 to 8.8 ppm). Dissolved oxygen affects growth, survival, distribution, behaviour and physiology of aquatic organisms (Solis, 1988). According to Bhatnagar and Singh (2010), DO level of > 5 ppm is essential to support good fish production. Oxygen depletion in water leads to poor feeding, starvation, reduced growth and fish mortality (Bhatnagar and Garg, 2000). Free carbon dioxide was detected only on a few days at low levels, the highest level recorded being 1.4 ppm. Santhosh and Singh (2007) opined that for supporting good fish production, free carbon dioxide in water should be less than 5 mg/L. Alkalinity of 42 to 70 ppm was recorded in this study. According to Wurts and Durborow (1992), alkalinity between 75 to 200 mg/L, but not less than 20 mg/L is ideal in aquaculture.

The best growth of fish was recorded under 2 g G-Pro treatment, followed by 1 g; fish fed higher doses did not differ from the control. Higher growth of fish fed G-Pro can be attributed to the feed additive, since all other ingredients were common to the diets tested. Yeast and B-complex vitamins, the main constituents of G-Pro, must have been responsible for the higher growth. Survival and net production were also the best in the treatment that yielded the best growth. Dietary supplementation of commercial live yeast, Saccharomyces cerevisiae, improved growth and feed utilization in Israeli carp (Noh et al., 1994) and Nile tilapia (Abdel-Tawwab et al., 2008). Studies of Panigrahi et al. (2005) and Abo-State et al. (2009) revealed a positive effect of using viable microorganisms in probiotic mixtures in fish diets. Dhanaraj et al. (2010) found significant improvement in growth performance and gut microbial load in koi carp fed with 0.5% brewer’s yeast in the basal diet. Khalil et al. (2012) found improved fish growth, feed intake and nutrient utilization, as well as fish carcass composition in tilapia fed yeast incorporated diets. Heidarieh et al. (2013) reported increased feed intake, improved feed conversion ratio (FCR) and growth performance and significant increases in trypsin and amylase activities in rainbow trout fed fermented Saccharomyces cerevisiae. Mona et al. (2015) observed significant increase in the growth of African catfish (Clarias gariepinus) fed yeast through diet. Gumus et al. (2016) who replaced fishmeal in the practical diet of goldfish with graded levels of brewer’s yeast recorded higher weight gain, SGR, FCR and PER with 35% yeast, compared to other diets. Supplementation of diet with probiotics significantly improved growth performance, feed utilization and survival of Channa striata fingerlings (Munir et al., 2016).

Feed conversion efficiency was better in the treated groups compared to the control; the best conversion was obtained under 2 g treatment. This must have been the result of better utilization of nutrients. The highest RNA: DNA ratio and VSI were recorded in 2 g G-pro treated fish, reflecting higher metabolic activity. The higher RNA/DNA can be correlated with increased protein synthesis. RNA: DNA ratios have usually been related to the tissue growth rate (Perago´n et al., 2000). VSI and HSI are indicative of food value (Keri et al., 2014). Feeding probiotics had a positive influence on these body indices in Channa striata fingerlings (Munir et al., 2016). Digestibility of protein and fat improved with the addition of G-Pro. This can be attributed to higher digestive enzyme activity. Amylase activity was higher in the treated fish with the exception of the 1^st intestinal segment in which the control showed higher activity. Total amylase activity was higher in the proximal end of the intestine in all the groups as reported in catla, rohu and mrigal by Dhage (1968). Improved protease activity was seen in the treated fish, except in the 2^nd intestinal segment of fish from 2 g and 4 g treatments. Lipase activity was higher only in the pancreas of fish fed 1 g and 2 g G-Pro diets. These findings indicate that the digestive process was positively influenced by the feed additive. Activity of digestive enzymes can be correlated with the nature and composition of the food consumed (Deguara et al., 2003). The digestion of food and absorption of nutrients depends on the availability and efficiency of digestive enzymes (Furne et al., 2005). Probiotics are known to aid digestion by exoenzyme supply and establishment of beneficial microflora in the digestive tract (Sankar et al., 2016). A study conducted by Balcázar et al. (2006) suggested that probiotics have a beneficial effect on the digestive processes of aquatic animals because probiotic strains synthesize extracellular enzymes such as proteases, amylases and lipases as well as provide growth factors like vitamins, fatty acids, and amino acids. Therefore, nutrients are absorbed more efficiently when the feed is supplemented with probiotics (Haroun et al., 2006). It has been reported that the probiotic yeast Debaryomyces hansenii HF1 secretes amylase and trypsin enzymes that aid digestion in sea bass (Dicentrarchus labrax) larvae (Tovar et al., 2002). Hunt et al. (2014) reported improved digestive enzyme activity and proximate nutrient composition in trout juveniles fed diets containing yeast based nucleotides.

Protein, fat and ash content of fish carcass was significantly affected in 1 g and 2 g G-pro treatments that recorded significantly higher growth, reflecting increased protein and fat deposition and a decline in ash level in the muscle, following treatment with the feed additive. Changes in protein and lipid content in fish body could be linked with changes in their synthesis, deposition rate in muscle and/or different growth rate (Abdel-Tawwab et al., 2006). Abdel-Tawwab et al. (2008) and Mona et al. (2015) reported that yeast supplementation significantly affected the whole-fish body composition in Nile tilapia and African catfish respectively. Biochemical composition of fish muscle is influenced by many factors, primarily the diet (Zietler et al., 1984).

From the present findings, it could be concluded that G-Pro is useful at levels of 1 g and 2 g/kg diet for enhancing production performance of common carp. Using this probiotic through diet could result in enhancing economic efficiency of fish farming.

Acknowledgement

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

References