Effect of Dietary Gelatinized Starch Level and Rearing Temperature on Fatty Acid Profile and DNA:RNA Ratio of Labeo rohita (Hamilton) Fingerlings

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1 The Israeli Journal of Aquaculture - Bamidgeh, IJA_ , 7 pages The IJA appears exclusively as a peer-reviewed on-line open-access journal at To read papers free of charge, please register online at registration form. Sale of IJA papers is strictly forbidden. Effect of Dietary Gelatinized Starch Level and Rearing Temperature on Fatty Acid Profile and DNA:RNA Ratio of Labeo rohita (Hamilton) Fingerlings A. Ciji 1 *, N.P. Sahu 1, A.K. Pal 1, M.S. Akhtar 1,2, P. Viji 3 1 Division of Fish Nutrition, Biochemistry and Physiology, Central Institute of Fisheries Education, Fisheries University Road, Versova, Mumbai , India 2 Fish Nutrition Section, Directorate of Coldwater Fisheries Research, Bhimtal , Nainital, Uttarakhand, India 3 Central Institute of Fisheries Technology, Mumbai Centre, Mumbai, India (Received , Accepted ) Key words: gelatinized starch, fatty acid, Labeo rohita, temperature, DNA, RNA, n-3:n-6 ratio Abstract A 60-day feeding trial was conducted to delineate the effect of three dietary levels of gelatinized starch on fatty acid profiles and RNA and DNA contents of fingerlings of the Indian major carp, Labeo rohita, reared at ambient water temperature (26±0.8 C) or 32 C. Two hundred and sixteen fingerlings (6.5±0.3 g) were randomly distributed into six treatments in triplicate. Three semi-purified isonitrogenous diets were prepared with 40%, 50%, or 58% gelatinized starch. Liver and muscle RNA and RNA:DNA ratio were significantly (p<0.05) higher in fish fed the 40% or 50% diet and reared in 32 C. DNA content did not significantly differ between treatments (p>0.05). Total saturated fatty acids (SAFA) in muscle was highest in fish fed the 50% diet; SAFA in fish fed the 58% diet were higher than in those fed the 40% diet but lower than in those fed the 50% diet. Polyunsaturated fatty acids (PUFA) were highest in fish fed the 40% diet. Total n-3 and n-6 fatty acids generally decreased as the level of starch increased; likewise, higher levels of gelatinized starch led to a reduced n-3:n-6 ratio. Rearing temperature had no significant effect on fatty acid composition of the muscle. RNA content and RNA:DNA ratio in the liver and muscle decreased gradually as the starch level increased. * Corresponding author. Tel.: , fax: , cijialex83@gmail.com

2 2 Ciji et al. Introduction A major production cost in aquaculture is expensive high protein diets. Finding low cost feeds is necessary for the sustainable development of aquaculture. The protein-sparing effects of dietary lipids is well known, but excessive dietary lipids can cause deterioration in flesh quality through high lipid accumulation, which in turn induces physiological disorders (Nakagawa et al., 2004). Fish are considered diabetic animals and generally have limited ability to utilize carbohydrates as sources of energy (Roberts, 1989; Lall, 1991). The herbivorous Labeo rohita has greater potential to utilize dietary carbohydrates than carnivores (Kumar et al., 2005; Misra et al., 2006; Alexander et al., 2011a). Labeo rohita fry can utilize up to 40% dietary gelatinized carbohydrate without detrimental effects on health (Mohapatra et al., 2003). In Catla catla, fish fed higher levels (45-50%) of gelatinized corn starch had higher carbohydrate digestibility (83%), indicating better utilization of carbohydrate (Yengkokpam et al., 2007). The fatty acid composition of fish tissues is generally influenced by the fatty acid composition of the dietary lipids and the ability of the species to modify dietary inputs by catabolism and conversion-including desaturation and elongation (Henderson and Tocher, 1987; Sargent et al., 2002). The metabolic pathways of lipogenesis and lipolysis in fish are essentially the same as in mammals (Alvarez et al., 2000). Increased dietary carbohydrate energy can be deposited as fat (Shimeno et al., 1979; Alliot et al., 1984). Dietary carbohydrates may stimulate lipogenesis and accelerate lipid deposition in rainbow trout (Brauge et al., 1995a). The influence of rearing temperature and dietary nutrients such as carbohydrates on tissue fatty acid composition may be a useful strategy for improving flesh quality. Elvers of Anguilla anguilla fed diets with a high percentage of carbohydrate (glucose or starch) and maintained at 27 C had a higher percentage of lipids than those maintained at 23 C (Gad and Dan, 1987). The pentose phosphate shunt in fish is usually considered in low or reduced temperatures (Walsh et al., 1985). For example, in carp Cyprinus carpio, lowering the water temperature from 27 C to 17 C led to activation of pentose phosphate dehydrogenase and accelerated fatty acid synthesis and fat deposition (Shikata et al., 1993; Shimeno and Shikata, 1993). In C. carpio and Oreochromis niloticus, activity of the pentose pathway that provides reduced forms of nicotinamide adenine dinucleotide phosphate (NADPH) for lipid synthesis increased as the level of dietary carbohydrate increased (Shimeno and Shikata, 1993). Also in L. rohita, activity of hepatic glucose-6-phosphate dehydrogenase increased in fish fed a diet containing 50% gelatinized starch and reared at ambient water temperature, indicating an increase of reductive biosynthesis, i.e. lipogenesis, reflected by a high lipid content in the fish tissue (Alexander et al., 2011b). In fish, RNA and DNA contents can be used as a measure of tissue growth and protein deposition (Bastrop et al., 1992). Labeo rohita fed a diet containing a suboptimal level of protein and non-gelatinized corn supplemented with α amylase had improved cell size in the muscle, indicating more efficient utilization of starch (Kumar et al., 2006). The current trend towards health consciousness has turned the attention of fish nutritionists to flesh quality in addition to growth performance. More attention is now being paid to improving the n-3 and n-6 fatty acid contents of fish. Studies on Indian major carps suggest including higher levels of gelatinized starch (up to 45-50%) to reduce feed costs and maximize profits, as such dietary intervention does not compromise growth or immune status (Yengkokpam et al., 2007; Alexander et al., 2011ab). The aim of this study was to evaluate the effect of three dietary levels (40%, 50%, 58%) of gelatinized starch on the fatty acid profile and DNA:RNA ratio of L. rohita fingerlings reared at ambient water temperature (26±0.8 C) or 32 C. Materials and Methods Diets and experimental design. Three semi-purified isonitrogenous (30.79±0.15% crude protein) diets were prepared with 40%, 50%, or 58% gelatinized starch from gelatinized corn (Table 1). Dietary ingredients were finely ground, well mixed, pelletized through a

3 Percent fatty acid Effect of dietary starch and temperature on Labeo rohita fingerlings 3 Table 1. Composition and biochemical analyses of experimental diets for Labeo rohita fingerlings. Ingredient Gelatinized corn carbohydrate level 40% 50% 58% Gelatinized corn Casein Gelatin Sunflower:cod liver oil (2:1) Cellulose Vitamin-mineral mix Carboxymethyl cellulose Betaine chloride Vitamin C Butylated hydroxytoluene Proximate analyses (dry matter basis) Crude protein (g/kg) 309.5± ± ±0.29 Total carbohydrate (g/kg) 506.9± ± ±0.25 Ether extract (g/kg) 89.2± ± ±0.55 Ash (g/kg) 94.4± ± ±0.31 Digestible energy (KJ/g diet) 15.39± ± ± Fat free, 75% crude protein 2 96% crude protein 3 EMIX Plus (per 2.5 kg): vitamin A 5,500,000 IU; vitamin D 3 1,100,000 IU; vitamin B mg; vitamin E 750 mg; vitamin K 1000 mg; vitamin B mg; vitamin B 12 6 mg; calcium pantothenate 2500 mg; nicotinamide 10 g; choline chloride 150 g; Mn 27,000 mg; I 1000 mg; Fe 7500 mg; Zn 5000 mg; Cu 2000 mg; Co 450 mg; Ca 500 g; P 300 g; L-lysine 10 g; DL-methionine 10 g; selenium 50 ppm; Satwari 250 ppm (Lactobacillus units + yeast culture units) hand pelletizer (Ace Exports, Mumbai, India) with a 2-mm diameter, and analyzed for fatty acid content (Fig. 1). Fingerlings of Labeo rohita were procured from Prem Fisheries Consultancy, Gujarat, and transported to the experimental facilities at the Central Institute of Fisheries Education, Mumbai. After 10 days of acclimatization, 216 fingerlings (6.5±0.3 g) were randomly distributed into 18 plastic tanks (80 x 57 x 42 cm, 150 l capacity) in a completely randomized design with triplicates of each of the six treatments: three diets two temperatures (ambient or 32 C). Temperature was maintained by thermo-static water heaters (range up to 50 C, DTC-PID-50L x 51B x 52H, General Trading Corporation, Mumbai, India). Aeration was provided 24 h. Uneaten feed and fecal matter were siphoned out daily and daily water exchange was approximately 75%. Physicochemical water parameters were within optimum ranges: ph , dissolved oxygen mg/l, ammonia nitrogen mg/l, nitrite nitrogen mg/l, and nitrate nitrogen mg/l. Fish were fed 3% of their body weight for 60 days based on fortnightly weighings. The daily feed ration was divided into two: about two-thirds was given at 09:00 and the rest at 18:00. Sampling. At the end of the feeding trial, two fish from each replicate (six per treatment) were anesthetized with CIFECALM at 200 µl/l (CIFE, Mumbai, India). Muscle and liver samples were aseptically collected immediately. Fish were starved for 24 h before sampling. Extraction of tissue lipids and preparation of fatty acid methyl esters (FAME). Total lipid was extracted using a 2:1 chloroform:methanol mixture following Folch et al. (1957). The solvent was evaporated by a continuous flow of nitrogen and residues were weighed to quantify the amount of extracted lipid. The residue was redissolved in chloroform:methanol (2:1, v/v), then stored in a 25-ml conical flask with a glass stopper in nitrogen at -20 C until needed. FAME were prepared from the isolated lipids by the method of AOAC (1995). Fatty acid analysis by gas chromatography. FAME were analyzed by a quadruple mass-spectrometer (GS-MS QP2010) with ionization energy of 70 ev, equipped with a 25-mm db-wax column and 0.25-µm film, with helium as the Fatty acid Fig. 1. Fatty acid composition (% of total fatty acids) of total lipid in diets containing different levels of gelatinized starch.

4 4 Ciji et al. carrier gas. The sample was injected at the split mode injection port at a 1:15 split ratio and 250 C. Oven temperature was programmed to reach 230 C from 50 C at the rate of 10 C/min. Relative abundance of m/z ranged to Chromatographic data were recorded and integrated using GS-Classic Software (Shimadzu, Japan). Individual fatty acids were presented as percentages of the total identified fatty acids. Quantification of DNA and RNA. DNA and RNA of the nucleic acid extracts from the tissues were quantified by pentose analysis (Schneider, 1957). The DNA and RNA contents of the extract were determined by the following equations: μg DNA/ml = OD at 600 nm/0.019, where OD = optical density; μg RNA/ml = [(OD at 600 nm ) - (μg DNA/ml x 0.013)]/ Statistical analysis. Data were analyzed by one-way and two-way analyses of variance (ANOVA) and significant differences between treatments were determined by Duncan s Multiple Range test using SPSS, ver. 14, with a 0.05 level of significance. Results Total saturated fatty acids (SAFA) rose when dietary gelatinized starch rose from 40% to 50% (Table 2). The main fatty acids in all treatment groups were palmitic acid (16:0), Table 2. Effect of dietary carbohydrate level, temperature, and their interaction on percent saturated and unsaturated fatty acids of total fatty acids in muscle (means±se, n = 3) and DNA, RNA, and RNA:DNA in liver and muscle (means±se, n = 6) of Labeo rohita fingerlings. Effect of gelatinized starch level alone SAFA MUFA PUFA n-3 n-6 n-3:n-6 40% 33.39±0.43 a 24.74±0.38 a 42.29±0.64 c 9.83± ±0.15 b 0.33±0.01 b 50% 37.01±0.37 c 26.80±0.88 b 38.07±0.33 a 7.13±0.28 a 28.23±0.11 a 0.25±0.01 a 58% 35.09±0.39 b 26.36±0.27 b 39.38±0.05 b 6.73±0.29 a 29.39±0.26 b 0.23±0.01 a P value Effect of temperature ( C) alone Ambient 35.82± ± ± ± ± ± C 34.51± ± ± ± ± ±0.03 P value Effect of starch level and temperature 40%, ambient 34.11±0.21 b 25.02±0.23 b 41.78±0.15 c 9.16±0.10 d 29.75±0.03 c 0.31±0.01 d 40%, 32 C 32.67±0.08 a 24.46±0.11 a 42.79±0.28 d 10.51±0.18 e 29.25±0.14 b 0.36±0.01 e 50%, ambient 37.59±0.09 e 27.56±0.06 e 38.21±0.35 a 7.36±0.07 c 28.29±0.25 a 0.26±0.01 c 50%, 32 C 36.43±0.40 d c ±0.01 c 37.93±0.01 a 6.89±0.01 b 28.16±0.03 a 0.24±0.01 b 58%, ambient 35.76±0.07 c 26.13±0.01 c 39.36±0.01 b 6.98±0.06 b 28.94±0.06 b 0.24±0.01 b 58%, 32 C 34.42±0.03 b 26.59±0.03 d 39.41±0.04 b 6.49±0.01 a 29.84±0.03 c 0.22±0.01 a P value Effect of gelatinized starch level alone RNA-P (μg RNA/ml) DNA-P (μg DNA/ml) RNA:DNA Ratio Liver Muscle Liver Muscle Liver Muscle 40% 0.89±0.03 c 0.18±0.01 c 1.64± ± ±0.01 b 0.31±0.01 c 50% 0.78±0.05 b 0.14±0.02 b 1.63± ± ±0.03 b 0.25±0.03 b 58% 0.57±0.03 a 0.07±0.01 a 1.63± ± ±0.02 a 0.12±0.01 a P value Effect of temperature ( C) alone Ambient 0.67±0.05 a 0.11± ± ± ±0.02 a 0.19± C 0.83±0.04 b 0.15± ± ± ±0.02 b 0.26±0.03 P value Effect of starch level and temperature together 40%, ambient 0.87±0.02 c 0.17±0.01 c 1.64± ± ±0.02 c 0.29±0.01 c 40%, 32 C 0.92±0.05 c 0.19±0.01 d 1.63± ± ±0.02 c 0.32±0.02 d 50%, ambient 0.65±0.01 b 0.10±0.01 b 1.64± ± ±0.01 b 0.17±0.01 b 50%, 32 C 0.91±0.02 c 0.19±0.01 d 1.62± ± ±0.02 c 0.33±0.01 d 58%, ambient 0.48±0.02 a 0.07±0.01 a 1.63± ± ±0.02 a 0.12±0.01 a 58%, 32 C 0.65±0.01 b 0.07±0.01 a 1.63± ± ±0.01 b 0.12±0.01 a P value Different superscripts in a column under each category signify statistical differences (p<0.05).

5 Effect of dietary starch and temperature on Labeo rohita fingerlings 5 oleic acid (18:1n-9), and linoleic acid (18:2n-6). Palmitic acid was highest in the 50%/ambient temperature group and lowest in 40% groups, however, all groups except 40%/32 C and 58%/32 C had similar levels of stearic acid (18:0). The highest percentage of monounsaturated fatty acids (MUFA) was in the 50%/ambient temperature group and the lowest in the 40%/32 C group. Among MUFA, oleic acid was highest in 50%/32 C group, followed by the 58% groups. Polyunsaturated fatty acids (PUFA), n-3, and n-6 were higher in the 40% groups, while SAFA and MUFA were lowest. DNA contents of liver and muscle tissues were not affected by dietary starch level or temperature. However, RNA content and RNA:DNA of muscle and liver were influenced by dietary starch level. Temperature significantly affected RNA content and RNA:DNA of the liver, but there was no effect of temperature on muscle tissues. Discussion SAFA and MUFA are the end products of lipid biosynthesis in animals (Enser, 1984). In the present study, SAFA was lowest in fish fed the 40% diet and highest in fish fed the 50% diet because excess energy available from gelatinized starch is converted to SAFA. Likewise, the inclusion of gelatinized starch led to a lower concentration of n-3 fatty acids and higher accumulation of SAFA in rainbow trout and sea bass (Alvarez et al., 1999). Dietary starch activates hepatic lipogenesis in rainbow trout (Brauge et al., 1994, 1995a). In contrast, dietary lipid depresses lipogenesis in ayu (Takeuchi, 1978). Rainbow trout and European sea bass fed gelatinized starch diets consumed more energy than their physiological needs, so part of the energy was used for fat synthesis, leading to a greater accumulation of nonessential fatty acids (Alvarez et al., 1999). As the level of gelatinized starch in the diet of L. rohita fingerlings increased, total n-3 fatty acids increased and SAFA decreased (Kumar et al., 2010). G-6-PDH activity was significantly higher in groups fed 50% diets and reared at ambient temperature (Alexander et al., 2011b). This indicates that starch (and thus carbon units) that are available to fish stimulate the utilization of NADPH, which generates NADP and, in turn, enhances the pentose phosphate pathway (Hilton and Atkinson, 1982). It seems that the availability of starch in diets regulates the generation of reducing equivalents through G-6-PDH. The present study indicates that part of the available starch is utilized for lipogenesis. Hence, increased amounts of available starch favor lipogenesis at ambient temperature and lead to an increased proportion of saturated fat compared to polyunsaturated fatty acids. There was a positive correlation between G-6-PDH and accumulation of SAFA (Y = 0.126X , R 2 = 0.85) in L. rohita fingerlings (Kumar et al., 2010). RNA:DNA is a good index of the growth rate in teleosts (Bulow, 1987). The relationship between nucleic acid concentration (RNA concentration and RNA:DNA) and specific growth rate is strong (Bastrop et al., 1992). Ribosomal RNA (rrna) forms more than 50% of the total cellular RNA. The RNA:DNA ratio is a rough measure of the amount of ribosome and hence the protein synthesis rate. The eukaryotic cell growth rate may be accompanied by a change in the rate of ribosome production (Larson et al., 1991). Hence, the RNA:DNA ratio can indicate the protein synthesis capacity of a tissue. In our study, the lowest concentration of liver and muscle RNA and RNA:DNA was in the group fed the 58% diet and reared at ambient temperature. This correlates with earlier results where the lowest weight gain and SGR were obtained in the same group (Alexander et al., 2011b). The protein:dna ratio increases as the growth rate increases (Pelletier et al., 1995). Protein:DNA is an index of cell size. Muscle cell size increased in Atlantic salmon (Stickland et al., 1988) and trout (Kiessling et al., 1991) during maximal growth. In the present study, PUFA and n-3 fatty acids were higher in fish fed 40% gelatinized starch while 50% and 58% led to reduced n-3 and n-3:n-6. Further, SAFA and MUFA were higher in fish fed the higher starch levels. It could be concluded that at the higher temperature of 32 C, L. rohita can effectively utilize up to 50% of dietary gelatinized starch but flesh quality is compromised in terms of reduced n-3 fatty acids and n-3:n-6. Therefore, feed producers and aquaculturists should consider flesh quality while considering the incorporation of higher levels of gelatinized starch to reduce feed costs.

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