Effects of graded levels of dietary methionine hydroxy analogue on immune response and antioxidant status of immune organs in juvenile Jian carp (Cyprinus carpio var. Jian)

Fish & Shellfish Immunology 32 (2012) 629e636

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Effects of graded levels of dietary methionine hydroxy analogue on immune response and antioxidant status of immune organs in juvenile Jian carp (Cyprinus carpio var. Jian) Sheng-Yao Kuang a, d, Wei-Wei Xiao a, Lin Feng a, b, c, Yang Liu a, b, c, Jun Jiang a, b, c, Wei-Dan Jiang a, b, c, Kai Hu a, b, c, Shu-Hong Li a, Ling Tang a, d, Xiao-Qiu Zhou a, b, * a

Animal Nutrition Institute, Sichuan Agricultural University, Ya’an 625014, China Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Ya’an 625014, China Key Laboratory for Animal Disease-resistance Nutrition of China Ministry of Education, Ya’an 625014, China d Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 May 2011 Received in revised form 29 September 2011 Accepted 27 December 2011 Available online 10 January 2012

Immune response and antioxidant status of immune organs in juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of methionine hydroxy analogue (MHA) (0, 5.1, 7.6, 10.2, 12.7, 15.3 g kg
1 diet) for 60 days were investigated. Results indicated that head kidney index, spleen index, red and white blood cell counts significantly increased with increasing MHA levels up to a point (P < 0.05), whereupon decreased (P < 0.05). Glutathione reductase activity in head kidney and spleen, anti-hydroxy radical and glutathione-S-transferase activities in spleen, catalase activity and GSH content in head kidney significantly increased by MHA supplement, while malondialdehyde content, anti-superoxide anion, superoxide dismutase, glutathione peroxidase activities in head kidney and spleen, protein carbonyl content and catalase activity in spleen, anti-hydroxy radical activity in head kidney significantly decreased by MHA supplement. However, protein carbonyl content and glutathione-S-transferase activity in head kidney, GSH content in spleen remained unaffected. After 60-day feeding trial, a challenge study was conducted by injection of Aeromonas hydrophila for 17 days. Results showed that survival rate, leukocytes phagocytic activity, lysozyme activity, acid phosphatase activity, total iron-binding capacity, haemagglutination titre, complement 3, 4 and immunoglobulin M contents significantly increased by optimal dietary MHA supplement (P < 0.05). These data suggested that MHA affected antioxidant status of immune organs and promoted immune response in juvenile Jian carp. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Cyprinus carpio var. Jian Methionine hydroxy analogue Immune response Antioxidant status

1. Introduction Methionine is an essential dietary nutrient for normal growth of fish [1,2]. The fish growth rate is often related to the diseases resistance [3]. Studies indicated that methionine deficiency led to depressed survival rate, as well as reduced growth performance in Jian carp (Cyprinus carpio var. Jian) [4], rainbow trout (Oncorhynchus mykiss) [5] and juvenile red drum (Sciaenops ocellatus) [6]. Supplement methionine hydroxy analogue (MHA), a common used synthetic methionine source, to methionine-deficient diets increased survival rate and also improved growth performance of

* Corresponding author. Animal Nutrition Institute, Sichuan Agricultural University, Ya’an 625014, China. Tel.: þ86 835 2885157; fax: þ86 835 2885968. E-mail addresses: [email protected], [email protected] (X.-Q. Zhou). 1050-4648/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.12.012

rainbow trout [5] and juvenile red drum [6]. Animal disease resistance in general is associated with immune organs growth [7] and immune response [8]. However, little is known concerning the effect of MHA on immune organs growth and immune response in fish. A few studies found that MHA improved spleen weight, serum lysozyme activity and phagocytosis of peripheral blood lymphocyte in broiler chicken [9], and increased antibody titres to sheep red blood cells in white leghorn layer [10]. Furthermore, studies had demonstrated that MHA was converted into L-methionine for effective utilization in chicken liver, rat liver and hog kidney [11]. Our laboratory previous study found that dietary DL-methionine could improve serum lysozyme activity, total iron-binding capacity, haemagglutination titre, complement 3, 4 and immunoglobulin M (IgM) contents in Jian carp [4]. These appear that MHA can also affect fish immune response, which needs investigation.

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Head kidney and spleen are two important immune organs in carp [12]. Head kidney is the principal immune organ responsible for phagocytosis, antigen processing, formation of IgM and immune memory through melanomacrophagic centres [13], while spleen is mainly responsible for antibody formation and B cells differentiation [14]. Thus the normal structure and function of immune organs is correlated with fish immunity. Nevertheless, oxidative injury often led to alteration of structure and function in many organs [15]. The higher percentage of polyunsaturated fatty acids in membranes and frequently exposed to ROS produced as part of normal function lead to sensitively oxidative stress in immune cells [16], which may also result in sensitively oxidative stress in head kidney and spleen. It is, therefore, very important to keep normal antioxidant status in head kidney and spleen, but no studies about the effect of MHA on antioxidant status in immune organs had been conducted. Our previous study found that MHA improved antioxidant defense in intestine and hepatopancreas of Jian carp [17]. It appears that MHA can also affect antioxidant status in immune organs to regulate fish immunity, which warrants further investigation. This study was a part of further study that involved in the determination of the effects of MHA on growth performance in Jian carp using the same growth trial as the previous study [18]. The study was performed to investigate the possible effects of MHA on fish immune response and further investigate antioxidant status in immune organs. 2. Materials and methods 2.1. Fish Hatchery-reared juvenile Jian carp were obtained from the TongWei Hatchery (Sichuan, China). Before starting the experiment, the fish were acclimatized to the experimental conditions for 4 weeks. A total of 900 fish with an initial weight of 8.24  0.03 g were randomly assigned to each of 18 experimental aquaria (90 L  30 W  40 H cm). Each experimental diet was randomly assigned to triplicate aquaria. All the aquaria system, operation of the culture system, and water quality were the same as our previous study [18]. The fish were hand-fed with the respective diet to apparent satiation six times daily from day 1 to 30 and four times daily from day 31 to 60. Thirty minutes after the feeding, uneaten feed were removed by syphoning and then air dried. The experimental units were under a natural light and dark cycle. The water temperature, pH value and dissolved oxygen were 25  1  C, 7.0  0.3 and 5 mg L
1, respectively. 2.2. Rations The formulation of the basal diet is presented in Table 1. Except methionine, dietary components (amino acids, vitamins and minerals) were supplemented to meet the requirements of juvenile Jian carp according to our previous studies [19e27] and reported nutritional requirements for common carp [28]. Six experimental diets were formulated according to MHA supplementation: 0 (control), 5.1, 7.6, 10.2, 12.7 and 15.3 g MHA kg
1 diet. Liquid MHA product (measured with 879 g kg
1 active substance) (Sumitomo-chemical, Tokyo, Japan) was added to the test diets to provide different concentrations, and the amount of corn starch was reduced to compensate final amount. The methionine concentration in the basal (control) diet was 6.9 g kg
1 diet, which was determined by the method of Spindler et al. [29]. Experimental diets and the procedures for diet preparation and storage (
20  C) were the same as was previously reported in Ref. [18].

Table 1 Ingredients and composition of the basal diet.a Ingredients

g kg
1

Fish meal Soybean meal Rice gluten meal Cottonseed meal Rapeseed meal Wheat flour Fish oil Soybean oil Vitamin premixb Trace mineral premixc Ca (H2PO4)2 Choline chloride (50%) Carboxymethyl cellulose Ethoxyquin (30%) Threonine (98.5%) Lysine (78.8%) MHA premixd Proximate composition Moisture (%) Crude protein (% dm) Crude fat (% dm) Crude ash (% dm) Cysteine (%) Methionine (%)

75.0 75.0 100.0 147.8 295.6 165.7 22. 5 7.2 10.0 10.0 26.7 1.3 20.0 0.5 5.9 6.8 e 11.70 32.14 5.44 8.79 0.79 0.69

a Fish meal, soybean meal, rapeseed meal, cottonseed meal and rice gluten meal were used as dietary protein sources. Fish oil, soybean oil and wheat flour were used as dietary lipid and carbohydrate source respectively. Lysine, threonine, available phosphorus, n-3 and n-6 calculated contents were 20.0, 17.0, 6.0, 10.0 and 10.0 g kg
1 diet respectively according to NRC (1993) [28] and Bell (1984) [67]. b Per kilogram of vitamin premix (g kg
1 diet): retinyl acetate, 0.80 (500,000 IU g
1); cholecalciferol, 0.48 (500,000 IU g
1); DL-a-tocopherol acetate, 20.00 (50%); menadione, 0.20 (50%); cyanocobalamin, 0.01 (10%); D-biotin, 0.50 (20%); folic acid, 0.52 (96%); thiamin nitrate, 0.10 (98%); ascorhyl acetate, 7.23 (92%); niacin, 2.86 (98%); meso-inositol, 52.86 (98%); calcium-D-pantothenate, 2.51 (98%); riboflavine, 0.63 (80%); pyridoxine hydrochloride, 0.76 (98%). All ingredients were diluted with corn starch to 1 kg. c Per kilogram of mineral premix (g kg
1 diet): FeSO4$7H2O, 69.70 (19.7% Fe); CuSO4$5H2O, 1.20 (25.0% Cu); ZnSO4$7H2O, 21.64 (22.5% Zn); MnSO4$H2O, 4.09 (31.8% Mn); KI, 2.90 (3.8% I); NaSeO3, 2.50 (1.0% Se). All ingredients were diluted with CaCO3 to 1 kg. d MHA premix (30 g kg
1 diet) was added to obtain graded level of MHA, and the amount of corn starch was reduced to compensate. Six MHA premix were elaborated according to different proportion liquid MHA (g) and corn starch (g): 0/1000.0, 193.3/806.7, 290.0/710.0, 386.7/613.3, 483.3/516.7 and 580.0/420.0.

2.3. Red and white blood cell counts assay After the feeding trial, blood was collected from the caudal vein by the syringe with heparin as the anticoagulant from 3 fish of each aquarium for blood cell count. The red and white blood cell counts were determined by using Neubauer haemocytometer and diluting the blood sample with Hayem’s and Turke’s solution, respectively [30].

2.4. Antioxidant-related parameters assay 2.4.1. Sample and tissue preparation At the end of the feeding trial, fish were anaesthetized with benzocaine (50 mg L
1) 12 h after the last feeding. Head kidney and spleen of 15 fish from each aquarium were quickly removed, weighed and frozen in liquid nitrogen, and then stored at
70  C until analyzed. Tissue samples of 6 fish per tank were homogenized on ice in 10 volumes (w v
1) of ice-cold physiological saline (0.7 g mL
1) and centrifuged at 6000 g for 20 min at 4  C respectively, and then supernatants were stored at
20  C for antioxidant parameters analysis.

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2.4.2. Detection of lipid peroxidation and protein oxidation The malondialdehyde (MDA) content was assayed as described by Livingstone et al. [31] using the thiobarbituric acid reaction. The results were expressed as nmol mg
1 protein, and the protein concentration of samples was determined by the method of Bradford [32]. The protein carbonyl content was determined according to the method described by Armenteros et al. [33]. The protein carbonyl content was calculated from the peak absorbance at 370 nm, using an absorption coefficient of 21,000 (M  cm)
1 and expressed as nmol mg
1 protein. 2.4.3. Detection of O2
-scavenging ability and OH -scavenging ability  The anti-superoxide anion capacity (O2
-scavenging ability)  and anti-hydroxy radical capacity (OH -scavenging ability) were determined by the method described by Jiang et al. [34]. Antisuperoxide anion (ASA) capacity was determined by using the superoxide anion free radical detection Kit (Nanjing Jiancheng  Bioengineer Institute). Superoxide radicals ðO2
Þ were generated by the action of xanthine and xanthine oxidase. With the electron acceptor added, a colouration reaction is developed by using the Griess reagent. The colouration degree is directly proportional to the quantity of superoxide anion in the reaction. Tissue ASA capacity was expressed in mU mg
1 protein. One mU was defined as the amount that scavenged superoxide anion free radical in 40 min per milligram of tissue protein which equalled to per microgramme of vitamin C scavenging at the same condition. Anti-hydroxy radical (AHR) capacity was determined by using the hydroxyl free radical detection Kit (Nanjing Jiancheng Bioengineer Institute). It was on the basis of Fenton reaction   (Fe2þ þ H2O2 / Fe3þ þ OH
þ OH ). Hydroxyl radicals (OH ) are generated in the Fenton reaction. With the electron acceptor added, a colouration reaction is developed by using the Griess reagent. The colouration degree is directly proportional to the quantity of hydroxyl radicals in the reaction. Tissue AHR capacity was expressed in U mg
1 protein. One U was defined as the amount that decreased 1 mmol L
1 H2O2 in 1 min per milligram of tissue protein. 



2.4.4. Antioxidant enzyme activity analysis Superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities were assayed as described by Zhang et al. [35]. Tissues SOD activity was expressed in U mg
1 protein. One U means 50% of inhibition by SOD of nitric ion production. Tissues GPX activity was expressed in U mg
1 protein. One U was defined as the amount that reduced 1 mmol L
1 GSH in 1 min per milligram of tissue protein. Catalase (CAT) activity was determined by the decomposition of hydrogen peroxide [36]. The result was expressed in U mg
1 protein. One U was defined as the amount that decreased 1 mmol L
1 H2O2 in 1 s per milligram of tissue protein. GlutathioneS-transferase (GST) activity was measured by monitoring the formation of an adduct between GSH and 1-chloro-2,4-dinitrobenzene [37]. The result was expressed in U mg
1 protein. One U was defined as the amount that decreased 1 mmol L
1 GSH in 1 min per milligram of tissue protein. Glutathione reductase (GR) activity was measured according to described by Lora et al. [38] and given as mU mg
1 protein. One mU was defined as the amount that decreased 1 mmol L
1 NADPH in 1 min per milligram of tissue protein. 2.4.5. GSH assay GSH content was determined by the formation of 5-thio2-nitrobenzoate followed spectrophoto-metrically at 412 nm [39]. The amount of GSH in the extract was expressed as mg g
1 protein and commercial GSH was used as standard.

631

2.5. Challenge After the growth trial, 10 fish with similar body weight obtained from each replicate were moved to the respectively label new tank and acclimatized to the experimental condition for 5 days. At the end of acclimatize period, fish were injected by intraperitoneal injection with 1.6  105 cfu g
1 fish body weight of Aeromonas hydrophila, which was obtained from Huazhong Agricultural University in China. The injected bacterial number was a semilethal dosage which was enough to activate the immune system and consequently enable the investigation of effluent on reactivity against a threatening disease according to our preliminary study data. The challenge trial was conducted for 17 days at the time antibody content was highest according to data (unpublished data) in our laboratory. Experiment conditions were the same as the feeding trial. Dead fish were removed and the final fish number was recorded. 2.6. Leukocytes phagocytic activity assay After the challenge trial, the living fish were also anaesthetized with benzocaine (50 mg L
1) 12 h after the last feeding. Some blood of 3 fish from each replicate was drawn from the caudal veins by using a syringe with heparin as the anticoagulant. The blood was immediately used for leukocytes phagocytic activity (LPA) analysis by the method of Pulsford et al. [40,41]. 2.7. Serum immune parameters assay Immediately, blood of all the living fish was drawn from the caudal vein, stored at 4  C overnight, and then centrifuged at 3000 g at 4  C for 10 min. The serum was stored at
20  C for immune parameters analysis. Serum haemagglutination titre (HT) was determined by using haemagglutination assay which was modified from Barracco et al. [42]. Serial two-fold dilutions of the serum from all groups were diluted with phosphate buffered saline (PBS, pH 7.2) in U-shaped bottom microtitre wells, to which an equal volume of freshly prepared 2% rabbit erythrocyte suspension (in PBS) was added, and incubated for 2 h at 25  C. Titres were recorded as the reciprocal of the highest dilution showing agglutination. The lysozyme and acid phosphatase (ACP) activities were determined according to El-Boshy et al. [43] and Barka & Anderson [44], respectively. Total iron-binding capacity (TIBC) was determined by the method of Soldin et al. [45]. The contents of complement 3 and 4 were assayed according to the method of Welker et al. [46]. The serum immunoglobulin M (IgM) level was assayed according to the method of Takemura [47]. 2.8. Calculations and statistical analysis Data on survival after vaccination, head kidney index, spleen index and phagocytic activity of leukocytes were calculated: Survival rate ¼ 100  final fish number/initial fish number. Head kidney index (HKI) ¼ 100  wet head kidney weight/wet body weight. Spleen index (SI) ¼ 100  wet spleen weight/wet body weight. Leukocytes phagocytic activity (LPA) ¼ 100  number of phagocytosing cells/number of total cells. All data were subjected to one-way analysis of variance (ANOVA) followed by the Duncan method to determine significant differences among treatment groups. All results were expressed as

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mean  standard deviation. The parameters with significant differences were subjected to quadratic regression analysis with dietary MHA level. 3. Results

(YLPA ¼ 26.6980 þ 4.1484X
0.2692X2, R2 ¼ 0.896, P ¼ 0.033; YLA ¼ 6.0209 þ 0.2754X
0.0237X2, R2 ¼ 0.943, P ¼ 0.014; YACP ¼ 436.1300 þ 84.9460X
4.2650 X2, R2 ¼ 0.973, P ¼ 0.005; YC3 ¼ 132.3900 þ 9.1687X
0.4968X2, R2 ¼ 0.786, P ¼ 0.099; YHT ¼ 5.7117 þ 0.3499X
0.0237X2, R2 ¼ 0.711, P ¼ 0.155; YIgM ¼ 86.6540 þ 2.8690X
0.1630X2, R2 ¼ 0.847, P ¼ 0.060).

3.1. Survival rate after challenge 3.4. Antioxidant-related parameters of head kidney Effect of dietary MHA supplement on survival rate after challenge with A. hydrophila was shown in Table 2. Because of the pathogenicity of A. hydrophila, some fish could not resist and dead fish occurred among experimental groups. With MHA level up to 5.1 g kg
1 diet, survival rate showed a trend to a increase, and then a decrease when dietary MHA further up to 10.2 g kg
1 diet (P ¼ 0.06). Moreover, survival rate showed quadratic response with graded levels of MHA (Y ¼
0.1636X2 þ 1.6607X þ 61.8120, R2 ¼ 0.700, P ¼ 0.165). 3.2. Organs index and blood cell counts Effects of dietary MHA supplement on head kidney index (HKI), spleen index (SI), red blood cell (RBC) and white blood cell (WBC) counts were shown in Table 3. HKI in fish fed diet containing 7.6 g MHA kg
1 diet was significantly higher than that of fish fed the other diets (P < 0.05). SI was the lowest in fish fed MHA unsupplement diet. SI significantly increased with dietary MHA supplement and obtained maximum when MHA level was at 12.7 g kg
1 diet (P < 0.05). Both RBC and WBC improved with MHA level up to 5.1 g kg
1 diet, and then gradually decreased (P < 0.05). Quadratic regression analysis showed that SI, RBC and WBC were quadratic responses to the increasing dietary MHA levels (YSI ¼ 0.1385 þ 0.0070X
0.0002X2, R2 ¼ 0.770, P ¼ 0.111; YRBC ¼ 2.1675 þ 0.0366X
0.0028X2, R2 ¼ 0.811, P ¼ 0.082; YWBC ¼ 4.7448 þ 0.1231X
0.0083X2, R2 ¼ 0.959, P ¼ 0.008). 3.3. Serum immune parameters Effects of dietary MHA supplement on leukocytes phagocytic activity (LPA), lysozyme activity (LA), acid phosphatase activity (ACP), total iron-binding capacity (TIBC), haemagglutination titre (HT), complement 3 (C3), 4 (C4) and IgM contents were presented in Table 4. LPA significantly improved with dietary MHA up to 10.2 g kg
1 diet and then decreased (P < 0.05). Both LA and ACP increased with MHA supplement and obtained maximum when MHA level was at 5.1 and 7.6 g kg
1 diet respectively, and then decreased (P < 0.05). TIBC and HT increased with MHA supplement and obtained maximum when MHA level was at 7.6 g kg
1 diet (P < 0.05). C3 content in fish fed MHA supplement diets was higher than that of fish fed control diet (P < 0.05), but no significant difference was found among MHA supplement diets (P > 0.05). With dietary MHA level up to 5.1 g kg
1 diet, C4 content significantly improved (P < 0.05), after that gradually decreased (P < 0.05). IgM content significantly increased when MHA level was at 7.6 g kg
1 diet (P < 0.05), and then plateaued. Moreover, leukocytes phagocytic activity (LPA), lysozyme activity (LA), acid phosphatase activity (ACP), C3 content, haemagglutination titre (HT) and IgM content showed quadratic response with graded levels of MHA

Effects of dietary MHA supplement on MDA and protein carbonyl content, anti-superoxide anion and anti-hydroxy radical capacities in head kidney were shown in Table 5. With the increasing MHA level up to 7.6 g kg
1 diet, MDA significantly decreased (P < 0.05), and then significantly improved when dietary MHA further up to 12.7 and 15.3 g kg
1 diet (P < 0.05). Protein carbonyl content was not significantly affected by MHA supplement (P > 0.05). Both anti-superoxide anion and anti-hydroxy radical capacities decreased as MHA level was up to 7.6 g kg
1 diet (P < 0.05), and then plateaued. SOD, CAT, GST, GPX, GR activities and GSH content in head kidney affected by dietary MHA supplement were shown in Table 6. SOD activity decreased as MHA level was up to 7.6 g kg
1 diet (P < 0.05), and then plateaued. Similar trend was obtained for GPX activity. CAT activity was the lowest in fish fed MHA unsupplement diet and significantly increased with dietary MHA supplement (P < 0.05). When MHA level was up to 12.7 g kg
1 diet, CAT activity obtained maximum. With the increasing MHA level up to 5.1 g kg
1 diet, GSH content significantly increased, after that decreased (P < 0.05). GR activity increased with dietary MHA level up to 5.1 g kg
1 diet, and then gradually decreased (P < 0.05). However, no significant difference was obtained for GST activity among diets (P > 0.05). Regression analyses showed that CAT and GR activities, as well as GSH content were quadratic response with graded levels of MHA (YCAT ¼ 1.1005 þ 0.1532X
0.0097X2, R2 ¼ 0.673, P ¼ 0.187; YGSH ¼ 5.8087 þ 0.1245X
0.0144X2, R2 ¼ 0.745, P ¼ 0.129; YGR ¼ 24.1330 þ 1.6389 X
0.1037X2, R2 ¼ 0.801, P ¼ 0.089). 3.5. Antioxidant-related parameters of spleen Effects of dietary MHA supplement on MDA and protein carbonyl content, anti-superoxide anion and anti-hydroxy radical capacities in spleen were presented in Table 7. MDA content in fish fed MHA unsupplement diet was significantly higher than that of the other diets (P < 0.05). Protein carbonyl content decreased with dietary MHA level up to 7.6 g kg
1 diet, and then increased (P < 0.05). Similar trend was obtained for anti-superoxide anion capacity. Anti-hydroxy radical capacity improved with dietary MHA level up to 5.1 g kg
1 diet (P < 0.05), and then significantly decreased with MHA level further up to 15.3 g kg
1 diet (P < 0.05). Meanwhile, protein carbonyl content (PC), anti-superoxide anion (ASA) and anti-hydroxy radical capacities (AHR) capacities showed quadratic response with graded levels of MHA (YPC ¼ 1.5708
0.1848X þ 0.0089X2, R2 ¼ 0.908, P ¼ 0.028; YASA ¼ 10.9410
0.4576X þ 0.0405X2, R2 ¼ 0.847, P ¼ 0.060; YAHR ¼ 48.0240 þ 2.8206X
0.1825X2, R2 ¼ 0.960, P ¼ 0.008). SOD, CAT, GST, GPX, GR activities and GSH content in head kidney affected by dietary MHA supplement were shown in Table 8.

Table 2 Effect of dietary MHA supplement on survival rate (SR) after challenge with Aeromonas hydrophila of juvenile Jian carp*. MHA

0

5.1

7.6

10.2

12.7

15.3

SR

60.00  0.0bc

70.00  10.0d

66.67  5.7cd

60.00  0.0bc

50.00  0.0a

53.33  5.7ab

*Mean values of triplicate groups. Mean values within the same column with different superscripts presented difference (P ¼ 0.06).

S.-Y. Kuang et al. / Fish & Shellfish Immunology 32 (2012) 629e636

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Table 3 Effects of dietary MHA supplement on head kidney index (HKI), spleen index (SI), red blood cell (RBC  1012 L
1) count and white blood cell (WBC  1010 L
1) count of juvenile Jian carp*. MHA

0

HKI SI RBC WBC

0.2159 0.1399 2.15 4.72

5.1    

0.0205a 0.0128a 0.08a 0.19a

7.6

0.2166 0.1653 2.33 5.23

   

0.0222a 0.0164b 0.09b 0.24c

10.2

0.2836 0.1805 2.28 5.20

   

0.0292b 0.0199c 0.11b 0.27c

12.7

0.2256 0.1734 2.25 5.09

   

0.0190a 0.0144bc 0.07b 0.27bc

0.2220 0.2084 2.13 4.95

15.3    

0.0285a 0.0179d 0.07a 0.22ab

0.2217 0.1825 2.12 4.73

   

0.0163a 0.0167c 0.10a 0.21a

*Mean values of triplicate groups. Mean values within the same column with different superscripts are significantly different (P < 0.05).

Table 4 Effects of dietary MHA supplement on leukocytes phagocytic activity (LPA), lysozyme activity (LA, ug mL
1), acid phosphatase activity (ACP, U 100 mL
1), iron-binding capacity (TIBC, mg L
1), haemagglutination titre (HT, Log2X), complement 3 (C3, mg L
1), 4 (C4, mg L
1) and IgM (mg L
1) contents of juvenile Jian carp*. MHA

0

LPA LA ACP TIBC C3 C4 HT IgM

26.67 5.97 428.5 81.41 128.1 15.73 5.75 87.27

5.1        

2.64a 0.30bc 37.2a 4.61a 7.0a 0.86a 0.50a 8.13a

41.00 7.08 774.3 91.18 178.1 25.32 6.50 94.55

7.6        

2.53c 0.58d 29.2bc 5.15bc 14.0b 2.10c 0.58ab 8.13ab

41.33 6.50 857.9 93.78 175.0 20.71 7.50 100.00

10.2        

2.17c 0.51c 34.3d 5.35c 17.1b 2.10b 0.58c 9.09b

44.67 6.24 809.2 85.32 162.5 19.56 7.00 101.82

12.7        

2.17d 0.40bc 66.0cd 3.57ab 14.0b 0.86b 0.00bc 7.61b

32.33 5.89 840.3 86.62 165.6 16.49 5.75 94.55

15.3        

2.79b 0.41b 39.4d 8.17abc 14.0b 1.72a 0.50a 8.13ab

28.33 4.63 743.0 87.27 162.5 15.73 5.75 92.73

       

2.64a 0.25a 60.9b 5.83abc 14.0b 0.86a 0.50a 4.07ab

*Mean values of triplicate groups. Mean values within the same row with different superscripts are significantly different (P < 0.05).

SOD activity decreased as MHA level was up to 7.6 g kg
1 diet, and then increased (P < 0.05). CAT activity was the highest in fish fed MHA unsupplement diet and significantly decreased with dietary MHA supplement (P < 0.05). When MHA level was up to 10.2 g kg
1 diet, CAT activity obtained minimum. No significant difference was obtained for GSH content among diets (P > 0.05). With the increasing MHA level up to 7.6 g kg
1 diet, GST activity significantly increased, after that decreased (P < 0.05). Similar tread was obtained for GR activity. GPX activity decreased with dietary MHA level up to 10.2 g kg
1 diet (P < 0.05), and then plateaued. Regression analyses showed that SOD, CAT, GST and GR activities were quadratic response with graded levels of MHA (YSOD ¼ 2.5840
0.1074X þ 0.0095X2, R2 ¼ 0.851, P ¼ 0.058; YCAT ¼ 0.6770
0.0907X þ 0.0055X2, R2 ¼ 0.857, P ¼ 0.054; YGST ¼ 25.6340 þ 2.1617X
0.1411X2, R2 ¼ 0.655, P ¼ 0.203; YGR ¼ 15.0550 þ 1.8807X
0.1321X2, R2 ¼ 0.808, P ¼ 0.084).

4. Discussion Fish are held in aquaculture pens, live in an environment where they are constantly exposed to various stress factors such as handling, crowding and infection leading to immune depression and outbreaks of infections [43]. To improve aquatic animal health, feed additives such as vitamins, minerals and prebiotics are considered as promising options in aquaculture [48]. Therefore, in present study we try to investigate the effects of feed additive, MHA, on diseases resistance and immune response of fish. Survival rate can reflect disease resistance of fish, especially the survival rate after challenge [49]. The present study firstly indicated that dietary MHA could improve survival rate after challenge with A. hydrophila of juvenile Jian carp. As we known, head kidney and spleen are two important immune organs in carp [12]. Therefore, enhanced head kidney and spleen index in our study may partly attribute to the positive effect of MHA on survival rate of Jian carp. Disease resistance is also associated with immune response [8]. Erythrocyte can induce blood B cells to produce immunoglobulin through proliferation, differentiation and maturity [50,51], while leucocytes can migrate in response to inflammatory stimuli [52]. With optimal MHA supplement, RBC and WBC counts significantly enhanced. Meanwhile, regression analyses indicated that curve linear relationship was observed between dietary MHA levels and RBC or WBC counts. Agrawal & Mahajan [53] implied that head kidney and spleen are important haemopoietic tissues for fish. These may interpret that increased RBC and WBC counts is partly due to improved growth of haemopoietic tissues. Phagocytes such as macrophages and neutrophils play an important role in limiting

3.6. Optimal MHA supplement level The optimal supplemental level of MHA for lysozyme activity and immunoglobulin M contents was estimated by quadratic regression analysis (Fig. 1a and b). Based on lysozyme activity, the optimal supplemental level of MHA was estimated to be 5.8 g kg
1 diet. The regression equation was as follows: Y ¼ 6.0209 þ 0.2754X
0.0237X2, R2 ¼ 0.943, P ¼ 0.014 (Fig. 1a). Based on immunoglobulin M contents, the optimal supplemental level of MHA was estimated to be 8.8 g kg
1 diet. The regression equation was as follows: Y ¼ 86.6540 þ 2.8690X
0.1630X2, R2 ¼ 0.847, P ¼ 0.060 (Fig. 1b).

Table 5 Effects of dietary MHA supplement on malondialdehyde (MDA, nmol mg
1 protein) and protein carbonyl (PC, nmol mg
1 protein) content, anti-superoxide anion (ASA, U g
1 protein) and anti-hydroxy radical (AHR, U mg
1 protein) capacities in head kidney of juvenile Jian carp*. MHA

0

MDA PC ASA AHR

3.35 2.47 13.87 199.1

5.1    

0.25c 0.27a 0.65c 14.3b

3.03 2.37 11.51 214.4

7.6    

0.27b 0.29a 1.14b 14.4b

2.45 2.25 8.39 178.2

10.2    

0.13a 0.11a 0.21a 11.8a

2.57 2.22 8.80 177.6

*Mean values of triplicate groups. Mean values within the same column with different superscripts are significantly different (P < 0.05).

12.7    

0.19a 0.10a 0.40a 3.5a

3.05 2.38 8.82 171.3

15.3    

0.21b 0.21a 0.41a 3.9a

2.91 2.23 8.58 173.3

   

0.13b 0.21a 0.35a 10.0a

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S.-Y. Kuang et al. / Fish & Shellfish Immunology 32 (2012) 629e636

Table 6 Effects of dietary MHA supplement on superoxide dismutase (SOD, U mg
1 protein), catalase (CAT, U mg
1 protein), glutathione-S-transferase (GST, U mg
1 protein), glutathione peroxidase (GPX, U mg
1 protein), glutathione reductase (GR, U g
1 protein) activities and glutathione (GSH, mg g
1 protein) content in head kidney of juvenile Jian carp*. MHA

0

SOD CAT GSH GST GPX GR

3.28 1.10 5.71 51.61 885.7 23.42

5.1      

0.15c 0.12a 0.33b 1.11a 56.0b 2.23a

2.72 1.65 6.46 52.24 873.1 32.14

7.6      

0.27b 0.13c 0.31c 1.23a 49.4b 2.47d

1.99 1.58 5.52 48.60 695.3 30.05

10.2      

0.05a 0.07c 0.28b 1.53a 52.6a 3.07cd

2.08 1.94 5.94 51.46 699.5 28.37

12.7      

0.09a 0.07d 0.27b 6.02a 38.9a 1.83bc

2.09 1.24 4.59 51.40 684.1 28.17

15.3      

0.10a 0.04b 0.33a 1.08a 65.4a 2.17bc

2.03 1.26 4.56 49.53 730.2 25.60

     

0.08a 0.07b 0.28a 1.81a 70.7a 1.90ab

*Mean values of triplicate groups. Mean values within the same column with different superscripts are significantly different (P < 0.05).

Table 7 Effects of dietary MHA supplement on malondialdehyde (MDA, nmol mg
1 protein) and protein carbonyl (PC, nmol mg
1 protein) content, anti-superoxide anion (ASA, U g
1 protein) and anti-hydroxy radical (AHR, U mg
1 protein) capacities in spleen of juvenile Jian carp*. MHA

0

MDA PC ASA AHR

5.27 1.63 11.14 47.56

5.1    

0.35b 0.08c 0.89b 2.04a

4.62 0.71 9.34 58.74

7.6    

0.12a 0.04ab 0.46a 6.39c

4.27 0.65 9.12 59.59

10.2    

0.18a 0.04a 0.50a 1.97c

4.24 0.73 11.51 56.25

12.7    

0.15a 0.04ab 0.54b 1.15c

4.20 0.78 11.77 53.96

15.3    

0.30a 0.02b 0.13b 2.07bc

4.14 0.73 13.10 49.16

   

0.44a 0.06ab 0.85c 2.92ab

*Mean values of triplicate groups. Mean values within the same column with different superscripts are significantly different (P < 0.05).

the dissemination of infectious agents, and are responsible for the eventual destruction of phagocytosed pathogens [54]. In present study, maximum leukocytes phagocytic activity was observed in fish fed diet with 10.2 g kg
1 MHA. Although such study has not yet been conducted in fish, similar trend was observed in broiler chicken [9]. Challenge by A. hydrophila can induce the immune response involving the activation of effector cells such as phagocytes, lymphocytes and natural killer cells, as well as a subsequent production of cytokines and other mediators, mainly reactive oxygen species (ROS) [55], which can lead to sensitively oxidative stress in these cells [16]. Thus, the improved leukocytes phagocytic activity after challenge by MHA may be partly attributed to increased antioxidant status in leukocytes. However, further research needs to be carried out. Teleosts also have various humoral defense components such as haemagglutination, complements, lysozyme and metal ion binding proteins [56]. Haemagglutination can be directly against various saccharide moieties on cell surfaces, and thus involve in the extracellular recognition and opsonisation of bacteria and protozoans [57]. In our study, optimal MHA supplement significantly improved haemagglutination titre in serum of fish after challenge. Fish complement can lyse foreign cells and opsonize foreign organisms for destruction by phagocytes [58], while C3 and C4 play a pivotal in the activation of complement system [59]. We found that C3 and C4 contents were positively related to MHA level, but higher MHA level had negative effect on C4 content. Lysozyme can lyse certain Gram-positive bacteria even some Gram-negative

bacteria by attacking the b-1,4 glycosidic bond between N-acetymuramic acid and N-acetylglucosamine in the peptidoglycan of bacterial cell walls [3]. Our study found that serum lysozyme activity by challenge significantly increased with optimal MHA supplement. ACP activity has been used as an indicator of cell mediated immunity [60]. Serum ACP activity showed significantly improvement with MHA supplement in our study. Total ironbinding capacity (TIBC) can represent the concentration of ironbinding proteins in serum [45]. The result indicated that TIBC was enhanced by increasing levels of MHA followed challenge. Producing immunoglobulins is also an adaptive response after being stimulated by antigen in fish, and IgM class is the primary immunoglobulin in most teleost [61]. In the present study, IgM content after challenge was significantly affected by dietary MHA levels. Moreover, significant curve linear relationship was obtained between dietary MHA level and lysozyme activity, ACP activity, C3 content, haemagglutination titre or IgM content. Fish immunity is usually correlated with the normal structure and function of immune organs [13]. As we known, structure and function of many organs can be altered by oxidative injury [15]. Our previous study had found that MHA could improve structure and function of intestine and hepatopancreas by regulating antioxidant status in these organs [17,18]. Therefore, lipid and protein oxidation, as well as antioxidant status in head kidney and spleen from unchallenged fish were further investigated to provide partial theoretical evidence for the improvement of immune response by MHA. In present study, although protein carbonyl content in head

Table 8 Effects of dietary MHA supplement on superoxide dismutase (SOD, U mg
1 protein), catalase (CAT, U mg
1 protein), glutathione-S-transferase (GST, U mg
1 protein), glutathione peroxidase (GPX, U mg
1 protein), glutathione reductase (GR, U g
1 protein) activities and glutathione (GSH, mg g
1 protein) content in spleen of juvenile Jian carp*. MHA

0

SOD CAT GSH GST GPX GR

2.63 0.69 1.78 25.96 148.6 15.10

5.1      

0.21b 0.02d 0.09a 2.58a 10.2c 1.19a

2.21 0.32 1.75 30.04 138.6 19.74

7.6      

0.11a 0.01b 0.16a 1.80bc 9.4bc 2.01b

2.16 0.33 1.82 38.02 139.1 24.54

10.2      

0.12a 0.03b 0.08a 1.73d 3.4bc 1.93c

2.72 0.26 2.00 33.31 129.8 20.08

*Mean values of triplicate groups. Mean values within the same column with different superscripts are significantly different (P < 0.05).

12.7      

0.13b 0.02a 0.15a 2.20c 7.3ab 1.67b

2.78 0.51 1.83 27.20 121.4 15.62

15.3      

0.03b 0.05c 0.20a 1.27ab 4.8a 0.97a

3.10 0.52 1.76 27.00 123.5 13.96

     

0.20c 0.04c 0.07a 2.31ab 3.1a 0.93a

S.-Y. Kuang et al. / Fish & Shellfish Immunology 32 (2012) 629e636

635

MHA level, all other parameters were negatively related to MHA levels. These results suggested that the effect of MHA on antioxidant enzymes activities and GSH content exist discrepancy in different organs. Activities of SOD and GPX, which are respectively  responsible for clearing O2
and H2O2 [64,65], decreased with MHA supplement in head kidney and spleen. With the result of ASA and AHR, we presume that head kidney and spleen have higher  potential to produce O2
and H2O2. GR can catalyze the reduction of the glutathione disulfide back to GSH by the expense of the NADPH [66]. Based on our results, GR activity was increased in head kidney and spleen by MHA, but only GSH content in head kidney was improved. Further investigation needs to be conducted to study these differences between head kidney and spleen. In conclusion, MHA improved disease resistance by increasing the growth of immune organs and humoral immune response, thus promoted growth of Jian carp. MHA also depressed lipid or protein oxidation in head kidney and spleen to protect structure and function of immune organs. Furthermore, MHA affect antioxidant enzymes activities and GSH content in head kidney and spleen, which provide some evidence for the improvement of immune response by MHA in fish. Acknowledgements This study was financially supported by Sumitomo-chemical (Japan), supported by National Science Foundation of China (30771671 and 30871926), National Department Public Benefit (Agricultural) Research Foundation of China (201003020), Programs for New Century Excellent Talents in University (NCET08-0905) and the Key Project of Chinese Ministry of Education (208120). Authors would like to thank the personnel of these teams for their kind assistance. Fig. 1. Quadratic regression analysis of lysozyme activity LA (a) and immunoglobulin M (IgM) content (b) for juvenile Jian carp challenged with Aeromonas hydrophila for 17 days. Requirements derived with the quadratic regression analysis for LA and IgM were 5.8 mg kg
1 and 8.8 mg kg
1, respectively.

kidney was not significantly influenced by MHA, MDA content in head kidney and spleen and protein carbonyl content in spleen all significantly decreased with MHA supplement. It is suggested that MHA could protect structure and function of immune organs through depressing lipid or protein oxidation. Endotoxin injection often results in a significant increase of ROS production such as   superoxide anion ðO2
Þ, H2O2 and hydroxy radical (OH ), which are responsible for endotoxin clearance [55]. According to this, we study the effect of MHA on ASA and AHR in immune organs. Results indicated that both ASA and AHR in head kidney significantly decreased and then plateaued with MHA supplement, showing that   MHA supplement enhanced the ability of ðO2
Þ and OH production  in head kidney. Similar result was found for O2
production in neutrophils affected by glutamine [62]. Therefore, MHA might improve potential sterilizing ability of head kidney. Interestingly, ASA in spleen was decreased by MHA, while opposite trend was observed for spleen AHR. This may be due to the difference of main functions for spleen and head kidney. However, further study will be conducted to prove our presumption. Fish antioxidant defense systems also consist of low-molecularweight antioxidants and antioxidant enzymes [63]. In present study, antioxidant enzymes activities and GSH content response were further measured in head kidney and spleen from unchallenged fish. Results showed that GST activity in head kidney and GSH content in spleen were not influenced by MHA level. Only CAT and GR activities and GSH content in head kidney, as well as GST and GR activities in spleen were positively correlated with dietary

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