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Effects of ammonia stress, dietary linseed oil and Edwardsiella ictaluri challenge on juvenile darkbarbel catfish Pelteobagrus vachelli
Fish & Shellfish Immunology 38 (2014) 158e165
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Effects of ammonia stress, dietary linseed oil and Edwardsiella ictaluri challenge on juvenile darkbarbel catfish Pelteobagrus vachelli Ming Li a, b, Na Yu a, **, Jian G. Qin c, Erchao Li a, Zhenyu Du a, Liqiao Chen a, * a
School of Life Sciences, East China Normal University, Shanghai 200062, China School of Marine Sciences, Ningbo University, Ningbo 315211, China c School of Biological Sciences, Flinders University, Adelaide, SA 5001, Australia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 November 2013 Received in revised form 5 February 2014 Accepted 12 March 2014 Available online 19 March 2014
A two-stage study was carried out to test the response of juvenile darkbarbel catfish Pelteobagrus vachelli to ammonia stress, dietary lipid and bacterial challenge. At stage 1, the catfish (0.99 0.01 g) fed a commercial diet were exposed to 0.01 and 5.70 mg L1 total ammonia nitrogen in nine replicates for 14 days. At stage 2, all fish previously exposed to either low or high ammonia were separately transferred into low ammonia (<0.01 mg L1), and divided into three feeding groups. Fish were then fed three levels of linseed oil (0, 2 and 4%) in triplicate for 46 days. Fish growth performance and immune response were low in high ammonia at stage 1. At stage 2, fish growth and immune response were not significantly different between fish previously exposed to low and high ammonia in all diets. Fish fed 4% linseed oil showed the greatest weight gain, feed efficiency ratio, red blood cells, hemoglobin and hematocrit, and achieved higher lysozyme activity, phagocytic index, respiratory burst and total immunoglobulin than fish fed 0% linseed oil, but did not differ from fish fed 2% linseed oil regardless of previous ammonia exposure. After 14-day infection of Edwardsiella ictaluri, cumulative mortality of fish previously exposed to low ammonia was lower than that of fish exposed to high ammonia in all diets. Cumulative mortality of fish fed 0% linseed oil was highest, but the antibody titer of fish fed 4% linseed oil was highest regardless of previous ammonia treatments. This study indicates that ammonia stress has a lasting effect even after ammonia is lowed, but the adverse effect on fish can be mitigated through manipulation of dietary oil inclusion, especially under the challenge of pathogenic bacteria. Ó 2014 Published by Elsevier Ltd.
Keywords: Ammonia Linseed oil Growth performance Immune responses Disease resistance
1. Introduction Intensive aquaculture systems can easily cause chronic ammonia stress and sometimes transient peaks due to ammonification of feed residual and animal excretion [1e3]. Ammonia can be practically removed by biological filtration or water exchange, but a sudden and quick increase of ammonia may be detrimental to fish [4]. Excessive ammonia can cause fish growth reduction [5,6], tissue erosion and degeneration [7,8], immune suppression and high mortality [9]. After experiencing stress by environmental factors such as unfavorable temperature, low feed supply or high ammonia, compensatory growth may be observed when these stress factors are removed [10e12]. Although compensatory
* Corresponding author. Tel./fax: þ86 21 62233637. ** Corresponding author. Tel./fax: þ86 21 54341002. E-mail addresses: [email protected] (N. Yu), (L. Chen). http://dx.doi.org/10.1016/j.fsi.2014.03.015 1050-4648/Ó 2014 Published by Elsevier Ltd.
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growth in fish such as Atlantic halibut Hippoglossus hippoglossus has been reported following a prolonged period of exposure to high ammonia, physiological and immunological changes of fish after ammonia exposure have not been investigated [2]. The ability of fish to adapt to environmental stress may depend on fish nutritional status [13]. Dietary manipulation has been used as a tool to alleviate the damage of ammonia on fish [14] because environmental stress may increase the requirement of essential fatty acids in fish such as rainbow trout Oncorhynchus mykiss [15] and European sea bass Dicentrarchus labrax [16]. Under stress, deficiency of essential fatty acids in diet can further deteriorate the ability of fish for immune response and disease resistance [17]. Linolenic acid, an essential fatty acid for most catfish [18], plays important roles in cell-membrane function, eicosanoid synthesis and prostaglandin regulation [19,20], and triggers immunological response to activate antigens [21]. Our previous study indicates that the harmful effects of chronic low ammonia on fish can be mitigated when fish are fed on 2% linseed oil and 400 mg kg1 vitamin E [22]. However, it is not clear whether manipulation of linseed oil
M. Li et al. / Fish & Shellfish Immunology 38 (2014) 158e165
contents can mitigate the adverse effect of ammonia on fish following acute high ammonia exposure. This study used a sequential approach to examine the effect of ammonia exposure, dietary linseed oil manipulation and bacterial challenge on fish growth, immunity and disease resistance. We first set the upper level of stress high ammonia at 5.70 mg L1 total ammonia nitrogen (TA-N) as suggested by Colt and Tchobanoglous in a study on channel catfish Ictalurus punctatus where fish mortality increases significantly above 5.71 mg L1 TA-N [23]. To test if dietary nutrition can mitigate the post-effect of ammonia exposure on fish, all fish previously exposed to different ammonia were fed with different levels of dietary linseed oil. At last, according to the previous treatment history of ammonia exposure and diet supply, fish were challenged with pathogenic bacteria Edwardsiella ictaluri to test the disease resistance. The experiments with such a design would enable us to test the following hypotheses: (1) exposure to a high level of ammonia compromises fish growth and health, and (2) dietary lipid manipulation improves growth performance, hematology status, antioxidant enzyme activities, immune responses and resistance to E. ictaluri challenge on juvenile darkbarbel catfish Pelteobagrus vachelli.
159
Table 2 Proximate composition (g 100 g1 lipid) and fatty-acid composition (% by weight of total fatty acids) in experimental diets. Experimental diets (g 100 g1 diet) 0% Proximate composition (%) Moisture 10.87 Protein 42.39 Lipid 11.09 Ash 4.75 Fatty-acid composition C12:0 98.78 C16:0 0.65 C18:0 0.27 C18:1n9 e C18:2n6 e C18:3n3 e
2%
4%
10.13 41.98 10.87 4.81
10.98 42.03 11.12 4.78
86.86 0.93 0.58 3.05 0.94 9.27
74.07 1.77 1.15 6.15 1.58 19.13
Values are means of three replicates in each treatment.
meter (Hach Company, Colorado, USA). During the trial, water quality variables were maintained at 27e29 C, dissolved oxygen 7.81 0.13 mg L1, pH 6.4e6.6, nitrate <0.5 mg L1 (Griess reaction [24]) and 12 h light and 12 h dark.
2. Materials and methods 2.1. Experimental diets and animals
2.2. Experimental designs and sampling
Three experimental diets were formulated including three levels of linseed oil (enriched with linolenic acid at 0, 2 and 4%, Table 1). The diet proximate composition and fatty-acid compositions are given in Table 2. Diets were processed into 2-mm diameter pellets, dried at room temperature to <10% moisture, ground and sieved to appropriate size before storage at 20 C. Darkbarbel catfish (0.99 0.01 g, mean SD), obtained from a fish farm in Chengdu (China), were acclimatized with the commercial feed (42% protein, 11% lipid, 10% moisture and 5% ash) for 14 days prior to this study. Fish were randomly stocked into eighteen 300-L rectangular plastic tanks at 35 fish each. All tanks were cleaned, and fish in each tank were group-weighed and counted fortnightly. All tanks were supplied with dechlorinated tap water with a daily water exchange rate at 1/3 of the tank volume. Water was continuously aerated using air stones. Water temperature and dissolved oxygen were measured daily with a HACH HQ30d oxygen
Table 1 Percentage composition of the basal diet ingredients. Experimental diets (g 100 g1 diet)
Casein, vitamin-free Galatin Coil starch Carboxymethyl cellulose Linseed oila Lauric acid Vitamin mixtureb Mineral mixturec Celufil Antioxidant (ethoxyquin) a
0%
2%
4%
40.0 10.0 25.0 3.0 0.0 11.0 0.5 5.0 5.5 0.02
40.0 10.0 25.0 3.0 2.0 9.0 0.5 5.0 5.5 0.02
40.0 10.0 25.0 3.0 4.0 7.0 0.5 5.0 5.5 0.02
Linseed oil: purchased from Hebei Xinqidian Bio. Tech. Co. Ltd. Hebei, P.R. China. Vitamin premix (kg1 diets): vitamin A, 5500 IU; vitamin D3, 1000 IU; vitamin E, 50 IU; vitamin K, 10 mg; niacin, 100 mg; riboflavin, 20 mg; pyridoxine, 20 mg; thiamin, 20 mg; biotin, 0.1 mg; D-calcium pantothenate, 50 mg; folacin, 5 mg; B12, 20 mg; ascorbic acid, 100 mg; inositol, 100 mg. c Mineral premix (mg kg1 diets): NaCl, 500; MgSO4$7H2O, 4575.0; NaH2PO4$2H2O, 12,500.0; KH2PO4, 16,000.0; Ca(H2PO4)2$H2O, 6850.0; FeSO4, 1250.0; C6H10CaO6$5H2O, 1750.0; ZnSO4$7H2O, 111.0; MnSO4$4H2O, 61.4; CuSO4$5H2O, 15.5; CoSO4$6H2O, 0.5; KI, 1.5; Starch, 6385.1. b
This study was divided into two stages. At stage 1, the experiment consisted of a low ammonia treatment [total ammonia nitrogen (TA-N) 0.01 mg L1, un-ionized ammonia (UIA-N) 0.001 mg L1] and a high ammonia treatment (TA-N 5.70 mg L1, UIA-N 0.12 mg L1). Each treatment was in nine replicates. Fish were fed with a commercial diet twice daily (07:30e08:30 h and 15:00e16:00 h) to apparent satiation for 14 days. The amount of diet consumption was recorded daily. The desired ammonia concentrations were achieved by supplementing a solution of NH4Cl (10 g L1) every 7 h. TA-N levels were measured by nesslerization [25]. Percentage UIA-N was calculated using the equation of Johansson and Wedborg [26]. This equation gives the percentage UIA-N as a function of pH and temperature. Corrections for pH measurements with low-ionic strength buffers (i.e., conversion to the Hansson scale) were performed according to the method of Whitfield [27]. At stage 2, all fish previously exposed to either low or high ammonia were separately transferred into the low ammonia water (<0.01 mg L1), and divided into three feeding groups. Fish were then fed with three levels of linseed oil (0, 2 and 4%) in triplicate for 46 days. Each treatment was fed three experimental diets twice daily (07:30e08:30 h and 15:00e16:00 h) to apparent satiation. The amount of diet consumption was recorded daily. At the end of the two stages, fish were starved for 24 h, and then were anesthetized with tricaine methanesulfonate (MS-222) at 120 mg L1 before weighing and counting. Three fish tank1 were randomly sampled, minced, pooled and stored at 80 C for fatty acid analysis. Blood samples (three fish tank1) were collected from the heart with heparinized (100 IU mL1) tuberculin syringes for hematological assays. The blood of additional three fish tank1 was taken using nonheparinized tuberculin syringes and clotted at 4 C overnight. Serum samples were collected, centrifuged and stored at 80 C for immune assays. After being bled, the head kidneys of six fish were removed for macrophage separation. Livers were removed and individually weighed to determine the hepatosomatic index. The indexes for assessing growth performance were calculated as follows:
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M. Li et al. / Fish & Shellfish Immunology 38 (2014) 158e165
Weight gain (WG, g) ¼ final weight (g) initial weight (g) Feed efficiency ratio (FER) ¼ wet weight gain (g)/dry feed fed (g) Survival (%) ¼ 100 (final fish number)/(initial fish number) Hepatosomatic index (HSI%) ¼ liver mass (g) 100/body mass (g)
2.3. Biochemical composition analysis All experimental diets were analyzed in duplicate for proximate composition following the standard methods [28]. Moisture was determined by oven dry at 105 C to a constant weight. Samples used for dry matter were digested with nitric acid and incinerated in a muffle furnace at 600 C overnight for ash content determination. Protein was measured by the combustion method using an FP-528 nitrogen analyzer (Leco Corporation, St. Joseph, MI, USA). Lipid was determined by the ether extraction method using the Soxtec system (2055 Soxtec Avanti; Foss Tecator, Hoganas, Sweden). Total lipid of experimental diets and fish samples was extracted using chloroform: methanol (2:1, v/v) in duplicate according to the method of Folch et al. [29]. The saponifiable lipids were converted to methyl esters by using the standard boron tri-fluoride-methanol method [30]. Fatty acid methyl esters (FAME) were analyzed on an Agilent 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA), equipped with a flame ionization detector (FID) and a SP2560 fused silica capillary column (100 m, 0.25 mm i.d. and 0.20 mm film thick). Injector and detector temperatures were 270 and 280 C respectively. Column temperature was held at 120 C for 5 min then programmed to increase at 3 C min1 up to 240 C where it was maintained for 20 min. Carrier gas was helium (2 mL min1), and the split ratio was 30:1. Identification of fatty acids was carried out by comparing the sample FAME peak relative retention times with the SigmaeAldrich (St. Louis, MO, USA) standards. The individual fatty acid methyl esters were identified by comparing the retention times of the authentic standard mixtures and quantified the absolute content with the internal standard. 2.4. Hematology, enzyme activity and lipid peroxidation assays Red blood cell and white blood cell counts were performed in duplicate for each sample by diluting (1:10,000) the whole blood in phosphate buffer saline (PBS) solution and enumerating with a hemocytometer [17]. Hemoglobin was determined using a commercial kit (Sigma Chemical Co., St. Louis, MO, USA) and its values were adjusted, as described by Larsen [31], using a cyanomethemoglobin correction factor for channel catfish. Hematocrit was determined by centrifuging the whole blood in heparinized microhematocrit capillary tubes at 650 g for 10 min and measuring the packed cell volume [32]. The levels of enzyme activity and lipid per-oxidation were measured with commercial assay kits (Nanjing Jiancheng Institute, Nanjing, China) in accordance with the instructions of the manufacturer. The assays are briefly described as follows: Total superoxide dismutase activity was determined following the method of Beauchamp and Fridovich [33]. The ratio of autooxidation rates of the samples with or without serum was determined at 550 nm. One unit of SOD activity was calculated using the amount of superoxide dismutase required to inhibit the reduction of nitroblue tetrazolium by 50%. Catalase activity was determined by measuring the decrease of H2O2 concentration [34]. After 10 mL of serum was added to the
reagent, the sample was incubated for 60 s at 37 C. The absorbance of the samples was read at 405 nm. One unit of CAT activity was defined as the amount of CAT required to transform 1 mmol of H2O2 min1. Glutathione peroxidase activity was measured following the method of Flohé and Günzler [35]. Blood samples were treated with Drabkin’s reagent to avoid hemoglobin interferences. After the addition of 1 mM GSH (reduced glutathione) the NADPH-consumption rate was monitored at 412 nm. One unit of GPX activity was defined as the amount of GPX required to oxidize 1 mmol of NADPH min1. Lipid-peroxidation levels were determined based on the malondialdehyde levels generated by oxidation of polyunsaturated fatty acids. In the presence of thiobarbituric acid, malondialdehyde started producing colored thiobarbituric-acid-reacting substances (TBARS) that were measured at 532 nm [36]. 2.5. Lysozyme activity, phagocytic index, respiratory burst and immunoglobulin assays Serum lysozyme activity was determined through the turbidimetric method [37] using a lysozyme detection kit (Nanjing Jiancheng Institute, Nanjing, China). The assay was based on the lysis of a lysozyme-sensitive Gram-positive bacterium via the lysozyme present in the serum. The head kidney was removed and transferred to L-15 culture medium (100 IU mL1 penicillin, 100 mg mL1 streptomycin, 10 IU mL1 heparin, 2% fetal bovine serum) and then filtered through a 100 mm metal mesh. The resulting cell suspensions were enriched by centrifugation (600 g, 5 min, 4 C) on the 34%/51% Percoll density gradient. The cells were collected at the 34e51% interface and washed twice. The final cell concentration was adjusted to approximately 1 107 mL1 and the cell viability was >95%. The phagocytic index was determined according to the method by Pulsford et al. [38]. The respiratory burst of phagocytic cells was measured by the nitroblue tetrazolium reduction assay following the method of Secombes [39]. Total immunoglobulin was assayed using a commercial kit (Zhejiang Elikan Biological Technology Co., Ltd, Wenzhou, China). The methods are described by Wu and Shang [40] for the activity analysis including the measurement of turbidity increase after the immunity response and the increase of its antibody. 2.6. Bacterial challenge A frozen stock-culture of E. ictaluri was obtained from the Institute of Hydrobiology, the Chinese Academy of Science (Wuhan, China). The stock culture of E. ictaluri frozen at 80 C was inoculated in 250 mL of braineheart infusion broth and cultured for 24 h in a water bath shaker at 26 C [41]. To determine the optimum bacterial concentration for fish infection, a preliminary trial was conducted by injecting 20 fish tank1 with 0.1 mL of 0, 1 106, 2 106, 4 106, 8 106, or 1.6 107 E. ictaluri CFU mL1 in triplicate. Mortality was recorded twice daily for 14 d. The LC50 level (i.e., 1 105 CFU fish1) was then used for the experimental challenge. At the end of the stage 2, fifteen fish were randomly selected from each tank and intraperitoneally injected with 1 105 CFU fish1 of E. ictaluri using a tuberculin syringe. In the preliminary trial, such a density of E. ictaluri could have adverse impact on darkbarbel catfish. Subsequently, fish in each group were separately fed with the above three diets. Mortality was recorded twice daily during the period of 14-day trial and dead fish were removed.
M. Li et al. / Fish & Shellfish Immunology 38 (2014) 158e165
At the end of the E. ictaluri challenge, blood samples were collected from four randomly chosen live fish, and the serum was collected. The agglutinating antibody titers against E. ictaluri in the pre- and post-challenge serum samples were measured as described in Chen and Light [42]. 2.7. Statistical analysis Data from stage 1 of the experiment were used to compare the effects of the ammonia by a t-test. Data from the stage 2 of the experiment were used to detect interaction between linseed oil and ammonia levels by two-way ANOVA with the level of pre-exposure ammonia and the level of linseed oil as two factors. If a significant main effect was detected, differences between group means were compared by Duncan’s multiple range tests. The level of significance was set at P <0.05. All analyses were performed using the SPSS 18.0.0 (Chicago, USA) for Windows.
3.1. Effects of ammonia at stage 1 Fish survival was not affected by different levels of ambient ammonia (P > 0.05). Weight gain (WG) and feed efficiency ratio (FER) of fish in the treatment of low ammonia were higher than those of fish in the treatment of high ammonia (P < 0.05, Table 3). But the treatment of low ammonia showed lower hepatosomatic index (HSI) compared to that of fish in the treatment of high ammonia (P < 0.05). The content of saturated fatty acids (SAFAs) and n-3 polyunsaturated fatty acids (PUFAs) showed a similar trend to WG and FER. The hemoglobin of fish in the treatment of low ammonia was higher than that of fish in the treatment of high ammonia (P < 0.05, Table 4). The activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) showed a similar trend to hemoglobin. However, the treatment of low ammonia showed lower malondialdehyde (MDA) contents compared to that of fish in the Table 3 Growth performance and fatty acid composition of darkbarbel catfish fed commercial feed with high or low ammonia exposure for 14 days.
Low Growth performance WG (g) 0.892 FER 0.332 HSI (%) 0.131 Survival (%) 100% Fatty acid composition of fish 12:0 10.05 14:0 7.58 16:0 20.442 18:0 3.16 SAFA 41.232 18:1n9 40.13 20:1n9 1.91 MUFA 42.04 18:2n6 13.01 20:3n6 1.30 PUFA n-6 14.31 18:3n3 1.092 20:5n3 0.532 22:6n3 0.122 PUFA n-3 1.742
Table 4 Hematology, antioxidant enzyme activity and immune response of darkbarbel catfish fed commercial feed with high or low ammonia exposure for 14 days. Ammonia Low Hematology assay RBC 106 mL1 2.82 WBC 105 mL1 3.03 1 Hemoglobin (g dL ) 8.262 Hematocrit (%) 24.41 Antioxidant enzyme activity SOD (U mL1) 104.582 CAT (U mL1) 16.16 GPX (U mL1) 313.142 1 1.051 MDA (nmol mL ) Immune response Lysozyme (U mL1) 253.852 PI (%) 5.782 RB (%) 69.322 Ig (mg mL1) 1.562
Pooled SEM
P values
2.83 3.04 8.061 24.47
0.015 0.007 0.007 0.051
0.731 0.381 0.001 0.538
98.511 16.12 305.451 2.952
0.084 0.198 0.909 0.007
0.001 0.907 0.001 0.001
235.041 5.281 64.561 1.341
0.677 0.030 0.236 0.013
0.001 0.001 0.001 0.001
High
Different superscript numbers (1, 2) indicate a significant effect of ammonia (P < 0.05). RBC: red blood cell; WBC: white blood cell; SOD: superoxide dismutase; CAT: catalase; GPX: glutathione peroxidase; MDA: malondialdehyde; PI: phagocytic index; RB: respiratory burst; Ig: total immunoglobulin.
3. Results
Ammonia
161
Pooled SEM
P values
0.701 0.251 0.592 100%
0.013 0.005 0.013 e
0.001 0.001 0.001 e
10.42 7.83 20.691 3.19 42.131 40.21 2.09 42.30 13.86 1.32 15.18 0.091 0.001 0.001 0.091
0.021 0.018 0.014 0.009 0.103 0.024 0.019 0.089 0.005 0.014 0.081 0.002 0.008 0.003 0.012
0.001 0.876 0.673 0.744 0.012 0.042 0.812 0.008 0.768 0.876 0.554 0.001 0.001 0.001 0.001
High
Different superscript numbers (1, 2) indicate a significant effect of ammonia (P < 0.05). WG: weight gain; FER: feed efficiency ratio; HSI: hepatosomatic index; SAFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.
treatment of high ammonia (P < 0.05). The lysozyme activity, phagocytic index (PI), respiratory burst (RB) and total immunoglobulin (Ig) contents of fish in the treatment of low ammonia were higher than those of fish in the treatment of high ammonia (P < 0.05).
3.2. Effects of dietary linseed oil at stage 2 Fish fed 4% linseed oil showed higher WG and FER compared to those in other groups regardless of the level of previous ammonia exposure (P < 0.05, Table 5). However, HSI was highest when fish fed on 0% linseed oil (P < 0.05). In addition, the HSI of fish previously exposed to low ammonia was lower than that of fish exposed to high ammonia in all diets (P < 0.05). Significant interactions between linseed oil and previous ammonia exposure on FER were detected (P < 0.05). SAFAs and monounsaturated fatty acids of fish fed 0% linseed oil were highest irrespective of the previous level of ammonia exposure (P < 0.05, Table 6). But n-6 PUFAs and n-3 PUFAs of fish fed 4% linseed oil were greater than those of fish fed 2% and 0% linseed oil (P < 0.05), while these variables in fish fed 2% linseed oil were greater than those fed 0% linseed oil (P < 0.05). The RBC, hemoglobin and hematocrit of fish fed 4% linseed oil were highest (P < 0.05, Table 7), while these variables in fish fed 2% linseed oil were higher than those fed 0% linseed oil regardless of previous ammonia exposure (P < 0.05). Significant interactions between linseed oil and previous ammonia exposure on RBC, hemoglobin and hematocrit were detected (P < 0.05). Fish previously exposed to low ammonia showed lower SOD, CAT, GPX activities and MDA contents compared to those of fish previously exposed to high ammonia (P < 0.05, Table 8). The SOD, CAT and GPX activities and MDA contents of fish fed 4% linseed oil were highest regardless of previous ammonia exposure (P < 0.05). Significant interactions between linseed oil and previous ammonia exposure on MDA were detected (P < 0.05). Fish fed 4% linseed oil achieved higher levels of lysozyme activity, PI, RB and Ig than fish fed 0% linseed oil (P < 0.05, Table 9), but did not differ from those of fish fed 2% linseed oil irrespective of previous ammonia exposure (P > 0.05). Significant interactions between linseed oil and previous ammonia exposure on lysozyme activity were detected (P < 0.05).
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Table 5 Mean final weight gain (WG), feed efficiency ratio (FER), hepatosomatic index (HSI) and survival of darkbarbel catfish fed purified diets supplemented with various levels of linseed oil for 46 days.
WG (g) FER HSI (%) Survival (%)
Ammonia
Linseed oil 0%
2%
4%
Low High Low High Low High Low High
4.40a 3.60A 1.21a 0.88A 1.50b1 2.25B2 100% 100%
5.82b 5.54B 1.79b 1.69B 0.53a1 1.36A2 100% 100%
5.86c 5.98C 1.86c 2.30C 0.47a1 1.34A2 100% 100%
Pooled SEM
PUFA ammonia (P value)
0.096 0.289 0.079 0.033 0.021 0.062 e e
0.482
Table 6 Fatty acid composition (% by weight of total fatty acids) of darkbarbel catfish fed purified diets supplemented with various levels of linseed oil for 46 days. Ammonia
12:0 14:0 0.010 16:0 0.711 18:0 e
Different superscript numbers (1, 2) indicate a significant effect of ammonia (P < 0.05). Different lowercase letters (a, b, c) indicate a significant effect of linseed oil following low ammonia (P < 0.05). Different uppercase letters (A, B, C) indicate a significant effect of linseed oil following high ammonia (P < 0.05).
SAFA 18:1n9 20:1n9 MUFA
3.3. Effects of bacterial challenge Cumulative mortality of fish previously exposed to low ammonia was lower than that of fish exposed to high ammonia (P < 0.05, Table 10). Fish fed 4% linseed oil achieved lower levels of cumulative mortality than fish fed 0% linseed oil (P < 0.05), but did not differ from that of fish fed 2% linseed oil irrespective of previous ammonia exposure (P > 0.05). Antibody titer of fish fed 4% linseed oil was highest (P < 0.05).
18:2n6 20:3n6 PUFA n-6 18:3n3 20:5n3 22:6n3 PUFA n-3
4. Discussion 4.1. Effects of ammonia at stage 1 Growth reduction due to high ammonia exposure has been reported in several fish species, such as spotted wolfish Anarhichas minor [43], turbot Scophthalmus maximus [4] and Atlantic halibut H. hippoglossus [2]. There is a close correlation between feed efficiency ratio and the levels of ammonia exposure [44,45]. In this study, the weight gain of fish exposed to high ammonia was lower than that of fish exposed to low ammonia due to the low feed efficiency ratio. At high external ammonia, the plasma ammonia is higher in the fed fish than the unfed fish and the mortality is higher in the fed fish than the unfed fish [46]. In this study, fish survival was not affected by ammonia exposure suggesting that 5.70 mg L1 TA-N falls into the tolerant range for darkbarbel catfish. Colt and Tchobanoglous [23] found that the mortality of channel catfish I. punctatus increased significantly when total ammonia was >5.71 mg L1 and the tolerance of TA-N for snakehead Channa striatus is 10.73 mg L1 at pH 8.0 [9]. Li et al. [47] showed that yellow catfish Pelteobagrus fulvidraco juveniles could not tolerate high levels of ammonia, and recommended that the ammonia concentration be less than 6.72 mg L1 TA-N. High ambient ammonia seems to favor the accumulation of saturated fatty acid in fish [48]. In this study, saturated fatty acids of fish in low ammonia were higher than those of fish in high ammonia. However, it should be noted that the n-3 PUFA (especially 18:3n3) of fish exposed to high ammonia was lower than that of fish in low ammonia. Ammonia exposure leads to the formation of excessive reactive oxygen species (ROS) [49,50]. The reduction of 18:3n3 may be related to fatty acid peroxidation [51]. Our result shows that ammonia exposure leads to deficiency of essential fatty acids in darkbarbel catfish. In addition, the contents of n-3 HUFA (EPA 20:5n3 and DHA 22:6n3) of fish experienced high ammonia exposure were not detected, though the ability of inhibiting
Low High Low High Low High Low High Low High Low High Low High Low High Low High Low High Low High Low High Low High Low High Low High
Linseed oil 0%
2%
4%
17.03c 17.01C 6.16 6.06 19.09c 19.03C 3.69 3.65 43.97c 45.97C 41.94c 41.89C 1.98 1.94 43.92c 43.83C 7.98a 7.91A 1.14 1.11 9.12a 9.02A 0.32a 0.27A 0.13a 0.09A 0.28a 0.19A 0.73a 0.55A
15.65b 15.28B 6.32 6.29 17.13b 17.77B 3.88 3.75 42.98b 42.98B 39.81b 39.16B 1.93 1.89 41.74b 41.05B 9.74b 10.17B 1.09 1.11 10.83b 11.28B 2.21b 2.14B 0.28b 0.21B 1.25b 1.45B 3.74b 3.80B
12.93a 12.88A 6.93 6.88 14.88a 14.92A 3.76 3.63 40.55a 38.55A 36.21a 36.66A 1.98 1.91 39.19a 38.57A 10.87c 10.99C 1.12 1.08 11.99c 12.07C 6.92c 6.89C 0.59c 0.58C 3.66c 3.11C 11.17c 10.58C
Pooled SEM
PUFA ammonia (P value)
0.087 0.071 0.008 0.093 0.041 0.101 0.099 0.067 0.109 0.056 0.101 0.098 0.078 0.045 0.078 0.566 0.091 0.012 0.055 0.046 0.091 0.101 0.018 0.012 0.033 0.019 0.096 0.045 0.032 0.098
0.123 0.576 0.544 0.412 0.884 0.307 0.496 0.211 0.135 0.285 0.825 0.313 0.206 0.339 0.121
Different lowercase letters (a, b, c) indicate a significant effect of linseed oil following low ammonia (P < 0.05). Different uppercase letters (A, B, C) indicate a significant effect of linseed oil following high ammonia (P < 0.05). SAFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.
bioconversion of 18-carbon atom fatty acids to 20- or 22-carbon atoms at high ambient ammonia was not confirmed. As ammonia toxicity may cause a rapid transient increase of ROS and result in blood cell damage [50]. In this study, fish hemoglobin in low ammonia was higher than in high ammonia. Fish can prevent ROS generation through the production of counteracting enzymes, such as superoxide dismutase, catalase and glutathione peroxidase [32]. But in this study, superoxide dismutase and glutathione peroxidase activities of fish exposed to high ammonia were lower than those of fish in low ammonia. This indicates that those enzymes may have been inhibited as a transitory response to ambient ammonia, which may be related to malondialdehyde toxicity.
Table 7 Mean red blood cell count (RBC), white blood cell count (WBC), hemoglobin and hematocrit of darkbarbel catfish fed purified diets supplemented with various levels of linseed oil for 46 days. Ammonia Linseed oil 0% RBC 106 mL1 WBC 105 mL1 Hemoglobin (g dL1) Hematocrit (%)
Low High Low High Low High Low High
2%
4%
2.34a 2.39b 2.41c 2.21A 2.37B 2.49C 3.33 3.33 3.35 3.39 3.49 3.57 a b 8.33 8.35c 8.23 8.13A 8.28B 8.44C 25.41a 25.76b 25.84c 25.11A 25.64B 25.94C
Pooled PUFA ammonia SEM (P value) 0.011 0.024 0.011 0.043 0.011 0.015 0.026 0.042
0.023 0.884 0.001 0.019
Different lowercase letters (a, b, c) indicate a significant effect of linseed oil following low ammonia (P < 0.05). Different uppercase letters (A, B, C) indicate a significant effect of linseed oil following high ammonia (P < 0.05).
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163
Table 8 Mean superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) activities and malondialdehyde (MDA) counts of darkbarbel catfish fed purified diets supplemented with various levels of linseed oil for 46 days. Ammonia
SOD (U mL1) 1
CAT (U mL
)
GPX (U mL1) MDA (nmol mL1)
Low High Low High Low High Low High
Linseed oil 0%
2%
4%
101.92a1 130.63A2 14.85a1 19.02A2 294.50a1 306.54A2 1.37a1 1.96A2
113.04a1 137.16B2 17.26a1 22.68B2 304.66a1 315.39B2 1.53a1 2.84B2
131.79b1 144.60C2 18.67b1 26.81C2 311.29b1 318.50C2 1.94b1 3.25C2
Pooled SEM
PUFA ammonia (P value)
2.287 0.935 0.437 0.485 1.288 1.097 0.033 0.043
0.057 0.076 0.504 0.001
Numbers (1, 2) indicate a significant effect of ammonia (P < 0.05). Lowercase letters (a, b, c) indicate a significant effect of linseed oil following low ammonia (P < 0.05). Uppercase letters (A, B, C) indicate a significant effect of linseed oil following high ammonia (P < 0.05).
When fish are exposed to high ammonia, a number of immunosuppressive factors are generated in the peripheral lymph tissue and released into the blood stream [52,53]. It typically results in reduction of lymphocytes and phagocytes and an immune response [54]. In this study, the lysozyme activity, phagocytic index, respiratory burst and total immunoglobulin contents of fish exposed to high ammonia were lower than those of fish exposed to low ammonia, which is in agreement with the finding in Nile tilapia Oreochromis niloticus [55]. 4.2. Effects of dietary linseed oil at stage 2 Compensatory growth refers to a rapid growth that follows a period of suppressed growth due to nutritional or environmental factors [10]. At stage 2, all fish were exposed to <0.01 mg L1 TA-N but fish previously exposed low and high ammonia were not significantly different in weight gain and feed efficiency. The potential ability to compensate for weight loss following exposure to high ammonia was observed. Animal resilience to stress is coregulated by a variety of nutrients in the diet such as essential fatty acids being a source of energy for fish metabolism [56]. Li et al. [57] reported that the level of 1.29% linolenic acid of the diet is required for optimum growth of darkbarbel catfish. But in this study, weight gain of fish fed 4% linseed oil (enriched with 2.0% linolenic acid) was highest, indicating that compensatory growth may increase the requirement of essential fatty acids after exposure to high ammonia. Fatty acid composition in fish is a reflection of dietary fatty acids [58]. At the second stage of this study, fatty acid composition did not differ between fish previously exposed low and high ammonia. But the component of 18:3n3 in fish increased with the increasing levels of dietary linseed oil, suggesting that essential fatty acids were supplemented through the dietary source. The positive relationship between unsaturated fatty acids in feed and in the muscles has been found in several species, such as channel catfish I. punctatus [59], African catfish Claris gariepinus [60], rainbow trout O. mykiss [61], gilthead sea bream Sparus aurata [62] and Nile tilapia O. niloticus [63]. No significant difference between fish previously exposed to low and high ammonia was identified in red blood cells, hemoglobin and hematocrit. It indicates that the dietary linolenic acid may be able to adjust physiological disturbances caused by ammonia exposure. Especially, the values of blood parameters of fish fed 4% linseed oil were higher than those of fish fed 0% and 2% linseed oil. However, the linolenic acid was prone to peroxidation [64], which can activate antioxidant enzymes. In this study, superoxide dismutase, catalase and glutathione peroxidase activities increased with the increasing levels of dietary linseed oil. It should be noted that the activities of antioxidant enzymes in fish
previously exposed to high ammonia were higher than those of fish exposed to low ammonia. It is possible that linolenic acid may mitigate the adverse effect of ammonia in the antioxidant enzyme system. Montero et al. [65] reported that the negative effect of n-3 PUFA dietary deficiency on the serum hemolytic activity of gilthead sea bream S. aurata. Yildirim-Aksoy et al. [66] showed that the serum lysozyme activity of channel catfish I. punctatus fed 3% or 6% PUFA was significantly higher than that of fish fed 0% PUFA. The PUFA can keep more adhesive sites on cell membranes for antibodies to modify membrane viscosity, modulate immunological recognition and enhance immunity [21]. In this study, the lysozyme activity, phagocytic index, respiratory burst and total immunoglobulin contents were enhanced with the increase of dietary linseed oil levels regardless of previous ammonia exposure. However, no significant difference between fish previously exposed to low and high ammonia was identified. Our result indicates that the linolenic acid may also reduce the adverse effect of ammonia exposure on the immune response. 4.3. Effects of bacterial challenge Cumulative mortality of fish previously exposed to high ammonia was higher than that of fish exposed to low ammonia, possibly due to the change of hepatosomatic index. At stage 2, the hepatosomatic index of fish previously exposed to high ammonia was greater than that of fish exposed to low ammonia, but decreased with the increase of dietary linseed oil levels. Liver degeneration can lead to fish death and stress can enhance hepatosomatic index [67]. In this study, fish mortality decreased with the increase of dietary linseed oil, but antibody production against
Table 9 Mean lysozyme activity, phagocytic index (PI), respiratory burst (RB) and total immunoglobulin (Ig) content of darkbarbel catfish fed purified diets supplemented with various levels of linseed oil for 46 days. Ammonia Linseed oil 0% Lysozyme (U mL1) PI (%)
Low High Low High RB (%) Low High Ig (mg mL1) Low High
2%
4%
310.36a 342.83b 346.37b 260.69A 338.62B 348.15B 6.35a 6.62b 6.69b 6.18A 6.56B 6.72B 73.65a 77.01b 77.02b 72.99A 76.05B 77.38B 2.11a 2.24b 2.26b 2.04A 2.16B 2.28B
Pooled PUFA ammonia SEM (P value) 2.019 4.970 0.042 0.024 0.158 0.202 0.026 0.011
0.004 0.258 0.130 0.339
Different lowercase letters (a, b, c) indicate a significant effect of linseed oil following low ammonia (P < 0.05). Different uppercase letters (A, B, C) indicate a significant effect of linseed oil following high ammonia (P < 0.05).
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M. Li et al. / Fish & Shellfish Immunology 38 (2014) 158e165
Table 10 Mean number of cumulative mortality of darkbarbel catfish 14-day post challenge with Edwardsiella ictaluri and antibody production against the same bacterium. Ammonia
Cumulative mortality (%) Antibody titer (log10)
Low High Low High
Linseed oil 0%
2%
4%
42.22b1 62.22B2 1.93a 1.84A
15.56a1 35.5A2 1.98b 1.96B
13.33a1 24.44A2 2.07c 2.10C
Pooled SEM
PUFA ammonia (P value)
2.457 3.629 0.018 0.011
0.644 0.108
Different numbers (1, 2) indicate a significant effect of ammonia (P < 0.05). Different lowercase letters (a, b, c) indicate a significant effect of linseed oil following low ammonia (P < 0.05). Different uppercase letters (A, B, C) indicate a significant effect of linseed oil following high ammonia (P < 0.05).
E. ictaluri infection to fish increased. It is worth noting that high ammonia exposure caused fish morality, but the ability of fish resistance to pathogens was improved as the level of dietary linseed oil increased. In summary, growth performance and immune response of juvenile darkbarbel catfish were restrained by acute ammonia exposure. The high levels of dietary linolenic acid could mitigate the adverse effect of high ammonia exposure on fish performance. Based on fish growth performance, immune responses and resistance to the E. ictaluri challenge, the level of 4% linseed oil (rich in 2% linolenic acid) in the diet is recommended in darkbarbel catfish especially when fish are likely to encounter acute ammonia stress. Acknowledgments This research was supported by grants from the National Basic Research Program (973 Program, No. 2014CB138603, 2009CB118702), “the Twelfth Five-year-plan” in National Science and Technology for the Rural Development in China (2012BAD25B03), the Special Fund for Agro-scientific Research in the Public Interest (No. 201003020, 201203065), National Natural Science Foundation of China (No. 31172422, 31001098), and partly by the E-Institute of Shanghai Municipal Education Commission (No. E03009). References [1] Leung KMY, Chu JCW, Wu RSS. Effects of body weight, water temperature and ration size on ammonia excretion by the areolated grouper (Epinephelus areolatus) and mangrove snapper (Lutjanus argentimaculatus). Aquaculture 1999;170:215e27. [2] Paust LO, Foss A, Imsland AK. Effects of chronic and periodic exposure to ammonia on growth, food conversion efficiency and blood physiology in juvenile Atlantic halibut (Hippoglossus hippoglossus L.). Aquaculture 2011;315: 400e6. [3] Randall DJ, Tsui TKN. Ammonia toxicity in fish. Mar Pollut Bull 2002;45:17e 23. [4] Foss A, Albert KI, Bjørn R, Edward S, Sigurd OS. Effects of chronic and periodic exposure to ammonia on growth and blood physiology in juvenile turbot (Scophthalmus maximus). Aquaculture 2009;296:45e50. [5] Atwood HL, Tomasso JR, Ronan PJ, Barton BA, Renner KJ. Brain monoamine concentrations as predictors of growth inhibition in channel catfish exposed to ammonia. J Aquat Anim Health 2000;12:69e73. [6] El-Shafai SA, El-Gohary FA, Nasr FA, Van Der Steen NP, Gijzen HJ. Chronic ammonia toxicity to duckweed-fed tilapia (Oreochromis niloticus). Aquaculture 2004;232:117e27. [7] Benli ACK, Köksal G, Özkul A. Sublethal ammonia exposure of Nile tilapia (Oreochromis niloticus L.): effects on gill, liver and kidney histology. Chemosphere 2008;72:1355e8. [8] Dong XY, Zhang XM, Qin JG, Zong SB. Acute ammonia toxicity and gill morphological changes of Japanese flounder Paralichthys olivaceus in normal versus supersaturated oxygen. Aquac Res 2013;44:1752e9. [9] Qin JG, Fast AW, Kai A. Lethal effects of ammonia and pH on snakehead (Channa striatus). J World Aquac Soc 1997;28:87e90. [10] Ali M, Nicieza A, Wootton RJ. Compensatory growth in fishes: a response to growth depression. Fish Fish 2003;4:147e90. [11] Tian XL, Qin JG. Effects of previous ration restriction on compensatory growth in barramundi Lates calcarifer. Aquaculture 2004;235:273e83. [12] Wang Z, Mai K, Liufu Z, Ma H, Xu W, Ai Q, et al. Effect of high dietary intakes of vitamin E and n-3 HUFA on immune responses and resistance to Edwardsiella tarda challenge in Japanese flounder (Paralichthys olivaceus, Temminck and Schlegel). Aquac Res 2006;37:681e92.
[13] Salte R, Thomassen MS, Wold K. Do high levels of dietary polyunsaturated fatty acids (EPA/DHA) prevent diseases associated with membrane degeneration in farmed Atlantic salmon at low water temperature? Bull Eur Assoc Fish Pathol 1988;8:63e6. [14] Lall SP. Nutrition and health of fish. In: Cruz-Suárez L, Ricque-Marie D, TapiaSalazar M, editors. Avances en Nutrición Acuicola V. Mérida: Memorias del V Simposium Internacional de Nutrición Acuícol; 2000. 13ep.23. [15] Calabretti A, Cateni F, Procida G, Favretto LG. Influence of environmental temperature on composition of lipids in edible flesh of rainbow trout (Oncorhynchus mykiss). J Sci Food Agric 2003;83:1493e8. [16] Person-Le Ruyet A, Skallib B, Dulaua N, Le Bayona H, Le Dellioua JHR. Does dietary n-3 highly unsaturated fatty acids level influence the European sea bass (Dicentrarchus labrax) capacity to adapt to a high temperature? Aquaculture 2004;242:571e88. [17] Lim C, Yildirim-Aksoy M, Li MH, Welker TL, Klesius PH. Influence of dietary levels of lipid and vitamin E on growth and resistance of Nile tilapia to Streptococcus iniae challenge. Aquaculture 2009;298:76e82. [18] National Research Council. Nutrient requirements of fish and shrimp. Washington, DC, USA: National Academies Press; 2011. [19] Kinsella JE. a-Linolenic acid: functions and effects on linolenic acid metabolism and eicosanoid-mediated reactions. Adv Food Nutr Res 1991;35:1e18. [20] March BE. Essential fatty acids in fish physiology. Can J Physiol Pharmacol 1992;71:685e9. [21] Obach A, Ouentel C, Laurencin FB. Effects of alpha-tocopherol and dietary oxidized fishoil on the immune response of sea bass Dicentrarchus labrax. Dis Aquat Org 1993;15:175e85. [22] Li M, Chen LQ, Qin JG, Li EC, Yu N, Du ZY. Growth performance, antioxidant status and immune response in darkbarbel catfish Pelteobagrus vachelli fed different PUFA/vitamin E dietary levels and exposed to high or low ammonia. Aquaculture 2013;406:18e27. [23] Colt J, Tchobanoglous G. Chronic exposure of channel catfish, Ictalurus punctatus, to ammonia: effects on growth and survival. Aquaculture 1978;15:353e73. [24] Siikavuopio SI, Sæther B. Effects of chronic nitrite exposure on growth in juvenile Atlantic cod, Gadus morhua. Aquaculture 2006;255:351e6. [25] Hegazi MM, Attia ZI, Ashour OA. Oxidative stress and antioxidant enzymes in liver and white muscle of Nile tilapia juveniles in chronic ammonia exposure. Aquat Toxicol 2010;99:118e25. [26] Johansson O, Wedborg M. Ammoniaeammonium equilibrium in seawater at temperatures between 5 and 25 degrees. C J Chem Soc 1980;9:37e44. [27] Whitfield M. The hydrolysis of ammonia ions in sea water e a theoretical study. J Mar Biol Assoc UK 1974;54:565e80. [28] AOAC. Official methods of analysis. 18th ed. Gaithersburg, MD, USA: Association of Official Analytical Chemists; 2005. [29] Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:497e 509. [30] Morrison WR, Smith LM. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J Lipid Res 1964;53: 600e8. [31] Larsen HN. Comparison of various methods of hemoglobin detection of channel catfish blood. Prog Fish Cult 1964;26:11e5. [32] Trenzado CE, Morales AE, Palma JM, Higuer M. Blood antioxidant defenses and hematological adjustments in crowded/uncrowded rainbow trout (Oncorhynchus mykiss) fed on diets with different levels of antioxidant vitamins and HUFA. Aquaculture 2009;149:440e7. [33] Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971;44:276e87. [34] Aebi H. Catalase in vitro. Methods Enzymol 1984;105:121e6. [35] Flohé L, Günzler WA. Assay of glutathione peroxidase. Methods Enzymol 1984;105:115e21. [36] Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978;52: 302e10. [37] Hultmark D, Steiner H, Rasmuson T, Boman HG. Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem 1980;106:7e16. [38] Pulsford AL, Crampe M, Langston A, Glynn PJ. Modulatory effects of disease, stress, copper, TBT and vitamin E on the immune system of flatfish. Fish Shellfish Immunol 1995;5:631e43. [39] Secombes CJ. Isolation of salmonid macrophages and analysis of their killing activity. In: Stolen JS, Fletcher TC, Anderson BS, Van Muiswinkel WB, editors.
M. Li et al. / Fish & Shellfish Immunology 38 (2014) 158e165
[40]
[41] [42] [43]
[44]
[45]
[46] [47]
[48] [49]
[50]
[51]
[52]
[53] [54]
Techniques in fish immunology. New Jersey: SOS Publications; 1990. pp. 137e 44. Wu J, Shang H. Clinical immunology and in section. In: Ye Y, Wang Y, Shen Z, editors. National guide to clinical laboratory procedures. Nanjing: Southeast University Press; 2006. pp. 595e606. Klesius PH. Carrier state of channel catfish infected with Edwardsiella ictaluri. J Aquat Anim Health 1992;4:227e30. Chen MF, Light TS. Specificity of the channel catfish antibody to Edwardsiella ictaluri. J Aquat Anim Health 1994;6:226e70. Foss A, Evensen TH, Vollen T, Øiestad V. Effects of chronic ammonia exposure on growth and food conversion efficiency in juvenile spotted wolffish. Aquaculture 2003;228:215e24. Dosdat A, Person-Le Ruyet J, Covès D, Dutto G, Gasset E, Le Roux A, et al. Effect of chronic exposure to ammonia on growth, food utilization and metabolism of the European sea bass (Dicentrarchus labrax). Aquat Living Resour 2003;16: 509e20. Rasmussen RS, Korsgaard B. The effect of external ammonia on growth and food utilization of juvenile turbot (Scophthalmus maximus L.). J Exp Mar Biol Ecol 1996;205:35e48. Wicks BJ, Randall DJ. The effect of feeding and fasting on ammonia toxicity in juvenile rainbow trout, Oncorhynchus mykiss. Aquat Toxicol 2002;59:71e82. Li B, Fan QX, Yang K, Zhang L, Guo HX, Wang QY, et al. Effects of chronic ammonia stress on to raging, growth, and haematological parameters of yellow catfish (Pelteobagrus fulvidraco) juveniles. Chin J Appl Environ Biol 2011;17:824e9. Iwama G. Stress in fish. Ann N Y Acad Sci 1998;851:304e10. Marcaida G, Felipo V, Hermenegildo C, Minana MD, Grisolia S. Acute ammonia toxicity is mediated by NMDA type of glutamate receptors. FEBS Lett 1992;296:67e8. Lemarié G, Dosdat A, Covès D, Dutto G, Gasset G, Person-Le Ruyet J. Effect of chronic ammonia exposure on growth of European seabass (Dicentrarchus labrax) juveniles. Aquaculture 2004;229:479e91. Fracalossi DM, Lovell RT. Dietary lipid sources influence responses of channel catfish, Ictalurus punctatus to challenge with the pathogen Edwardsiella ictaluri. Aquaculture 1994;119:287e98. Barton BA, Iwama GK. Physiological changes in fish from stress in aquaculture with emphasis on the response and effects of corticosteroids. Ann Rev Fish Dis 1991;1:3e26. Bonga SEW. The stress response of fish. Physiol Rev 1997;77:591e625. Mock A, Peters G. Lysozyme activity in rainbow trout, Oncorhynchus mykiss (Walbaum), stressed by handling, transport and water pollution. J Fish Biol 1990;37:873e85.
165
[55] Chen JZ, Zhang XL, Hu GD, Qu JH, Fan LM, Song C. The immune response of GIFT Oreochromis niloticus and its susceptibility to Streptococcus iniae under stress in different ammonia. Ecol Environ Sci 2011;20:629e34. [56] Watanabe T. Lipid nutrition in fish. Comp Biochem Physiol B 1982;73:3e15. [57] Li M, Chen LQ, Li EC, Yu N, Ding ZL, Chen YL, et al. Growth, immune response and resistance to Aeromonas hydrophila of darkbarbel catfish, Pelteobagrus vachelli (Richardson), fed diets with different linolenic acid levels. Aquac Res; 2013. http://dx.doi.org/10.1111/are.12236. [58] Francis DS, Turchini GM, Jones PL, De Silva SS. Effects of dietary oil source on growth and fillet fatty acid composition of Murray cod, Maccullochella peelii peelii. Aquaculture 2006;253:547e56. [59] Stickney RR, Andrews JW. Effects of dietary lipids on growth, food conversion, lipid and fatty acid composition of channel catfish. J Nutr 1972;102:249e58. [60] Ng WK, Lim PK, Boey PL. Dietary lipid and palm oil source affects growth, fatty acid composition and muscle a-tocopherol concentration in African catfish, Claris gariepinus. Aquaculture 2003;215:229e43. [61] Yu TC, Sinnhuber RO. Effect of dietary u3 and u6 fatty acids on growth and feed conversion efficiency of coho salmon (Oncorhynchus kisutch). Aquaculture 1979;16:31e8. [62] Kalogeropoulos N, Zlexis MN, Henderson RJ. Effects of dietary soybean and cod-liver oil levels on growth and body composition of gilthead bread (Sparus aurata). Aquaculture 1992;104:293e308. [63] Tonial IB, Stevanato FB, Matsushita M, De Souza NE, Furuya WM, Visentainer JV. Optimization of flaxseed oil feeding time length in adult Nile tilapia (Oreochromis niloticus) as a function of muscle omega-3 fatty acid composition. Aquac Nutr 2009;15:564e8. [64] Mourente G, Díaz-Salvago E, Tocher DR, Bell JG. Effects of dietary polyunsaturated fatty acid/vitamin E (PUFA)/tocopherol ratio on antioxidant defence mechanisms of juvenile gilthead sea bream (Sparus aurata L., Osteichthyes, Sparidae). Fish Physiol Biochem 2000;23:337e51. [65] Montero D, Tort L, Izquierdo MS, Robaina L, Vergara JM. Depletion of serum alternative complement pathway activity in gilthead seabream caused by alpha-tocopherol and n-3 HUFA dietary deficiencies. Fish Physiol Biochem 1998;18:399e407. [66] Yildirim-Aksoy M, Lim C, Shelby R, Klesius PH. Increasing fish oil levels in commercial diets influences hematological and immunological responses of channel catfish, Ictalurus punctatus. J World Aquac Soc 2009;40:76e86. [67] Mourento G, Díaz-Salvago E, Bell JG, Tocher DR. Increased activities of hepatic antioxidant defense enzymes in juvenile gilthead sea bream (Sprausaurata L., Osteichthyes, Sparide) fed dietary oxidized oil: attenuation by dietary vitamin E. Aquaculture 2002;214:343e61.
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Full length article
Effects of ammonia stress, dietary linseed oil and Edwardsiella ictaluri challenge on juvenile darkbarbel catfish Pelteobagrus vachelli Ming Li a, b, Na Yu a, **, Jian G. Qin c, Erchao Li a, Zhenyu Du a, Liqiao Chen a, * a
School of Life Sciences, East China Normal University, Shanghai 200062, China School of Marine Sciences, Ningbo University, Ningbo 315211, China c School of Biological Sciences, Flinders University, Adelaide, SA 5001, Australia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 November 2013 Received in revised form 5 February 2014 Accepted 12 March 2014 Available online 19 March 2014
A two-stage study was carried out to test the response of juvenile darkbarbel catfish Pelteobagrus vachelli to ammonia stress, dietary lipid and bacterial challenge. At stage 1, the catfish (0.99 0.01 g) fed a commercial diet were exposed to 0.01 and 5.70 mg L1 total ammonia nitrogen in nine replicates for 14 days. At stage 2, all fish previously exposed to either low or high ammonia were separately transferred into low ammonia (<0.01 mg L1), and divided into three feeding groups. Fish were then fed three levels of linseed oil (0, 2 and 4%) in triplicate for 46 days. Fish growth performance and immune response were low in high ammonia at stage 1. At stage 2, fish growth and immune response were not significantly different between fish previously exposed to low and high ammonia in all diets. Fish fed 4% linseed oil showed the greatest weight gain, feed efficiency ratio, red blood cells, hemoglobin and hematocrit, and achieved higher lysozyme activity, phagocytic index, respiratory burst and total immunoglobulin than fish fed 0% linseed oil, but did not differ from fish fed 2% linseed oil regardless of previous ammonia exposure. After 14-day infection of Edwardsiella ictaluri, cumulative mortality of fish previously exposed to low ammonia was lower than that of fish exposed to high ammonia in all diets. Cumulative mortality of fish fed 0% linseed oil was highest, but the antibody titer of fish fed 4% linseed oil was highest regardless of previous ammonia treatments. This study indicates that ammonia stress has a lasting effect even after ammonia is lowed, but the adverse effect on fish can be mitigated through manipulation of dietary oil inclusion, especially under the challenge of pathogenic bacteria. Ó 2014 Published by Elsevier Ltd.
Keywords: Ammonia Linseed oil Growth performance Immune responses Disease resistance
1. Introduction Intensive aquaculture systems can easily cause chronic ammonia stress and sometimes transient peaks due to ammonification of feed residual and animal excretion [1e3]. Ammonia can be practically removed by biological filtration or water exchange, but a sudden and quick increase of ammonia may be detrimental to fish [4]. Excessive ammonia can cause fish growth reduction [5,6], tissue erosion and degeneration [7,8], immune suppression and high mortality [9]. After experiencing stress by environmental factors such as unfavorable temperature, low feed supply or high ammonia, compensatory growth may be observed when these stress factors are removed [10e12]. Although compensatory
* Corresponding author. Tel./fax: þ86 21 62233637. ** Corresponding author. Tel./fax: þ86 21 54341002. E-mail addresses: [email protected] (N. Yu), (L. Chen). http://dx.doi.org/10.1016/j.fsi.2014.03.015 1050-4648/Ó 2014 Published by Elsevier Ltd.
[email protected]
growth in fish such as Atlantic halibut Hippoglossus hippoglossus has been reported following a prolonged period of exposure to high ammonia, physiological and immunological changes of fish after ammonia exposure have not been investigated [2]. The ability of fish to adapt to environmental stress may depend on fish nutritional status [13]. Dietary manipulation has been used as a tool to alleviate the damage of ammonia on fish [14] because environmental stress may increase the requirement of essential fatty acids in fish such as rainbow trout Oncorhynchus mykiss [15] and European sea bass Dicentrarchus labrax [16]. Under stress, deficiency of essential fatty acids in diet can further deteriorate the ability of fish for immune response and disease resistance [17]. Linolenic acid, an essential fatty acid for most catfish [18], plays important roles in cell-membrane function, eicosanoid synthesis and prostaglandin regulation [19,20], and triggers immunological response to activate antigens [21]. Our previous study indicates that the harmful effects of chronic low ammonia on fish can be mitigated when fish are fed on 2% linseed oil and 400 mg kg1 vitamin E [22]. However, it is not clear whether manipulation of linseed oil
M. Li et al. / Fish & Shellfish Immunology 38 (2014) 158e165
contents can mitigate the adverse effect of ammonia on fish following acute high ammonia exposure. This study used a sequential approach to examine the effect of ammonia exposure, dietary linseed oil manipulation and bacterial challenge on fish growth, immunity and disease resistance. We first set the upper level of stress high ammonia at 5.70 mg L1 total ammonia nitrogen (TA-N) as suggested by Colt and Tchobanoglous in a study on channel catfish Ictalurus punctatus where fish mortality increases significantly above 5.71 mg L1 TA-N [23]. To test if dietary nutrition can mitigate the post-effect of ammonia exposure on fish, all fish previously exposed to different ammonia were fed with different levels of dietary linseed oil. At last, according to the previous treatment history of ammonia exposure and diet supply, fish were challenged with pathogenic bacteria Edwardsiella ictaluri to test the disease resistance. The experiments with such a design would enable us to test the following hypotheses: (1) exposure to a high level of ammonia compromises fish growth and health, and (2) dietary lipid manipulation improves growth performance, hematology status, antioxidant enzyme activities, immune responses and resistance to E. ictaluri challenge on juvenile darkbarbel catfish Pelteobagrus vachelli.
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Table 2 Proximate composition (g 100 g1 lipid) and fatty-acid composition (% by weight of total fatty acids) in experimental diets. Experimental diets (g 100 g1 diet) 0% Proximate composition (%) Moisture 10.87 Protein 42.39 Lipid 11.09 Ash 4.75 Fatty-acid composition C12:0 98.78 C16:0 0.65 C18:0 0.27 C18:1n9 e C18:2n6 e C18:3n3 e
2%
4%
10.13 41.98 10.87 4.81
10.98 42.03 11.12 4.78
86.86 0.93 0.58 3.05 0.94 9.27
74.07 1.77 1.15 6.15 1.58 19.13
Values are means of three replicates in each treatment.
meter (Hach Company, Colorado, USA). During the trial, water quality variables were maintained at 27e29 C, dissolved oxygen 7.81 0.13 mg L1, pH 6.4e6.6, nitrate <0.5 mg L1 (Griess reaction [24]) and 12 h light and 12 h dark.
2. Materials and methods 2.1. Experimental diets and animals
2.2. Experimental designs and sampling
Three experimental diets were formulated including three levels of linseed oil (enriched with linolenic acid at 0, 2 and 4%, Table 1). The diet proximate composition and fatty-acid compositions are given in Table 2. Diets were processed into 2-mm diameter pellets, dried at room temperature to <10% moisture, ground and sieved to appropriate size before storage at 20 C. Darkbarbel catfish (0.99 0.01 g, mean SD), obtained from a fish farm in Chengdu (China), were acclimatized with the commercial feed (42% protein, 11% lipid, 10% moisture and 5% ash) for 14 days prior to this study. Fish were randomly stocked into eighteen 300-L rectangular plastic tanks at 35 fish each. All tanks were cleaned, and fish in each tank were group-weighed and counted fortnightly. All tanks were supplied with dechlorinated tap water with a daily water exchange rate at 1/3 of the tank volume. Water was continuously aerated using air stones. Water temperature and dissolved oxygen were measured daily with a HACH HQ30d oxygen
Table 1 Percentage composition of the basal diet ingredients. Experimental diets (g 100 g1 diet)
Casein, vitamin-free Galatin Coil starch Carboxymethyl cellulose Linseed oila Lauric acid Vitamin mixtureb Mineral mixturec Celufil Antioxidant (ethoxyquin) a
0%
2%
4%
40.0 10.0 25.0 3.0 0.0 11.0 0.5 5.0 5.5 0.02
40.0 10.0 25.0 3.0 2.0 9.0 0.5 5.0 5.5 0.02
40.0 10.0 25.0 3.0 4.0 7.0 0.5 5.0 5.5 0.02
Linseed oil: purchased from Hebei Xinqidian Bio. Tech. Co. Ltd. Hebei, P.R. China. Vitamin premix (kg1 diets): vitamin A, 5500 IU; vitamin D3, 1000 IU; vitamin E, 50 IU; vitamin K, 10 mg; niacin, 100 mg; riboflavin, 20 mg; pyridoxine, 20 mg; thiamin, 20 mg; biotin, 0.1 mg; D-calcium pantothenate, 50 mg; folacin, 5 mg; B12, 20 mg; ascorbic acid, 100 mg; inositol, 100 mg. c Mineral premix (mg kg1 diets): NaCl, 500; MgSO4$7H2O, 4575.0; NaH2PO4$2H2O, 12,500.0; KH2PO4, 16,000.0; Ca(H2PO4)2$H2O, 6850.0; FeSO4, 1250.0; C6H10CaO6$5H2O, 1750.0; ZnSO4$7H2O, 111.0; MnSO4$4H2O, 61.4; CuSO4$5H2O, 15.5; CoSO4$6H2O, 0.5; KI, 1.5; Starch, 6385.1. b
This study was divided into two stages. At stage 1, the experiment consisted of a low ammonia treatment [total ammonia nitrogen (TA-N) 0.01 mg L1, un-ionized ammonia (UIA-N) 0.001 mg L1] and a high ammonia treatment (TA-N 5.70 mg L1, UIA-N 0.12 mg L1). Each treatment was in nine replicates. Fish were fed with a commercial diet twice daily (07:30e08:30 h and 15:00e16:00 h) to apparent satiation for 14 days. The amount of diet consumption was recorded daily. The desired ammonia concentrations were achieved by supplementing a solution of NH4Cl (10 g L1) every 7 h. TA-N levels were measured by nesslerization [25]. Percentage UIA-N was calculated using the equation of Johansson and Wedborg [26]. This equation gives the percentage UIA-N as a function of pH and temperature. Corrections for pH measurements with low-ionic strength buffers (i.e., conversion to the Hansson scale) were performed according to the method of Whitfield [27]. At stage 2, all fish previously exposed to either low or high ammonia were separately transferred into the low ammonia water (<0.01 mg L1), and divided into three feeding groups. Fish were then fed with three levels of linseed oil (0, 2 and 4%) in triplicate for 46 days. Each treatment was fed three experimental diets twice daily (07:30e08:30 h and 15:00e16:00 h) to apparent satiation. The amount of diet consumption was recorded daily. At the end of the two stages, fish were starved for 24 h, and then were anesthetized with tricaine methanesulfonate (MS-222) at 120 mg L1 before weighing and counting. Three fish tank1 were randomly sampled, minced, pooled and stored at 80 C for fatty acid analysis. Blood samples (three fish tank1) were collected from the heart with heparinized (100 IU mL1) tuberculin syringes for hematological assays. The blood of additional three fish tank1 was taken using nonheparinized tuberculin syringes and clotted at 4 C overnight. Serum samples were collected, centrifuged and stored at 80 C for immune assays. After being bled, the head kidneys of six fish were removed for macrophage separation. Livers were removed and individually weighed to determine the hepatosomatic index. The indexes for assessing growth performance were calculated as follows:
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Weight gain (WG, g) ¼ final weight (g) initial weight (g) Feed efficiency ratio (FER) ¼ wet weight gain (g)/dry feed fed (g) Survival (%) ¼ 100 (final fish number)/(initial fish number) Hepatosomatic index (HSI%) ¼ liver mass (g) 100/body mass (g)
2.3. Biochemical composition analysis All experimental diets were analyzed in duplicate for proximate composition following the standard methods [28]. Moisture was determined by oven dry at 105 C to a constant weight. Samples used for dry matter were digested with nitric acid and incinerated in a muffle furnace at 600 C overnight for ash content determination. Protein was measured by the combustion method using an FP-528 nitrogen analyzer (Leco Corporation, St. Joseph, MI, USA). Lipid was determined by the ether extraction method using the Soxtec system (2055 Soxtec Avanti; Foss Tecator, Hoganas, Sweden). Total lipid of experimental diets and fish samples was extracted using chloroform: methanol (2:1, v/v) in duplicate according to the method of Folch et al. [29]. The saponifiable lipids were converted to methyl esters by using the standard boron tri-fluoride-methanol method [30]. Fatty acid methyl esters (FAME) were analyzed on an Agilent 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA), equipped with a flame ionization detector (FID) and a SP2560 fused silica capillary column (100 m, 0.25 mm i.d. and 0.20 mm film thick). Injector and detector temperatures were 270 and 280 C respectively. Column temperature was held at 120 C for 5 min then programmed to increase at 3 C min1 up to 240 C where it was maintained for 20 min. Carrier gas was helium (2 mL min1), and the split ratio was 30:1. Identification of fatty acids was carried out by comparing the sample FAME peak relative retention times with the SigmaeAldrich (St. Louis, MO, USA) standards. The individual fatty acid methyl esters were identified by comparing the retention times of the authentic standard mixtures and quantified the absolute content with the internal standard. 2.4. Hematology, enzyme activity and lipid peroxidation assays Red blood cell and white blood cell counts were performed in duplicate for each sample by diluting (1:10,000) the whole blood in phosphate buffer saline (PBS) solution and enumerating with a hemocytometer [17]. Hemoglobin was determined using a commercial kit (Sigma Chemical Co., St. Louis, MO, USA) and its values were adjusted, as described by Larsen [31], using a cyanomethemoglobin correction factor for channel catfish. Hematocrit was determined by centrifuging the whole blood in heparinized microhematocrit capillary tubes at 650 g for 10 min and measuring the packed cell volume [32]. The levels of enzyme activity and lipid per-oxidation were measured with commercial assay kits (Nanjing Jiancheng Institute, Nanjing, China) in accordance with the instructions of the manufacturer. The assays are briefly described as follows: Total superoxide dismutase activity was determined following the method of Beauchamp and Fridovich [33]. The ratio of autooxidation rates of the samples with or without serum was determined at 550 nm. One unit of SOD activity was calculated using the amount of superoxide dismutase required to inhibit the reduction of nitroblue tetrazolium by 50%. Catalase activity was determined by measuring the decrease of H2O2 concentration [34]. After 10 mL of serum was added to the
reagent, the sample was incubated for 60 s at 37 C. The absorbance of the samples was read at 405 nm. One unit of CAT activity was defined as the amount of CAT required to transform 1 mmol of H2O2 min1. Glutathione peroxidase activity was measured following the method of Flohé and Günzler [35]. Blood samples were treated with Drabkin’s reagent to avoid hemoglobin interferences. After the addition of 1 mM GSH (reduced glutathione) the NADPH-consumption rate was monitored at 412 nm. One unit of GPX activity was defined as the amount of GPX required to oxidize 1 mmol of NADPH min1. Lipid-peroxidation levels were determined based on the malondialdehyde levels generated by oxidation of polyunsaturated fatty acids. In the presence of thiobarbituric acid, malondialdehyde started producing colored thiobarbituric-acid-reacting substances (TBARS) that were measured at 532 nm [36]. 2.5. Lysozyme activity, phagocytic index, respiratory burst and immunoglobulin assays Serum lysozyme activity was determined through the turbidimetric method [37] using a lysozyme detection kit (Nanjing Jiancheng Institute, Nanjing, China). The assay was based on the lysis of a lysozyme-sensitive Gram-positive bacterium via the lysozyme present in the serum. The head kidney was removed and transferred to L-15 culture medium (100 IU mL1 penicillin, 100 mg mL1 streptomycin, 10 IU mL1 heparin, 2% fetal bovine serum) and then filtered through a 100 mm metal mesh. The resulting cell suspensions were enriched by centrifugation (600 g, 5 min, 4 C) on the 34%/51% Percoll density gradient. The cells were collected at the 34e51% interface and washed twice. The final cell concentration was adjusted to approximately 1 107 mL1 and the cell viability was >95%. The phagocytic index was determined according to the method by Pulsford et al. [38]. The respiratory burst of phagocytic cells was measured by the nitroblue tetrazolium reduction assay following the method of Secombes [39]. Total immunoglobulin was assayed using a commercial kit (Zhejiang Elikan Biological Technology Co., Ltd, Wenzhou, China). The methods are described by Wu and Shang [40] for the activity analysis including the measurement of turbidity increase after the immunity response and the increase of its antibody. 2.6. Bacterial challenge A frozen stock-culture of E. ictaluri was obtained from the Institute of Hydrobiology, the Chinese Academy of Science (Wuhan, China). The stock culture of E. ictaluri frozen at 80 C was inoculated in 250 mL of braineheart infusion broth and cultured for 24 h in a water bath shaker at 26 C [41]. To determine the optimum bacterial concentration for fish infection, a preliminary trial was conducted by injecting 20 fish tank1 with 0.1 mL of 0, 1 106, 2 106, 4 106, 8 106, or 1.6 107 E. ictaluri CFU mL1 in triplicate. Mortality was recorded twice daily for 14 d. The LC50 level (i.e., 1 105 CFU fish1) was then used for the experimental challenge. At the end of the stage 2, fifteen fish were randomly selected from each tank and intraperitoneally injected with 1 105 CFU fish1 of E. ictaluri using a tuberculin syringe. In the preliminary trial, such a density of E. ictaluri could have adverse impact on darkbarbel catfish. Subsequently, fish in each group were separately fed with the above three diets. Mortality was recorded twice daily during the period of 14-day trial and dead fish were removed.
M. Li et al. / Fish & Shellfish Immunology 38 (2014) 158e165
At the end of the E. ictaluri challenge, blood samples were collected from four randomly chosen live fish, and the serum was collected. The agglutinating antibody titers against E. ictaluri in the pre- and post-challenge serum samples were measured as described in Chen and Light [42]. 2.7. Statistical analysis Data from stage 1 of the experiment were used to compare the effects of the ammonia by a t-test. Data from the stage 2 of the experiment were used to detect interaction between linseed oil and ammonia levels by two-way ANOVA with the level of pre-exposure ammonia and the level of linseed oil as two factors. If a significant main effect was detected, differences between group means were compared by Duncan’s multiple range tests. The level of significance was set at P <0.05. All analyses were performed using the SPSS 18.0.0 (Chicago, USA) for Windows.
3.1. Effects of ammonia at stage 1 Fish survival was not affected by different levels of ambient ammonia (P > 0.05). Weight gain (WG) and feed efficiency ratio (FER) of fish in the treatment of low ammonia were higher than those of fish in the treatment of high ammonia (P < 0.05, Table 3). But the treatment of low ammonia showed lower hepatosomatic index (HSI) compared to that of fish in the treatment of high ammonia (P < 0.05). The content of saturated fatty acids (SAFAs) and n-3 polyunsaturated fatty acids (PUFAs) showed a similar trend to WG and FER. The hemoglobin of fish in the treatment of low ammonia was higher than that of fish in the treatment of high ammonia (P < 0.05, Table 4). The activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) showed a similar trend to hemoglobin. However, the treatment of low ammonia showed lower malondialdehyde (MDA) contents compared to that of fish in the Table 3 Growth performance and fatty acid composition of darkbarbel catfish fed commercial feed with high or low ammonia exposure for 14 days.
Low Growth performance WG (g) 0.892 FER 0.332 HSI (%) 0.131 Survival (%) 100% Fatty acid composition of fish 12:0 10.05 14:0 7.58 16:0 20.442 18:0 3.16 SAFA 41.232 18:1n9 40.13 20:1n9 1.91 MUFA 42.04 18:2n6 13.01 20:3n6 1.30 PUFA n-6 14.31 18:3n3 1.092 20:5n3 0.532 22:6n3 0.122 PUFA n-3 1.742
Table 4 Hematology, antioxidant enzyme activity and immune response of darkbarbel catfish fed commercial feed with high or low ammonia exposure for 14 days. Ammonia Low Hematology assay RBC 106 mL1 2.82 WBC 105 mL1 3.03 1 Hemoglobin (g dL ) 8.262 Hematocrit (%) 24.41 Antioxidant enzyme activity SOD (U mL1) 104.582 CAT (U mL1) 16.16 GPX (U mL1) 313.142 1 1.051 MDA (nmol mL ) Immune response Lysozyme (U mL1) 253.852 PI (%) 5.782 RB (%) 69.322 Ig (mg mL1) 1.562
Pooled SEM
P values
2.83 3.04 8.061 24.47
0.015 0.007 0.007 0.051
0.731 0.381 0.001 0.538
98.511 16.12 305.451 2.952
0.084 0.198 0.909 0.007
0.001 0.907 0.001 0.001
235.041 5.281 64.561 1.341
0.677 0.030 0.236 0.013
0.001 0.001 0.001 0.001
High
Different superscript numbers (1, 2) indicate a significant effect of ammonia (P < 0.05). RBC: red blood cell; WBC: white blood cell; SOD: superoxide dismutase; CAT: catalase; GPX: glutathione peroxidase; MDA: malondialdehyde; PI: phagocytic index; RB: respiratory burst; Ig: total immunoglobulin.
3. Results
Ammonia
161
Pooled SEM
P values
0.701 0.251 0.592 100%
0.013 0.005 0.013 e
0.001 0.001 0.001 e
10.42 7.83 20.691 3.19 42.131 40.21 2.09 42.30 13.86 1.32 15.18 0.091 0.001 0.001 0.091
0.021 0.018 0.014 0.009 0.103 0.024 0.019 0.089 0.005 0.014 0.081 0.002 0.008 0.003 0.012
0.001 0.876 0.673 0.744 0.012 0.042 0.812 0.008 0.768 0.876 0.554 0.001 0.001 0.001 0.001
High
Different superscript numbers (1, 2) indicate a significant effect of ammonia (P < 0.05). WG: weight gain; FER: feed efficiency ratio; HSI: hepatosomatic index; SAFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.
treatment of high ammonia (P < 0.05). The lysozyme activity, phagocytic index (PI), respiratory burst (RB) and total immunoglobulin (Ig) contents of fish in the treatment of low ammonia were higher than those of fish in the treatment of high ammonia (P < 0.05).
3.2. Effects of dietary linseed oil at stage 2 Fish fed 4% linseed oil showed higher WG and FER compared to those in other groups regardless of the level of previous ammonia exposure (P < 0.05, Table 5). However, HSI was highest when fish fed on 0% linseed oil (P < 0.05). In addition, the HSI of fish previously exposed to low ammonia was lower than that of fish exposed to high ammonia in all diets (P < 0.05). Significant interactions between linseed oil and previous ammonia exposure on FER were detected (P < 0.05). SAFAs and monounsaturated fatty acids of fish fed 0% linseed oil were highest irrespective of the previous level of ammonia exposure (P < 0.05, Table 6). But n-6 PUFAs and n-3 PUFAs of fish fed 4% linseed oil were greater than those of fish fed 2% and 0% linseed oil (P < 0.05), while these variables in fish fed 2% linseed oil were greater than those fed 0% linseed oil (P < 0.05). The RBC, hemoglobin and hematocrit of fish fed 4% linseed oil were highest (P < 0.05, Table 7), while these variables in fish fed 2% linseed oil were higher than those fed 0% linseed oil regardless of previous ammonia exposure (P < 0.05). Significant interactions between linseed oil and previous ammonia exposure on RBC, hemoglobin and hematocrit were detected (P < 0.05). Fish previously exposed to low ammonia showed lower SOD, CAT, GPX activities and MDA contents compared to those of fish previously exposed to high ammonia (P < 0.05, Table 8). The SOD, CAT and GPX activities and MDA contents of fish fed 4% linseed oil were highest regardless of previous ammonia exposure (P < 0.05). Significant interactions between linseed oil and previous ammonia exposure on MDA were detected (P < 0.05). Fish fed 4% linseed oil achieved higher levels of lysozyme activity, PI, RB and Ig than fish fed 0% linseed oil (P < 0.05, Table 9), but did not differ from those of fish fed 2% linseed oil irrespective of previous ammonia exposure (P > 0.05). Significant interactions between linseed oil and previous ammonia exposure on lysozyme activity were detected (P < 0.05).
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Table 5 Mean final weight gain (WG), feed efficiency ratio (FER), hepatosomatic index (HSI) and survival of darkbarbel catfish fed purified diets supplemented with various levels of linseed oil for 46 days.
WG (g) FER HSI (%) Survival (%)
Ammonia
Linseed oil 0%
2%
4%
Low High Low High Low High Low High
4.40a 3.60A 1.21a 0.88A 1.50b1 2.25B2 100% 100%
5.82b 5.54B 1.79b 1.69B 0.53a1 1.36A2 100% 100%
5.86c 5.98C 1.86c 2.30C 0.47a1 1.34A2 100% 100%
Pooled SEM
PUFA ammonia (P value)
0.096 0.289 0.079 0.033 0.021 0.062 e e
0.482
Table 6 Fatty acid composition (% by weight of total fatty acids) of darkbarbel catfish fed purified diets supplemented with various levels of linseed oil for 46 days. Ammonia
12:0 14:0 0.010 16:0 0.711 18:0 e
Different superscript numbers (1, 2) indicate a significant effect of ammonia (P < 0.05). Different lowercase letters (a, b, c) indicate a significant effect of linseed oil following low ammonia (P < 0.05). Different uppercase letters (A, B, C) indicate a significant effect of linseed oil following high ammonia (P < 0.05).
SAFA 18:1n9 20:1n9 MUFA
3.3. Effects of bacterial challenge Cumulative mortality of fish previously exposed to low ammonia was lower than that of fish exposed to high ammonia (P < 0.05, Table 10). Fish fed 4% linseed oil achieved lower levels of cumulative mortality than fish fed 0% linseed oil (P < 0.05), but did not differ from that of fish fed 2% linseed oil irrespective of previous ammonia exposure (P > 0.05). Antibody titer of fish fed 4% linseed oil was highest (P < 0.05).
18:2n6 20:3n6 PUFA n-6 18:3n3 20:5n3 22:6n3 PUFA n-3
4. Discussion 4.1. Effects of ammonia at stage 1 Growth reduction due to high ammonia exposure has been reported in several fish species, such as spotted wolfish Anarhichas minor [43], turbot Scophthalmus maximus [4] and Atlantic halibut H. hippoglossus [2]. There is a close correlation between feed efficiency ratio and the levels of ammonia exposure [44,45]. In this study, the weight gain of fish exposed to high ammonia was lower than that of fish exposed to low ammonia due to the low feed efficiency ratio. At high external ammonia, the plasma ammonia is higher in the fed fish than the unfed fish and the mortality is higher in the fed fish than the unfed fish [46]. In this study, fish survival was not affected by ammonia exposure suggesting that 5.70 mg L1 TA-N falls into the tolerant range for darkbarbel catfish. Colt and Tchobanoglous [23] found that the mortality of channel catfish I. punctatus increased significantly when total ammonia was >5.71 mg L1 and the tolerance of TA-N for snakehead Channa striatus is 10.73 mg L1 at pH 8.0 [9]. Li et al. [47] showed that yellow catfish Pelteobagrus fulvidraco juveniles could not tolerate high levels of ammonia, and recommended that the ammonia concentration be less than 6.72 mg L1 TA-N. High ambient ammonia seems to favor the accumulation of saturated fatty acid in fish [48]. In this study, saturated fatty acids of fish in low ammonia were higher than those of fish in high ammonia. However, it should be noted that the n-3 PUFA (especially 18:3n3) of fish exposed to high ammonia was lower than that of fish in low ammonia. Ammonia exposure leads to the formation of excessive reactive oxygen species (ROS) [49,50]. The reduction of 18:3n3 may be related to fatty acid peroxidation [51]. Our result shows that ammonia exposure leads to deficiency of essential fatty acids in darkbarbel catfish. In addition, the contents of n-3 HUFA (EPA 20:5n3 and DHA 22:6n3) of fish experienced high ammonia exposure were not detected, though the ability of inhibiting
Low High Low High Low High Low High Low High Low High Low High Low High Low High Low High Low High Low High Low High Low High Low High
Linseed oil 0%
2%
4%
17.03c 17.01C 6.16 6.06 19.09c 19.03C 3.69 3.65 43.97c 45.97C 41.94c 41.89C 1.98 1.94 43.92c 43.83C 7.98a 7.91A 1.14 1.11 9.12a 9.02A 0.32a 0.27A 0.13a 0.09A 0.28a 0.19A 0.73a 0.55A
15.65b 15.28B 6.32 6.29 17.13b 17.77B 3.88 3.75 42.98b 42.98B 39.81b 39.16B 1.93 1.89 41.74b 41.05B 9.74b 10.17B 1.09 1.11 10.83b 11.28B 2.21b 2.14B 0.28b 0.21B 1.25b 1.45B 3.74b 3.80B
12.93a 12.88A 6.93 6.88 14.88a 14.92A 3.76 3.63 40.55a 38.55A 36.21a 36.66A 1.98 1.91 39.19a 38.57A 10.87c 10.99C 1.12 1.08 11.99c 12.07C 6.92c 6.89C 0.59c 0.58C 3.66c 3.11C 11.17c 10.58C
Pooled SEM
PUFA ammonia (P value)
0.087 0.071 0.008 0.093 0.041 0.101 0.099 0.067 0.109 0.056 0.101 0.098 0.078 0.045 0.078 0.566 0.091 0.012 0.055 0.046 0.091 0.101 0.018 0.012 0.033 0.019 0.096 0.045 0.032 0.098
0.123 0.576 0.544 0.412 0.884 0.307 0.496 0.211 0.135 0.285 0.825 0.313 0.206 0.339 0.121
Different lowercase letters (a, b, c) indicate a significant effect of linseed oil following low ammonia (P < 0.05). Different uppercase letters (A, B, C) indicate a significant effect of linseed oil following high ammonia (P < 0.05). SAFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.
bioconversion of 18-carbon atom fatty acids to 20- or 22-carbon atoms at high ambient ammonia was not confirmed. As ammonia toxicity may cause a rapid transient increase of ROS and result in blood cell damage [50]. In this study, fish hemoglobin in low ammonia was higher than in high ammonia. Fish can prevent ROS generation through the production of counteracting enzymes, such as superoxide dismutase, catalase and glutathione peroxidase [32]. But in this study, superoxide dismutase and glutathione peroxidase activities of fish exposed to high ammonia were lower than those of fish in low ammonia. This indicates that those enzymes may have been inhibited as a transitory response to ambient ammonia, which may be related to malondialdehyde toxicity.
Table 7 Mean red blood cell count (RBC), white blood cell count (WBC), hemoglobin and hematocrit of darkbarbel catfish fed purified diets supplemented with various levels of linseed oil for 46 days. Ammonia Linseed oil 0% RBC 106 mL1 WBC 105 mL1 Hemoglobin (g dL1) Hematocrit (%)
Low High Low High Low High Low High
2%
4%
2.34a 2.39b 2.41c 2.21A 2.37B 2.49C 3.33 3.33 3.35 3.39 3.49 3.57 a b 8.33 8.35c 8.23 8.13A 8.28B 8.44C 25.41a 25.76b 25.84c 25.11A 25.64B 25.94C
Pooled PUFA ammonia SEM (P value) 0.011 0.024 0.011 0.043 0.011 0.015 0.026 0.042
0.023 0.884 0.001 0.019
Different lowercase letters (a, b, c) indicate a significant effect of linseed oil following low ammonia (P < 0.05). Different uppercase letters (A, B, C) indicate a significant effect of linseed oil following high ammonia (P < 0.05).
M. Li et al. / Fish & Shellfish Immunology 38 (2014) 158e165
163
Table 8 Mean superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) activities and malondialdehyde (MDA) counts of darkbarbel catfish fed purified diets supplemented with various levels of linseed oil for 46 days. Ammonia
SOD (U mL1) 1
CAT (U mL
)
GPX (U mL1) MDA (nmol mL1)
Low High Low High Low High Low High
Linseed oil 0%
2%
4%
101.92a1 130.63A2 14.85a1 19.02A2 294.50a1 306.54A2 1.37a1 1.96A2
113.04a1 137.16B2 17.26a1 22.68B2 304.66a1 315.39B2 1.53a1 2.84B2
131.79b1 144.60C2 18.67b1 26.81C2 311.29b1 318.50C2 1.94b1 3.25C2
Pooled SEM
PUFA ammonia (P value)
2.287 0.935 0.437 0.485 1.288 1.097 0.033 0.043
0.057 0.076 0.504 0.001
Numbers (1, 2) indicate a significant effect of ammonia (P < 0.05). Lowercase letters (a, b, c) indicate a significant effect of linseed oil following low ammonia (P < 0.05). Uppercase letters (A, B, C) indicate a significant effect of linseed oil following high ammonia (P < 0.05).
When fish are exposed to high ammonia, a number of immunosuppressive factors are generated in the peripheral lymph tissue and released into the blood stream [52,53]. It typically results in reduction of lymphocytes and phagocytes and an immune response [54]. In this study, the lysozyme activity, phagocytic index, respiratory burst and total immunoglobulin contents of fish exposed to high ammonia were lower than those of fish exposed to low ammonia, which is in agreement with the finding in Nile tilapia Oreochromis niloticus [55]. 4.2. Effects of dietary linseed oil at stage 2 Compensatory growth refers to a rapid growth that follows a period of suppressed growth due to nutritional or environmental factors [10]. At stage 2, all fish were exposed to <0.01 mg L1 TA-N but fish previously exposed low and high ammonia were not significantly different in weight gain and feed efficiency. The potential ability to compensate for weight loss following exposure to high ammonia was observed. Animal resilience to stress is coregulated by a variety of nutrients in the diet such as essential fatty acids being a source of energy for fish metabolism [56]. Li et al. [57] reported that the level of 1.29% linolenic acid of the diet is required for optimum growth of darkbarbel catfish. But in this study, weight gain of fish fed 4% linseed oil (enriched with 2.0% linolenic acid) was highest, indicating that compensatory growth may increase the requirement of essential fatty acids after exposure to high ammonia. Fatty acid composition in fish is a reflection of dietary fatty acids [58]. At the second stage of this study, fatty acid composition did not differ between fish previously exposed low and high ammonia. But the component of 18:3n3 in fish increased with the increasing levels of dietary linseed oil, suggesting that essential fatty acids were supplemented through the dietary source. The positive relationship between unsaturated fatty acids in feed and in the muscles has been found in several species, such as channel catfish I. punctatus [59], African catfish Claris gariepinus [60], rainbow trout O. mykiss [61], gilthead sea bream Sparus aurata [62] and Nile tilapia O. niloticus [63]. No significant difference between fish previously exposed to low and high ammonia was identified in red blood cells, hemoglobin and hematocrit. It indicates that the dietary linolenic acid may be able to adjust physiological disturbances caused by ammonia exposure. Especially, the values of blood parameters of fish fed 4% linseed oil were higher than those of fish fed 0% and 2% linseed oil. However, the linolenic acid was prone to peroxidation [64], which can activate antioxidant enzymes. In this study, superoxide dismutase, catalase and glutathione peroxidase activities increased with the increasing levels of dietary linseed oil. It should be noted that the activities of antioxidant enzymes in fish
previously exposed to high ammonia were higher than those of fish exposed to low ammonia. It is possible that linolenic acid may mitigate the adverse effect of ammonia in the antioxidant enzyme system. Montero et al. [65] reported that the negative effect of n-3 PUFA dietary deficiency on the serum hemolytic activity of gilthead sea bream S. aurata. Yildirim-Aksoy et al. [66] showed that the serum lysozyme activity of channel catfish I. punctatus fed 3% or 6% PUFA was significantly higher than that of fish fed 0% PUFA. The PUFA can keep more adhesive sites on cell membranes for antibodies to modify membrane viscosity, modulate immunological recognition and enhance immunity [21]. In this study, the lysozyme activity, phagocytic index, respiratory burst and total immunoglobulin contents were enhanced with the increase of dietary linseed oil levels regardless of previous ammonia exposure. However, no significant difference between fish previously exposed to low and high ammonia was identified. Our result indicates that the linolenic acid may also reduce the adverse effect of ammonia exposure on the immune response. 4.3. Effects of bacterial challenge Cumulative mortality of fish previously exposed to high ammonia was higher than that of fish exposed to low ammonia, possibly due to the change of hepatosomatic index. At stage 2, the hepatosomatic index of fish previously exposed to high ammonia was greater than that of fish exposed to low ammonia, but decreased with the increase of dietary linseed oil levels. Liver degeneration can lead to fish death and stress can enhance hepatosomatic index [67]. In this study, fish mortality decreased with the increase of dietary linseed oil, but antibody production against
Table 9 Mean lysozyme activity, phagocytic index (PI), respiratory burst (RB) and total immunoglobulin (Ig) content of darkbarbel catfish fed purified diets supplemented with various levels of linseed oil for 46 days. Ammonia Linseed oil 0% Lysozyme (U mL1) PI (%)
Low High Low High RB (%) Low High Ig (mg mL1) Low High
2%
4%
310.36a 342.83b 346.37b 260.69A 338.62B 348.15B 6.35a 6.62b 6.69b 6.18A 6.56B 6.72B 73.65a 77.01b 77.02b 72.99A 76.05B 77.38B 2.11a 2.24b 2.26b 2.04A 2.16B 2.28B
Pooled PUFA ammonia SEM (P value) 2.019 4.970 0.042 0.024 0.158 0.202 0.026 0.011
0.004 0.258 0.130 0.339
Different lowercase letters (a, b, c) indicate a significant effect of linseed oil following low ammonia (P < 0.05). Different uppercase letters (A, B, C) indicate a significant effect of linseed oil following high ammonia (P < 0.05).
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Table 10 Mean number of cumulative mortality of darkbarbel catfish 14-day post challenge with Edwardsiella ictaluri and antibody production against the same bacterium. Ammonia
Cumulative mortality (%) Antibody titer (log10)
Low High Low High
Linseed oil 0%
2%
4%
42.22b1 62.22B2 1.93a 1.84A
15.56a1 35.5A2 1.98b 1.96B
13.33a1 24.44A2 2.07c 2.10C
Pooled SEM
PUFA ammonia (P value)
2.457 3.629 0.018 0.011
0.644 0.108
Different numbers (1, 2) indicate a significant effect of ammonia (P < 0.05). Different lowercase letters (a, b, c) indicate a significant effect of linseed oil following low ammonia (P < 0.05). Different uppercase letters (A, B, C) indicate a significant effect of linseed oil following high ammonia (P < 0.05).
E. ictaluri infection to fish increased. It is worth noting that high ammonia exposure caused fish morality, but the ability of fish resistance to pathogens was improved as the level of dietary linseed oil increased. In summary, growth performance and immune response of juvenile darkbarbel catfish were restrained by acute ammonia exposure. The high levels of dietary linolenic acid could mitigate the adverse effect of high ammonia exposure on fish performance. Based on fish growth performance, immune responses and resistance to the E. ictaluri challenge, the level of 4% linseed oil (rich in 2% linolenic acid) in the diet is recommended in darkbarbel catfish especially when fish are likely to encounter acute ammonia stress. Acknowledgments This research was supported by grants from the National Basic Research Program (973 Program, No. 2014CB138603, 2009CB118702), “the Twelfth Five-year-plan” in National Science and Technology for the Rural Development in China (2012BAD25B03), the Special Fund for Agro-scientific Research in the Public Interest (No. 201003020, 201203065), National Natural Science Foundation of China (No. 31172422, 31001098), and partly by the E-Institute of Shanghai Municipal Education Commission (No. E03009). References [1] Leung KMY, Chu JCW, Wu RSS. Effects of body weight, water temperature and ration size on ammonia excretion by the areolated grouper (Epinephelus areolatus) and mangrove snapper (Lutjanus argentimaculatus). Aquaculture 1999;170:215e27. [2] Paust LO, Foss A, Imsland AK. Effects of chronic and periodic exposure to ammonia on growth, food conversion efficiency and blood physiology in juvenile Atlantic halibut (Hippoglossus hippoglossus L.). Aquaculture 2011;315: 400e6. [3] Randall DJ, Tsui TKN. Ammonia toxicity in fish. Mar Pollut Bull 2002;45:17e 23. [4] Foss A, Albert KI, Bjørn R, Edward S, Sigurd OS. Effects of chronic and periodic exposure to ammonia on growth and blood physiology in juvenile turbot (Scophthalmus maximus). Aquaculture 2009;296:45e50. [5] Atwood HL, Tomasso JR, Ronan PJ, Barton BA, Renner KJ. Brain monoamine concentrations as predictors of growth inhibition in channel catfish exposed to ammonia. J Aquat Anim Health 2000;12:69e73. [6] El-Shafai SA, El-Gohary FA, Nasr FA, Van Der Steen NP, Gijzen HJ. Chronic ammonia toxicity to duckweed-fed tilapia (Oreochromis niloticus). Aquaculture 2004;232:117e27. [7] Benli ACK, Köksal G, Özkul A. Sublethal ammonia exposure of Nile tilapia (Oreochromis niloticus L.): effects on gill, liver and kidney histology. Chemosphere 2008;72:1355e8. [8] Dong XY, Zhang XM, Qin JG, Zong SB. Acute ammonia toxicity and gill morphological changes of Japanese flounder Paralichthys olivaceus in normal versus supersaturated oxygen. Aquac Res 2013;44:1752e9. [9] Qin JG, Fast AW, Kai A. Lethal effects of ammonia and pH on snakehead (Channa striatus). J World Aquac Soc 1997;28:87e90. [10] Ali M, Nicieza A, Wootton RJ. Compensatory growth in fishes: a response to growth depression. Fish Fish 2003;4:147e90. [11] Tian XL, Qin JG. Effects of previous ration restriction on compensatory growth in barramundi Lates calcarifer. Aquaculture 2004;235:273e83. [12] Wang Z, Mai K, Liufu Z, Ma H, Xu W, Ai Q, et al. Effect of high dietary intakes of vitamin E and n-3 HUFA on immune responses and resistance to Edwardsiella tarda challenge in Japanese flounder (Paralichthys olivaceus, Temminck and Schlegel). Aquac Res 2006;37:681e92.
[13] Salte R, Thomassen MS, Wold K. Do high levels of dietary polyunsaturated fatty acids (EPA/DHA) prevent diseases associated with membrane degeneration in farmed Atlantic salmon at low water temperature? Bull Eur Assoc Fish Pathol 1988;8:63e6. [14] Lall SP. Nutrition and health of fish. In: Cruz-Suárez L, Ricque-Marie D, TapiaSalazar M, editors. Avances en Nutrición Acuicola V. Mérida: Memorias del V Simposium Internacional de Nutrición Acuícol; 2000. 13ep.23. [15] Calabretti A, Cateni F, Procida G, Favretto LG. Influence of environmental temperature on composition of lipids in edible flesh of rainbow trout (Oncorhynchus mykiss). J Sci Food Agric 2003;83:1493e8. [16] Person-Le Ruyet A, Skallib B, Dulaua N, Le Bayona H, Le Dellioua JHR. Does dietary n-3 highly unsaturated fatty acids level influence the European sea bass (Dicentrarchus labrax) capacity to adapt to a high temperature? Aquaculture 2004;242:571e88. [17] Lim C, Yildirim-Aksoy M, Li MH, Welker TL, Klesius PH. Influence of dietary levels of lipid and vitamin E on growth and resistance of Nile tilapia to Streptococcus iniae challenge. Aquaculture 2009;298:76e82. [18] National Research Council. Nutrient requirements of fish and shrimp. Washington, DC, USA: National Academies Press; 2011. [19] Kinsella JE. a-Linolenic acid: functions and effects on linolenic acid metabolism and eicosanoid-mediated reactions. Adv Food Nutr Res 1991;35:1e18. [20] March BE. Essential fatty acids in fish physiology. Can J Physiol Pharmacol 1992;71:685e9. [21] Obach A, Ouentel C, Laurencin FB. Effects of alpha-tocopherol and dietary oxidized fishoil on the immune response of sea bass Dicentrarchus labrax. Dis Aquat Org 1993;15:175e85. [22] Li M, Chen LQ, Qin JG, Li EC, Yu N, Du ZY. Growth performance, antioxidant status and immune response in darkbarbel catfish Pelteobagrus vachelli fed different PUFA/vitamin E dietary levels and exposed to high or low ammonia. Aquaculture 2013;406:18e27. [23] Colt J, Tchobanoglous G. Chronic exposure of channel catfish, Ictalurus punctatus, to ammonia: effects on growth and survival. Aquaculture 1978;15:353e73. [24] Siikavuopio SI, Sæther B. Effects of chronic nitrite exposure on growth in juvenile Atlantic cod, Gadus morhua. Aquaculture 2006;255:351e6. [25] Hegazi MM, Attia ZI, Ashour OA. Oxidative stress and antioxidant enzymes in liver and white muscle of Nile tilapia juveniles in chronic ammonia exposure. Aquat Toxicol 2010;99:118e25. [26] Johansson O, Wedborg M. Ammoniaeammonium equilibrium in seawater at temperatures between 5 and 25 degrees. C J Chem Soc 1980;9:37e44. [27] Whitfield M. The hydrolysis of ammonia ions in sea water e a theoretical study. J Mar Biol Assoc UK 1974;54:565e80. [28] AOAC. Official methods of analysis. 18th ed. Gaithersburg, MD, USA: Association of Official Analytical Chemists; 2005. [29] Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:497e 509. [30] Morrison WR, Smith LM. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J Lipid Res 1964;53: 600e8. [31] Larsen HN. Comparison of various methods of hemoglobin detection of channel catfish blood. Prog Fish Cult 1964;26:11e5. [32] Trenzado CE, Morales AE, Palma JM, Higuer M. Blood antioxidant defenses and hematological adjustments in crowded/uncrowded rainbow trout (Oncorhynchus mykiss) fed on diets with different levels of antioxidant vitamins and HUFA. Aquaculture 2009;149:440e7. [33] Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971;44:276e87. [34] Aebi H. Catalase in vitro. Methods Enzymol 1984;105:121e6. [35] Flohé L, Günzler WA. Assay of glutathione peroxidase. Methods Enzymol 1984;105:115e21. [36] Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978;52: 302e10. [37] Hultmark D, Steiner H, Rasmuson T, Boman HG. Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem 1980;106:7e16. [38] Pulsford AL, Crampe M, Langston A, Glynn PJ. Modulatory effects of disease, stress, copper, TBT and vitamin E on the immune system of flatfish. Fish Shellfish Immunol 1995;5:631e43. [39] Secombes CJ. Isolation of salmonid macrophages and analysis of their killing activity. In: Stolen JS, Fletcher TC, Anderson BS, Van Muiswinkel WB, editors.
M. Li et al. / Fish & Shellfish Immunology 38 (2014) 158e165
[40]
[41] [42] [43]
[44]
[45]
[46] [47]
[48] [49]
[50]
[51]
[52]
[53] [54]
Techniques in fish immunology. New Jersey: SOS Publications; 1990. pp. 137e 44. Wu J, Shang H. Clinical immunology and in section. In: Ye Y, Wang Y, Shen Z, editors. National guide to clinical laboratory procedures. Nanjing: Southeast University Press; 2006. pp. 595e606. Klesius PH. Carrier state of channel catfish infected with Edwardsiella ictaluri. J Aquat Anim Health 1992;4:227e30. Chen MF, Light TS. Specificity of the channel catfish antibody to Edwardsiella ictaluri. J Aquat Anim Health 1994;6:226e70. Foss A, Evensen TH, Vollen T, Øiestad V. Effects of chronic ammonia exposure on growth and food conversion efficiency in juvenile spotted wolffish. Aquaculture 2003;228:215e24. Dosdat A, Person-Le Ruyet J, Covès D, Dutto G, Gasset E, Le Roux A, et al. Effect of chronic exposure to ammonia on growth, food utilization and metabolism of the European sea bass (Dicentrarchus labrax). Aquat Living Resour 2003;16: 509e20. Rasmussen RS, Korsgaard B. The effect of external ammonia on growth and food utilization of juvenile turbot (Scophthalmus maximus L.). J Exp Mar Biol Ecol 1996;205:35e48. Wicks BJ, Randall DJ. The effect of feeding and fasting on ammonia toxicity in juvenile rainbow trout, Oncorhynchus mykiss. Aquat Toxicol 2002;59:71e82. Li B, Fan QX, Yang K, Zhang L, Guo HX, Wang QY, et al. Effects of chronic ammonia stress on to raging, growth, and haematological parameters of yellow catfish (Pelteobagrus fulvidraco) juveniles. Chin J Appl Environ Biol 2011;17:824e9. Iwama G. Stress in fish. Ann N Y Acad Sci 1998;851:304e10. Marcaida G, Felipo V, Hermenegildo C, Minana MD, Grisolia S. Acute ammonia toxicity is mediated by NMDA type of glutamate receptors. FEBS Lett 1992;296:67e8. Lemarié G, Dosdat A, Covès D, Dutto G, Gasset G, Person-Le Ruyet J. Effect of chronic ammonia exposure on growth of European seabass (Dicentrarchus labrax) juveniles. Aquaculture 2004;229:479e91. Fracalossi DM, Lovell RT. Dietary lipid sources influence responses of channel catfish, Ictalurus punctatus to challenge with the pathogen Edwardsiella ictaluri. Aquaculture 1994;119:287e98. Barton BA, Iwama GK. Physiological changes in fish from stress in aquaculture with emphasis on the response and effects of corticosteroids. Ann Rev Fish Dis 1991;1:3e26. Bonga SEW. The stress response of fish. Physiol Rev 1997;77:591e625. Mock A, Peters G. Lysozyme activity in rainbow trout, Oncorhynchus mykiss (Walbaum), stressed by handling, transport and water pollution. J Fish Biol 1990;37:873e85.
165
[55] Chen JZ, Zhang XL, Hu GD, Qu JH, Fan LM, Song C. The immune response of GIFT Oreochromis niloticus and its susceptibility to Streptococcus iniae under stress in different ammonia. Ecol Environ Sci 2011;20:629e34. [56] Watanabe T. Lipid nutrition in fish. Comp Biochem Physiol B 1982;73:3e15. [57] Li M, Chen LQ, Li EC, Yu N, Ding ZL, Chen YL, et al. Growth, immune response and resistance to Aeromonas hydrophila of darkbarbel catfish, Pelteobagrus vachelli (Richardson), fed diets with different linolenic acid levels. Aquac Res; 2013. http://dx.doi.org/10.1111/are.12236. [58] Francis DS, Turchini GM, Jones PL, De Silva SS. Effects of dietary oil source on growth and fillet fatty acid composition of Murray cod, Maccullochella peelii peelii. Aquaculture 2006;253:547e56. [59] Stickney RR, Andrews JW. Effects of dietary lipids on growth, food conversion, lipid and fatty acid composition of channel catfish. J Nutr 1972;102:249e58. [60] Ng WK, Lim PK, Boey PL. Dietary lipid and palm oil source affects growth, fatty acid composition and muscle a-tocopherol concentration in African catfish, Claris gariepinus. Aquaculture 2003;215:229e43. [61] Yu TC, Sinnhuber RO. Effect of dietary u3 and u6 fatty acids on growth and feed conversion efficiency of coho salmon (Oncorhynchus kisutch). Aquaculture 1979;16:31e8. [62] Kalogeropoulos N, Zlexis MN, Henderson RJ. Effects of dietary soybean and cod-liver oil levels on growth and body composition of gilthead bread (Sparus aurata). Aquaculture 1992;104:293e308. [63] Tonial IB, Stevanato FB, Matsushita M, De Souza NE, Furuya WM, Visentainer JV. Optimization of flaxseed oil feeding time length in adult Nile tilapia (Oreochromis niloticus) as a function of muscle omega-3 fatty acid composition. Aquac Nutr 2009;15:564e8. [64] Mourente G, Díaz-Salvago E, Tocher DR, Bell JG. Effects of dietary polyunsaturated fatty acid/vitamin E (PUFA)/tocopherol ratio on antioxidant defence mechanisms of juvenile gilthead sea bream (Sparus aurata L., Osteichthyes, Sparidae). Fish Physiol Biochem 2000;23:337e51. [65] Montero D, Tort L, Izquierdo MS, Robaina L, Vergara JM. Depletion of serum alternative complement pathway activity in gilthead seabream caused by alpha-tocopherol and n-3 HUFA dietary deficiencies. Fish Physiol Biochem 1998;18:399e407. [66] Yildirim-Aksoy M, Lim C, Shelby R, Klesius PH. Increasing fish oil levels in commercial diets influences hematological and immunological responses of channel catfish, Ictalurus punctatus. J World Aquac Soc 2009;40:76e86. [67] Mourento G, Díaz-Salvago E, Bell JG, Tocher DR. Increased activities of hepatic antioxidant defense enzymes in juvenile gilthead sea bream (Sprausaurata L., Osteichthyes, Sparide) fed dietary oxidized oil: attenuation by dietary vitamin E. Aquaculture 2002;214:343e61.