Effect of dietary carbohydrate on non-specific immune response, hepatic antioxidative abilities and disease resistance of juvenile golden pompano (Trachinotus ovatus)

Fish & Shellfish Immunology 41 (2014) 183e190

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Effect of dietary carbohydrate on non-specific immune response, hepatic antioxidative abilities and disease resistance of juvenile golden pompano (Trachinotus ovatus) Chuanpeng Zhou a, b, c, Xianping Ge c, *, Heizhao Lin a, *, Jin Niu a a

Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture, the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, PR China South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China c Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 June 2014 Received in revised form 16 August 2014 Accepted 18 August 2014 Available online 27 August 2014

The present study was conducted to investigate the effects of dietary carbohydrate (CHO) levels on nonspecific immune responses, hepatic antioxidative status and disease resistance of juvenile golden pompano. Fish were fed six isonitrogenous and isoenergetic diets containing various CHO levels for 8 weeks. After the feeding trial, fish were challenged by Vibrio harveyi and survival rate was recorded for the next 12 days. Plasma total protein and albumin content, respiratory burst activity, alkaline phosphatase, slightly increased with dietary starch level from 0% to 16.8%, but significantly decreased at dietary starch levels of 16.8%e28%. Plasma lysozyme, complement 3 and complement 4 levels increased with increasing dietary carbohydrate up to 11.2% and then declined (P < 0.05). Contrary to glutamicoxalacetic transaminase and triiodothyronine, plasma cortisol content increased with increasing dietary carbohydrate up to 22.4%, and then levelled off. The hepatic total antioxidative capacity, reduced glutathione and catalase levels reached the peak at the fish fed diet with 16.8% carbohydrate (P < 0.05). This also held true for hepatic superoxide dismutase activities, whereas the hepatic malondialdehyde content of fish fed dietary starch level of 16.8% was significantly lower than that of fish fed no CHO diet, but showed little difference (P > 0.05) with those of the other treatments. After challenge, fish fed 11.2% and 16.8% dietary CHO showed higher survival rate than that of fish in 0% CHO group (P < 0.05). However, survival rate showed little difference among 0%, 5.6%, 22.4% and 28% CHO groups (P > 0.05). The results of this study suggest that ingestion of 11.2e16.8% dietary CHO can enhance the non-specific immune responses, increase the hepatic antioxidant abilities, and improve resistance to V. harveyi infection of juvenile golden pompano. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Trachinotus ovatus Dietary carbohydrate level Non-specific immune responses Hepatic antioxidative abilities Bacterial challenge

1. Introduction Recent achievements in immunonutrition studies suggest the close link between nutritional conditions and immune status of fish [1e6]. This has drawn the attention of fish nutritionists to the immune-protection of fish beside the growth. Despite many research into the effects of nutrition on stress and immune functions of fish, few trials have examined the changes associated with dietary macronutrient composition [4,7]. The carbohydrates as one of non* Corresponding authors. E-mail addresses: [email protected] [email protected] (H. Lin).

(C.

Zhou),

http://dx.doi.org/10.1016/j.fsi.2014.08.024 1050-4648/© 2014 Elsevier Ltd. All rights reserved.

[email protected]

(X.

Ge),

nitrogenous energy sources for fish, is commonly incorporated in fish diets to maximize the use of dietary protein for growth [8]. For the production of low cost feed, more carbohydrate has to be included in fish diets due to its abundant availability and low cost [9]. However, fish especially carnivorous species generally has a limited ability to digest, absorb and metabolize carbohydrates and hence, excess dietary carbohydrate may cause metabolic load and pathological conditions of fish [10], and has been speculated to influence the stress responses of fish [11,12], which might lead to suppressed immune functions and increase the susceptibility to infectious diseases [13]. Although some preliminary work has been conducted previously [2,14,15], studies concerning the health implications of high dietary carbohydrate in fish are still limited.

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Golden pompano (Trachinotus ovatus) belongs to family carangidae, genus Trachinotus [16]. Trachinotus ovatus is a carnivorous fish which mainly prey on some zooplankton and small crustacea, shellfish and fish [17]. It is a widely distributed species in China, Japan, Australia and other countries [18]. As one of the most popular seafood fishes, and golden pompano commands a significantly higher price than many other marine and freshwater species [16]. Recently, there is an increasing interest in commercial culture of golden pompano in China and the south-east Asian countries such as Malaysia and Singapore [19,20]. Limited research has been conducted on the correlation between dietary macronutrient and fish health status for T. ovatus [21,22]. To our knowledge, no information has been published to evaluate dietary carbohydrate and its implication on immunological responses for T. ovatus. Therefore, the objectives of the present study were to evaluate the effect of dietary carbohydrate levels on non-specific immune responses, hepatic antioxidative abilities and disease resistance of juvenile golden pompano. These results may be useful for better understanding of the health implications of dietary carbohydrate for similar species.

2.3. Plasma and liver sample collection

2. Materials and methods 2.1. Experimental design and diets Six isonitrogenous (43% crude protein) and isoenergetic (ca. 16.8 MJ gross energy kg
1) semi-purified diets were formulated to contain graded levels of corn starch (uncooked) from 0 to 28% (Table 1). Fish meal and soy protein concentrate were used as protein sources, and fish oil was used as the lipid source. Isoenergetic diets were made by adjusting the lipid and cellulose content. Diet ingredients were ground through a 60-mesh screen. Vitamins and minerals were mixed by the progressive enlargement method [23]. Lipid and distilled water (35e40%, v/w) were added to the premixed dry ingredients and thoroughly mixed until homogenous in a Hobart-type mixer. The 1-mm diameter pellets were wet-extruded by a pelletizer (Institute of Chemical Engineering, South China University of Technology, Guangzhou, China), and then air-dried, sealed in plastic bags and stored frozen at
20  C until used. 2.2. Experimental fish and experimental conditions Juvenile golden pompano (Trachinotus ovatus) were obtained from Shenzhen Experimental Station of South China Sea Fisheries Research Institute of CAFS. Prior to the start of the trial, animals Table 1 Ingredient and chemical composition of the experimental diets. Ingredient

Dietary carbohydrate levels 0

5.6

11.2

16.8

22.4

28.0

71.00 0.00 15.50 13.50

71.00 5.60 12.40 11.00

71.00 11.20 9.30 8.50

71.00 16.80 6.20 6.00

71.00 22.40 3.10 3.50

71.00 28.00 0.00 1.00

Proximate composition (% dry matter) Crude protein 43.00 43.02 Crude lipid 16.77 14.33 Ash 8.75 8.77 b Carbohydrate 0.25 5.75
1 Gross energy (MJ kg ) 16.84 16.82

43.03 11.89 8.79 11.25 16.80

43.05 9.45 8.81 16.76 16.79

43.07 7.01 8.83 22.26 16.77

43.08 4.57 8.85 27.77 16.75

a

Basal mixture Corn starch Microcrystalline cellulose Fish oil

were acclimated to a commercial diet (containing 42% crude protein and 5% crude lipid) for 2 weeks and were fed twice daily to apparent satiation. At the beginning of the feeding trial, the fish were starved for 24 h, weighed, and then the fish with similar size (initial body weight 9.24 ± 0.03 g) were randomly allotted into 18 sea cages (1.0 m  1.0 m  1.5 m; three cages per treatment) and each cage stocked with 20 fish. Each experimental diet was randomly assigned to three cages. Juvenile golden pompano were fed twice daily (08:00 h and 16:00 h) at a rate of 3e4% wet body weight for 56 days. To prevent the waste of pellets, fish were slowly hand-fed to satiation, based on visual observation of their feeding behaviour. Feed consumption was recorded for each cage every day. Water quality parameters were monitored daily. During the feeding trial, water temperature ranged from 27.5 to 31.5  C, salinity from 27 to 30 psu, pH from 7.5 to 8.0. Dissolved oxygen was not less than 6.0 mg L
1 and ammonia nitrogen was maintained lower than 0.03 mg L
1. At the end of experiment, the fish were fasted for 24 h and fish in each cage were weighed.

a Basal mixture includes the following ingredients (%): fish meal (65.00% CP), 40.00; Soy protein concentrate (68.00% CP), 25.00; calcium biphosphate, 1.00; vitamin and mineral premix [24], 2.00; carboxymethyl cellulose, 2.00; choline chloride, 0.5; Lecithin, 0.5%. b Determined by the 3,5-dinitro salicylic acid method [25].

At the end of the trial, nine individuals from each group (3 cages, 3 fish per cage) were anesthetized with diluted eugenol (1:10,000; Shanghai Reagent Corp., China) and sampled. In fed fish, gut contents were systematically checked to make sure that the fish had actually consumed the test diets. Blood was sampled from the caudal vein using 2 ml heparinized syringes, centrifuged for 10 min (4  C, 3000 g), and kept frozen until analysis. After collection, 50 ml whole blood was used for analysis of respiratory burst activity. The liver was excised, frozen in liquid nitrogen, and stored at
80  C until analysis. 2.4. Haemato-immunological indexes assays Plasma total protein content was tested by ROCHE-P800 automatic biochemical analyzer (Roche, Basel, Switzerland). Bovine albumin was used as standard (BSA: 66 kDa; Nanjing Jiancheng Biological Engineering Research Institute of China) [8,23]. Plasma globulin was determined by subtracting plasma albumin from total protein. The respiratory burst activity (RBA) of phagocytes was determined by the nitro-blue-tetrazolium (NBT; Sigma, USA) assay described by Anderson and Siwicki [26] with some modifications by Kumari and Sahoo [27]. Dimethylformamide was used as the blank, and the optical density of supernatant was measured at 540 nm. The lysozyme (LYZ) activity was measured using turbidimetric assay according to Muona and Soivio [28]. The plasma complement 3 (C3) and complement 4 (C4) levels were determined using immune turbidimetric method described by Sun et al. [29], using commercial test kits (Zhejiang Elikan Biological Technology Co., Ltd.,China). Plasma tumour necrosis factor a (TNF-a) was estimated by validated radioimmunoassay (RIA) methods as described by previous studies [30]. The TNF-a was tested by a GC2016 gamma RIA counter using commercial kits (Beifang Biotech Research Institute, Beijing, China). 2.5. Plasma biochemical parameters assays Plasma glutamic-oxalacetic transaminase (AST), glutamicpyruvic transaminase (ALT) and alkaline phosphatase (AKP) activities were all tested by ROCHE-P800 automatic biochemical analyzer (Roche, Basel, Switzerland) [31,32].

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version 19.0 (SPSS Inc., Michigan Avenue, Chicago, IL, USA) for Windows.

2.6. Plasma hormones assays Plasma cortisol (COR), triiodothyronine (T3) and thyroxine (T4) were estimated by validated radioimmunoassay (RIA) methods as described by previous studies [33,34]. The COR, T3 and T4 were tested by a GC2016 gamma RIA counter using commercial kits (Beifang Biotech Research Institute, Beijing, China). 2.7. Hepatic antioxidant status analysis Hepatic samples were homogenized in ice-cold phosphate buffer (1:10 dilution) (phosphate buffer: 0.064 M, pH 6.4). The homogenate was then centrifuged for 20 min (4  C, 3000 g) and aliquots of the supernatant were used to quantify hepatic total antioxidative capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), reduced glutathione (GSH), glutathioneS-transferase (GST) and catalase (CAT). T-AOC was measured by the method described in the other study [35] using commercial kits (Jiancheng Institute of Biotechnology, Nanjing, China). Hepatic SOD activity and MDA content were measured using a xanthine oxides [36] and barbituric acid reaction chronometry [37], respectively. GSH activity was assayed as described by Jiang et al. [38]. GST activity was measured by monitoring the formation of an adduct between GSH and 1-chloro-2,4-dinitrobenzene (CDNB) [39]. CAT activity was determined by the decomposition of hydrogen peroxide [40]. We measured the hepatic protein content using the Folin method [41], with bovine serum albumin as the standard. 2.8. Challenge test Vibrio harveyi obtained from South China Sea Fisheries Research Institute (Guangzhou, China) was activated twice following the methods described by Alexander et al. [14]. After 1 week of initial sampling, all fish (10 per cage) were injected intraperitoneally with 0.1 ml bacterial suspension (4  108 CFU ml
1) [22] using medical syringes. Fish continued to receive their assigned diets after the injection. Fish were carefully monitored and mortality was recorded twice daily over a period of 12 days. Survival rate was determined and V. harveyi was confirmed after reisolating it from the dead fish. 2.9. Statistical analysis Results are presented as means ± SEM of three replicates. All data were subjected to one-way analysis of variance (ANOVA). When there were significant differences, the group means were further compared with Duncan's multiple-range test [42]. All statistical analyses were performed using the SPSS programme

3. Results 3.1. Effects of dietary carbohydrate levels on plasma immunerelated indexes Haemato-immunological indexes of juvenile golden pompano were presented in Table 2. Plasma total protein and albumin content were significantly affected by the dietary carbohydrate levels, with the highest plasma total protein and albumin content occurring at the 16.8% dietary starch level (P < 0.05). Plasma total protein and albumin content slightly increased with dietary starch level from 0% to 16.8%. However, plasma total protein and albumin content significantly decreased at dietary starch levels of 16.8%e 28%. The plasma globulin content increased with increasing dietary carbohydrate up to 16.8%, and thereafter declined (P > 0.05). Compared with the control, the 5.6, 11.2, 16.8 and 22.4% CHO groups had a tendency to increase, however the other group supplemented with 28% had a tendency to decrease in plasma globulin content. There were no significant difference in A/G ratios (albumin/globulin) among the dietary treatments (P > 0.05). RBA of fish fed the 16.8% dietary carbohydrate level was significantly higher than those of fish fed 5.6% and 28% dietary starch, but showed little difference (P > 0.05) with those of the other treatments. TNF-a activities of fish increased with increasing dietary carbohydrate up to 16.8% and then declined, though without statistical differences among all the treatments. LYZ activity increased with increasing dietary carbohydrate up to 11.2% and then declined (P < 0.05). The LYZ activity of fish fed the highest dietary carbohydrate level was significantly lower than those of fish fed 11.2% and 16.8% dietary carbohydrate, respectively (P < 0.05). The plasma C3 content increased with increasing dietary carbohydrate up to 11.2%, and thereafter remained nearly the same. Compared with 5.6% and 11.2% CHO groups, the 22.4% and 28% CHO groups had significantly decreased plasma C3 content (P < 0.05). The plasma C4 content showed an upward trend with the increasing dietary carbohydrate levels. The plasma C4 content reached the peak at the fish fed diet with 11.2% carbohydrate. The plasma C4 content of fish fed the highest dietary carbohydrate level was significantly lower than those of fish fed 5.6% and 11.2% dietary carbohydrate, respectively (P < 0.05). 3.2. Effects of dietary carbohydrate levels on plasma biochemical indices Plasma biochemistry analyzes were summarized in Table 3. There were no differences in AST activities among treatments,

Table 2 Haemato-immunological indexes of Trachinotus ovatus fed diets differing in carbohydrate levels. Dietary carbohydrate levels 0 Total protein (g L
1) Albumin (g L
1) Globulin (g L
1) A/G RBA (540 nm) TNF-a (fmol ml
1) LYZ (U ml
1) C3 (g L
1) C4 (g L
1)

20.43 5.93 14.50 0.41 0.58 7.39 54.07 12.24 10.31

5.6 ± ± ± ± ± ± ± ± ±

2.86ab 1.01ab 1.89a 0.03a 0.06abc 0.46a 6.15ab 1.99ab 0.99bc

22.73 5.73 17.00 0.35 0.52 7.73 55.32 17.03 13.77

11.2 ± ± ± ± ± ± ± ± ±

2.65ab 0.19ab 2.59a 0.05a 0.04bc 0.33a 5.63ab 2.99a 1.99ab

26.90 6.97 19.93 0.35 0.63 8.84 70.49 17.69 16.51

16.8 ± ± ± ± ± ± ± ± ±

2.90ab 0.96ab 1.97a 0.02a 0.08abc 0.37a 7.29a 1.50a 0.57a

28.30 7.50 20.80 0.37 0.76 10.62 58.14 12.11 11.45

22.4 ± ± ± ± ± ± ± ± ±

3.07a 0.40a 2.68a 0.03a 0.04a 1.73a 1.42a 0.29ab 0.31bc

22.03 5.63 16.40 0.35 0.67 9.05 54.40 10.79 12.81

28.0 ± ± ± ± ± ± ± ± ±

2.34ab 0.23ab 2.15a 0.04a 0.06ab 0.67a 3.49ab 1.57b 1.36bc

18.80 5.07 13.73 0.38 0.48 8.03 38.28 10.90 9.82

± ± ± ± ± ± ± ± ±

1.21b 0.19b 1.39a 0.05a 0.04c 1.26a 5.47b 0.66b 0.54c

Values are means ± SEM of three replications. Means in the same line with different superscripts are significantly different (P < 0.05). A, albumin; G, globulin; RBA, respiratory burst activity; LYZ, lysozyme; C3, complement 3; C4, complement 4.

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Table 3 Plasma biochemical parameters of Trachinotus ovatus fed diets differing in carbohydrate levels. Dietary carbohydrate levels 0
1

AST (U L ) ALT (U L
1) ALT/AST AKP (U L
1)

39.00 43.33 1.12 16.33

5.6 ± ± ± ±

a

6.66 8.19a 0.14a 0.88b

28.67 36.33 1.28 22.00

11.2 ± ± ± ±

ab

0.88 1.76a 0.10a 1.73ab

34.00 33.00 1.01 26.67

16.8 ± ± ± ±

ab

6.81 1.53a 0.14a 4.41a

22.4

24.07 33.67 1.42 27.67

± ± ± ±

b

0.58 0.67a 0.14a 3.48a

23.37 35.00 1.55 23.33

28.0 ± ± ± ±

b

0.86 4.00a 0.30a 2.91ab

26.00 37.00 1.46 22.67

± ± ± ±

1.53ab 4.16a 0.27a 2.03ab

Values are means ± SEM of three replications. Means in the same line with different superscripts are significantly different (P < 0.05). ALT, alanine aminotransferase; AST, aspartate aminotransferase; AKP, alkaline phosphatase.

except for fish fed the 16.8% and 22.4% starch diets which had lower activities than that of fish fed 0% CHO group (P < 0.05). The ALT had the same tendency as AST activities, but without statistical difference among all the treatments (P > 0.05). No significant differences were observed in ALT/AST ratios among all dietary treatments (P > 0.05). Contrary to AST activities, the AKP activities significantly increased with dietary starch level from 0% to 16.8% (P < 0.05), whereas slightly decreased at dietary carbohydrate levels of 16.8%e 28%.

treatments. The hepatic GSH contents increased with increasing dietary carbohydrate up to 16.8%, and thereafter declined. There were no differences in hepatic GSH activities among treatments, except for fish fed the 11.2% and 16.8% CHO diets which had higher contents than those fed other CHO diets (P < 0.05). Compared with the no CHO group, the 5.6% and 28% CHO groups had a tendency of decrease, and the other groups supplemented with 11.2%, 16.8% and 22.4% had a tendency of increase, in hepatic GST activities (P > 0.05). The hepatic CAT activities showed an increasing trend with the increasing dietary carbohydrate levels. The CAT activity reached the peak at the fish fed diet with 16.8% carbohydrate (P < 0.05).

3.3. Effects of dietary carbohydrate levels on plasma hormones Plasma COR content, T3 and T4 concentrations, T3/T4 ratio were provided in Table 4. The plasma COR content increased with increasing dietary carbohydrate up to 22.4%, and then levelled off. There were no differences in plasma COR contents among treatments, except for fish fed the 22.4% and 28% starch diets which had higher activities than those fed dietary carbohydrate levels from 0% to 11.2% (P < 0.05). The plasma T3 concentrations decreased with an increase of dietary carbohydrate levels. The lowest values were observed for fish fed highest dietary carbohydrate (P < 0.05). No significantly difference in T4 concentrations was observed among dietary treatments. The T3/T4 ratio reflecting thyroid hormone activity varied in accordance with changes in T3 concentrations. Plasma T3/T4 ratio in fish fed dietary CHO levels of 0% and 5.6% were significantly higher (P < 0.05) than that in fish from the 28% CHO group, respectively.

3.5. Challenge test The challenge test showed that the highest survival rate was observed in fish fed a CHO level of 16.8% (Fig. 1). The survival rate of fish in 11.2% and 16.8% CHO group were significantly higher than that of fish in 0% CHO group, respectively (P < 0.05). However, survival rate showed little difference among 0%, 5.6%, 22.4% and 28% CHO groups (P > 0.05). 4. Discussions Scientific evidences gathered over recent decades indicate that dietary nutrients as well as additives could stimulate the immune system of fish and help to fend off diseases [43,44]. In the present study, six isonitrogenous diets with different carbohydrate levels were formulated to evaluate the capability of a carnivorous seawater golden pompano Trachinotus ovatus against infection and disease. The results observed here indicated that dietary carbohydrate level may affect the immune response of golden pompano. It is generally assumed that the optimal dietary digestible CHO level is <20% for carnivorous fish but much higher (30e40%) for herbivorous fish [45]. Excessive dietary CHO can impact plasma metabolites such as protein. Proteins are the most important compounds in the plasma with albumin and globulin being the major plasma proteins [8]. The hemolymph protein content is used as an immune parameter that can indicate whether or not a fish is healthy [46]. In this study, the 16.8% CHO group showed the highest level of plasma total protein and albumin content.

3.4. Hepatic oxidative status of juvenile golden pompano fed different diets for 8 weeks The effect of dietary carbohydrate on hepatic oxidative status was shown in Table 5. The hepatic T-AOC showed an upward trend with the increasing dietary carbohydrate levels. The hepatic T-AOC reached the peak at the fish fed diet with 16.8% carbohydrate (P < 0.05). There were no differences in hepatic SOD activities among treatments, except for fish fed the 28% CHO diets which had lower activities than those fed 11.2%, 16.8% and 22.4% CHO diets (P < 0.05). The hepatic MDA content of fish fed dietary starch level of 16.8% was significantly lower than that of fish fed no CHO diet, but showed little difference (P > 0.05) with those of the other Table 4 Plasma hormones of Trachinotus ovatus fed diets differing in carbohydrate levels. Dietary carbohydrate levels 0 COR (ng ml
1) T3 (ng ml
1) T4 (ng ml
1) T3/T4 ratio

31.00 0.48 22.38 0.021

5.6 ± ± ± ±

6.05c 0.11a 0.46a 0.005a

47.09 0.42 22.05 0.019

11.2 ± ± ± ±

4.69c 0.06a 0.51a 0.003a

46.83 0.25 22.13 0.011

16.8 ± ± ± ±

6.04c 0.09ab 1.03a 0.004ab

49.13 0.29 20.64 0.014

22.4 ± ± ± ±

3.47bc 0.06ab 0.59a 0.040ab

83.24 0.34 22.01 0.015

28.0 ± ± ± ±

9.57a 0.03ab 0.83a 0.001ab

67.28 0.15 22.96 0.007

± ± ± ±

3.98ab 0.02b 1.91a 0.002b

Values are means ± SEM of three replications. Means in the same line with different superscripts are significantly different (P < 0.05). COR, cortisol; T3, triiodothyronine; T4, thyroxine; TNF-a, tumour necrosis factor a.

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Table 5 Hepatic antioxidative status of Trachinotus ovatus fed diets differing in carbohydrate levels. Dietary carbohydrate levels 0
1

T-AOC (U mg prot) SOD (U mg
1 prot) MDA (nmol mg
1 prot) GSH (mg g
1 prot) GST (U/mg prot) CAT (U mg
1 prot)

0.44 125.48 16.19 24.47 21.26 11.07

5.6 ± ± ± ± ± ±

b

0.04 14.76ab 1.14a 0.80b 1.05a 0.38ab

0.46 141.27 13.19 36.87 19.75 10.75

11.2 ± ± ± ± ± ±

b

0.08 12.01ab 0.68ab 2.66b 2.63a 1.04ab

16.8

0.76 160.25 13.82 83.87 21.39 12.14

± ± ± ± ± ±

a

0.07 1.45a 1.92ab 9.13a 6.07a 1.97ab

0.77 156.29 12.19 93.89 26.31 13.74

22.4 ± ± ± ± ± ±

a

0.06 13.17a 0.80b 14.25a 1.20a 1.31a

0.63 158.32 12.37 35.86 22.28 9.46

28.0 ± ± ± ± ± ±

ab

0.10 18.13a 0.71ab 4.89b 3.05a 1.01ab

0.67 110.80 12.59 30.67 18.62 9.02

± ± ± ± ± ±

0.09ab 12.78b 1.26ab 3.97b 2.64a 1.39b

Values are means ± SEM of three replications. Means in the same line with different superscripts are significantly different (P < 0.05). T-AOC, total antioxidative capacity; SOD, superoxide dismutase; MDA, malondialdehyde; GSH, reduced glutathione; GST, glutathione-S-transferase; CAT, catalase.

RBA, the release of reactive oxygen species (ROS) by phagocytes, is a crucial step in the innate immune and antioxidant responses and an important mechanism by which potential pathogens and parasites are eliminated following phagocytosis [47]. An important microbicidal response of phagocytes is the production of ROS [48]. Results in the present study showed that the RBA was increased with increasing dietary carbohydrate level, but declined slightly when the dietary carbohydrate level was beyond 16.8%. Same results were reported in other species such as European sea bass and Wuchang bream [4,23]. Lysozyme is an important component of the immune defense in fish species, which is wildly used as humoral immune indicators in fish [39,40,49,50]. It is responsible for breaking down the polysaccharide wall of bacteria and thus prevents pathologic infection and disease [51]. Lysozyme levels reportedly decreased in Epinephelus malabaricus with an increase in dietary protein [12]. Similarly, plasma lysozyme activity declined with an increase in dietary CHO [23,52]. A relative relationship between serum lysozyme activity and total protein concentration was obtained in Limanda limanda [53]. In this study, the fish fed the highest dietary CHO had the lowest plasma LYZ activity, which indicated that the high-CHO diet may reduce the immune ability of T. ovatus to some degree. Complement is the major humoral component of the innate immune responses and thus plays an essential role in alerting the host immune system of the presence of potential pathogens as well as their clearance [54,55]. Complement is initiated by one or a combination of three pathways, namely the classical, alternative, and lectin. C3 is involved in all the three pathways which merge and proceed through a terminal pathway that leads to the formation of a membrane attack complex, directly lysing pathogenic cells

Fig. 1. Effects of dietary carbohydrate levels on the survival rate after V. harveyi (4  108 CFU ml
1) infection of T. ovatus on 12th day. Data are expressed as the mean ± SEM (n ¼ 9). Values not sharing a common superscript are significantly different (P < 0.05).

[54]. In previous studies on grouper Epinephelus coioides, plasma C3 and C4 levels in probiotic groups significantly increased after 30 days of feeding [29]. In wuchang bream Megalobrama amblycephala, higher dietary CHO group showed lower plasma C3 content [23,56]. Similarly, in this study, compared with 22.4% and 28% CHO groups, the 5.6% and 11.2% CHO groups had significantly increased serum complement C3 levels. C4 is a key component of both classical and lectin pathways [54]. In our study, compared with the lowest and highest CHO groups, the 11.2% CHO group had significantly increased serum complement C4 levels. However, in Wuchang bream, diets containing different dietary CHO levels ranging from 0 to 47% have been reported to produce fish with no significant difference in C4 levels [23]. AST and ALT are ubiquitous aminotransferases in the mitochondrion of fish, and are important parameters for diagnosis of hepatopancreas function and damage [23,57,58]. In the present study, the lowest plasma AST activities were found in fish fed 16.8%e22.4% carbohydrate content. Moreover the activities of both enzymes had tendency of increase in the fish fed with control diet and highest CHO diet, suggesting that carbohydrate free diet and high dietary carbohydrate level might both impair its hepatic functions because of the heavy metabolic load on this species [59]. Similar results were also observed in top-mouth culter (Erythroculter ilishaeformis Bleeker) [52], juvenile grass carp (Ctenopharyngodon idella) [60] and Wuchang bream (Megalobrama amblycephala) [23]. AKP is an important regulative enzyme which has been associated with a number of essential functions in all living organisms. In Drosophila virilis, the AKP activity has been shown to decrease upon heat stress [61]. AKP activity in high glucose group (40% CHO) was significantly lower than that in low glucose group (20% CHO) in Mylopharyngodon piceus Richardson [62]. In Wuchang bream, compared with no CHO diet, the proper dietary CHO level had significantly increased serum AKP activity [23]. Similarly, in our study, the highest plasma AKP activities were obtained in fish fed with 11.2% and 16.8% CHO diets, indicating that dietary carbohydrate deprives or excessive caused stress in fish, leading to lower health status. Hypercortisolemia is a classic stress response in fish [63]. Increase of the blood cortisol level is widely used as an indication that a fish is under stress [64]. Dietary intake of high CHO was shown to improve the plasma cortisol level in Atlantic salmon (Salmo salar L.) [2], whitefish (Coregonus lavaretus) [65] and top-mouth culter (Erythroculter ilishaeformis Bleeker) [52], whereas decreased plasma cortisol level in whitefish [12]. Adding to the confusion, dietary carbohydrate content imposed no influence on plasma cortisol in cod [66] and rainbow trout [67]. Inconsistent response of plasma cortisol to dietary CHO level exists implied that the stress response induced by dietary CHO level may vary among species. In addition, our results showed that plasma cortisol contents of golden pompano fed dietary starch levels of 22.4% were

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significantly higher than that of fish fed dietary CHO levels ranging from 0% to 16.8%, but showed little difference with that in 28% CHO treatment. This implies that juvenile golden pompano can accept a diet containing at least up to 16.8% carbohydrate without exhibiting symptoms of stress. According to some authors, thyroid hormones and their activities are good biological markers of health status in fish [68e70]. There has been considerable controversy regarding the influence of dietary carbohydrate content on thyroidal status in fish. Even for the same fish species, dietary CHO can induce lower, unchanged or higher thyroidal status [70]. In the current study, the plasma T3 concentrations decreased with an increase of dietary carbohydrate levels. A similar result was observed in barramundi [71]. However, in rainbow trout, dietary carbohydrate either exerted no effect on circulating T3 level [72,73] or increased the rate of T3 formation [69]. In this study, no significantly difference in T4 concentrations was observed among dietary treatments. Similar result was found in silver sea bream [70]. However, carbohydrate intake appears to be regulating T4 [74] as rainbow trout fed a carbohydrate-rich diet [72]. The body's defense system of the antioxidation ability strong and the weak is closely related to health [75]. In addition, multiple types of antioxidants (e.g., T-AOC, CAT, SOD and MDA) are needed to maintain the complex immune system of fish [76,77]. The antioxidant capacity includes enzymatic and non-enzymatic antioxidant activities. Antioxidant enzymes include T-AOC, CAT, SOD and MDA that constitute the first line of enzymatic defence mechanism against free radicals in organisms. In general, antioxidant defenses in fish somehow depend on nutritional factors [78]. The T-AOC ability is the comprehensive reflection of the body, the enzyme antioxidant system and antioxidant enzyme system to complete the antioxidant effect [75]. For fish, the T-AOC level could reflect the oxidation resistance capability and is associated with the health status [79]. In the present study, the hepatic T-AOC showed an upward trend with the increasing dietary carbohydrate levels, and reached the peak at the fish fed diet with 16.8% carbohydrate. Similar results were found in previous studies on Wuchang bream and top-mouth culter [23,52]. The coordinate actions of various cellular antioxidants in mammalian cells are critical for the effective detoxification of free radicals [80]. SOD and GSH are major antioxidants, which involve in defense mechanism against lipid peroxidation in biological system and convert active oxygen molecules into non-toxic compounds [81]. A reduction in SOD and GSH is associated with the accumulation of high-living free radical, leading to injury of cell function [82]. In the present study, treatments of fish fed with 11.2% and 16.8% dietary CHO increased the hepatic antioxidant capacity as evidenced by the increased GSH level and activities of SOD. Free radicals under the function of superoxide dismutase would produce oxygen molecule and hydrogen peroxide [58]. CAT catalyses breakdown of hydrogen peroxide to water and molecular oxygen, so as to clean free radicals to reduce lipidic superoxide damage [83]. In the current study, the hepatic CAT activities decreased significantly as dietary CHO levels increased from 16.8 to 28%, which is consistent with above result of hepatic SOD activities of golden pompano. MDA production was a notable oxidation process resulting from the peroxidation of membrane polyunsaturated fatty acid, influencing cell membrane fluidity as well as the integrity of biomolecules and was an important indicator of lipid peroxidation [84]. In the present study, Golden pompano fed dietary 16.8% CHO diet reduced hepatic MDA level. The study showed that appropriate dietary CHO level could improve the total antioxidant response and reduce the speed of lipid peroxidation reaction, clear the MDA of body in the commutation period and avoid the body damage. Similar results were also observed in previous study [52].

Bacterial challenge tests have often been used as a final indicator of fish health status after nutrition trials [22,60]. V. harveyi is a halophilic Gram-negative bacterium that has been wildly used in fish immune-nutrition studies, because it is known to cause disease to fish, shrimp and shellfish either in the culture systems or in the wild aquatic environments, thus resulting in heavy losses and causes economic loss to fish farmers [85e87]. Previous studies showed that higher mortality in the high CHO fed groups in L. rohita juveniles [6,9,23]. The results of this study indicate that resistance to V. harveyi infection could be enhanced in juvenile golden pompano (T. ovatus) by supplementing their diet with 16.8% CHO. 5. Conclusions It is concluded that appropriate level of dietary carbohydrate could improve the non-specific immunity and oxidation ability of juvenile golden pompano. The diet supplemented with the 11.2e16.8% CHO seems to be optimum for improving immunity in T. ovatus. This report may be helpful for the nutritionists to recast their feed formulation. The further studies concerning relationships between dietary nutrients and health status of T. ovatus are required. Acknowledgements The research was supported by Open Fund of Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization of the Ministry of Agriculture (KF201311), Special Scientific Research Funds for Central Non-profit Institutes, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (2014YD01) and Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences (2013A08XK04). References [1] Oliva-Teles A. Nutrition and health of aquaculture fish. J Fish Dis 2012;35: 83e108. [2] Waagbø R, Glette J, Sandnes K, Hemre GI. Influence of dietary carbohydrate on blood chemistry, immunity and disease resistance in Atlantic salmon, Salmo salar L. J Fish Dis 1994;17:245e58. [3] Lygren B, Waagbø R. Nutritional impacts on the chemiluminescent response of Atlantic salmon (Salmo salar L.) head kidney phagocytes, in vitro. Fish Shellfish Immunol 1999;9:445e56. rez-Sa nchez J. Diet related changes in non-specific im[4] Sitj
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