Fish Physiol Biochem https://doi.org/10.1007/s10695-018-0480-6
Nutritional history does not modulate hepatic oxidative status of European sea bass (Dicentrarchus labrax) submitted to handling stress Carolina Castro & Amalia Peréz-Jiménez & Filipe Coutinho & Geneviève Corraze & Stéphane Panserat & Helena Peres & Aires Oliva Teles & Paula Enes
Received: 31 October 2017 / Accepted: 7 February 2018 # Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract The aim of the present study was to assess the impact of an acute handling stress on hepatic oxidative status of European sea bass (Dicentrarchus labrax) juveniles fed diets differing in lipid so urce and carbohydrate content. For that purpose, four diets were formulated with fish oil (FO) and vegetable oils (VO) as lipid source and with 20 or 0% gelatinized starch as carbohydrate source. Triplicate groups of fish with 74 g were fed each diet during 13 weeks and then subjected to an acute handling stress. Stress exposure decreased hematocrit (Ht) and hemoglobin (Hb) levels. Independent of dietary treatment, stress exposure increased hepatic lipid peroxidation (LPO). Stressed fish exhibited lower glucose 6-phosphate dehydrogenase (G6PD), catalase (CAT), and superoxide dismutase (SOD) activities, independent of previous nutritional history. In the VO groups, stress exposure
C. Castro (*) : A. Peréz-Jiménez : F. Coutinho : H. Peres : A. O. Teles : P. Enes CIMAR/CIIMAR - Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal e-mail:
[email protected] A. Peréz-Jiménez Departamento de Zoología, Facultad de Ciencias, Universidad de Granada, Campus Fuentenueva s/n, 18071 Granada, Spain G. Corraze : S. Panserat INRA, Univ Pau & Pays de l’Adour, UMR1419 Nutrition Metabolism Aquaculture, 64310 Saint-Pée-sur-Nivelle, France
increased glutathione peroxidase (GPX) activity. Diet composition had no effect on Ht and Hb levels. In contrast, dietary carbohydrate decreased hepatic LPO and CAT activity and increased glutathione reductase (GR) and G6PD activities. Dietary lipids had no effect on LPO. Fish fed the VO diets exhibited higher G6PD activity than fish fed the FO diets. In conclusion, dietary carbohydrates contributed to the reduction of oxidative stress in fish. However, under the imposed handling stress conditions, liver enzymatic antioxidant mechanisms were not enhanced, which may explain the overall increased oxidative stress. Keywords Antioxidant enzymes . Carbohydrates . Handling stress . Lipid oxidative damage . Vegetable oils
Introduction Under intensive rearing conditions, fish are often exposed to husbandry-related stressors such as acute water temperature changes, deterioration in water quality, crowding, handling, transportation, or confinement. Exposure to these stressors is known to induce a generalized stress response that includes changes in endocrine, metabolic, cellular, hematological, antioxidant, and immune functions (Barton 2002; Tort 2013). As a result, animal performancerelated conditions, such as feeding, growth, reproduction, and disease resistance, may be compromised leading to considerable economic losses for fish farmers.
Fish Physiol Biochem
Fish antioxidant response to stressors can be modulated by its nutritional history, since nutritional factors were reported to differently affect fish oxidative status. Accordingly, dietary substitution of lipid sources with high unsaturation indexes (fish oil, FO) by low unsaturated ones (vegetable oils, VO) was reported to decrease fish hepatic and muscular lipid peroxidation (LPO) susceptibility (Menoyo et al. 2004; Lin and Shiau 2007; Peng et al. 2008; Gao et al. 2012; Ng et al. 2013; Castro et al. 2016). Dietary carbohydrates were also reported to protect tissues against oxidative damage (Pérez-Jiménez et al. 2009, 2017; Wang et al. 2014; Castro et al. 2015a, 2016). However, studies assessing the potential interactions between nutrients on fish oxidative status are limited (Castro et al. 2012, 2015a, 2016). Overall, aquafeeds allowing fish to cope with husbandryrelated stressful conditions would be of value to avoid the negative effects related to oxidative stress and to improve fish health and welfare in the context of the development of eco-friendly aquafeeds, rich in nonfisheries-related feedstuffs such as VO and carbohydrates. Thus, the aim of the present study was to assess the impact of an acute handling stress—a common practice in fish farming—on hepatic oxidative status of European sea bass juveniles fed diets differing in lipid source (FO or a blend of VO) and carbohydrate content (0 and 20% gelatinized starch).
Table 1 Ingredients and chemical composition of the experimental diets Experimental diets Lipid source
FO
Carbohydrates
CH−
CH+
CH−
VO CH+
Fish meala
86.5
64.5
86.5
64.5
Gelatinized maize starchb
0
20
0
20
Cod liver oilc
10
12
0
0
Vegetable oil blendd
0
0
10
12
Vitaminse
1.5
1.5
1.5
1.5
Mineralsf
1.0
1.0
1.0
1.0
Binderg
1.0
1.0
1.0
1.0
90.4
91.3
91.0
Ingredients (% dry weight)
Proximate analyses (% dry matter) Dry matter (DM)
89.5
Crude protein (CP)
62.4
46.6
62.4
47.1
Crude lipid (CL)
18.4
18.4
18.2
18.3
Gross energy (kJ g−1)
26.2
25.8
26.7
25.6
Starch
0.8
18.7
0.8
17.5
Ash
17.6
14.0
17.6
13.8
Fatty acid composition of the experimental diets are presented in Castro et al. (2015) FO, fish oil; VO, blend of vegetable oils; carbohydrate content, 0% (CH−) or 20% (CH+) gelatinized maize starch a
Steam dried LT fish meal, Pesquera Diamante, Perú (CP 71.1% DM; CL 8.8% DM)
b
C-Gel Instant-12018, Cerestar, Mechelen, Belgium
c
Labchem, Laborspirit Lda., Lisboa, Portugal
d
Materials and methods Experimental diets Four isolipidic diets differing in carbohydrate content (0 and 20% gelatinized starch, diets CH− and CH+, respectively) and lipid source (FO or a VO blend, diets FO and VO, respectively) were formulated (FOCH−, FOCH+, VOCH−, VOCH+), with the dietary carbohydrate content being increased at the expense of dietary protein (Table 1). The VO blend was composed of 20% rapeseed, 50% linseed, and 30% palm oils and replaced approximately 70% of dietary lipids, which were provided by cod liver oil and fish meal. All dietary ingredients were thoroughly mixed and dry-pelleted in a laboratory pellet mill (California Pellet Mill, CPM Crawfordsville, IN, USA), through a 3.0-mm die. The pellets were air-dried for 24 h and stored in a refrigerator (4 °C) until use.
30% palm oil (Colmi, Malasia), 50% linseed oil (Sociedade Portuense de Drogas, S.A.,Portugal), and 20% rapeseed oil (Huilerie Emile Noël S.A.S., France) Vitamins (mg kg−1 diet): retinol acetate, 18,000 (IU kg−1 diet); cholecalciferol, 2000 (IU kg−1 diet); alpha tocopherol acetate, 35; sodium menadione bisulphate, 10; thiamin-HCl, 15; riboflavin, 25; calcium pantothenate, 50; nicotinic acid, 200; pyridoxine HCl, 5; folic acid 10; cyanocobalamin, 0.02; biotin, 1.5; ascorbic acid, 50; inositol, 400. Premix, Viana do Castelo, Portugal e
Minerals (mg kg−1 diet): cobalt sulphate, 1.91; copper sulphate, 19.6; iron sulphate, 200; sodium fluoride, 2.21; potassium iodide, 0.78; magnesium oxide, 830; manganese oxide, 26; sodiumselenite, 0.66; zinc oxide, 37.5; dibasic calcium phosphate, 5.93 (g kg−1 diet); potassium chloride, 1.15 (g kg−1 diet); sodium chloride, 0.40 (g kg−1 diet). Premix, Viana do Castelo, Portugal f
g Aquacube (guar gum, polymethyl carbamide, manioc starch blend, hydrate calcium sulphate). Agil, England
Chemical analyses of the diets were performed following the Association of Official Analytical Chemists methods (AOAC 2016). Starch content was analyzed according to Beutler (1984).
Fish Physiol Biochem
Fish, experimental conditions, and sampling This experiment was directed by accredited scientists (following FELASA category C recommendations) and conducted according to the European Union Directive (2010/63/EU) on the protection of animals for scientific purposes. European sea bass (Dicentrarchus labrax) juveniles were obtained from a commercial fish farm (Maresa S.A., Ayamonte, Huelva, Spain) and transported to the Marine Zoology Station’s facilities (Porto University, Porto, Portugal). After transport, fish were submitted to a quarantine period of 2 weeks during which they were fed a commercial diet (Skretting, Stavanger, Norway) containing 16% of lipids and 47% of protein. Thereafter, groups of 20 fish with an initial mean body weight of 74.0 ± 1.5 g were distributed in 12 fiberglass tanks of 300 L water capacity and each experimental diet was randomly assigned to triplicate groups. The trial lasted 13 weeks, and during that period, fish were fed by hand, twice a day, 6 days a week until visual satiation. Utmost care was taken to assure that all feed supplied was consumed. The trial was conducted in a recirculating aquaculture system (RAS), thermoregulated to 25.4 ± 0.5 °C, and supplied with continuous flow of seawater (35.0 ± 1.0 g L−1 salinity, circa 7 mg L−1 oxygen). The photoperiod regime was the natural one for June–August. At the end of the trial, three fish from each tank were randomly sampled 18 h after the last meal. Blood was collected from the caudal vein using heparinized syringes and placed in heparinized tubes for hematocrit (Ht) and hemoglobin (Hb) determination. After blood collection, fish were euthanized with a sharp blow in the head. Livers were removed, immediately frozen in liquid nitrogen, and stored at − 80 °C until measurement of enzyme activities and LPO levels. To induce handling stress, at the end of the sampling procedures, six fish from each tank were transferred to a RAS equipped with 12 fiberglass tanks of 100 L water capacity. One hour later, fish were further subjected to water shaking with a stick for 30 s, each 20 min, during 5 h. Then, blood and liver from three fish per tank were sampled as described above. Hematological analysis Fresh heparinized blood was used for Ht and Hb determination. Ht value was determined by
microcentrifugation (10,000g for 10 min, at room temperature) and Hb was determined using Drabkin’s solution (Spinreact, ref. 1001230; Girona, Spain). Enzymatic activity Liver samples were homogenized on ice in five volumes of ice-cold 100-mM Tris-HCl buffer containing 0.1 mM EDTA and 0.1% (v/v) Triton X-100 (pH 7.8). Homogenates were centrifuged at 30,000g for 30 min at 4 °C, and the resultant supernatants were separated in aliquots and stored at − 80 °C until use. The optimal substrate and protein concentrations for measurement of maximal activity for each enzyme were established by preliminary assays. Superoxide dismutase (SOD, EC1.15.1.1), catalase (CAT, EC1.11.1.6), glutathione peroxidase (GPX, EC1.11.1.9), glutathione reductase (GR; EC1.6.4.2), and glucose 6-phosphate dehydrogenase (G6PD, EC1.1.1.49) activities were determined as described in Castro et al. (2015a). Lipid peroxidation Concentration of malondialdehyde (MDA) was determined as a marker for LPO following the methodology described by Buege and Aust (1978). An aliquot of supernatant from the homogenate (100 μL) was mixed with 500 μL of a previously prepared solution containing 15% (w/v) TCA, 0.375% (w/v) thiobarbituric acid (TBA), 80% (v/v) HCl 0.25 N, and 0.01% (w/v) butylated hydroxytoluene. The mixture was heated to 100 °C for 15 min. After being cooled to room temperature and centrifuged at 1500g for 10 min, the absorbance was measured at 535 nm in the supernatant. Concentration was expressed as nanomole MDA per gram of tissue, calculated from a calibration curve. Statistical analysis Data are presented as mean values and standard deviations. Data were analyzed by factorial analysis of variance (ANOVA) with carbohydrate level, lipid source, and stress as factors using SPSS 21 software package (SPSS® Inc.). Previous to ANOVA, data were tested for normality and homogeneity (Shapiro-Wilk and Levene’s tests, respectively) and when necessary transformed to achieve ANOVA assumptions. When significant interaction between factors was found, one-way
Fish Physiol Biochem
ANOVA was performed for each factor. For all data, the probability level for rejection of the null hypotheses was 0.05.
in unstressed fish. Stress also promoted an increase in GPX activity in VO groups (Table 3).
Discussion Results Growth performance of fish during the 13 weeks rearing period was not the aim of this study, and results were presented elsewhere (Castro et al. 2015b). Dietary composition had no effect on Ht and Hb values (Table 2). In contrast, stress exposure decreased both parameters. Dietary lipid source also did not affect LPO or the activity of oxidative stress enzymes, whereas dietary carbohydrate affected strongly LPO values and the activity of GR, CAT, and G6PD (Table 3). Regardless of the stress condition, LPO damage and CAT activity were lower and GR and G6PD activities were higher in the CH+ groups (Table 3). In the VO groups, but not in the FO groups, CH+ induced a decrease in SOD activity. G6PD activity was higher in the VO than that in the FO groups. Stress increased hepatic LPO damage in all dietary treatments. Under stress conditions, G6PD, CAT, and SOD activities were lower than Table 2 Hematocrit (Ht; %) and hemoglobin (Hb; g dL−1) of European sea bass under non-stressful (NS) and stressful (S) conditions Non-stress/stress
Ht NS
FO VO
Hb S
NS
S
CH−
30.7 ± 3.3 24.8 ± 3.4 8.0 ± 0.9 6.5 ± 0.6
CH+
31.0 ± 1.5 24.9 ± 2.6 7.4 ± 0.4 6.4 ± 0.5
CH−
29.1 ± 2.7 26.0 ± 4.5 7.4 ± 0.8 6.8 ± 0.7
CH+
29.4 ± 2.5 24.8 ± 2.0 7.5 ± 0.6 6.6 ± 0.5
Three-way p value ANOVA factor Carbohydrate 0.868 (CH) Lipid source (LS) 0.515
p value 0.219 0.959
Stress
˂ 0.001
˂ 0.001
CH × LS
0.695
0.323
CH × stress
0.557
0.797
LS × stress
0.163
0.102
CH × LS × stress 0.677
0.191
Values present as means ± standard deviation (± SD) (n = 9) FO, fish oil; VO, blend of vegetable oils; carbohydrate content, 0% (CH−) or 20% (CH+) gelatinized maize starch
A relation between fish previous nutritional status and response efficiency to stress situations, such as thermic (Ibarz et al. 2010; Kumar et al. 2011; Castro et al. 2012), hypoxia (Henrique et al. 1998; McKenzie et al. 2008; Pérez-Jiménez et al., 2012), salt water (Hemre et al. 2002), and confinement (Ruane et al. 2001, 2002) stress, has been described. However, to the best of our knowledge, the effects of diet composition on antioxidant mechanisms to counteract stressful conditions were scarcely evaluated (Kumar et al. 2011; Castro et al. 2012; Pérez-Jiménez et al. 2012; Magnoni et al. 2017). In Labeo rohita fingerlings, high-protein diets (≥ 40%) lead to an increase in liver SOD and CAT activities after an acute thermal shock (Kumar et al. 2011). In Senegalese sole juveniles (Solea senegalensis), nutritional status modulated lipid oxidative damage susceptibility to thermal stress, with fish fed a high-protein diet (55%) being more vulnerable than fish fed a low-protein diet (45%) to an acute exposition to warmer or colder temperatures (Castro et al. 2012). In contrast, in the present study, nutritional status did not affect oxidative stress response in fish exposed to handling stress. Enzymatic antioxidant mechanisms failed counteract the increased oxidative stress condition, leading to increased LPO levels in all groups. Indeed, stress exposure even decreased SOD, CAT, and G6PD activities. The response to stress in this and the abovementioned studies is however difficult to compare, as the cause of stress and diet composition differ between studies. In the present study, Ht and Hb also decreased in the stressed group. This was somewhat unexpectable as an increase of Hb levels was previously observed in fish submitted to crowding and handling stress (Montero et al. 1999; Benfey and Biron 2000; Trenzado et al. 2009). An increase of Hb levels has been described as a strategy for increasing oxygen-carrying capacity of blood during periods of high-energy demand. On the other hand, decreased oxygen-carrying capacity in response to oxygen-rich environments was described as a physiological adaptation to a reduction in the need for oxygen transport and/or to limit the amount of oxygen being delivered to the tissues (Caldwell and Hinshaw 1994; Hosfeld et al. 2010; Lushchak 2011). In the
7.6 ± 2.2 7.0 ± 1.7 617.9 ± 89.6 543.7 ± 72.7 232.2 ± 29.0 9.5 ± 2.9 9.7 ± 2.5 517.7 ± 68.0 441.7 ± 54.6 219.5 ± 57.8
55.6b ± 14.3
S
0.014 0.892
0.275
0.464
0.462
CH × stress
LS × stress
CH × LS × stress
0.103
0.851
0.592
0.366
0.801
0.360
0.392
˂ 0.001
p value
˂ 0.001
0.558
0.854
0.524
0.499
0.074
0.157
0.025
˂ 0.001 0.701
0.275
0.120
p value
0.370
˂ 0.001
p value
0.317
0.209
0.163
0.673
0.002
0.530
˂ 0.001
p value
FO, fish oil; VO, blend of vegetable oils; carbohydrate content, 0% (CH−) or 20% (CH+) gelatinized maize starch
Values present as means ± standard deviation (± SD) (n = 9). If interaction was significant, one-way ANOVA was performed for each factor: means in the same column with different capital and small letters (a, b; A, B) indicate significant differences (p < 0.05) between the two tested lipid sources and two CH levels, respectively; means in the same line with different letters (x, z) indicate significant differences between the non-stressful (NS) and stressful (S) conditions; means with no letters are not significantly different (p > 0.05)
0.068
0.957
0.022
0.050
0.025
0.316
0.625
˂ 0.001
CH × LS
p value
p value
9.3 ± 1.3
508.5 ± 59.3 48.6z ± 20.2 76.3ax ± 21.4 9.1 ± 1.4 8.9 ± 1.7 551.6 ± 55.6 450.2 ± 68.9 178.2B ± 44.8 162.4B ± 32.9 8.7 ± 0.7
9.9 ± 2.4
CH+ 571.5 ± 55.9
9.0 ± 1.6
10.6 ± 1.0 13.6 ± 2.0
NS
255.8 ± 45.8 55.6z ± 21.3 65.5ax ± 17.0 7.6 ± 3.0 6.2 ± 1.8 619.8 ± 78.4 558.9 ± 63.6 220.3A ± 39.9 190.8A ± 29.1 11.5 ± 2.3 12.5 ± 2.4
183.0 ± 39.9
157.2 ± 20.3
S
LPO
CH− 294.7 ± 33.7
NS
CH+ 539.3 ± 153.9 515.7 ± 87.3 51.9 ± 14.8
S
45.5b ± 16.9
CH− 269.3 ± 43.5
NS
SOD
220.9 ± 39.5 57.2 ± 15.4
S
CAT
NS
GR S
NS
NS
S
GPX
G6PD
Three-way ANOVA factor Carbohydrates (CH) Lipid source (LS) Stress
VO
FO
Non-stress/stress
Table 3 Specific activities of glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GPX), glutathione reductase (GR) (mU mg protein−1), catalase (CAT), superoxide dismutase (SOD) (U mg protein−1), and lipid peroxidation (LPO) (nmol malondialdehyde g tissue−1) in the liver of European sea bass under non-stressful (NS) and stressful (S) conditions
Fish Physiol Biochem
Fish Physiol Biochem
current study, handling stress was promoted by water shaking with a stick which might have increased oxygenation of water and consequently the oxygen supply. Therefore, it is not possible to exclude that the hematological alterations as well as the changes in key markers of oxidative stress and redox-activated antioxidant defense were a consequence of higher environmental oxygen levels. Under such circumstances, an increase in oxygen levels is expected to promote generation of ROS due to enhanced probability of electrons escaped from electron-transport chains to combine with molecular oxygen (Lushchak and Bagnyukova 2006; Lushchak 2011). In accordance with the higher LPO levels observed in the stressed groups, enhanced LPO due to exposure to higher oxygen levels was also reported in the liver of Atlantic salmon Salmo salar (Lygren et al. 2000) and goldfish Carassius auratus (Lushchak et al. 2005). However, in those studies, the increase in LPO levels did not trigger an effective response of antioxidant defenses since no changes or a decrease in the activities of several antioxidant enzymes (SOD, catalase, GPX, and GR) was detected (Lygren et al. 2000; Lushchak et al. 2005). As in those studies, the cause behind the lack of activation in the antioxidant response could not be completely addressed in the present study. However, as pointed out by Lushchak et al. (2005), the susceptibility of antioxidant enzymes undergoing inactivation due to ROS attack should be considered and evaluated in future studies. Although dietary carbohydrate level or lipid source had no effect on attenuating oxidative stress under the imposed handling stress, data on liver LPO content and enzymatic antioxidant mechanisms indicate that CH+ groups were less prone to oxidative damage. This can be related with the glucose molecule per se, since it also acts as free radical scavenger, or to the increased activity of the pentose phosphate pathway and glutathione redox status (Sagone et al. 1983; Alvarez et al. 1999; Lygren and Hemre 2001; Pérez-Jiménez et al. 2009, 2017; Castro et al. 2015a, 2016). Accordingly, higher G6PD and GR activities were observed in the CH+ groups. Regarding lipid sources, the higher GPX (under stress conditions) and G6PD activities observed in VO groups may be related with the presence of phytochemicals in VO. Vegetable-related feedstuffs are rich in a group of phenolic phytochemicals that have antioxidant properties and may protect against oxidative damage, for example by increasing the capacity of endogenous antioxidant defenses (Rice-Evans 2001; Moskaug et al. 2005;
Procházková et al. 2011). This fact along with the lower unsaturation index of VO diets was expected to decrease the susceptibility to oxidation in the VO groups. However, LPO levels indicate that VO and FO groups were equally susceptible to oxidative damage. In conclusion, dietary carbohydrates contributed to a reduction of liver oxidative stress in European sea bass juveniles. However, previous nutritional history did not modulate liver oxidative status under the imposed stress conditions. Liver enzymatic antioxidant mechanisms were not enhanced during handling stress, which may explain the overall increased oxidative stress. Funding information This work was partially supported by the FCT (Foundation for Science and Technology), Portugal (project PTDC/MAR-BIO/4107/2012) and co-financed by the European Regional Development Fund (ERDF) through the COMPETE Operational Competitiveness Programme and national funds through FCT, under the project “PEst-C/MAR/LA0015/2011”. C. Castro, A. Peréz-Jiménez, F. Coutinho, and P. Enes were supported by grants (SFRH/BD/76297/2011; SFRH/BPD/64684/ 2009; SFRH/BD/86799/2012; SFRH/BPD/101012/2014, respectively) from FCT. Compliance with ethical standards This experiment was directed by accredited scientists (following FELASA category C recommendations) and conducted according to the European Union Directive (2010/63/EU) on the protection of animals for scientific purposes.
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