Indian J. Anim. Res., 51 (3) 2017 : *Corresponding author s

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1 Indian J. Anim. Res., 51 (3) 2017 : Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Effect of dietary intake of acid ensiled fish waste on the growth, feed utilization, hematology and serum biochemistry of rohu Labeo rohita, (Hamilton, 1822) fingerlings S. Tanuja*, Anil Kumar and S.K. Nayak Central Institute for Women in Agriculture, Opposite Kalinga studio, Baramunda Post, Bhubaneswar , Odisha, India. Received: Accepted: DOI: /ijar.v0i0f.3792 ABSTRACT Acid ensilage was prepared from the gut and gills of freshwater carps. Isonitrogenous diets containing 0%, 2.5%, 5% and 10% acid ensilage was fed to rohu (Labeo rohita) fingerlings to study the effect on the growth performance, feed utilization, blood biochemistry and hematology of the fingerlings. The results of the study for 60 days indicated no significant difference (p>0.05) in the weight gain, specific growth rate (SGR), percentage survival and feed conversion ratio (FCR) in L. rohita fingerlings by the dietary intake of acid ensilage. The apparent digestibility coefficient (ADC) of dry matter and protein was significantly higher (p<0.05) for feed with 10% silage. Dietary intake of acid ensilage resulted in significantly higher (p<0.05) fat and protein in the tissues of the fingerlings. In blood hematology, total leucocyte count in the blood of fishes fed acid fish ensilage was found to be significantly higher (p<0.05). Significant increase (p<0.05) in total serum protein was observed in 2.5% silage and 5% silage fed fishes. The serum glucose, triglyceride and total cholesterol was not altered significantly by the intake of acid ensilage. The inclusion of fish silage at 5% level resulted in a 14.31% reduction in feed cost/kg weight gain in rohu fingerlings. Key words: Acid ensiled fish waste, Growth performance, Hematology, Labeo rohita, Serum biochemistry. INTRODUCTION Freshwater aquaculture in India, contributes over 95% of the total aquaculture production. Feed cost accounts for over 50 per cent of the production cost in aquaculture. Fish meal, a high quality animal protein source, has been a key ingredient in most fish feeds, accounting for between 5 and 50 per cent of feed weight for carps, salmon, trout, marine fish, and shrimp feeds. In recent years, increased costs of energy, El Nino effects and increasing demand have resulted in a global increase in fishmeal price and a decrease in supply of good quality fishmeal. The increasing cost of aquafeeds has been adversely affecting the economic viability of smallscale aquaculture. Under such circumstances, it is necessary to identify low cost feed ingredients which will reduce the overall production cost of feed. Fish silage is one such alternative that could find a potential place as a fish meal alternative. Fish silage is a liquid product produced from the whole fish or parts of it, to which acids, enzymes or lacticacid-producing bacteria are added, with the liquefaction of the mass provoked by the action of enzymes from the fish. It has high nutritional value protein with all the essential amino acids (Vidotti et al., 2003; Ramírez-Ramírez, 2009). Due to the similarity of this protein source with the raw material, silage has a high potential for use in aquaculture (Vidotti et al., 2003; Goddard and Perret, 2005). The aim of the present study was to utilize the gut and gills of carps that would otherwise go as waste, for the production of acid ensilage and to study the effect of varying levels of the silage on the growth performance, feed utilization, blood biochemistry and hematology of Labeo rohita fingerlings. MATERIALS AND METHODS Acid ensilage was prepared from the gut and gills of freshwater carps procured from a local market in Bhubaneswar. The waste was washed in potable water, chopped and ground using meat grinder into paste for silage preparation. 1.5% per cent formic acid (v/w) and 1.5 per cent conc. hydrochloric acid (HCl) (v/w) was added to the visceral waste and mixed well. Synthetic antioxidant Butyl Hydroxy Toleune (BHT) (200ppm) was added to prevent the autooxidation in the silage. To prevent the mould growth, 0.1 per cent (w/w) potassium sorbate was added. The silage was stored in plastic containers with lids at 30 0 C for the liquefaction to take place. It was stirred twice daily for uniform distribution of the acid. A total of 180 fingerlings of rohu (L. rohita) of average weight of g were distributed in four treatment groups with three replicates each. Fibre reinforced *Corresponding author s tanujasomarajan@gmail.com.

2 502 INDIAN JOURNAL OF ANIMAL RESEARCH plastic (FRP) tanks of 500 litre capacity were used for the experiment. Water level in the tanks was maintained at 300 litres with weekly coping up for the evaporation loss. Fifteen fish with near uniform biomass were stocked randomly into triplicate tanks. The fishes were acclimatized by feeding control feed at the rate of 10 per cent of their body weight for two weeks. Thereafter, the fishes were fed with a ration of 5 per cent of their body weight daily with the treatment feeds in two split doses given at 08:00 and 17:00 hours. Fishes were weighed every fortnight and the daily rations were adjusted accordingly. Water samples were analysed daily for ph and temperature and weekly for dissolved oxygen, hardness, ammonia and nitrate. Final sampling for proximate analysis, liver weight, hematological and serum biochemical analysis were done at the end of 60 days feeding. The fishes were fed isonitrogenous diets containing 0% fish silage, 2.5% fish silage, 5% fish silage and 10% fish silage as a replacement of fish meal (Table 1). Proximate composition of the diets and fish tissue were determined using AOAC (2000) procedures. Apparent nutrient digestibility co-efficient of the diets were studied using 10g titanium dioxide (TiO 2 ) /kg of feed as marker during the last 30 days of experiment. Two fishes each from each treatment group was stocked in aquarium tanks of capacity 25l. Round the clock aeration was provided in the tanks. Measured amount of marker added feed were given to the fish in two split doses at 08:00 and 17:00 hours. The unconsumed feed and fresh faeces were collected after 2 hours of feeding. The feed and faeces were dried at C in a hot air oven. The oven dried feaces were stored at C until analysis. Titanium dioxide in the feed and faeces was determined using the method described by Richter et al. (2003) with some modifications. The quantity of TiO 2 (mg/ml) in the 1millilitre aliquot was computed by using the following equation Marker TiO 2 (µg/ml) =108.1(Abs 405) The Apparent Digestibility Co-efficient (ADC) of feed were calculated as follows. ADC of dry matter of diet (%) = 100 x [1-(dietary TiO 2 /feacal TiO 2 )] ADC of protein of diet (%) = 100 x [1-(dietary TiO 2 /Feacal TiO 2 )x(fecal protein concentration/dietary protein concentration)] The growth parameters of rohu fingerlings were assessed by taking their body weight and length on a fortnightly basis. The fishes were kept starved for eight hours before the measurement of body weight. The growth performance was assessed using the following standard formulae for different growth parameters and body indices Weight gain % = (Final weight Initial weight)/(initial weight)x 100 Specific growth rate (SGR) =100 (log e Average final weight - log e Average initial weight) /number of culture days Feed conversion ratio (FCR) = Total dry feed intake (g) / wet weight gain (g). Protein efficiency ratio (PER)= Wet weight gain (g) / Protein intake (g) Survival (%) = (Total number of fishes harvested/total number of fishes stocked) X 100 Feed cost/kg gain in weight = Cost of diet x Feed conversion ratio (cost of feed was calculated based on the market price of feed ingredients) Hepatosomatic index (HSI)= [Weight of liver (g) / Weight of fish (g)] x 100 Gastrosomatic Index (GSI)= [Weight of digestive tract (g) / weight of fish (g)] x 100 Table 1: Percent ingredients and composition of experimental diets for L.rohita fingerlings Feed Ingredients (g/kg) Ground nut oil cake Soyabean meal Fish silage Fish meal Rice bran Tapioca Vit and mineral mix Crude Protein (%) Ether Extract (%) Crude Fibre (%) Total carbohydrate (%) Ash (%) Moisture (%) Digestive energy (Kcal/100gm) Feed cost/kg weight gain (Rs) Decrease in feed cost (%)

3 For collection of blood from rohu fingerlings, the body surface of the fishes were cleaned with blotting paper and blood was collected from the heart. The total red blood cell count (TRBC), total leucocyte count (TLC) and hemoglobin (Hb) were estimated for hematological analysis. In serum biochemistry, the total protein, albumen, cholesterol, triglyceride, alkaline phosphatase, serum glucose, urea and creatinine were estimated as per standard colorimetric method using commercial reagent kits in a spectrophotometer. Parameter wise data sets were analysed using Analysis of Variance (ANOVA) and all possible pair wise treatments were compared using Tukey s Honestly Significant Difference (HSD). The data were statistically analysed by statistical package SAS version 9.3. RESULTS AND DISCUSSION During the experimental period, dissolved oxygen (DO) and ph ranged from 6.1 to 7.2 ppm and 6.5 to 7.5, respectively. The ammonia and nitrate levels varied between ppm and ppm, respectively. Water temperature varied from 26.9 to C. The carbonate hardness was found to range from 236 to 245 mg /L during the experimental period. The results indicate a non significant effect of dietary intake of acid ensilage on the growth of rohu (Table 2). The apparent digestibility of feed with 10% acid ensilage was significantly higher (p<0.05) than that of the other experimental feeds (Table 3). Fishes fed with 2.5% and 10% silage had significantly higher crude protein and fat in their tissues when compared to the control (Fig 1). All the hematological parameters studied (Hb, TLC and TRBC) showed significant variation (p<0.05) among the treatments. Table 2: Growth and feed utilization parameters of L.rohita fed acid ensiled fish waste Volume 51 Issue 3 (2017) 503 Among the serum biochemical parameters studied, serum glucose and albumen concentration did not vary significantly between the treatments. The non significant effect of dietary intake on growth parameters and feed utilization corroborates with the findings of Gullu (2014). Although non significant (p>0.05), the fishes fed with silage showed a higher weight gain than that of the control group (0% silage). Rama subharayan et al. (2013) have reported a significant increase in growth caused by feeding of formic acid added silage in Cyprinus carpio fingerlings. Non significant change in FCR has been reported by Siddique (2009) when European sea bass was fed with formic acid fish silage. Hardy et al. (1984) have observed a decrease in fish growth with dietary inclusion of acid ensilage due to the acidity of the diets. In the present study, the non significant effect of acid added silage based diets on the growth of rohu fingerlings may be because of the near neutral ph ( ) attained in the experimental feeds. Body indices like HSI or GSI were not affected significantly by the dietary intake of acid ensilage. Liver being the main site of detoxification, the presence of any toxicant in the feed will stimulate synthesis of hepatic detoxification enzymes in the liver increasing its size and thus the HSI. The results indicate the non toxicity posed by acid ensilage on the fishes. Digestibility studies are one of the best methods of evaluating the capacity of a species to use the nutrients of a particular feed. In general, higher the digestibility of feed, greater is the growth performance of the fish. The apparent digestibility coefficient (ADC) of dry matter and protein was similar for all the feeds with significantly higher value for Parameters Experimental feeds P value Initial weight (g) 2.67± ± ± ±0.67 Final weight (g) 4.83± ± ± ±0.65 Weight gain(g) 2.17±0.37 a 2.3±0.18 a 2.78±0.13 a 2.61±0.44 a Weight gain % 81.0±8.97 a 87.95±10.05 a ±8.16 a 98.59±21.26 a GSI a a a a HSI a a a a SGR a a a a 0.29 FCR 4.19±0.8 a 4.05±0.39 a 3.64±0.25 a 3.71±0.43 a 0.51 PER a a a a Survival (%) 84.45±3.85 a 82.23±10.18 a 86.67±11.55 a 97.78±3.85 a 0.17 Table 3: Apparent digestibility coefficients (ADC) of dry matter and protein for L.rohita fingerlings fed acid ensiled fish waste Apparent Digestibility (%) Experimental feeds P value Dry matter 52.78±0.24 b 42.94±0.85 c 52.65±1.69 b 58.4±0.48 a < Protein 70.50±0.17 b 64.36±0.5 c 70.40±1.06 b 74±0.3 a <0.0001

4 504 INDIAN JOURNAL OF ANIMAL RESEARCH Fig 1: Proximate composition of L. rohita fingerlings fed acid ensiled fish waste feed with 10% silage. Ali et al. (1994) have also reported a higher apparent protein digestibility of fish silage based diet compared to fish meal diets when fed to carp and rainbow trout, respectively. The ADC values of dry matter and protein obtained in the present investigation were lower than that reported by Mamun et al. (2007) for Oreochromis niloticus feed. This might be because of the low proportion of fish meal used in the feed and the rice bran which is used as a filler in the feed. Siddique (2009) has reported a low dry matter digestibility of 58.03% for formic acid fish silage based feed fed to European sea bass. Vidotti et al. (2002) have also reported a lower ADC ( %) for pacu (Piaractus mesopotamicus) fed fish silage. The increase in ADC dry matter with increase in percentage inclusion of silage in feed corroborates with the findings of Vidotti et al. (2002) and Ali et al. (1994). According to Soltan and Tarwat (2006) exogenous and endogenous factors determine the body composition of fish. As the other factors were kept constant, the only determining factor for variation in body composition will be the feed. The increased protein content and fat content in silage fed fishes might be due to the better nutrient assimilation from the diet and higher fat content in their diets, respectively (Ramasubharayan et al., 2013). On the contrary Soltan et al. (2008) had reported a decrease in crude protein and crude fat content in Clarias garipenus when fed with 25%, 50% 75% and 100% fermented fish silage. A decrease in protein, lipid and ash content was also noticed in rainbow trout when the percentage inclusion of silage in their diets increased from 0 to 60 percentage (Gullu, 2014). The blood characteristics and physiological condition of the fish were closely related. The Hb concentration was higher than that reported by Manas et al. (2002) for tank reared Labeo rohita fingerlings. Najim et al. (2014) have reported an increase in total red blood cell count (TRBC) with increase in percentage inclusion of fish bio silage in the diet. But in the present study, TRBC count was found to be significantly lower in fishes fed with 10% fish silage than those fed with control diet (Table 4). Total leucocyte count (TLC) in the blood of fishes fed acid fish ensilage was found to be significantly higher than that of control (0% acid fish ensilage). The effect of bio-active peptides in fish silage may be a contributing factor for increase in TLC. The total serum protein plays a significant role in the innate immune response. Significant increase in total serum protein was observed in 2.5% silage and 5% silage fed fishes (Table 4). Cho et al. (2007) reported that when the animals were fed high quality protein sources, they had higher concentrations of total protein in serum. Increased glucose level is a well-known stress indicator in fish and nutritional status can affect the glucose response in fish (Martínez Porchas et al., 2009). There was no significant change in the blood glucose level in the blood of fishes of the different treatments (Table 4). Serum cholesterol was within the range as reported by Manas et al. (2002). Cholesterol in fishes fed 10% acid silage was significantly lower (p<0.05) than that of control (0% acid ensilage). The triglyceride content in the blood of fishes did not show a definite trend in accordance with the proportion of acid ensilage in the diet. Najim et al. (2014) have reported a decrease in triglyceride and cholesterol level in the blood of Table 4: Hematological and serum biochemical parameters of L.rohita fingerlings fed acid ensiled fish waste Hematological parameters Experimental feeds P value Hb(mg%) 11.23±0.06 b a 11.07±1 b 11.03±0.06 b TLC (mm 3 ) 3300 d ±57.74 c 6400 b ±57.74 a < TRBC(x 10 6 /mm 3 ) 4.07±0.12 a 4.23±0.06 a 3.73±0.23 ab 3.2±0.35 a Serum Biochemistry Serum glucose (mg/dl) 98±7.55 a ±6.63 a ±8.73 a 92±7.21 a Cholesterol(mg/dl) 179±16.15 a 161±5.57 a 148.8±12.83 ab 126.9±7.79 b Trigyceride(mg/dl) ±22.64 bc ±36.56 a 290.9±5.8 b ±7.79 bc Total protein(g/dl) 6.13±0.12 b 8.3±0.36 a 8 a 6.13±0.12 b < Albumen(g/dl) 3.73±0.46 a 3.8±0.53 a 3.6±0.69 a 3.6±0.69 a Globulin(g/dl) 2.13±0.12 b 4.5±0.87 a 4.4±0.69 a 2.53±0.64 b 0.003

5 Cyprinus carpio fingerlings fed with fish bio silage. This could be due to an altered cholesterol metabolism caused due to the diet. Based on the results it was concluded that acid ensiled fish waste could be used as a protein supplement in diet of rohu fingerlings upto a level of 10% without affecting their growth performance, feed utilisation and survival. Volume 51 Issue 3 (2017) 505 Moreover, the 14.31% reduction in feed cost/kg weight gain by replacing fish meal with 5% fish silage will result in increased profit margin to the fish farmers. The use of acid ensiled fish waste in aquafeeds will not only help in reduction of feed cost but will also protect the environment from the pollution that is being caused by the piling heaps of fishery wastes. REFERNCES Ali, M.Z., Gheyasuddin, S., Zaher, M., Hossain, M.A. and Islam, M.N. (1994). Evaluation of fish silage prepared from under-utilised marine fishes as protein sources in the diet of major carp (Cirrhinus mrigala). J. Aquacult.Trop., 9: AOAC. (2000). Official Methods of Analysis. 16th ed, Association of Official Analytical Chemists, Washington D.C. pp Cho, J.H., Min, B.J., Chen, Y.J., Yoo, Y.S., Wang, Q., Kim, J.D. and Kim, I.H. (2007). Evaluation of FSP (fermented soy protein) to replace soybean meal in weaned pigs: growth performance, blood urea nitrogen and total protein concentrations in serum and nutrient digestibility. Asian Australian J. Anim. Sci., 20: Goddard, J.S. and Perret, J.S.M. (2005). Co-drying fish silage for use in aquafeeds. Anim. Feed Sci. Technol., 118: Gullu, K., Acar, U., Tezel, R. and Yozukmaz, A. (2014). Replacement of Fish Meal with Fish Processing by-product Silage in diets for the Rainbow Trout, Oncorhynchus mykiss. Pakistan J. Zool., 46: Hardy, R.W., Shearer, K.D. and Spinelli, J. (1984). The nutritional propet1ies of eo-dried fish silage in rainbow trout (Salmo gairdneri) dry diets. Aquacult., 38: Mamun, S.M., Focken, U. and Becker, K. (2007). Comparison of metabolic rates and feed nutrient digestibility in conventional, genetically improved (GIFT) and genetically male (GMNT) Nile tilapia, Oreochromis niloticus (L.). Comp. Biochem. Physiol. Part A: Molecular and Integrative Physiology, 148: Manas, K.D., Das, R.K. and Mondal, S.K. (2002). Some stress sensitive parameters of young major carp, Labeo rohita (Hamilton-Buchanan). Indian J.Fish., 49: Martínez-Porchas, M., Martínez-Córdova, L.R. and Ramos-Enriquez, R. (2009). Cortisol and glucose: reliable indicators of fish stress. Panamjas., 4: Najim, S.M., Al-Noor, S.S. and Jasim, B.M. (2014). Effects of fish meal replacement with fish biosilage on some haematological and biochemical parameters in common carp Cyprinus carpio fingerlings. Int. J. Res. Fish. Aquacult., 4: Ramasubburayan, R., Iyapparaj, P., Subhashini, K.J., Chandran, M.N., Palavesam, A. and Immanuel, G. (2013). Characterization and Nutritional Quality of Formic Acid Silage Developed from Marine Fishery Waste and their Potential Utilization as Feed Stuff for Common Carp Cyprinus carpio Fingerlings. Turk. J. Fish. Aquat. Sci., 13: Ramírez-Ramírez, J.C. (2009). Utilization of shrimp bycatch and fishery byproducts through fish silage production. PhD. Thesis submitted to the Universidad Autónoma Metropolitana, Mexico City, Mexico. Richter, H., Luckstadt, C., Focken, U. and Becker, K. (2003). Evacuation of pelleted feed and the suitability of titanium (IV) oxide as a feed marker for gut kinetics in Nile tilapia. J. Fish Biol., 63: Siddique Milad El-Ajnaf. (2009). Development of fermented fish silage derived from pelagic sardine and apple pomace by-product for inclusion in diets for the European seabass (Dicentrarchus labrax). Ph.D. Thesis submitted to the University of Plymouth. Soltan, M.A. and Tharwat, A.A. (2006). Use of fish silage for partial or complete replacement offish meal in diets of Nile tilapia (Oreochromis niloticus) and African catfish (Clarias gariepinus). Egypt. J. Nutr. Feed., 9: Soltan, M.A., Hanafy, M.A. and Wafa, M.I.A. (2008). An evaluation of fermented silage made from fish by-products as a feed ingredient for African catfish (Clarias gariepinus). Global Vet., 2: Vidotti, R. M., Viegas, E. M. M. and Carneiro, D. J. (2003). Amino acid composition of processed fish silage using diferent raw materials. Anim. Feed Sci. Technol., 105: Vidotti, R.M., Dalton, J.C. and Viegas, E.M.M. (2002). Acid and fermented silage characterization and determination of apparent digestibility coefficient of crude protein for pacu, Piaractus mesopotamicus. J. World Aquacult. Soc., 33: