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Aquaculture 448 (2015) 98–104

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Effects of dietary zinc on the growth, digestive enzyme activities, muscle biochemical compositions, and antioxidant status of the giant freshwater prawn Macrobrachium rosenbergii T. Muralisankar a,⁎, P. Saravana Bhavan a, S. Radhakrishnan a, C. Seenivasan a, V. Srinivasan a, P. Santhanam b a b

Crustacean Biology Laboratory, Department of Zoology, Bharathiar University, Coimbatore, Tamil Nadu, India Marine Planktonology and Aquaculture Laboratory, Department of Marine Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India

a r t i c l e

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Article history: Received 5 February 2015 Received in revised form 28 May 2015 Accepted 30 May 2015 Available online 3 June 2015 Keywords: Macrobrachium rosenbergii Zinc Digestive enzymes Biochemical compositions Antioxidants Metabolic enzymes

a b s t r a c t Current study was made to assess the dietary zinc (Zn) on the growth, activity of digestive enzymes, muscle biochemical compositions, antioxidant and metabolic enzyme status of the freshwater prawn, Macrobrachium rosenbergii. Zn was supplemented at 0, 10, 20, 40, 60, and 80 mg kg−1 with the basal diet, and the concentrations of Zn in Zn supplemented diets were 31.45, 42.10, 53.80, 65.70, 92.80, and 113.50 mg kg−1 respectively. M. rosenbergii were fed these Zn supplemented diets for a period of 90 days. Results showed that prawns fed with 10–60 mg kg−1 Zn supplemented diets attained significant (P b 0.05) improvement in survival, growth, digestive enzyme activities, and muscle biochemical compositions. However, the prawns fed with 80 mg Zn kg−1 showed poor performance. The enzymatic antioxidants, metabolic enzymes and lipid peroxidation status in muscle and hepatopancreas showed no significant (P N 0.05) alterations in prawns fed with 10–60 mg Zn kg−1 supplemented diets. However, prawns fed 80 mg of Zn kg−1 supplemented feed PL showed significant (P b 0.05) alterations in these antioxidant and metabolic enzymes activities suggesting toxic effects of this dose. Hence, the present study suggests that 60 mg Zn kg−1 can be supplemented for regulating better survival, growth and production of M. rosenbergii. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Aquaculture is one of the largest food producing sectors next to agriculture. The freshwater prawn, Macrobrachium rosenbergii is an edible crustacean species among the aquatic cultivable organisms in the world due to its rapid growth, larger size, better meat quality and delicious taste. Minerals play an essential role in biological, physiological, and immunological responses of an organism. Among the minerals, the trace element zinc (Zn) is an essential mineral for stabilizing cellular membranes and a component of all tissues and fluids of organs of an organism (Yamaguchi, 1998). It also has a vital role in growth, cell division, protein synthesis, metabolism of carbohydrate, fertility, and immune system of all organisms. Zn is crucial to several enzymes and transcription factors that regulate key cellular functions such as response to oxidative damage, DNA duplication, DNA repair, and cell cycle regulation (Dong et al., 1999; Ho et al., 2003; Witkiewicz-Kucharczyk and Bal, 2006). Zn based physiological functions are influenced by its transport ⁎ Corresponding author at: Crustacean Biology Laboratory, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore, Tamil Nadu, India. E-mail address: [email protected] (T. Muralisankar).

http://dx.doi.org/10.1016/j.aquaculture.2015.05.045 0044-8486/© 2015 Elsevier B.V. All rights reserved.

and storage. It is metabolized by hepatic metallothionein proteins. A single metalloprotein like metallothionein-1 is capable of binding seven atoms of Zn (Gammanpila et al., 2007; Herland and Cooper, 2011; Tan and Mai, 2001). The dietary requirement of Zn is species dependent. The role of Zn in better survival, growth, muscle composition, immune response, antimicrobial activities, and stress tolerance has been reported in some fish and crustaceans (Davis et al., 1993; Gammanpila et al., 2007; Herland and Cooper, 2011; Shiau and Jiang, 2006; Tan and Mai, 2001). However, Zn deficiency can suppress the function of signaling molecules and proteins directly involved in DNA replication and repair of an organism. The excess absorption of Zn suppresses the reproductive performance, growth, muscle composition and utilization of other minerals in fishes and crustaceans (Gatlin and Wilson, 1983; Jeng and Sun, 1981; Rani et al., 2012; Shiau and Jiang, 2006). Hence, the current study evaluated the influence of dietary Zn supplementation on survival, growth, activities of digestive enzymes, muscle biochemical compositions, carcass mineral contents, activities of antioxidant such as superoxide dismutase (SOD), catalase (CAT), and lipid peroxidation (LPO), and activities of metabolic enzymes such as glutamate–oxaloacetate transaminase (GOT) and glutamate–pyruvate transaminase (GPT) in M. rosenbergii post larvae (PL).

T. Muralisankar et al. / Aquaculture 448 (2015) 98–104

2. Materials and methods 2.1. Feed formulation Diets were prepared at the laboratory and composed of locally available feed ingredients (Table 1). Fishmeal, soybean meal, wheat bran, tapioca flour, and eggs were purchased from local markets. Cod liver oil and vitamin mix were purchased from local medicinal shops. For these diets, fishmeal and soybean meal were used as protein sources, wheat flour and tapioca flour were used as carbohydrate sources, cod liver oil was used as a lipid source, tapioca flour and egg albumin served as binding agents and vitamin B complex with vitamin C were also added. Zn free mineral mix was also prepared (Muralisankar et al., 2014) and added (Table 1). The graded concentrations of dietary Zn were designed according to dietary Zn requirements in crustaceans (Gammanpila et al., 2007). In the present study, 99.99% pure Zn (Sigma-Aldrich, product no. 324930, purchased from Sigma-Aldrich Chemicals Pvt. Limited, Bangalore, India) was supplemented with the basal diets at concentrations of 0, 10, 20, 40, 60, and 80 mg kg−1 and the analyzed content of Zn in these supplemented diets were found to be 31.45, 42.10, 53.80, 65.70, 92.80, and 113.50 mg kg−1 respectively (Table 2). The 3.0 ± 0.97 mm diets were prepared and stored until use for the feeding experiments according to the method of Muralisankar et al. (2014). The analyzed proximate compositions of formulated feeds are given in Table 1 (AOAC, 1995). 2.2. Experimental prawns M. rosenbergii PL (PL-5) were procured from Aqua Hatchery, Koovathur, Kanchipuram District, Tamil Nadu, India. They were safely transported to the laboratory in plastic bags half filled with hatchery water and well-oxygenated. They were acclimatized to ambient laboratory conditions for 3 weeks in a large cement tank (1000 L) with ground water (temperature 28 °C; pH, 7.11 ± 0.20; total dissolved solids, 0.95 ± 0.02 g L−1; dissolved oxygen, 7.24 ± 0.311 mg L− 1; BOD, 10.60 ± 1.15 mg L− 1; COD, 65.0 ± 2.00 mg L− 1; ammonia, 0.018 ± 0.003 mg L−1; zinc level in the water flowing into the rearing system was 27.00 ± 2.41 μg L− 1). The prawns were provided adequate Table 1 Ingredients and proximate biochemical composition of basal diet. Ingredients

Weight (g kg−1)

Fish meal Soybean meal Wheat bran Tapioca flour Egg albumin Cod liver oil Vitamin mixa Zn free Mineral mixb

400 200 180 150 30 20 10 10

Proximate composition (g kg−1) Protein Carbohydrate Fiber Lipid Ash (%) Moisture (%) Energy (kJ g−1)

410.78 290.37 50.10 60.29 10.46 7.00 14.76

a Becosules capsules (manufactured by Pfizer), each capsule contains Thiamine Mononitrate 1P 10 mg; Riboflavin 1P 10 mg; Pyridoxine Hydrochloride 1P 3 mg; Vitamin B12 (as tablets 1:100) 1P 15 mcg Niacinamide 1P 100 mg; Calcium pantothenate 1P 50 mg; Folic acid 1P 1.5 mg; Biotin USP 100 mcg; Ascorbic acid 1P 150 m. b Zinc free mineral mix contains CuSO4.5H2O, 6 mg; CaCO3, 164 mg; NaH2 PO 4 2H2 O, 148 mg; KH 2 PO 4 2H 2 O, 337.6 mg; CaCl 2 , 66.64 mg; MgSO 4 , 7H 2 O 80 mg; KCl, 22.40 mg; AlCl 3 . 6H 2 O, 0.96 mg; MnSO 4 H 2 O, 11.45 mg; FeSO 4 .7H 2 O, 90 mg; COCl 2 6H 2 O , 1.41 mg; KI 1.81 mg; cellulose, 69.74 mg per gram.

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aeration. During acclimatization period they were fed with boiled egg albumin, Artemia nauplii (Artemia salina) and control feed prepared with basal ingredients alternatively three times (at 6 pm, 6 am, and 12 pm respectively) per day, and 80% of aquarium water was renewed daily at 6 am. 2.3. Feeding experiment In the study, six groups of M. rosenbergii PL (1.42 ± 0.35 cm length; 0.18 ± 0.02 g weight) were assigned for this experiment in triplicate for 90 days. One group served as control and was fed with ‘0’ concentration of Zn supplemented diet. The remaining five groups were fed with 10, 20, 40, 60, and 80 mg kg−1 Zn supplemented diets respectively. Each group consisted of 40 PL in an aquarium maintained with 40 L of ground water. The water medium was changed every 24 h by siphoning method with minimum disturbance to the prawns and aerated adequately. The experimental prawns were fed with these feeds at 10% of body weight twice per day. During the feeding trial, the unconsumed feed, feces, and molts were removed on a daily basis while renewing the aquarium water. 2.4. Analysis of survival, growth, and food indices At the end of the feeding trial, the survival rate, growth (length gain and weight gain) and other food index parameters such as feed intake, specific growth rate, feed conversion ratio, and protein efficiency ratio were individually calculated by the following equations (Tekinay and Davies, 2001): Survival rate ð%Þ ¼ no: of live prawns=no: of prawns introduced  100; Length gain ðcmÞ ¼ final length ðcmÞ–initial length ðcmÞ; Weight gainðgÞ ¼ final  weight ðgÞ–initial weight ðgÞ; ‐1 ¼ feed eaten ðgÞ=total number of days; Feed intake g day   ‐1 Specific growth rate % day ¼log final weight ðgÞ –log initial weight ðgÞ = total number of days  100; Feed conversion ratio ¼ feed intake ðgÞ=weight gain ðgÞ; Protein efficiency ratio ðgÞ ¼ weight gain ðgÞ=protein intake ðgÞ:

2.5. Assay of digestive enzymes Activities of digestive enzymes (protease, amylase, and lipase) were assayed on the initial and final days of the feeding experiment. The whole digestive tract and hepatopancreas were homogenized in ice cold double distilled water and centrifuged at 9300 g under 4 °C for 20 min. The supernatant was used as a crude enzyme source. Total protease activity was determined by the casein-hydrolysis method of Furne et al. (2005) where 1 unit of enzyme activity represents the amount of enzyme required to liberate 1 μg of tyrosine per minute under assay conditions. Amylase activity was determined by the starch-hydrolysis method. The specific activity of amylase was calculated as milligrams of maltose liberated per gram of protein per hour (Bernfeld, 1955). Lipase activity was analyzed by the method of Furne et al. (2005). One unit of lipase activity was defined as the amount of free fatty acid released from triacylglycerol per unit of time estimated by the amount of NaOH required to maintain pH constant and represented as mille equivalents of alkali consumed. 2.6. Estimation of muscle biochemical composition and carcass mineral contents Analysis of total nitrogen, crude protein, moisture, and ash contents was performed according to the standard procedures of AOAC (1995). Dry matter was obtained by drying at 105 °C until a constant weight was achieved. Ash content was obtained by burning in a muffle furnace

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Table 2 Mineral compositions (mg kg−1) of formulated diets. Minerals

Controla

Ca Cu Fe K Mg Na Zn

1.74 ± 0.01a 1.73 ± 0.01a 2.13 ± 0.03a 1.34 ± 0.01a 2.04 ± 0.02a 1.45 ± 0.04a 31.45 ± 1.42f

Zn concentrations (mg kg−1) 10

20

40

60

80

P value

1.71 ± 0.04a 1.74 ± 0.03a 2.11 ± 0.01a 1.32 ± 0.01a 2.05 ± 0.03a 1.42 ± 0.03a 42.10 ± 1.81e

1.73 ± 0.02a 1.72 ± 0.01a 2.12 ± 0.02a 1.32 ± 0.03a 2.02 ± 0.02a 1.44 ± 0.02a 53.80 ± 2.10d

1.72 ± 0.02a 1.75 ± 0.02a 2.12 ± 0.02a 1.34 ± 0.02a 2.03 ± 0.01a 1.45 ± 0.02a 65.70 ± 1.65c

1.71 ± 0.02a 1.75 ± 0.01a 2.13 ± 0.04a 1.31 ± 0.02a 2.06 ± 0.02a 1.42 ± 0.03a 92.80 ± 2.41b

1.72 ± 0.01a 1.74 ± 0.02a 2.14 ± 0.03a 1.34 ± 0.03a 2.06 ± 0.03a 1.46 ± 0.05a 113.50 ± 2.87a

0.55 0.35 0.79 0.39 0.24 0.60 0.00

Mean values within the same row sharing the same superscript are not significantly different (P N 0.05). a Zn free diet; n = 3 (three samples from each treatment), mean ± SD.

at 600 °C for 12 h. Total nitrogen and crude protein (N ∗ 6.25) were analyzed after single acid digestion using a Kjeldhal apparatus (model: Kelplus DISTYL-BS, manufactured by Pelican Equipments Pvt. Ltd. Chennai, India). Concentrations of muscle total protein (Lowry et al., 1951), total amino acids (Moore and Stein, 1948) and total carbohydrate (Roe, 1955) were analyzed following standard methods. The total lipid was extracted by the method of Folch et al. (1957) and estimated by the method of Barnes and Black Stock (1973). The carcass mineral contents such as Cu, Zn, Fe, Ca, Mg, Na, and K were analyzed using the Atomic Absorption Spectrophotometer (AAS) (Perkin-Elmer; Model 2380) in air acetylene flame by adopting the triple acid digestion method (AOAC, 1995). To achieve this, sacrificed prawns were digested in 9:3:1 ratio of HNO3, H2SO4, and HClO4 using a hotplate at 400 °C for 2 h. The digested samples were allowed to cool to room temperature and diluted with double distilled water. 2.7. Activities of enzymatic antioxidants and lipid peroxidation Muscles and hepatopancreas of test prawns were individually homogenized (10% w/v) in ice-cold 50 mM Tris buffer (pH 7.4), centrifuged at 9300 g for 20 min at 4 °C and the supernatant was used to assay the enzyme activities. Soluble protein concentration was determined by the method of Lowry et al. (1951). Superoxide dismutase (SOD) activity was measured using pyrogallol (10 mM) autoxidation in Tris buffer (50 mM, pH 7.0) (Marklund and Marklund, 1974) and the specific activity of the enzyme was expressed in U/mg protein. Catalase (CAT) activity was measured using H2O2 as the substrate in phosphate buffer (Sinha, 1972) and the activity of catalase was expressed as μmoles of hydrogen peroxide consumed/min/mg protein. Lipid peroxidation (LPO) in the tissue homogenates was measured by estimating the formation of thiobarbituric acid reactive substances (TBARS) (Ohkawa et al., 1979) and the TBARS was expressed as nmoles of malondialdehyde (MDA)/mg protein.

For GPT analysis, Buffered L-Alanine, 2-Oxoglutarate substrate (500 μL; pH 7.4) was added to a 100 μL sample and incubated at 37 °C for 20 min. With this, 500 μL of 2, 4-dinitrophenyl hydrazine was added and allowed to stand at room temperature for 30 min followed by the addition of 3 mL of freshly prepared 4 N sodium hydroxide solution. The color development was read at 505 nm using a spectrophotometer within 15 min. Sodium pyruvate (170 U/L) was used as a calibrator. The activity of GPT was expressed as U/L. 2.9. Statistical analysis The data were analyzed by one-way analysis of variance (ANOVA) using SPSS (16.0), followed by Duncan's multiple range test (DMRT) to compare the differences among treatments where significant differences (P b 0.05) were observed. Data were expressed as mean ± S.D. Dietary zinc requirements of M. rosenbergii were estimated by broken-line regression analysis (Robbins, 1986; Robbins et al., 1979). 3. Results 3.1. Survival, growth and food indices The survival, growth, and other food indices parameters such as feed intake, specific growth rate, and protein efficiency ratio were significantly elevated (P b 0.05) in prawns fed with 10–60 mg Zn kg−1 supplemented diets when compared with the control diet fed prawns. Among these concentrations, 60 mg Zn kg−1 showed better performance. However, 80 mg Zn kg−1 supplemented diet fed prawns attained poor performance at these parameters. The feed conversion ratio was found to be decreased in prawns fed with 10–60 Zn kg−1 supplemented diets (Table 3). The breakpoint in the regression line, 92.48 and 92.51 mg Zn kg− 1 diets was considered to be the optimum dietary level for better response for weight gain and specific growth rate of M. rosenbergii respectively (Fig. 1).

2.8. Analysis of metabolic enzymes activity 3.2. Activity of digestive enzymes The metabolic enzymes such as glutamic oxaloacetate transaminase (GOT) and glutamic pyruvate transaminase (GPT) were analyzed according to the method of Reitman and Frankel (1957) using a med source kit (Medsource Ozone Biomedicals Pvt. Ltd. Haryana, India). 100 mg of muscle and hepatopancreas tissues were homogenized in 0.25 M sucrose and centrifuged at 3300 g for 20 min in a high speed cooling centrifuge at 4 °C. The supernatant was used as the enzyme source. For GOT analysis, the substrate solution, L-Aspartic acid (500 μL; pH 7.4) was added to a 100 μL sample and incubated at 37 °C for 1 h. Further, 500 μL of 2, 4-dinitrophenyl hydrazine was added and allowed to stand for 20 min at room temperature. Then 3 mL of freshly prepared 4 N sodium hydroxide solution was added to the above solution. The color development was read at 505 nm using spectrophotometer within 15 min. Sodium pyruvate (160 U/L) was used as a calibrator. The activity of GOT was expressed as U/L.

Activities of digestive enzymes such as protease, amylase, and lipase were significantly increased (P b 0.05) in prawns fed with 60 mg Zn kg− 1 diets when compared with control. In contrast, in the case of amylase activity, there was no significant difference found between 10, 20, 40, and 80 mg Zn kg−1 supplemented diet fed prawns. The non-significant (P N 0.05) difference was also found in lipase activity when prawns fed with 10 and 80 mg Zn kg−1 supplemented diets when compared to the control (Table 4). 3.3. Muscle biochemical compositions and carcass minerals The percentage of total nitrogen and crude protein levels were found to be gradually elevated in 10–60 mg Zn kg−1 supplementation when compared with control (P b 0.05), whereas, their levels were found to have declined in 80 mg Zn kg− 1 supplemented diet fed prawns.

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Table 3 Survival, growth and food indices of M. rosenbergii fed with Zn supplemented diets. Parameters

SR (%) Length (cm) Weight (g) LG (cm) WG (g) FI (g day−1) DM (no. of molts d−1) SGR (% day−1) FCR PER

Controla

75.83 ± 1.84d 3.64 ± 0.28c 0.58 ± 0.13c 3.29 ± 0.32f 0.40 ± 0.01e 0.39 ± 0.01e 2.50 ± 0.11c 0.55 ± 0.03d 2.69 ± 0.35a 1.22 ± 0.06e

Zn concentrations (mg kg−1)

P value

10

20

40

60

80

76.66 ± 1.44c 4.84 ± 0.47b 0.89 ± 0.23bc 4.15 ± 0.15e 0.71 ± 0.03d 0.44 ± 0.01d 2.53 ± 0.09c 0.76 ± 0.04c 1.40 ± 0.15b 2.53 ± 0.10d

77.50 ± 2.50bc 4.98 ± 0.49ab 0.98 ± 0.30bc 4.49 ± 0.23d 0.80 ± 0.04c 0.48 ± 0.01c 2.56 ± 0.06c 0.81 ± 0.02c 1.35 ± 0.10b 2.66 ± 0.11d

81.66 ± 3.81b 5.24 ± 0.87ab 1.29 ± 0.43ab 4.89 ± 0.21c 1.11 ± 0.06b 0.53 ± 0.01b 2.91 ± 0.07ab 0.94 ± 0.03b 0.94 ± 0.11c 3.26 ± 0.13c

85.83 ± 1.44a 5.90 ± 0.80a 1.68 ± 0.59a 5.55 ± 0.26a 1.50 ± 0.07a 0.60 ± 0.01a 3.13 ± 0.12a 1.06 ± 0.08a 0.91 ± 0.09c 3.94 ± 0.20a

70.00 ± 2.50c 5.60 ± 0.82ab 1.28 ± 0.51ab 5.25 ± 0.41b 1.10 ± 0.07b 0.44 ± 0.01d 2.64 ± 0.10bc 0.92 ± 0.09b 0.95 ± 0.07c 3.64 ± 0.24b

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Mean values within the same row sharing the same superscript are not significantly different (P N 0.05). Initial length and weight were 1.42 ± 0.35 cm and 0.18 ± 0.02 respectively. SR, survival rate; LG, length gain; WG, weight gain; FI, feed intake; DM, daily molt; SGR, specific growth rate; FCR, feed conversion ratio; PER, protein efficiency ratio. a Zn free diet; n = 3 (three samples from each treatment), mean ± SD.

Likewise, the muscle biochemical compositions such as of total protein, total amino acids, total carbohydrate, total lipid, and ash were significantly elevated (P b 0.05) in prawns fed with 20–60 mg Zn kg−1 supplemented diets (Table 5) when compared with control, whereas, their levels were found to have decreased in 80 mg Zn kg−1 supplementation diet fed prawns. The contents of mineral salts, such as Cu, Fe, Ca, Mg, Na, and K, were almost similar in the formulated diets (Table 2), whereas, the levels of these mineral salts were significantly increased (P b 0.05) in prawns fed with 10–60 mg Zn kg−1 supplemented diets when compared with control. However, Zn content was significantly (P b 0.05) increased in 10–80 mg Zn kg−1 supplemented diet fed prawns (Table 5). 3.4. Activities of SOD, CAT, LPO, GOT, and GPT In the present study, no significant changes in activities of SOD, CAT, LPO, GOT, and GPT were recorded in muscle and hepatopancreas of prawns fed with 10–60 mg Zn kg−1 supplemented diets throughout the sampling period. In the case of 80 mg Zn kg−1 supplemented diet fed prawns, these antioxidants (SOD, CAT, and LPO) and metabolic enzyme (GOT and GPT) activities showed significant elevations

(P b 0.05) when compared with those of control and other diets (Tables 6 and 7). 4. Discussion Aquatic organisms have the ability to absorb some zinc from water, but the diet is the predominant uptake route. The dietary zinc requirements have been established for a number of different aquatic species fed semi-purified diets (NRC, 2011). Zinc plays an essential role in growth, development and maintenance of physiological activities, and functions as a cofactor of many enzymes and an integral part of more than 20 metalloenzymes including alkaline phosphatase, alcohol dehydrogenase, and carbonic anhydrase (Watanabe et al., 1997). However, Zn deficiency affects the digestibility and metabolism of protein and carbohydrates due to the reduced activity of carboxypeptidase. The optimum dietary requirement of Zn has been quantified for a few fish and crustaceans for regulating proper production (Davis et al., 1993; Gammanpila et al., 2007; Herland and Cooper, 2011; Li et al., 2010; Rani et al., 2012; Shiau and Jiang, 2006). In the present study, the improved survival, growth (length and weight gain), feed intake, specific growth rate, and protein efficiency

Fig. 1. Regression of weight gain (WG) and specific growth rate (SGR) on dietary zinc levels and breakpoints (bkpt) in the lines for M. rosenbergii fed diets containing graded levels of zinc for 90 days. The term “Criteria values” represents the values of a selected parameter, such as WG and SGR.

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Table 4 Activities of digestive enzymes (U/mg protein) in M. rosenbergii fed with Zn supplemented diets.

Protease Amylase Lipaseb

Zn concentrations (mg kg−1)

Controla

Enzymes

Initial Final Initial Final Initial Final

0.27 ± 0.06 1.00 ± 0.12b 0.16 ± 0.04 0.75 ± 0.11b 0.74 ± 0.05 0.22 ± 0.02d

P value

10

20

40

60

80

0.27 ± 0.06 1.22 ± 0.32b 0.16 ± 0.04 0.78 ± 0.10b 0.74 ± 0.05 0.28 ± 0.01d

0.27 ± 0.06 1.30 ± 0.11b 0.16 ± 0.04 0.83 ± 0.13b 0.74 ± 0.05 0.34 ± 0.04c

0.27 ± 0.06 1.48 ± 0.23ab 0.16 ± 0.04 0.88 ± 0.12b 0.74 ± 0.05 0.42 ± 0.03b

0.27 ± 0.06 1.87 ± 0.42a 0.16 ± 0.04 1.70 ± 0.14a 0.74 ± 0.05 0.48 ± 0.06a

0.27 ± 0.06 1.12 ± 0.11b 0.16 ± 0.04 0.75 ± 0.10b 0.74 ± 0.05 0.23 ± 0.01d

0.01 0.00 0.00

Mean values within the same row sharing the same superscript are not significantly different (P N 0.05). a Zn free diet. b ×102; n = 3 (three samples from each treatment), mean ± SD.

ratio indicates that 10–60 mg Zn kg−1 have the ability to promote the survival, feed intake, and growth of M. rosenbergii. However, the broken-line model indicated that the optimum dietary concentration for better responses in weight gain and specific growth rate for prawn species were 92.48 and 92.51 mg Zn kg−1 respectively. The lower feed conversion ratio recorded in 60 mg Zn kg−1 indicates the better quality of the feed. Zn supplemented diet fed fishes such as Oreochromis niloticus, Oreochromis mykiss, Gadus morhua, Ictalurus punctatus, Carassius auratus, and crustaceans such as the shrimp, Penaeus monodon, and the Chinese mitten crab, Eriochei sinensis showed increases in survival, feed intake, and growth which have been reported (Davis et al., 1993; Gammanpila et al., 2007; Gatlin and Wilson, 1983; Herland and Cooper, 2011; Li et al., 2010; Rani et al., 2012; Sarker and Satoh, 2007; Shiau and Jiang, 2006; Tan et al., 2011). The poor growth performance in control and 80 mg Zn kg−1 diet fed prawns suggests deficiency and excess of Zn in the diet respectively. Similarly, Satoh et al. (1987) reported that Zn deficiency leads to slow growth and increased mortality in fish. An excess of dietary Zn suppressing survival and growth have also been reported in C. auratus, P. monodon, I. punctatus, Cyprinus carpio, Cirrhinus mrigala, and E. sinensis (Gatlin and Wilson, 1983; Jeng and Sun, 1981; Mohanty et al., 2009; Rani et al., 2012; Shiau and Jiang, 2006; Tan et al., 2011). The digestive enzyme in crustaceans plays an essential role in nutritional physiology and regulates the growth and molt cycle (Lovett and Felder, 1990). In this study, the elevated digestive enzyme activity

indicates that supplemented Zn has influence on digestive enzyme secretion in M. rosenbergii. Similarly, Zn nanoparticles supplemented feed fed M. rosenbergii produced significant improvements in digestive enzyme (protease, amylase, and lipase) secretion, which has been reported (Muralisankar et al., 2014). The dietary supplementation of Zn can promote these digestive enzyme secretion has been reported in tilapia, O. niloticus, Oreochromis aureus, and C. carpio (Li et al., 2007; Tan et al., 2011). Similarly, Zn supplemented feed fed rats and pigs showed increases in the activity of protease, amylase, maltase, pepsin, and protease (Hedemann et al., 2006; Jing et al., 2009). The decreased digestive enzyme activities recorded in 80 mg Zn kg−1 supplemented feed fed prawns indicates its toxicity due to excess dosage. It has also been reported in M. rosenbergii fed with 80 mg kg−1 Zn nanoparticle supplemented diet (Muralisankar et al., 2014). Zn plays a vital role in lipid, protein, carbohydrate, and nucleic acid metabolism (Lall, 2002). The elevations recorded in muscle biochemical compositions, such as total nitrogen, protein, amino acid, carbohydrate, lipid, and ash suggests that dietary Zn has influence on nutrient absorption and increases the synthesis and storage of protein, amino acids, carbohydrate, and lipid in M. rosenbergii. The increased protein, lipid, fatty acid, and ash contents have also been reported in Zn supplemented diet fed G. morhua, O. niloticus and E. sinensis (Gammanpila et al., 2007; Herland and Cooper, 2011; Li et al., 2010). However, the decreased level of these biochemical constituents recorded in 80 mg Zn kg−1 supplemented diet fed prawn group due to the excess level of Zn in their

Table 5 Proximate muscle biochemical composition and whole body minerals of M. rosenbergii fed with Zn supplemented diets. Controla

Parameters

Total nitrogen (% dry wt.) Crude protein (% dry wt.) Total protein (mg g−1 wet wt.) Amino acid (mg g−1 wet wt.) Carbohydrate (mg g−1 wet wt.) Lipid (mg g−1 wet wt.) Ash (%) Moisture (%) Whole body minerals content (μg g−1)

Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Ca Cu Fe K Mg Na Zn

4.10 ± 0.18 8.17 ± 0.36d 25.66 ± 1.16 51.10 ± 2.29d 46.64 ± 2.14 148.79 ± 2.41e 25.33 ± 2.30 90.00 ± 3.46d 18.07 ± 1.07 32.07 ± 1.88d 10.68 ± 0.74 18.03 ± 2.69c 10.40 ± 0.69 15.50 ± 1.37a 78.33 ± 1.52 75.00 ± 4.00a 32.85 ± 2.07f 37.05 ± 1.28d 26.25 ± 2.01d 146.28 ± 3.01c 78.50 ± 3.11f 124.65 ± 2.44e 19.75 ± 1.30f

Zn concentrations (mg kg−1)

P value

10

20

40

60

80

4.10 ± 0.18 8.52 ± 0.22cd 25.66 ± 1.16 53.25 ± 1.40cd 46.64 ± 2.14 163.53 ± 4.25d 25.33 ± 2.30 100.00 ± 4.00cd 18.07 ± 1.07 38.65 ± 3.64c 10.68 ± 0.74 21.28 ± 2.35bc 10.40 ± 0.69 16.70 ± 1.80a 78.33 ± 1.52 73.66 ± 3.05a 72.55 ± 3.06d 51.80 ± 2.89c 34.50 ± 2.64c 152.48 ± 2.73b 97.85 ± 2.76e 132.56 ± 2.49d 45.25 ± 2.11e

4.10 ± 0.18 8.91 ± 0.45bc 25.66 ± 1.16 55.70 ± 2.85bc 46.64 ± 2.14 172.21 ± 3.48c 25.33 ± 2.30 105.33 ± 3.05c 18.07 ± 1.07 42.72 ± 3.73bc 10.68 ± 0.74 23.84 ± 3.07ab 10.40 ± 0.69 17.23 ± 1.56a 78.33 ± 1.52 72.23 ± 1.96a 84.55 ± 4.10c 68.65 ± 2.21b 41.35 ± 2.57b 157.31 ± 2.35b 115.90 ± 4.08d 154.92 ± 2.51c 78.75 ± 3.02d

4.10 ± 0.18 9.46 ± 0.21b 25.66 ± 1.16 59.15 ± 1.36b 46.64 ± 2.14 190.60 ± 2.68b 25.33 ± 2.30 118.40 ± 3.41b 18.07 ± 1.07 47.06 ± 3.17ab 10.68 ± 0.74 25.64 ± 2.56ab 10.40 ± 0.69 17.43 ± 1.50a 78.33 ± 1.52 70.00 ± 3.00a 94.60 ± 3.11b 71.90 ± 2.54b 42.35 ± 3.21b 163.25 ± 2.91b 134.91 ± 2.97c 158.43 ± 3.06c 84.35 ± 1.06c

4.10 ± 0.18 10.15 ± 0.56a 25.66 ± 1.16 63.46 ± 3.50a 46.64 ± 2.14 221.85 ± 3.44a 25.33 ± 2.30 135.46 ± 4.38a 18.07 ± 1.07 52.34 ± 1.73a 10.68 ± 0.74 28.14 ± 1.96a 10.40 ± 0.69 17.56 ± 1.25a 78.33 ± 1.52 68.23 ± 3.35a 139.45 ± 3.03a 91.75 ± 3.67a 59.80 ± 2.44a 167.92 ± 3.02a 168.50 ± 2.50a 194.61 ± 2.38a 87.10 ± 2.22b

4.10 ± 0.18 8.81 ± 0.31abc 25.66 ± 1.16 55.12 ± 1.95abc 46.64 ± 2.14 155.64 ± 5.81de 25.33 ± 2.30 97.26 ± 7.97cd 18.07 ± 1.07 45.30 ± 1.73a 10.68 ± 0.74 21.74 ± 2.20bc 10.40 ± 0.69 15.83 ± 1.19a 78.33 ± 1.52 73.90 ± 4.00a 54.30 ± 2.11e 90.55 ± 3.65a 24.55 ± 3.01d 163.26 ± 2.48b 146.50 ± 2.92b 183.46 ± 3.29b 103.60 ± 4.05a

Mean values within the same row sharing the same superscript are not significantly different (P N 0.05). a Zn free diet; n = 3 (three samples from each treatment), mean ± SD.

0.00 0.00 0.00 0.00 0.00 0.00 0.43 0.17 0.00 0.00 0.00 0.00 0.00 0.00 0.00

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103

Table 6 Activities of antioxidant and metabolic enzymes in the muscle of M. rosenbergii fed with Zn supplemented diets. Controla

Parameters

Initial

30 days

60 days

90 days

SOD (μmol/min/mg protein) CAT (U/mg protein) LPO (nmol MDA/mg protein) GOT (U/L) GPT (U/L) SOD (μmol/min/mg protein) CAT (U/mg protein) LPO (nmol MDA/mg protein) GOT (U/L) GPT (U/L) SOD (μmol/min/mg protein) CAT (U/mg protein) LPO (nmol MDA/mg protein) GOT (U/L) GPT (U/L) SOD (μmol/min/mg protein) CAT (U/mg protein) LPO (nmol MDA/mg protein) GOT (U/L) GPT (U/L)

4.81 ± 1.01 11.20 ± 1.01 0.13 ± 0.01 6.32 ± 0.67 7.95 ± 1.01 8.28 ± 1.17 b 20.90 ± 1.23 b 0.63 ± 0.02 b 8.53 ± 1.11b 9.21 ± 1.08 b 8.27 ± 1.24 b 20.92 ± 1.32 b 0.63 ± 0.01 b 8.53 ± 1.12 b 9.22 ± 0.83 b 8.30 ± 1.46 b 20.97 ± 1.44 b 0.63 ± 0.02 b 8.54 ± 1.13 b 9.23 ± 1.13 b

Zn concentrations (mg kg−1)

P value

10

20

40

60

80

4.81 ± 1.01 11.20 ± 1.01 0.13 ± 0.01 6.32 ± 0.67 7.95 ± 1.01 8.28 ± 1.21 b 20.83 ± 1.21 b 0.63 ± 0.01 b 8.51 ± 0.55 b 9.21 ± 1.07 b 8.27 ± 1.29 b 21.19 ± 1.41 b 0.64 ± 0.02 b 8.52 ± 0.53 b 9.22 ± 1.10 b 8.44 ± 1.50 b 21.43 ± 1.35 b 0.63 ± 0.01 b 8.52 ± 0.55 b 9.23 ± 1.38 b

4.81 ± 1.01 11.20 ± 1.01 0.13 ± 0.01 6.32 ± 0.67 7.95 ± 1.01 8.28 ± 1.89 b 20.84 ± 1.75 b 0.63 ± 0.01 b 8.52 ± 0.62 b 9.22 ± 1.03 b 8.31 ± 1.17 b 21.40 ± 1.56 b 0.64 ± 0.02 b 8.52 ± 0.66 b 9.22 ± 1.17 b 8.51 ± 1.14 b 21.43 ± 1.53 b 0.63 ± 0.03 b 8.53 ± 0.56 b 9.23 ± 1.32 b

4.81 ± 1.01 11.20 ± 1.01 0.13 ± 0.01 6.32 ± 0.67 7.95 ± 1.01 8.31 ± 1.00 b 20.92 ± 1.22 b 0.64 ± 0.02 b 8.53 ± 1.20 b 9.22 ± 2.12 b 8.40 ± 1.03 b 21.25 ± 1.33 b 0.65 ± 0.01 b 8.52 ± 1.28 b 9.21 ± 0.86 b 8.62 ± 1.23 b 21.55 ± 2.00 b 0.64 ± 0.02 b 8.53 ± 1.22 b 9.21 ± 1.29 b

4.81 ± 1.01 11.20 ± 1.01 0.13 ± 0.01 6.32 ± 0.67 7.95 ± 1.01 8.34 ± 1.33 b 20.94 ± 1.43 b 0.66 ± 0.01 b 8.52 ± 1.21 b 9.21 ± 1.06 b 8.40 ± 1.25 b 21.27 ± 1.34 b 0.65 ± 0.01 b 8.53 ± 1.11 b 9.22 ± 1.42 b 8.67 ± 1.42 b 21.82 ± 2.01 b 0.66 ± 0.01 b 8.53 ± 1.15 b 9.26 ± 1.20 b

4.81 ± 1.01 11.20 ± 1.01 0.13 ± 0.01 6.32 ± 0.67 7.95 ± 1.01 11.57 ± 1.38 a 24.14 ± 2.14 a 1.31 ± 0.02 a 11.65 ± 1.28a 11.94 ± 1.47 a 13.62 ± 1.18 a 24.54 ± 1.67 a 1.48 ± 0.19 a 12.32 ± 2.38 a 13.04 ± 2.00 a 14.04 ± 1.35 a 27.32 ± 2.35 a 1.93 ± 0.02 a 14.91 ± 1.92 a 14.14 ± 3.38 a

0.06 0.11 0.00 0.01 0.15 0.00 0.07 0.00 0.02 0.01 0.00 0.00 0.00 0.00 0.02

Mean values within the same row sharing the same superscript are not significantly different (P N 0.05). a Zn free diet; n = 3 (three samples from each treatment), mean ± SD.

diet. This observation agrees with earlier reports on G. morhua and Haliotis discus hannai fed with over-optimized concentration of Zn supplemented diets which showed poor performance in muscle biochemical compositions (Herland and Cooper, 2011; Tan and Mai, 2001). In the present study, the analyzed mineral contents clearly showed that the 10–60 mg kg− 1 of dietary Zn promotes mineral utilization. Increased iron, phosphorus, sodium, and copper utilization has been reported in H. discus hannai, and O. mykiss due to supplementation of dietary Zn (Ramseyer et al., 1999; Sarker and Satoh, 2007; Tan and Mai, 2001). A study on I. punctatus has reported that the dietary supplementation Zn promotes the serum and bone Zn, and Ca levels (Gatlin and Wilson, 1983). The Cu, Zn, and K supplemented diet fed G. morhua showing higher utilization of Cu, Zn, K, Mg, and Ca has also been reported (Herland and Cooper, 2011). In the present study, 80 mg Zn kg− 1 supplemented diet fed prawns showed poor absorption of minerals when compared to 60 mg Zn kg − 1

supplemented diet fed prawns. It indicates the fact that mineral utilization of prawns was based on the level of Zn supplementation. Zn supplemented feed fed C. auratus showed significant elevations in Fe and Cu content up to 60 mg Zn kg− 1 and decreased at higher concentrations of dietary Zn supplementation (Rani et al., 2012). The antioxidant enzymes, SOD and CAT are responsible for scavenging reactive species to avoid cellular damage. In this study, 10– 60 mg Zn kg−1 did not produce any significant alterations in the activities of SOD, CAT, GOT, and GPT, along with the level of LPO suggesting that 10–60 mg kg−1 concentrations of Zn can be taken as a safe dietary level for M. rosenbergii. In the present study, 80 mg Zn kg−1 supplementation has produced some significant elevations in SOD and CAT activities, which led to suppression of the growth performance, activities of digestive enzymes, and muscle composition of M. rosenbergii due to toxic effects. The elevated activities of LPO, GOT, and GPT in 80 mg Zn kg−1 supplemented feed fed M. rosenbergii further confirms

Table 7 Activities of antioxidant and metabolic enzymes in the hepatopancreas of M. rosenbergii fed with Zn supplemented diets. Controla

Parameters

Initial

30 days

60 days

90 days

SOD (μmol/min/mg protein) CAT (U/mg protein) LPO (nmol MDA/mg protein) GOT (U/L) GPT (U/L) SOD (μmol/min/mg protein) CAT (U/mg protein) LPO (nmol MDA/mg protein) GOT (U/L) GPT (U/L) SOD (μmol/min/mg protein) CAT (U/mg protein) LPO (nmol MDA/mg protein) GOT (U/L) GPT (U/L) SOD (μmol/min/mg protein) CAT (U/mg protein) LPO (nmol MDA/mg protein) GOT (U/L) GPT (U/L)

5.81 ± 1.01 14.20 ± 1.01 0.14 ± 0.010 8.32 ± 0.67 9.15 ± 1.01 14.49 ± 1.43 b 26.24 ± 2.33 b 1.89 ± 0.11 b 13.85 ± 1.12 b 14.32 ± 0.86 b 14.49 ± 1.21 b 26.51 ± 2.00 b 1.88 ± 0.21 b 13.86 ± 1.31 b 14.33 ± 0.88 b 14.49 ± 2.09 b 26.56 ± 2.11 b 1.89 ± 0.12 b 13.86 ± 1.92 b 14.32 ± 0.93 b

Zn concentrations (mg kg−1)

P value

10

20

40

60

80

5.81 ± 1.01 14.20 ± 1.01 0.13 ± 0.010 8.32 ± 0.67 9.15 ± 1.01 14.49 ± 1.76 b 26.40 ± 2.13 b 1.89 ± 0.20 b 13.85 ± 1.18 b 14.32 ± 0.55 b 14.53 ± 1.36 b 26.53 ± 2.10 b 1.88 ± 0.14 b 13.86 ± 1.30 b 14.33 ± 0.56 b 14.56 ± 2.02 b 26.68 ± 2.23 b 1.90 ± 0.17 b 13.85 ± 2.91 b 14.33 ± 0.49 b

5.81 ± 1.01 14.20 ± 1.01 0.13 ± 0.010 8.32 ± 0.67 9.15 ± 1.01 14.53 ± 1.24 b 26.49 ± 2.44 b 1.90 ± 0.12 b 13.84 ± 1.49 b 14.32 ± 0.99 b 14.87 ± 1.09 b 26.54 ± 1.98 b 1.88 ± 0.18 b 13.85 ± 1.07 b 14.32 ± 0.94 b 14.66 ± 1.38 b 26.70 ± 1.87 b 1.92 ± 0.13 b 13.85 ± 2.89 b 14.33 ± 0.99 b

5.81 ± 1.01 14.20 ± 1.01 0.13 ± 0.010 8.32 ± 0.67 9.15 ± 1.01 14.58 ± 1.65 b 26.86 ± 1.89 b 1.93 ± 1.19 b 13.86 ± 1.25 b 14.31 ± 0.93 b 14.87 ± 1.62 b 26.51 ± 1.56 b 1.90 ± 0.12 b 13.85 ± 1.33 b 14.33 ± 0.84 b 14.71 ± 2.18 b 26.72 ± 2.00 b 1.93 ± 0.10 b 13.86 ± 1.92 b 14.32 ± 0.98 b

5.81 ± 1.01 14.20 ± 1.01 0.13 ± 0.010 8.32 ± 0.67 9.15 ± 1.01 14.72 ± 1.23 b 27.01 ± 3.00 b 1.94 ± 0.16 b 13.85 ± 1.30 b 14.33 ± 0.60 b 14.91 ± 1.37 b 26.59 ± 2.57 b 1.91 ± 0.21 b 13.85 ± 1.42 b 14.33 ± 0.90 b 14.78 ± 1.65 b 26.95 ± 2.43 b 2.06 ± 0.24 b 13.86 ± 0.77 b 14.32 ± 0.91 b

5.81 ± 1.01 14.20 ± 1.01 0.13 ± 0.01 8.32 ± 0.67 9.15 ± 1.01 18.73 ± 1.56 a 33.51 ± 3.15 a 2.97 ± 0.18 a 16.47 ± 1.07 a 17.15 ± 2.75 a 18.84 ± 1.44 a 30.49 ± 2.34 a 3.02 ± 0.150 a 18.71 ± 1.42 a 18.03 ± 1.45 a 18.69 ± 2.54 a 31.16 ± 2.55 a 4.45 ± 0.26 a 18.15 ± 1.47 a 20.52 ± 3.31 a

Mean values within the same row sharing the same superscript are not significantly different (P N 0.05). a Zn free diet; n = 3 (three samples from each treatment), mean ± SD.

0.02 0.02 0.12 0.11 0.11 0.01 0.15 0.00 0.00 0.00 0.14 0.14 0.00 0.14 0.00

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the Zn toxicity. It has been reported that dietary supplementation of Zn at 60 mg kg−1 did not produce any significant alterations in liver SOD activities on C. auratus whereas exceeding this 60 mg Zn kg−1 supplementation has produced some significant elevation in SOD activity (Rani et al., 2012). It has also been reported that higher concentrations of dietary Zn produced significant alterations in SOD, CAT and LPO activities in C. carpio (Feng et al., 2011). 5. Conclusion The optimum dietary requirement of Zn should be optimized to achieve the maximum production without harmful effects to the cultured species. The present study showed that 10–60 mg Zn kg−1 produced better performance in survival, growth, digestive enzymes, and muscle biochemical compositions with unaltered antioxidants and metabolic enzymes. Among these Zn supplementations, 60 mg Zn kg−1 produced better performance in M. rosenbergii, suggesting that 60 mg Zn kg− 1 supplementation is optimal for production of the giant prawn M. rosenbergii. Acknowledgments Bharathiar University, Coimbatore, Tamil Nadu, India is gratefully acknowledged for the financial support provided in the form of University Research Fellowship to the first author. The University Grants Commission, Government of India, New Delhi is also gratefully acknowledged for the acquired laboratory facility by the second author through a Major Research Project operated (2009–2012) on prawn nutrition. References AOAC, 1995. Official Methods of Analysis. 16th ed. AOAC International Publishers, Arlington VA. Barnes, H., Black Stock, J., 1973. Estimation of lipids in marine animals and tissues detailed investigation of the Sulphophosphovanillin method for total lipids. J. Exp. Mar. Biol. Ecol. 12, 103–118. Bernfeld, P., 1955. Amylases. In: Colowick, S.P., Kaplan, N.O. (Eds.), Methods in Enzymology. Academic Press, New York, pp. 149–158. Davis, D.A., Lawrence, A.L., Galtin III, D.M., 1993. Evaluation of the dietary zinc requirement of Penaeus vannamei and effects of phytic acid on zinc and phosphorus bioavailability. J. World Aquacult. Soc. 24, 40–47. Dong, J., Park, J.S., Lee, S.H., 1999. In vitro analysis of the zinc-finger motif in human replication protein A. Biochem. J. 337, 311–317. Feng, L., Tan, L.N., Liu, Y., Jiang, J., Jiang, W.D., Hu, K., Li, S.H., Zhou, X.Q., 2011. Influence of dietary zinc on lipid peroxidation protein oxidation and antioxidant defence of juvenile Jain carp (Cyprinus carpio var Jain). Aquacult. Nutr. 17, e875–e882. Folch, J., Lees, M., Bloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 266, 497–509. Furne, M., Hidalgo, M.C., Lopez, A., Garcia-Gallego, M., Morales, A.E., Domenzain, A., Domezain, J., Sanz, A., 2005. Digestive enzyme activities in Adriatic sturgeon Acipenser naccarii and rainbow trout Oncorhynchus mykiss. A comparative study. Aquaculture 250, 391–398. Gammanpila, M., Age, A.Y., Bart, A.N., 2007. Evaluation of the effects of dietary vitamin C, E and Zinc supplementation on reproductive performance of Nile tilapia (Oreochromis niloticus). Sri Lanka J. Aquat. Sci. 12, 39–60. Gatlin III, D.M., Wilson, R.P., 1983. Dietary zinc requirement of fingerling channel catfish. J. Nutr. 113, 630–635. Hedemann, M.S., Jensen, B.B., Poulsen, H.D., 2006. Influence of dietary zinc and copper on digestive enzyme activity and intestinal morphology in weaned pigs. J. Anim. Sci. 84, 3310–3320. Herland, H., Cooper, M., 2011. Effects of dietary mineral supplementation on quality of fresh and salt-cured fillets from farmed Atlantic cod Gadus morhua. J. World Aquacult. Soc. 42, 261–267. Ho, E., Courtemanche, C., Ames, B.N., 2003. Zinc deficiency induce oxidative DNA damage and increases p53 expression in human lung fibroblasts. J. Nutr. 133, 2543–2548.

Jeng, S.S., Sun, L.T., 1981. Effects of dietary zinc levels on zinc concentrations in tissues of common carp. J. Nutr. 111, 134–140. Jing, M.Y., Sun, J.Y., Weng, X.Y., Wang, J.F., 2009. Effects of zinc levels on activities of gastrointestinal enzymes in growing rats. J. Anim. Physiol. Anim. Nutr. 93, 606–612. Lall, S.P., 2002. The minerals. In: Halver, J.E., Hardy, R.D. (Eds.), Fish Nutrition, 3rd ed. Academic Press, New York, New York, pp. 259–308. Li, J.S., Li, J.L., Wu, T.T., 2007. The effects of copper, iron and zinc on digestive enzyme activity in the hybrid tilapia Oreochromis niloticus (L) Oreochromis aureus (Steindachner). J. Fish Biol. 71, 1788–1798. Li, W.W., Gong, Y.N., Jin, X.K., He, L., Jiang, H., Ren, F., Wang, Q., 2010. The effect of dietary zinc supplementation on the growth hepatopancreas fatty acid composition and gene expression in the Chinese mitten crab Eriocheir sinensis (H Milne-Edwards) (Decapoda, Grapsidae). Aquacult. Res. 41, 828–837. Lovett, D.L., Felder, D.L., 1990. Ontogenic change in digestive enzyme activity of larval and postlarval white shrimp Penaeus setiferus (Crustacea, Decapoda, Penaeidae). Biol. Bull. 178, 144–159. Lowry, O.H., Rosenbrough, W.J., Fair, A.L., Randall, R.J., 1951. Protein measurement with the folinphenol reagent. J. Biol. Chem. 193, 265–275. Marklund, S., Marklund, G., 1974. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47, 469–474. Mohanty, M., Adhikari, S., Mohanty, P., Sarangi, N., 2009. Effect of waterborne zinc on survival, growth, and feed intake of Indian major carp, Cirrhinus mrigala (Hamilton). Water Air Soil Pollut. 201, 3–7. Moore, S., Stein, W.H., 1948. Photometric ninhydrin method for use in the chromatography of amino acid. J. Biol. Chem. 176, 367–388. Muralisankar, T., Bhavan, P.S., Radhakrishnan, S., Seenivasan, C., Manickam, N., Srinivasan, V., 2014. Dietary supplementation of zinc nanoparticles and its influence on biology physiology and immune responses of the freshwater prawn Macrobrachium rosenbergii. Biol. Trace Elem. Res. 160, 56–66. NRC, 2011. Nutrient Requirements of Fish and Shrimp. National Academies Press, Washington D.C. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Ramseyer, L., Garling, D., Hill, G., Link, J., 1999. Effect of dietary zinc supplementation and phytase pre-treatment of soybean meal or corn gluten meal on growth, zinc status and zinc-related metabolism in rainbow trout, Oncorhynchus mykiss. Fish Physiol. Biochem. 20, 251–261. Rani, B., Hasnat, A., Kohli, M.P.S., Chandraprakash, G., 2012. Zinc supplementation and its effect on thermal stress resistance in Carassius auratus Fry. Isr. J. Aquacult. Bamidgeh 64, 779–786. Reitman, S., Frankel, S., 1957. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28, 56–63. Robbins, K.R., 1986. A method, SAS program, and examples for fitting the broken line to growth data. Univ. Tenn. Res. Rep. Univ. Tenn Agric. Exp. Sta, Knoxville, TN, pp. 86–90. Robbins, K.R., Norton, H.W., Baker, D.H., 1979. Estimation of nutrient requirements from growth data. J. Nutr. 109, 1710–1714. Roe, J.H., 1955. The determination of sugar and blood and spinal fluid with anthrone reagent. J. Biol. Chem. 212, 335–343. Sarker, M.S., Satoh, S., 2007. Influence of dietary phosphorus and zinc levels on whole body mineral liver mineral and liver vitamin-C contents of fingerling rainbow trout Oncorhynchus mykiss. J. Agric. Rural Dev. 5, 135–142. Satoh, S., Izume, K., Takeuchi, T., Watanabe, T., 1987. Availability to rainbow trout of zinc contained in various types of fish meals. Nippon Suisan Gakkaishi 53, 1861–1866. Shiau, S.Y., Jiang, L.C., 2006. Dietary zinc requirements of grass shrimp, Penaeus monodon, and effects on immune responses. Aquaculture 254, 476–482. Sinha, A.K., 1972. Colorimetric assay of catalase. Anal. Biochem. 47, 389–394. Tan, B., Mai, K., 2001. Zinc methionine and zinc sulfate as sources of dietary zinc for juvenile abalone, Haliotis discus hannai Ino. Aquaculture 192, 67–84. Tan, L.N., Feng, L., Liu, Y., Jiang, J., Jiang, W.D., Hu, K., Li, S.H., Zhou, X.Q., 2011. Growth body composition and intestinal enzyme activities of juvenile Jain carp (Cyprinus carpio var Jain) fed graded level of dietary zinc. Aquacult. Nutr. 17, 338–345. Tekinay, A.A., Davies, S.J., 2001. Dietary carbohydrate level influencing feed intake, nutrient utilization and plasma glucose concentration in the rainbow trout, Oncorhynchus mykiss. Turk. J. Vet. Anim. Sci. 25, 657–666. Watanabe, T., Kiron, V., Satoh, S., 1997. Trace minerals in fish nutrition. Aquaculture 151, 185–207. Witkiewicz-Kucharczyk, A., Bal, W., 2006. Damage of zinc fingers in DNA repair proteins, a novel molecular mechanism in carcinogenesis. Toxicol. Lett. 162, 29–42. Yamaguchi, M., 1998. Role of zinc in bone formation and bone resorption. J. Trace Elem. Exp. Med. 1, 119–135.