Fish Physiol Biochem DOI 10.1007/s10695-013-9875-6
The effects of dietary thiamin on oxidative damage and antioxidant defence of juvenile fish Xue-Yin Li • Hui-Hua Huang • Kai Hu • Yang Liu • Wei-Dan Jiang • Jun Jiang • Shu-Hong Li • Lin Feng • Xiao-Qiu Zhou
Received: 31 December 2012 / Accepted: 3 October 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The present study explored the effects of thiamin on antioxidant capacity of juvenile Jian carp (Cyprinus carpio var. Jian). In a 60-day feeding trial, a total of 1,050 juvenile Jian carp (8.20 ± 0.02 g) were fed graded levels of thiamin at 0.25, 0.48, 0.79, 1.06, 1.37, 1.63 and 2.65 mg thiamin kg-1 diets. The results showed that malondialdehyde and protein carbonyl contents in serum, hepatopancreas, intestine and muscle were significantly decreased with increasing dietary thiamin levels (P \ 0.05). Conversely, the anti-superoxide anion capacity and anti-hydroxyl radical capacity in serum, hepatopancreas, intestine and muscle were the lowest in fish fed the thiaminunsupplemented diet. Meanwhile, the activities of catalase (CAT), glutathione peroxidase, glutathione
X.-Y. Li H.-H. Huang K. Hu Y. Liu W.-D. Jiang J. Jiang S.-H. Li L. Feng (&) X.-Q. Zhou (&) Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China e-mail: [email protected] X.-Q. Zhou e-mail: [email protected]; [email protected]; [email protected] X.-Y. Li H.-H. Huang K. Hu Y. Liu W.-D. Jiang J. Jiang L. Feng X.-Q. Zhou Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China
S-transferase and glutathione reductase, and the contents of glutathione in serum, hepatopancreas, intestine and muscle were enhanced with increasing dietary thiamin levels (P \ 0.05). Superoxide dismutase (SOD) activity in serum, hepatopancreas and intestine followed a similar trend as CAT (P \ 0.05). However, SOD activity in muscle was not affected by dietary thiamin level (P [ 0.05). The results indicated that thiamin could improve antioxidant defence and inhibit lipid peroxidation and protein oxidation of juvenile Jian carp. Keywords Cyprinus carpio var. Jian Thiamin Oxidative damage Antioxidant Antioxidant enzyme
Introduction Thiamin has been demonstrated to be an essential dietary nutrient for fish (Aoe et al. 1969). Thiamin pyrophosphate is the major coenzyme form of thiamin, which is involved in several enzymatic steps in energy production, including oxidative decarboxylation of aketo acids and transketolation reactions in the pentose phosphate pathway (NRC 2011). Thiamin deficiency caused muscle atrophy in Chinook salmon (Halver 1957) and hemorrhage of fins in eel (Hashimoto et al. 1970). Researchers had reported that the thiamin requirement of common carp (Aoe et al. 1969),
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channel catfish (Murai and Andrews 1978) and rainbow trout (Morito et al. 1986) were 0.5, 1.0 and 1.0 mg kg-1 diet, respectively, based on weight gain or absence of deficiency signs. A previous study from our laboratory demonstrated that thiamin deficiency decreased growth and survival, induced congestion of the fins and skin, caused atrophy of intestine and hepatopancreas and decreased the activities of digestive and brush border enzymes of juvenile Jian carp (Cyprinus carpio var. Jian) (Huang et al. 2011). The disturbance of growth and abnormal digestive and absorptive function may be related to the structural integrity of cells in fish (Lushchak et al. 2001). The cell membrane integrity and functionality is associated with the oxidant–antioxidant balance (Knight 2000). However, only few studies have been reported about the relationship between thiamin and antioxidant defence in fish. Thiamin effectively inhibited lipid peroxidation of liver in mouse and fish (Huang et al. 2007; Lukienko et al. 2000), suggesting that dietary thiamin may be related to antioxidant status of animal, which needs further investigation. Superoxide anions (O2 ) and hydroxyl radicals ( OH) are the most reactive oxygen radicals and capable of damaging cellular macromolecules, such as membrane lipids, proteins and nuclei acids (Nordberg and Arne´r 2001; Calingasan et al. 1999). Like all aerobic organisms, antioxidant systems in fish contain antioxidant enzymes and low molecular weight antioxidants (Kohen and Nyska 2002). The enzymatic antioxidant includes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), and non-enzymatic antioxidants, such as glutathione (GSH) (Knight 2000). In vitro, thiamin can scavenge O2 and OH directly (Jung and Kim 2003). Furthermore, it has been reported that thiamin interacted with free radicals and hydroperoxides through transferring (2H? ? 2e-) from the NH2 group of the pyrimidine ring to radicals in vitro (Lukienko et al. 2000). In rats, thiamin increased insulin secretion of pancreas, which resulted in an increase in Cu/Zn-SOD and CAT activities in liver (Rathanaswami et al. 1991; Sindhu et al. 2004). Meanwhile, the regeneration of reduced GSH required NADPH providing reducing equivalents. Thiamin is a cofactor of transketolase, which is a key enzyme of pentose phosphate pathway for the production of NADPH in rat (Shangari et al. 2007). These
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observations lead to the idea that the improvement of structure and function of fish tissues by thiamin may be partly related to the antioxidant defence, which needs further investigation. To date, few studies have been conducted to investigate the effects of thiamin on lipid peroxidation, protein oxidation and the antioxidant enzyme activities in fish. Therefore, the objective of this study was to explore the effects of dietary thiamin on the antioxidant capacity in hepatopancreas, intestines and muscle of juvenile Jian carp. This study was a part of a larger study that involved in the determination of the effects of thiamin on growth performance, digestive and absorptive abilities of fish (Huang et al. 2011). The graded levels of dietary thiamin were designed according to dietary thiamin requirement (0.5–1.0 mg kg-1) of finfish (Aoe et al. 1969; Morito et al. 1986; Murai and Andrews 1978). The study can partly provide theoretical evidence for the effects of thiamin on growth, digestive and absorptive ability in fish. Materials and methods Experimental design and diets Formulation of basal diet is present in Table 1. Experimental diets and the procedures for diet preparation were the same as Huang et al. (2011). The basal diet was formulated to meet the nutrient requirements of Jian carp based on our laboratory’s studies (He et al. 2009; Jiang et al. 2009b; Li et al. 2010; Wen et al. 2009; Zhou et al. 2008) and contained 307.8 g crude protein kg-1 diet and 64.8 g crude lipid kg-1 diet. The basal diet contained 0.21 mg thiamin kg-1 diet, which was calculated according to the analyzed thiamin concentrations of ingredients. Thiamin hydrochloride (Sigma, St Louis, USA) was added to the test diets to provide graded concentrations of thiamin. The thiamin concentrations of the seven diets were determined by the fluorometric method of AOAC (1990) to be 0.25, 0.48, 0.79, 1.06, 1.37, 1.63 and 2.65 mg thiamin kg-1 diet, respectively. After preparation, the diets were stored at -20 °C until used according to Hu et al. (2008). Feeding management Juvenile Jian carp obtained from the Ya’an fisheries were acclimatized to the experimental environment
Fish Physiol Biochem Table 1 Composition of the basal diet Ingredients
g kg-1
Nutrients content
g kg-1
Fish meal
226.5
Crude protein
307.8
Casein
148.5
Gelatin
60.0
Crude lipid
64.8
Lys
22.9
a-Starch
283.0
Methionine ? cystine
13.6
Corn starch
190.3
n-3
10.0
Fish oil
10.9
n-6
10.0
Soybean oil
18.9
DL-Met
3.2
Cellulose
20.0
Ca (H2PO4)2
16.9
Choline chloride Ethoxyquin
and reduce ammonia concentrations. Water temperature was maintained at 25.0 ± 1 °C. Additional incandescent lighting provided a diurnal light/dark cycle of 12:12 h, which was similar to Deng et al. (2010). For the feeding trial, each of seven experimental diets was fed to triplicate groups of fish six times every day. Thirty minutes after the feeding, uneaten feed was removed by siphoning. The feeding experiment lasted 60 days. Feeding management was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Animal Nutrition Institute, Sichuan Agricultural University. Sample collection and analysis
1.3 0.5
Vitamin mixture(thiamin free)a
10.0
Mineral mixtureb
10.0
Thiamin premixc
–
Analyzed chemical composition of basal diet (g kg-1): crude protein, 307.8; lipid, 64.8; ash, 65.8 a
Vitamin mixture (g kg-1 mixture): retinyl acetate (500,000 IU g-1), 0.800 g; cholecalciferol (500,000 IU g-1), 0.480 g; DL-a-tocopherol acetate (500 g kg-1), 20.000 g; menadione (500 g kg-1), 0.200 g; riboflavin (800 g kg-1), 0.625 g; pyridoxine hydrochloride (980 g kg-1), 0.755 g; 0.010 g; ascorbyl-2cyanocobalamin (100 g kg-1), 19.029 g; calcium-Dpolyphosphate (350 g kg-1), pantothenate (980 g kg-1), 2.511 g; niacin (980 g kg-1), 2.857 g; D-biotin (200 g kg-1), 0.500 g; meso-inositol (980 g kg-1), 52.857 g; folic acid (960 g kg-1), 0.521 g. All ingredients were diluted with corn starch to 1 kg b
Mineral mixture (g kg-1 mixture): FeSO47H2O -1 Fe), 76.147 g; CuSO45H2O (250.0 g kg-1 (197.0 g kg Cu), 1.20 g; MnSO4H2O (318.0 g kg-1 Mn), 4.59 g; ZnSO47H2O (225.0 g kg-1 Zn), 13.34 g; KI (38.0 g kg-1 I), 2.90 g; NaSeO3 (10.0 g kg-1 Se), 2.50 g. All ingredients were diluted with CaCO3 to 1 kg c
Thiamin premix: premix was added to obtain graded levels of thiamin. The analyzed thiamin levels of the diets were: 0.25, 0.48, 0.79, 1.06, 1.37, 1.63 and 2.65 mg kg-1 diet, respectively
for 4 weeks. At the end of acclimatization period, a total of 1,050 carp with an initial weight of 8.20 ± 0.02 g were randomly distributed into each of 21 glass aquaria (90 9 30 9 40 cm). The aquarium was connected to a closed recirculating water system and oxygen auto-supplemented system. Water circulated at 1.2 L min-1 in each aquarium, and the water was drained through biofilters to remove impurities
Fish in each cage were weighed and counted at the initiation and termination of the feeding trial. At the end of the feeding trial, 15 fish from each aquarium were anaesthetized with 50 mg L-1 benzocaine as described by Berdikova Bohne et al. (2007) after 12 h of the last feeding. Blood of 15 fish from each aquarium was drawn from caudal vein, stored at 4 °C overnight and centrifuged at 3,0009g for 10 min. The serum was obtained for antioxidant parameters analysis. The intestine, hepatopancreas and muscle were quickly removed, weighed, frozen in liquid nitrogen and finally stored at -70 °C. Tissue samples were homogenized on ice in 10 volumes (w v-1) of ice-cold physiological saline and centrifuged at 6,0009g for 20 min at 4 °C; then, the supernatant was collected for antioxidant parameters analysis. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan Agricultural University. The protein concentration of samples was determined by the method of Bradford (1976). Malondialdehyde (MDA) content was analyzed as described by Livingstone et al. (1990) using the thiobarbituric acid (TBA) reaction. The content of protein carbonyl was determined by the formation of protein hydrazones using 2,4-dinitrophenylhydrazine (DNPH) (Halliwell and Whiteman 2009). The anti-superoxide anion (ASA) capacity and the anti-hydroxyl radical (AHR) capacity were assayed as described by Jiang et al. (2009a). Superoxide anion radicals were generated by the action of xanthine and xanthine oxidase. With the electron acceptor added, a coloration reaction is developed using the Griess reagent. The coloration degree is directly proportional to the quantity of superoxide anion in the reaction. Hydroxyl radicals are
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Fish Physiol Biochem
generated in a Fenton reaction. With the electron acceptor added, a coloration reaction is developed using the Griess reagent. The coloration degree is directly proportional to the quantity of hydroxyl radicals in the reaction. The activities of SOD and GPx were assayed as described by Zhang et al. (2008). CAT activity was determined by the decomposition of hydrogen peroxide (Aebi 1984). Glutathione S-transferase (GST) activity was measured by monitoring the formation of adduct between GSH and 1-chloro-2,4dinitrobenzene (Lushchak et al. 2001). GR activity was measured according to a procedure described by Lora et al. (2004). Reduced GSH content was quantitated according to the method of Vardi et al. (2008). Calculation and statistical analysis The data were presented as mean ± SD. All data were subjected to one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test to determine significant differences among treatment groups at the level of P \ 0.05 through SPSS 13.0 (SPSS Inc., Chicago, IL, USA) (Fig. 1).
Results MDA and PC content in serum, hepatopancreas, intestine and muscle The MDA and PC content in serum, hepatopancreas, intestine and muscle of juvenile Jian carp were significantly affected by dietary thiamin level (Table 2). MDA content in serum was lower in fish fed C0.79 mg thiamin kg-1 diet than fish fed the diet with thiamin-unsupplemented group (P \ 0.05). The contents of MDA were significantly decreased with increasing dietary thiamin levels up to 1.06 mg kg-1 diet in hepatopancreas, intestine and muscle (P \ 0.05), and no significant differences were observed with further increase in thiamin levels (P [ 0.05). PC contents in serum and hepatopancreas were significantly decreased with increasing dietary thiamin levels up to 1.06 mg kg-1 diet (P \ 0.05) and then plateaued (P [ 0.05). PC contents in intestine and muscle followed the same trend as that in serum and obtained the lowest values in fish fed the diets with the levels of thiamin from 0.79 to 2.65 mg kg-1 diet (P \ 0.05). MDA content in serum (R2 = 0.932;
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Fig. 1 Broken-line regression analysis of malondialdehyde (MDA) content (a) and protein carbonyl (PC) content (b) in serum for juvenile Jian carp (C. carpio var. Jian) fed diets containing graded levels of thiamin for 60 days
P \ 0.01), hepatopancreas (R2 = 0.926; P \ 0.01), intestine (R2 = 0.865; P \ 0.05) and muscle (R2 = 0.954; P \ 0.01) (Fig. 2), and PC content in serum (R2 = 0.883; P \ 0.05), hepatopancreas (R2 = 0.811; P \ 0.05), intestine (R2 = 0.897; P \ 0.05) and muscle (R2 = 0.918; P \ 0.01) (Fig. 3) showed significantly quadratic response to increasing dietary levels of thiamin. ASA and AHR capacities in serum, hepatopancreas, intestine and muscle ASA and AHR capacities in serum, hepatopancreas, intestine and muscle are presented in Table 3. Both in serum and hepatopancreas, ASA capacity was significantly increased with increasing dietary thiamin levels up to 0.79 mg kg-1 diet (P \ 0.05), and there were no significant differences with further increment in thiamin levels (P [ 0.05). ASA capacity in intestine and muscle was significantly increased with
Fish Physiol Biochem Table 2 Malondialdehyde content (MDA, nmol mL-1) and protein carbonyl content (PC, nmol mL-1) in serum, MDA content (nmol mg-1 protein) and PC content (nmol mg-1
protein) in hepatopancreas, intestine and muscle of Jian carp (C. carpio var. Jian) fed diet with graded levels of thiamin for 60 days
Dietary thiamin levels (mg kg-1 diet) 0.25
0.48
0.79
1.06
1.37
1.63
2.65
Serum MDA PC
15.2 ± 0.39b
14.5 ± 0.54b
d
c
11.6 ± 0.22
14.2 ± 0.47ab
9.69 ± 0.23
7.49 ± 0.33
b
13.4 ± 1.0a
13.4 ± 0.60a ab
7.24 ± 0.26
7.35 ± 0.24
ab
13.5 ± 0.71a
13.3 ± 0.91a
a
7.09 ± 0.18a
7.13 ± 0.12
Hepatopancreas MDA PC
12.1 ± 0.32d
6.39 ± 0.21b c
4.97 ± 0.14a b
4.79 ± 0.11a ab
2.08 ± 0.107a
2.06 ± 0.095a
1.93 ± 0.103a
a
a
1.26 ± 0.173
1.20 ± 0.171a
1.94 ± 0.156a
1.93 ± 0.143a
1.58 ± 0.146
1.55 ± 0.087
Intestine MDA 4.30 ± 0.321d
2.99 ± 0.140c
2.32 ± 0.138b
2.12 ± 0.125ab
c
b
a
1.93 ± 0.129
ab
4.73 ± 0.21a 1.43 ± 0.137a
1.62 ± 0.073
a
4.90 ± 0.15a 1.50 ± 0.140
1.78 ± 0.076
PC
2.39 ± 0.091
8.62 ± 0.22c d
1.59 ± 0.110
1.35 ± 0.055
1.34 ± 0.106
1.28 ± 0.056
3.21 ± 0.255c
2.74 ± 0.206b
2.16 ± 0.140a
1.97 ± 0.137a
ab
Muscle MDA PC
4.37 ± 0.218d 1.79 ± 0.07
c
b
1.53 ± 0.06
1.23 ± 0.11
a
a
1.18 ± 0.10
1.16 ± 0.14
a
a
1.14 ± 0.13
1.11 ± 0.15a
Values are mean ± SD of three groups of fish, with 5 fish in each group. Mean values with the different superscripts in the same row are significantly different (P \ 0.05)
Fig. 2 Regression analysis and significance of graded level of dietary thiamin and malondialdehyde content (MDA) of Jian carp (C. carpio var. Jian)
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Fig. 3 Regression analysis and significance of graded level of dietary thiamin and protein carbonyl content (PC) of Jian carp (C. carpio var. Jian) Table 3 Anti-superoxide anion capacities (ASA, U mL-1) and anti-hydroxyl radical capacities (AHR, U mL-1) in serum, ASA capacities (U mg-1 protein) and AHR capacities (U mg-1
protein) in hepatopancreas, intestine and muscle of Jian carp (C. carpio var. Jian) fed diet with graded levels of thiamin for 60 days
Dietary thiamin levels (mg kg-1 diet) 0.25
0.48
0.79
1.06
1.37
1.63
2.65
Serum 357 ± 10.6a
ASA AHR
1,963 ± 151
a
460 ± 10.3b
500 ± 20.2c
513 ± 22.2c
508 ± 19.3c
b
b
2,291 ± 87.5
2,300 ± 83.6
b
b
184.4 ± 7.2c
181.7 ± 4.0c
2,319 ± 78.2
521 ± 27.1c b
502 ± 21.8c
2,366 ± 62.7
2,412 ± 122
2,291 ± 87.5b
179.8 ± 7.6c
181.9 ± 5.9c
185.3 ± 8.9c
Hepatopancreas ASA
153.6 ± 9.5a
169.3 ± 10.5b
AHR
a
ab
128.4 ± 8.9
142.6 ± 8.3
ab
142.2 ± 7.6
150.4 ± 3.4
b
b
b
145.3 ± 15
149.9 ± 16b
154.1 ± 14
Intestine 145 ± 13.9a
ASA AHR
109.6 ± 8.2
a
168 ± 8.0b 131.0 ± 12.7
167 ± 7.3b b
169 ± 11.4b b
136.0 ± 12.6
133.8 ± 6.2
b
173 ± 11.1b b
136.1 ± 8.3
172 ± 6.7b 129.1 ± 11.3
163 ± 6.8b b
134.5 ± 12.3b
Muscle ASA
76.6 ± 8.5a
AHR
128 ± 17.0
119 ± 9.8b a
137 ± 13.4
123 ± 10.5b ab
ab
142 ± 9.7
121 ± 8.2b 152 ± 10.9
b
126 ± 10.2b
128 ± 11.5b
b
b
151 ± 13.9
154 ± 16.2
132 ± 9.7b 153 ± 14.5b
Values are mean ± SD of three groups of fish, with 5 fish in each group. Mean values with the different superscripts in the same row are significantly different (P \ 0.05)
increasing dietary thiamin levels up to 0.48 mg kg-1 diet (P \ 0.05) and then plateaued (P [ 0.05). AHR capacities in serum and intestine were improved with increasing dietary thiamin levels up to 0.48 mg kg-1
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diet (P \ 0.05), and no significant differences were found with further increase in thiamin levels (P [ 0.05). In hepatopancreas and muscle, AHR capacity was the highest for fish fed diet containing
Fish Physiol Biochem
Fig. 4 Regression analysis and significance of graded level of dietary thiamin and the anti-hydroxyl radical capacities (AHR) of Jian carp (C. carpio var. Jian)
thiamin C1.06 mg kg-1 diet and higher than fish fed thiamin-unsupplemented group (P \ 0.05). There were significantly quadratic regression relationship between the AHR capacity in hepatopancreas (R2 = 0.814; P \ 0.05) and muscle (R2 = 0.964; P \ 0.01) and the increased dietary concentrations of thiamin (Fig. 4).
activities in serum and hepatopancreas were significantly enhanced with increasing dietary thiamin levels up to 0.79 and 0.48 mg kg-1 diet, respectively (P \ 0.05), and plateaued thereafter (P [ 0.05). The activity of SOD in intestine followed a similar pattern to that in hepatopancreas. Dietary thiamin had no effects on SOD activity in muscle (P [ 0.05). CAT activity in serum was significantly increased with increasing dietary thiamin levels up to 0.79 mg kg-1 diet (P \ 0.05), and no differences were found with further increase in thiamin levels (P [ 0.05). The activities of CAT in hepatopancreas, intestine and muscle were the lowest for fish fed 0.25 mg thiamin kg-1 diet
SOD and CAT activities in serum, hepatopancreas, intestine and muscle SOD and CAT activities in serum, hepatopancreas, intestine and muscle are shown in Table 4. SOD Table 4 The activities superoxide dismutase (SOD, U mL-1) and catalase (CAT, U mL-1) in serum, SOD activities (U mg-1 protein) and CAT activities (U mg-1 protein) in
hepatopancreas, intestine and muscle of Jian carp (C. carpio var. Jian) fed diet with graded levels of thiamin for 60 days
Dietary thiamin levels (mg kg-1 diet) 0.25
0.48
0.79
1.06
1.37
1.63
2.65
Serum SOD
33.2 ± 3.83a
44.4 ± 3.72b
76.3 ± 3.81c
77.2 ± 5.89c
81.5 ± 5.81c
78.0 ± 6.54c
78.9 ± 8.29c
CAT
a
b
c
c
c
c
17.6 ± 0.60
18.5 ± 0.62c
10.4 ± 0.95
15.6 ± 0.93
17.8 ± 0.94
18.1 ± 1.13
18.3 ± 0.92
90.9 ± 3.18b
91.4 ± 4.02b
93.5 ± 4.37b
91.2 ± 2.58b
92.1 ± 2.66b
91.3 ± 2.92b
b
b
b
b
32.53 ± 1.73b
Hepatopancreas SOD
83.8 ± 4.91a
CAT
a
28.65 ± 2.98
ab
30.67 ± 2.01
32.73 ± 1.44
31.86 ± 2.24
32.52 ± 2.12
32.19 ± 2.64
Intestine SOD
71.1 ± 5.91a
81.5 ± 6.25b
84.6 ± 3.11b
83.8 ± 3.10b
83.3 ± 2.34b
82.0 ± 3.27b
84.4 ± 4.84b
CAT
a
b
b
b
b
b
23.9 ± 1.52
23.6 ± 1.87b
18.4 ± 1.98
23.5 ± 1.11
23.6 ± 1.54
23.8 ± 1.59
24.0 ± 2.01
Muscle SOD
37.93 ± 3.74a
38.78 ± 2.87a
40.20 ± 2.07a
39.56 ± 1.64a
39.23 ± 1.91a
38.77 ± 1.61a
39.84 ± 2.72a
CAT
a
b
b
b
b
b
20.8 ± 1.70b
14.5 ± 1.71
18.9 ± 2.23
19.6 ± 1.98
20.5 ± 2.88
20.6 ± 1.82
21.1 ± 1.15
Values are mean ± SD of three groups of fish, with 5 fish in each group. Mean values with the different superscripts in the same row are significantly different (P \ 0.05)
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Fish Physiol Biochem
Fig. 5 Regression analysis and significance of graded level of dietary thiamin and superoxide dismutase (SOD) and catalase (CAT) of Jian carp (C. carpio var. Jian)
(P \ 0.05), and no significant difference were observed in other groups (P [ 0.05). The activity of SOD in serum (R2 = 0.888; P \ 0.05) and CAT in muscle (R2 = 0.840; P \ 0.05) showed significantly quadratic response to graded levels of dietary thiamin (Fig. 5). GPx, GST and GR activities in serum, hepatopancreas, intestine and muscle The activities of GPx, GST and GR in serum, hepatopancreas, intestine and muscle are shown in Table 5. GPx activity in serum was the highest for fish fed diets containing 2.65 mg thiamin kg-1 diet and the lowest for fish fed the thiamin-unsupplemented diet (P \ 0.05). The activity of GPx in intestine followed a similar pattern to that in serum. Both in hepatopancreas and muscle, GPx activities were significantly increased with increasing dietary thiamin levels up to 0.79 mg kg-1 diet (P \ 0.05), and no differences were observed with further increase in thiamin levels (P [ 0.05). The activities of GST in serum, hepatopancreas and muscle were higher in fish fed diet supplemented with thiamin than those fed the control diet (P \ 0.05). GST activity in intestine was significantly improved with increasing dietary thiamin levels up to 0.48 mg kg-1 diet (P \ 0.05) and plateaued thereafter (P [ 0.05). GR activities in serum and hepatopancreas were significantly improved with increasing dietary thiamin levels up to 0.79 and 1.06 mg kg-1 diet, respectively (P \ 0.05), and there were no differences with further increase in thiamin levels (P [ 0.05). The activities of GR in intestine and muscle also followed the same pattern to that in serum. The activity of GR in serum (R2 = 0.904; P \ 0.01)
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and hepatopancreas (R2 = 0.899; P = 0.01); GPx activity in serum (R2 = 0.894; P \ 0.05), intestine (R2 = 0.936; P \ 0.01), hepatopancreas (R2 = 0.842; P \ 0.05) and muscle (R2 = 0.809; P \ 0.05); and GST activity in serum (R2 = 0.885; P \ 0.05), hepatopancreas (R2 = 0.891; P \ 0.05) and muscle (R2 = 0.855; P \ 0.05) showed significantly quadratic response to graded levels of dietary thiamin (Fig. 6). GSH content in serum, hepatopancreas, intestine and muscle GSH contents in serum, hepatopancreas, intestine and muscle are shown in Table 6. GSH contents in serum and muscle were significantly increased with increasing dietary thiamin levels up to 1.06 and 0.79 mg kg-1 diet, respectively (P \ 0.05), and then plateaued (P [ 0.05). The levels of GSH in hepatopancreas and intestine also followed a similar pattern to that in serum. The GSH content in serum (R2 = 0.965; P \ 0.01), hepatopancreas (R2 = 0.960; P \ 0.01) and intestine (R2 = 0.978; P \ 0.01) showed significantly quadratic response to graded levels of dietary thiamin (Fig. 7). Thiamin requirement As shown in Fig. 1a, the dietary thiamin levels estimated by the broken-line model based on serum MDA content was 1.07 mg kg-1 diet. The regression equation was as follows: Y = -2.0624x ? 15.655, R2 = 0.9604; Ymax = 13.4 (Fig. 1a). The PC content in serum to dietary levels of thiamin relationship was
Fish Physiol Biochem protein), GST (U mg-1 protein) and GR (U g-1 protein) in hepatopancreas, intestine and muscle of Jian carp (C. carpio var. Jian) fed diet with graded levels of thiamin for 60 days
Table 5 The activities of glutathione peroxidase (GPx, U 0.1 mL-1), glutathione S-transferase (GST, U mL-1) and glutathione reductase (GR, U L-1) in serum, GPx (U mg-1 Dietary thiamin levels (mg kg-1 diet) 0.25
0.48
0.79
1.06
1.37
1.63
2.65
Serum GPx
327 ± 14.9a
433 ± 10.7b
568 ± 12.0c
GST
a
201 ± 11.5
a
b
185 ± 22.0
b
GR
198.4 ± 11.7
a
145 ± 22.1
229 ± 10.8
c
580 ± 18.8
cd
302 ± 23.4
cd c
241 ± 28.4
250 ± 34.0
544 ± 16.5c
584 ± 11.2
cd
c
293 ± 10.9
c
257 ± 21.7
590 ± 22.3
cd
c
600 ± 29.7d 314 ± 14.7d
294 ± 5.8
c
257 ± 21.9c
558 ± 15.8c
564 ± 20.6c
249.0 ± 33.5
Hepatopancreas GPx
438 ± 16.9a
481 ± 21.8b
554 ± 18.0c
GST GR
a
133 ± 7.36 92.2 ± 7.65a
b
162 ± 7.87 136 ± 10.5b
b
168 ± 10.0 151 ± 15.0c
204 ± 10.5 170 ± 11.5d
192 ± 4.37 173 ± 10.2d
GPx
382 ± 21.0a
413 ± 19.5b
499 ± 17.8c
500 ± 15.4c
517 ± 16.3
GST
a
203 ± 7.61
b
b
213 ± 9.61
b
97.6 ± 7.52
139 ± 7.91
b
171 ± 16.1
169 ± 14.4
c
GPx
405 ± 29.1a
463 ± 24.7b
557 ± 20.1c
547 ± 17.5c
GST
a
97.1 ± 7.63
109 ± 10.7
GR
49.4 ± 10.0a
109 ± 11.1b
cd
541 ± 16.0c c
cd
207 ± 11.0d 171 ± 16.0d
cd
536 ± 19.5d
199 ± 8.46 168 ± 7.43d
Intestine
GR
139 ± 15.6
a
204 ± 8.24
c
cd
b
525 ± 16.6
b
207 ± 12.1b
179 ± 10.5
c
181 ± 10.7
178 ± 6.67c
545 ± 12.8c
545 ± 12.6c
568 ± 23.3c
206 ± 10.5
c
199 ± 8.23
Muscle ab
bcd
bcd
115 ± 11.8
122 ± 10.3
148 ± 10.9c
138 ± 10.3c
bc
cd
114 ± 8.82
123 ± 6.23
139 ± 10.1c
150 ± 15.3c
129 ± 11.3d 149 ± 20.9c
Values are mean ± SD of three groups of fish, with 5 fish in each group. Mean values with the different superscripts in the same row are significantly different (P \ 0.05)
described by broken-line model [Y = -5.5517x ? 12.586, R2 = 0.920; Ymax = 7.20 (Fig. 1b)]. Based on the above equation, the optimum thiamin level was estimated to be 0.95 mg kg-1 diet.
Discussion The present research used the same animal trial as our previous study by Huang et al. (2011). Our previous study has estimated thiamin requirement of juvenile Jian carp (1.02 mg kg-1 diet) based on percent weight gain and showed that thiamin promoted growth, protein deposition and enhanced digestive and absorption capacity of fish (Huang et al. 2011). As we known, the function of digestive tract and growth of tissues is partly dependent on the structural integrity of fish (Lushchak et al. 2001; Enesco and Leblond 1962). The high degree of unsaturation x-3 fatty acids in fish cell membrane and muscle predisposes them to oxidative stress (Martinez-Alvarez et al. 2005). A number of studies have shown that ascorbic acid (Lee and Dabrowski 2003), myo-inositol (Jiang et al. 2009b)
and pyridoxine (Hu et al. 2011) could improve antioxidant defence in fish. Study in grouper has showed that thiamin decreased the liver lipid peroxidation (Huang et al. 2007). Therefore, the present study investigated the effects of dietary thiamin on oxidative stress and antioxidant responses in serum, hepatopancreas, intestine and muscle of juvenile Jian carp. Malondialdehyde (MDA) content is known to reflect lipid peroxidation, which can be defined as an important consequence of oxidative deterioration of lipids (Lee and Dabrowski 2003). Meanwhile, protein damage can also be directly induced by reactive oxygen species (ROS) (Nordberg and Arne´r 2001). Protein carbonyl (PC) content is regarded as a marker of protein oxidation (Armenteros et al. 2009). The present study demonstrated that MDA and PC contents in serum, hepatopancreas, intestine and muscle were gradually decreased with increasing dietary thiamin levels up to certain values and then remained constant, which was in agreement with the result in juvenile grouper (Huang et al. 2007). This may attribute that thiamin is a water-soluble vitamin, and fish can
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Fish Physiol Biochem
123
Fish Physiol Biochem b Fig. 6 Regression analysis and significance of graded level of
thiamin inhibiting lipid peroxidation and protein oxidation may be that it blocked generation of detrimental intermediary metabolite in fish. Glyoxal promoted lipid peroxidation in hepatocytes of rats (Shangari and O’Brien 2004). In human red blood cells, thiamin decreased the formation of glyoxal through increase the activity of transketolase (Thornalley et al. 2001). Besides, advanced glycation end (AGE) products increased MDA content in vessel wall
dietary thiamin and glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) of Jian carp (Cyprinus carpio var. Jian)
maintain the homeostasis through increasing excretion after feeding an overdose of thiamin (Lall and Tibbetts 2009). However, little information is available about the effects of thiamin on the lipid peroxidation and protein oxidation in fish. The benefit effects of dietary
Table 6 Reduced glutathione (GSH, mg L-1) contents in serum, GSH (mg g-1 protein) contents in hepatopancreas, intestine and muscle of Jian carp (C. carpio var. Jian) fed diet with graded levels of thiamin for 60 days Dietary thiamin levels (mg kg-1 diet) 0.25 Serum Hepatopancreas Intestine Muscle
0.48
0.79
1.06
1.37
1.63
2.65
62.8 ± 2.16a
79.4 ± 4.86b
97.2 ± 2.94c
108 ± 2.96d
110 ± 2.65d
111 ± 2.16d
112 ± 2.62d
a
b
c
d
d
d
27.4 ± 0.42d
d
22.6 ± 0.49d 19.3 ± 0.67c
18.5 ± 0.82
a
15.8 ± 0.77 14.8 ± 1.26a
20.3 ± 0.90
b
18.0 ± 0.56 16.8 ± 0.89b
24.3 ± 0.60
c
19.4 ± 0.71 18.6 ± 0.44c
27.3 ± 0.49
d
22.0 ± 0.64 18.5 ± 0.48c
27.0 ± 0.34
d
22.5 ± 0.41 18.5 ± 0.35c
27.7 ± 0.48
22.7 ± 0.34 18.9 ± 0.26c
Values are mean ± SD of three groups of fish, with 5 fish in each group. Mean values with the different superscripts in the same row are significantly different (P \ 0.05)
Fig. 7 Regression analysis and significance of graded level of dietary thiamin and reduced glutathione content (GSH) of Jian carp (C. carpio var. Jian)
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Fish Physiol Biochem
of bovine (Yan et al. 1994). In human endothelial cells, thiamin in its active form can inhibit AGE formation (Beltramo et al. 2008). Calingasan and Gibson (2000) reported that nitric oxide (NO) lead to protein oxidation in mice brain. In humans, thiamin decreased production of NO in endothelial cells (Hazell and Butterworth 2009). Therefore, the mechanism by which dietary thiamin alleviates oxidative damage of lipid and protein needs further investigation. Based on serum MDA and PC content data, the dietary thiamin requirements of juvenile Jian carp were estimated to be 1.07 and 0.95 mg kg-1 diet, respectively, which were similar to thiamin requirement (1.02 mg kg-1) based on percent weight gain reported by Huang et al. (2011). Oxidative injury of lipids and proteins is mostly induced by cellular ROS in vertebrate animals (Knight 2000). Superoxide anions and hydroxyl radicals are the most potent ROS and can cause a wide range of oxidative damage within the cell (Kohen and Nyska 2002). In our study, with the increasing of dietary thiamin levels, the scavenging ability against the superoxide anion and the hydroxyl radical was improved with increasing thiamin levels up to certain values and then plateaued in serum, hepatopancreas, intestine and muscle, suggesting thiamin promoted the elimination of ROS in fish. Thiamin may act as superoxide anion and hydroxyl radical scavenger directly or indirectly in fish. Firstly, thiamin interacts directly with superoxide and peroxide radicals in vitro (Jung and Kim 2003; Lukienko et al. 2000). The scavenging effects of thiamin are probably related to successive transfer of (2H? ? 2e-) from the NH2 group of the pyrimidine ring to radicals (Lukienko et al. 2000). Secondly, thiamin may scavenge free radicals through inhibiting heme oxygenase-1 activity and AGE products accumulation in fish. Heme oxygenase-1 induces formation of hydroxyl radicals through the Fenton reaction in rat mesangial cells (Sandau et al. 1998). Calingasan et al. (1999) reported that thiamin prevented heme oxygenase-1 accumulation in brain of rat. Besides, AGE products enhanced the generation of reactive oxygen intermediates in vessel wall of bovine (Yan et al. 1994). Thiamin in its active form inhibited AGE formation on bovine serum albumin (Beltramo et al. 2008). Thirdly, thiamin may inhibit glutamate N-methyl-D-aspartate (NMDA)
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receptor activation for free radical production in fish. It has been shown that activation of NMDA receptor in neuronal cultures led to the formation of superoxide radicals (Langlais et al. 1997). In rat, thiamin inhibited glutamate NMDA receptors activation via preventing glutamate transports in brain (Bondy and Lee 1993; Hazell et al. 2001). Fish are susceptible to the attack of reactive ROS and have developed antioxidant defence system, including enzymatic and non-enzymatic defences (Martinez-Alvarez et al. 2005). A major component of the antioxidant system in fish consists of antioxidant enzymes, such as SOD, CAT, GPx and GST (Zhou et al. 2011). SOD can dismutate two O2 into H2O2 and O2 and can protect cells from the damage induced by O2 (Zhou et al. 2012). Data in the present study showed that with dietary thiamin levels up to 0.79, 0.48 and 0.48 mg kg-1, SOD activities were markedly increased in serum, hepatopancreas and intestine, while activity of SOD in muscle was not affected by thiamin. The end product of the dismutation reaction, H2O2, can be removed by enzymes CAT and GPx (Kohen and Nyska 2002). GST is an enzymatic multifamily that is able to detoxify xenobiotics containing reactive electrophilic compounds to facilitate their excretion from cells (Lushchak et al. 2001). The present study also showed that the activities of CAT, GPx and GST were increased with increasing dietary thiamin levels up to certain values and then remained constant in serum, hepatopancreas, intestine and muscle. The results suggested that the increases in the activities of these enzymes, noticed in the certain thiamin levels, were sufficient to prevent the rise of lipid peroxidation and protein oxidation. As we known, few studies have yet investigated the effect of thiamin on antioxidant enzyme activities in fish. Huang et al. (2007) reported that the SOD activity was not affected by thiamin in liver of grouper. This may be attributed that SOD is not a main factor affecting oxidative stress in grouper liver. In mammals, thiamin can affect antioxidant enzyme activities in several ways. Firstly, thiamin may correct GPx activity via blockade of triosephosphate accumulation. In bovine erythrocytes, the activity of GPx was inactivated by methylglyoxal, which is derived from triosephosphates (Park et al. 2003). Shangari et al. (2003) reported that thiamin inhibited methylglyoxal
Fish Physiol Biochem
generation via disposal of excess triosephosphates in rat hepatocytes. Furthermore, in rat liver, insulin administration resulted in an increase in Cu/Zn-SOD and CAT activities (Sindhu et al. 2004). Rathanaswami et al. (1991) reported that thiamin elevated insulin secretion in pancreatic islets of rats. Thirdly, thiamin may increase antioxidant enzyme activities by suppression of the expression of the transcription factor p53. It has been reported that p53 repressed the antioxidant gene transcription by binding to the antioxidant response cis elements (AREs) in mouse Hepa1-6 cells (Faraonio et al. 2006). Yang et al. (2004) reported that thiamin could decrease the p53 expression in retina neurons of rats. Taken together, dietary thiamin stimulation of antioxidant enzyme activities of fish may be partly related to these pathways, which needs further investigation. As non-enzymatic antioxidant, reduced GSH is critical to resistance of oxidative stress in organisms (Vardi et al. 2008). In the present study, dietary supplementation of thiamin improved GSH contents levels up to certain values in serum, hepatopancreas, intestine and muscle. To our knowledge, few studies have been reported on the relationship between dietary thiamin and GSH contents in fish. In rats, thiamin increased reduced GSH contents in hepatocytes (Shangari et al. 2007). On the one hand, thiamin may promote GSH synthesis via elevating glutamate concentration. In rat brain, thiamin deficiency caused impairment of glutamate transport and decreased glutamate uptake (Hazell et al. 2001). Glutamate is a constituent amino acid of GSH and necessary for GSH synthesis in enteron of piglets (Stoll et al. 1998). On the other hand, thiamin may promote reduced GSH regeneration from oxidized GSH (GSSG). In goldfish, GR catalyzes the reduction in the oxidized form of glutathione, at the expense of the NADPH (Lushchak et al. 2001). In our study, GR activities were enhanced by dietary thiamin levels up to certain values and then plateaued in serum, hepatopancreas, intestine and muscle. Correlation analysis showed that GSH content was positively correlated with GR activity in serum (r = ?0.986; P \ 0.01), hepatopancreas (r = ?0.948; P \ 0.01), intestine (r = ?0.929; P \ 0.01) and muscle (r = ?0.986; P \ 0.01), respectively. Meanwhile, thiamin increased the activity transketolase, a rate-limiting enzyme of the pentose phosphate pathway, which plays a major role in the production of NADPH for maintaining GSH levels in rat
(Shangari et al. 2007). Nevertheless, the underlying mechanism needs further study. In conclusion, our results show that dietary thiamin supplementation inhibits lipid peroxidation and protein oxidation in fish. The protective effect of thiamin may be attributed to the enhanced free radical scavenging ability or to improved enzymatic and non-enzymatic antioxidant defences. However, the mechanism of thiamin functioned as an antioxidant in fish needs further investigation. Acknowledgments This research was financially supported by the National Department Public Benefit Research Foundation (Agriculture) of China (201003020) and Science and Technology Support Program of Sichuan Province (2011NZ0071). The authors would like to thank the personnel of these teams for their kind assistance.
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The effects of dietary thiamin on oxidative damage and antioxidant defence of juvenile fish Xue-Yin Li • Hui-Hua Huang • Kai Hu • Yang Liu • Wei-Dan Jiang • Jun Jiang • Shu-Hong Li • Lin Feng • Xiao-Qiu Zhou
Received: 31 December 2012 / Accepted: 3 October 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The present study explored the effects of thiamin on antioxidant capacity of juvenile Jian carp (Cyprinus carpio var. Jian). In a 60-day feeding trial, a total of 1,050 juvenile Jian carp (8.20 ± 0.02 g) were fed graded levels of thiamin at 0.25, 0.48, 0.79, 1.06, 1.37, 1.63 and 2.65 mg thiamin kg-1 diets. The results showed that malondialdehyde and protein carbonyl contents in serum, hepatopancreas, intestine and muscle were significantly decreased with increasing dietary thiamin levels (P \ 0.05). Conversely, the anti-superoxide anion capacity and anti-hydroxyl radical capacity in serum, hepatopancreas, intestine and muscle were the lowest in fish fed the thiaminunsupplemented diet. Meanwhile, the activities of catalase (CAT), glutathione peroxidase, glutathione
X.-Y. Li H.-H. Huang K. Hu Y. Liu W.-D. Jiang J. Jiang S.-H. Li L. Feng (&) X.-Q. Zhou (&) Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China e-mail: [email protected] X.-Q. Zhou e-mail: [email protected]; [email protected]; [email protected] X.-Y. Li H.-H. Huang K. Hu Y. Liu W.-D. Jiang J. Jiang L. Feng X.-Q. Zhou Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China
S-transferase and glutathione reductase, and the contents of glutathione in serum, hepatopancreas, intestine and muscle were enhanced with increasing dietary thiamin levels (P \ 0.05). Superoxide dismutase (SOD) activity in serum, hepatopancreas and intestine followed a similar trend as CAT (P \ 0.05). However, SOD activity in muscle was not affected by dietary thiamin level (P [ 0.05). The results indicated that thiamin could improve antioxidant defence and inhibit lipid peroxidation and protein oxidation of juvenile Jian carp. Keywords Cyprinus carpio var. Jian Thiamin Oxidative damage Antioxidant Antioxidant enzyme
Introduction Thiamin has been demonstrated to be an essential dietary nutrient for fish (Aoe et al. 1969). Thiamin pyrophosphate is the major coenzyme form of thiamin, which is involved in several enzymatic steps in energy production, including oxidative decarboxylation of aketo acids and transketolation reactions in the pentose phosphate pathway (NRC 2011). Thiamin deficiency caused muscle atrophy in Chinook salmon (Halver 1957) and hemorrhage of fins in eel (Hashimoto et al. 1970). Researchers had reported that the thiamin requirement of common carp (Aoe et al. 1969),
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channel catfish (Murai and Andrews 1978) and rainbow trout (Morito et al. 1986) were 0.5, 1.0 and 1.0 mg kg-1 diet, respectively, based on weight gain or absence of deficiency signs. A previous study from our laboratory demonstrated that thiamin deficiency decreased growth and survival, induced congestion of the fins and skin, caused atrophy of intestine and hepatopancreas and decreased the activities of digestive and brush border enzymes of juvenile Jian carp (Cyprinus carpio var. Jian) (Huang et al. 2011). The disturbance of growth and abnormal digestive and absorptive function may be related to the structural integrity of cells in fish (Lushchak et al. 2001). The cell membrane integrity and functionality is associated with the oxidant–antioxidant balance (Knight 2000). However, only few studies have been reported about the relationship between thiamin and antioxidant defence in fish. Thiamin effectively inhibited lipid peroxidation of liver in mouse and fish (Huang et al. 2007; Lukienko et al. 2000), suggesting that dietary thiamin may be related to antioxidant status of animal, which needs further investigation. Superoxide anions (O2 ) and hydroxyl radicals ( OH) are the most reactive oxygen radicals and capable of damaging cellular macromolecules, such as membrane lipids, proteins and nuclei acids (Nordberg and Arne´r 2001; Calingasan et al. 1999). Like all aerobic organisms, antioxidant systems in fish contain antioxidant enzymes and low molecular weight antioxidants (Kohen and Nyska 2002). The enzymatic antioxidant includes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), and non-enzymatic antioxidants, such as glutathione (GSH) (Knight 2000). In vitro, thiamin can scavenge O2 and OH directly (Jung and Kim 2003). Furthermore, it has been reported that thiamin interacted with free radicals and hydroperoxides through transferring (2H? ? 2e-) from the NH2 group of the pyrimidine ring to radicals in vitro (Lukienko et al. 2000). In rats, thiamin increased insulin secretion of pancreas, which resulted in an increase in Cu/Zn-SOD and CAT activities in liver (Rathanaswami et al. 1991; Sindhu et al. 2004). Meanwhile, the regeneration of reduced GSH required NADPH providing reducing equivalents. Thiamin is a cofactor of transketolase, which is a key enzyme of pentose phosphate pathway for the production of NADPH in rat (Shangari et al. 2007). These
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observations lead to the idea that the improvement of structure and function of fish tissues by thiamin may be partly related to the antioxidant defence, which needs further investigation. To date, few studies have been conducted to investigate the effects of thiamin on lipid peroxidation, protein oxidation and the antioxidant enzyme activities in fish. Therefore, the objective of this study was to explore the effects of dietary thiamin on the antioxidant capacity in hepatopancreas, intestines and muscle of juvenile Jian carp. This study was a part of a larger study that involved in the determination of the effects of thiamin on growth performance, digestive and absorptive abilities of fish (Huang et al. 2011). The graded levels of dietary thiamin were designed according to dietary thiamin requirement (0.5–1.0 mg kg-1) of finfish (Aoe et al. 1969; Morito et al. 1986; Murai and Andrews 1978). The study can partly provide theoretical evidence for the effects of thiamin on growth, digestive and absorptive ability in fish. Materials and methods Experimental design and diets Formulation of basal diet is present in Table 1. Experimental diets and the procedures for diet preparation were the same as Huang et al. (2011). The basal diet was formulated to meet the nutrient requirements of Jian carp based on our laboratory’s studies (He et al. 2009; Jiang et al. 2009b; Li et al. 2010; Wen et al. 2009; Zhou et al. 2008) and contained 307.8 g crude protein kg-1 diet and 64.8 g crude lipid kg-1 diet. The basal diet contained 0.21 mg thiamin kg-1 diet, which was calculated according to the analyzed thiamin concentrations of ingredients. Thiamin hydrochloride (Sigma, St Louis, USA) was added to the test diets to provide graded concentrations of thiamin. The thiamin concentrations of the seven diets were determined by the fluorometric method of AOAC (1990) to be 0.25, 0.48, 0.79, 1.06, 1.37, 1.63 and 2.65 mg thiamin kg-1 diet, respectively. After preparation, the diets were stored at -20 °C until used according to Hu et al. (2008). Feeding management Juvenile Jian carp obtained from the Ya’an fisheries were acclimatized to the experimental environment
Fish Physiol Biochem Table 1 Composition of the basal diet Ingredients
g kg-1
Nutrients content
g kg-1
Fish meal
226.5
Crude protein
307.8
Casein
148.5
Gelatin
60.0
Crude lipid
64.8
Lys
22.9
a-Starch
283.0
Methionine ? cystine
13.6
Corn starch
190.3
n-3
10.0
Fish oil
10.9
n-6
10.0
Soybean oil
18.9
DL-Met
3.2
Cellulose
20.0
Ca (H2PO4)2
16.9
Choline chloride Ethoxyquin
and reduce ammonia concentrations. Water temperature was maintained at 25.0 ± 1 °C. Additional incandescent lighting provided a diurnal light/dark cycle of 12:12 h, which was similar to Deng et al. (2010). For the feeding trial, each of seven experimental diets was fed to triplicate groups of fish six times every day. Thirty minutes after the feeding, uneaten feed was removed by siphoning. The feeding experiment lasted 60 days. Feeding management was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Animal Nutrition Institute, Sichuan Agricultural University. Sample collection and analysis
1.3 0.5
Vitamin mixture(thiamin free)a
10.0
Mineral mixtureb
10.0
Thiamin premixc
–
Analyzed chemical composition of basal diet (g kg-1): crude protein, 307.8; lipid, 64.8; ash, 65.8 a
Vitamin mixture (g kg-1 mixture): retinyl acetate (500,000 IU g-1), 0.800 g; cholecalciferol (500,000 IU g-1), 0.480 g; DL-a-tocopherol acetate (500 g kg-1), 20.000 g; menadione (500 g kg-1), 0.200 g; riboflavin (800 g kg-1), 0.625 g; pyridoxine hydrochloride (980 g kg-1), 0.755 g; 0.010 g; ascorbyl-2cyanocobalamin (100 g kg-1), 19.029 g; calcium-Dpolyphosphate (350 g kg-1), pantothenate (980 g kg-1), 2.511 g; niacin (980 g kg-1), 2.857 g; D-biotin (200 g kg-1), 0.500 g; meso-inositol (980 g kg-1), 52.857 g; folic acid (960 g kg-1), 0.521 g. All ingredients were diluted with corn starch to 1 kg b
Mineral mixture (g kg-1 mixture): FeSO47H2O -1 Fe), 76.147 g; CuSO45H2O (250.0 g kg-1 (197.0 g kg Cu), 1.20 g; MnSO4H2O (318.0 g kg-1 Mn), 4.59 g; ZnSO47H2O (225.0 g kg-1 Zn), 13.34 g; KI (38.0 g kg-1 I), 2.90 g; NaSeO3 (10.0 g kg-1 Se), 2.50 g. All ingredients were diluted with CaCO3 to 1 kg c
Thiamin premix: premix was added to obtain graded levels of thiamin. The analyzed thiamin levels of the diets were: 0.25, 0.48, 0.79, 1.06, 1.37, 1.63 and 2.65 mg kg-1 diet, respectively
for 4 weeks. At the end of acclimatization period, a total of 1,050 carp with an initial weight of 8.20 ± 0.02 g were randomly distributed into each of 21 glass aquaria (90 9 30 9 40 cm). The aquarium was connected to a closed recirculating water system and oxygen auto-supplemented system. Water circulated at 1.2 L min-1 in each aquarium, and the water was drained through biofilters to remove impurities
Fish in each cage were weighed and counted at the initiation and termination of the feeding trial. At the end of the feeding trial, 15 fish from each aquarium were anaesthetized with 50 mg L-1 benzocaine as described by Berdikova Bohne et al. (2007) after 12 h of the last feeding. Blood of 15 fish from each aquarium was drawn from caudal vein, stored at 4 °C overnight and centrifuged at 3,0009g for 10 min. The serum was obtained for antioxidant parameters analysis. The intestine, hepatopancreas and muscle were quickly removed, weighed, frozen in liquid nitrogen and finally stored at -70 °C. Tissue samples were homogenized on ice in 10 volumes (w v-1) of ice-cold physiological saline and centrifuged at 6,0009g for 20 min at 4 °C; then, the supernatant was collected for antioxidant parameters analysis. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan Agricultural University. The protein concentration of samples was determined by the method of Bradford (1976). Malondialdehyde (MDA) content was analyzed as described by Livingstone et al. (1990) using the thiobarbituric acid (TBA) reaction. The content of protein carbonyl was determined by the formation of protein hydrazones using 2,4-dinitrophenylhydrazine (DNPH) (Halliwell and Whiteman 2009). The anti-superoxide anion (ASA) capacity and the anti-hydroxyl radical (AHR) capacity were assayed as described by Jiang et al. (2009a). Superoxide anion radicals were generated by the action of xanthine and xanthine oxidase. With the electron acceptor added, a coloration reaction is developed using the Griess reagent. The coloration degree is directly proportional to the quantity of superoxide anion in the reaction. Hydroxyl radicals are
123
Fish Physiol Biochem
generated in a Fenton reaction. With the electron acceptor added, a coloration reaction is developed using the Griess reagent. The coloration degree is directly proportional to the quantity of hydroxyl radicals in the reaction. The activities of SOD and GPx were assayed as described by Zhang et al. (2008). CAT activity was determined by the decomposition of hydrogen peroxide (Aebi 1984). Glutathione S-transferase (GST) activity was measured by monitoring the formation of adduct between GSH and 1-chloro-2,4dinitrobenzene (Lushchak et al. 2001). GR activity was measured according to a procedure described by Lora et al. (2004). Reduced GSH content was quantitated according to the method of Vardi et al. (2008). Calculation and statistical analysis The data were presented as mean ± SD. All data were subjected to one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test to determine significant differences among treatment groups at the level of P \ 0.05 through SPSS 13.0 (SPSS Inc., Chicago, IL, USA) (Fig. 1).
Results MDA and PC content in serum, hepatopancreas, intestine and muscle The MDA and PC content in serum, hepatopancreas, intestine and muscle of juvenile Jian carp were significantly affected by dietary thiamin level (Table 2). MDA content in serum was lower in fish fed C0.79 mg thiamin kg-1 diet than fish fed the diet with thiamin-unsupplemented group (P \ 0.05). The contents of MDA were significantly decreased with increasing dietary thiamin levels up to 1.06 mg kg-1 diet in hepatopancreas, intestine and muscle (P \ 0.05), and no significant differences were observed with further increase in thiamin levels (P [ 0.05). PC contents in serum and hepatopancreas were significantly decreased with increasing dietary thiamin levels up to 1.06 mg kg-1 diet (P \ 0.05) and then plateaued (P [ 0.05). PC contents in intestine and muscle followed the same trend as that in serum and obtained the lowest values in fish fed the diets with the levels of thiamin from 0.79 to 2.65 mg kg-1 diet (P \ 0.05). MDA content in serum (R2 = 0.932;
123
Fig. 1 Broken-line regression analysis of malondialdehyde (MDA) content (a) and protein carbonyl (PC) content (b) in serum for juvenile Jian carp (C. carpio var. Jian) fed diets containing graded levels of thiamin for 60 days
P \ 0.01), hepatopancreas (R2 = 0.926; P \ 0.01), intestine (R2 = 0.865; P \ 0.05) and muscle (R2 = 0.954; P \ 0.01) (Fig. 2), and PC content in serum (R2 = 0.883; P \ 0.05), hepatopancreas (R2 = 0.811; P \ 0.05), intestine (R2 = 0.897; P \ 0.05) and muscle (R2 = 0.918; P \ 0.01) (Fig. 3) showed significantly quadratic response to increasing dietary levels of thiamin. ASA and AHR capacities in serum, hepatopancreas, intestine and muscle ASA and AHR capacities in serum, hepatopancreas, intestine and muscle are presented in Table 3. Both in serum and hepatopancreas, ASA capacity was significantly increased with increasing dietary thiamin levels up to 0.79 mg kg-1 diet (P \ 0.05), and there were no significant differences with further increment in thiamin levels (P [ 0.05). ASA capacity in intestine and muscle was significantly increased with
Fish Physiol Biochem Table 2 Malondialdehyde content (MDA, nmol mL-1) and protein carbonyl content (PC, nmol mL-1) in serum, MDA content (nmol mg-1 protein) and PC content (nmol mg-1
protein) in hepatopancreas, intestine and muscle of Jian carp (C. carpio var. Jian) fed diet with graded levels of thiamin for 60 days
Dietary thiamin levels (mg kg-1 diet) 0.25
0.48
0.79
1.06
1.37
1.63
2.65
Serum MDA PC
15.2 ± 0.39b
14.5 ± 0.54b
d
c
11.6 ± 0.22
14.2 ± 0.47ab
9.69 ± 0.23
7.49 ± 0.33
b
13.4 ± 1.0a
13.4 ± 0.60a ab
7.24 ± 0.26
7.35 ± 0.24
ab
13.5 ± 0.71a
13.3 ± 0.91a
a
7.09 ± 0.18a
7.13 ± 0.12
Hepatopancreas MDA PC
12.1 ± 0.32d
6.39 ± 0.21b c
4.97 ± 0.14a b
4.79 ± 0.11a ab
2.08 ± 0.107a
2.06 ± 0.095a
1.93 ± 0.103a
a
a
1.26 ± 0.173
1.20 ± 0.171a
1.94 ± 0.156a
1.93 ± 0.143a
1.58 ± 0.146
1.55 ± 0.087
Intestine MDA 4.30 ± 0.321d
2.99 ± 0.140c
2.32 ± 0.138b
2.12 ± 0.125ab
c
b
a
1.93 ± 0.129
ab
4.73 ± 0.21a 1.43 ± 0.137a
1.62 ± 0.073
a
4.90 ± 0.15a 1.50 ± 0.140
1.78 ± 0.076
PC
2.39 ± 0.091
8.62 ± 0.22c d
1.59 ± 0.110
1.35 ± 0.055
1.34 ± 0.106
1.28 ± 0.056
3.21 ± 0.255c
2.74 ± 0.206b
2.16 ± 0.140a
1.97 ± 0.137a
ab
Muscle MDA PC
4.37 ± 0.218d 1.79 ± 0.07
c
b
1.53 ± 0.06
1.23 ± 0.11
a
a
1.18 ± 0.10
1.16 ± 0.14
a
a
1.14 ± 0.13
1.11 ± 0.15a
Values are mean ± SD of three groups of fish, with 5 fish in each group. Mean values with the different superscripts in the same row are significantly different (P \ 0.05)
Fig. 2 Regression analysis and significance of graded level of dietary thiamin and malondialdehyde content (MDA) of Jian carp (C. carpio var. Jian)
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Fish Physiol Biochem
Fig. 3 Regression analysis and significance of graded level of dietary thiamin and protein carbonyl content (PC) of Jian carp (C. carpio var. Jian) Table 3 Anti-superoxide anion capacities (ASA, U mL-1) and anti-hydroxyl radical capacities (AHR, U mL-1) in serum, ASA capacities (U mg-1 protein) and AHR capacities (U mg-1
protein) in hepatopancreas, intestine and muscle of Jian carp (C. carpio var. Jian) fed diet with graded levels of thiamin for 60 days
Dietary thiamin levels (mg kg-1 diet) 0.25
0.48
0.79
1.06
1.37
1.63
2.65
Serum 357 ± 10.6a
ASA AHR
1,963 ± 151
a
460 ± 10.3b
500 ± 20.2c
513 ± 22.2c
508 ± 19.3c
b
b
2,291 ± 87.5
2,300 ± 83.6
b
b
184.4 ± 7.2c
181.7 ± 4.0c
2,319 ± 78.2
521 ± 27.1c b
502 ± 21.8c
2,366 ± 62.7
2,412 ± 122
2,291 ± 87.5b
179.8 ± 7.6c
181.9 ± 5.9c
185.3 ± 8.9c
Hepatopancreas ASA
153.6 ± 9.5a
169.3 ± 10.5b
AHR
a
ab
128.4 ± 8.9
142.6 ± 8.3
ab
142.2 ± 7.6
150.4 ± 3.4
b
b
b
145.3 ± 15
149.9 ± 16b
154.1 ± 14
Intestine 145 ± 13.9a
ASA AHR
109.6 ± 8.2
a
168 ± 8.0b 131.0 ± 12.7
167 ± 7.3b b
169 ± 11.4b b
136.0 ± 12.6
133.8 ± 6.2
b
173 ± 11.1b b
136.1 ± 8.3
172 ± 6.7b 129.1 ± 11.3
163 ± 6.8b b
134.5 ± 12.3b
Muscle ASA
76.6 ± 8.5a
AHR
128 ± 17.0
119 ± 9.8b a
137 ± 13.4
123 ± 10.5b ab
ab
142 ± 9.7
121 ± 8.2b 152 ± 10.9
b
126 ± 10.2b
128 ± 11.5b
b
b
151 ± 13.9
154 ± 16.2
132 ± 9.7b 153 ± 14.5b
Values are mean ± SD of three groups of fish, with 5 fish in each group. Mean values with the different superscripts in the same row are significantly different (P \ 0.05)
increasing dietary thiamin levels up to 0.48 mg kg-1 diet (P \ 0.05) and then plateaued (P [ 0.05). AHR capacities in serum and intestine were improved with increasing dietary thiamin levels up to 0.48 mg kg-1
123
diet (P \ 0.05), and no significant differences were found with further increase in thiamin levels (P [ 0.05). In hepatopancreas and muscle, AHR capacity was the highest for fish fed diet containing
Fish Physiol Biochem
Fig. 4 Regression analysis and significance of graded level of dietary thiamin and the anti-hydroxyl radical capacities (AHR) of Jian carp (C. carpio var. Jian)
thiamin C1.06 mg kg-1 diet and higher than fish fed thiamin-unsupplemented group (P \ 0.05). There were significantly quadratic regression relationship between the AHR capacity in hepatopancreas (R2 = 0.814; P \ 0.05) and muscle (R2 = 0.964; P \ 0.01) and the increased dietary concentrations of thiamin (Fig. 4).
activities in serum and hepatopancreas were significantly enhanced with increasing dietary thiamin levels up to 0.79 and 0.48 mg kg-1 diet, respectively (P \ 0.05), and plateaued thereafter (P [ 0.05). The activity of SOD in intestine followed a similar pattern to that in hepatopancreas. Dietary thiamin had no effects on SOD activity in muscle (P [ 0.05). CAT activity in serum was significantly increased with increasing dietary thiamin levels up to 0.79 mg kg-1 diet (P \ 0.05), and no differences were found with further increase in thiamin levels (P [ 0.05). The activities of CAT in hepatopancreas, intestine and muscle were the lowest for fish fed 0.25 mg thiamin kg-1 diet
SOD and CAT activities in serum, hepatopancreas, intestine and muscle SOD and CAT activities in serum, hepatopancreas, intestine and muscle are shown in Table 4. SOD Table 4 The activities superoxide dismutase (SOD, U mL-1) and catalase (CAT, U mL-1) in serum, SOD activities (U mg-1 protein) and CAT activities (U mg-1 protein) in
hepatopancreas, intestine and muscle of Jian carp (C. carpio var. Jian) fed diet with graded levels of thiamin for 60 days
Dietary thiamin levels (mg kg-1 diet) 0.25
0.48
0.79
1.06
1.37
1.63
2.65
Serum SOD
33.2 ± 3.83a
44.4 ± 3.72b
76.3 ± 3.81c
77.2 ± 5.89c
81.5 ± 5.81c
78.0 ± 6.54c
78.9 ± 8.29c
CAT
a
b
c
c
c
c
17.6 ± 0.60
18.5 ± 0.62c
10.4 ± 0.95
15.6 ± 0.93
17.8 ± 0.94
18.1 ± 1.13
18.3 ± 0.92
90.9 ± 3.18b
91.4 ± 4.02b
93.5 ± 4.37b
91.2 ± 2.58b
92.1 ± 2.66b
91.3 ± 2.92b
b
b
b
b
32.53 ± 1.73b
Hepatopancreas SOD
83.8 ± 4.91a
CAT
a
28.65 ± 2.98
ab
30.67 ± 2.01
32.73 ± 1.44
31.86 ± 2.24
32.52 ± 2.12
32.19 ± 2.64
Intestine SOD
71.1 ± 5.91a
81.5 ± 6.25b
84.6 ± 3.11b
83.8 ± 3.10b
83.3 ± 2.34b
82.0 ± 3.27b
84.4 ± 4.84b
CAT
a
b
b
b
b
b
23.9 ± 1.52
23.6 ± 1.87b
18.4 ± 1.98
23.5 ± 1.11
23.6 ± 1.54
23.8 ± 1.59
24.0 ± 2.01
Muscle SOD
37.93 ± 3.74a
38.78 ± 2.87a
40.20 ± 2.07a
39.56 ± 1.64a
39.23 ± 1.91a
38.77 ± 1.61a
39.84 ± 2.72a
CAT
a
b
b
b
b
b
20.8 ± 1.70b
14.5 ± 1.71
18.9 ± 2.23
19.6 ± 1.98
20.5 ± 2.88
20.6 ± 1.82
21.1 ± 1.15
Values are mean ± SD of three groups of fish, with 5 fish in each group. Mean values with the different superscripts in the same row are significantly different (P \ 0.05)
123
Fish Physiol Biochem
Fig. 5 Regression analysis and significance of graded level of dietary thiamin and superoxide dismutase (SOD) and catalase (CAT) of Jian carp (C. carpio var. Jian)
(P \ 0.05), and no significant difference were observed in other groups (P [ 0.05). The activity of SOD in serum (R2 = 0.888; P \ 0.05) and CAT in muscle (R2 = 0.840; P \ 0.05) showed significantly quadratic response to graded levels of dietary thiamin (Fig. 5). GPx, GST and GR activities in serum, hepatopancreas, intestine and muscle The activities of GPx, GST and GR in serum, hepatopancreas, intestine and muscle are shown in Table 5. GPx activity in serum was the highest for fish fed diets containing 2.65 mg thiamin kg-1 diet and the lowest for fish fed the thiamin-unsupplemented diet (P \ 0.05). The activity of GPx in intestine followed a similar pattern to that in serum. Both in hepatopancreas and muscle, GPx activities were significantly increased with increasing dietary thiamin levels up to 0.79 mg kg-1 diet (P \ 0.05), and no differences were observed with further increase in thiamin levels (P [ 0.05). The activities of GST in serum, hepatopancreas and muscle were higher in fish fed diet supplemented with thiamin than those fed the control diet (P \ 0.05). GST activity in intestine was significantly improved with increasing dietary thiamin levels up to 0.48 mg kg-1 diet (P \ 0.05) and plateaued thereafter (P [ 0.05). GR activities in serum and hepatopancreas were significantly improved with increasing dietary thiamin levels up to 0.79 and 1.06 mg kg-1 diet, respectively (P \ 0.05), and there were no differences with further increase in thiamin levels (P [ 0.05). The activities of GR in intestine and muscle also followed the same pattern to that in serum. The activity of GR in serum (R2 = 0.904; P \ 0.01)
123
and hepatopancreas (R2 = 0.899; P = 0.01); GPx activity in serum (R2 = 0.894; P \ 0.05), intestine (R2 = 0.936; P \ 0.01), hepatopancreas (R2 = 0.842; P \ 0.05) and muscle (R2 = 0.809; P \ 0.05); and GST activity in serum (R2 = 0.885; P \ 0.05), hepatopancreas (R2 = 0.891; P \ 0.05) and muscle (R2 = 0.855; P \ 0.05) showed significantly quadratic response to graded levels of dietary thiamin (Fig. 6). GSH content in serum, hepatopancreas, intestine and muscle GSH contents in serum, hepatopancreas, intestine and muscle are shown in Table 6. GSH contents in serum and muscle were significantly increased with increasing dietary thiamin levels up to 1.06 and 0.79 mg kg-1 diet, respectively (P \ 0.05), and then plateaued (P [ 0.05). The levels of GSH in hepatopancreas and intestine also followed a similar pattern to that in serum. The GSH content in serum (R2 = 0.965; P \ 0.01), hepatopancreas (R2 = 0.960; P \ 0.01) and intestine (R2 = 0.978; P \ 0.01) showed significantly quadratic response to graded levels of dietary thiamin (Fig. 7). Thiamin requirement As shown in Fig. 1a, the dietary thiamin levels estimated by the broken-line model based on serum MDA content was 1.07 mg kg-1 diet. The regression equation was as follows: Y = -2.0624x ? 15.655, R2 = 0.9604; Ymax = 13.4 (Fig. 1a). The PC content in serum to dietary levels of thiamin relationship was
Fish Physiol Biochem protein), GST (U mg-1 protein) and GR (U g-1 protein) in hepatopancreas, intestine and muscle of Jian carp (C. carpio var. Jian) fed diet with graded levels of thiamin for 60 days
Table 5 The activities of glutathione peroxidase (GPx, U 0.1 mL-1), glutathione S-transferase (GST, U mL-1) and glutathione reductase (GR, U L-1) in serum, GPx (U mg-1 Dietary thiamin levels (mg kg-1 diet) 0.25
0.48
0.79
1.06
1.37
1.63
2.65
Serum GPx
327 ± 14.9a
433 ± 10.7b
568 ± 12.0c
GST
a
201 ± 11.5
a
b
185 ± 22.0
b
GR
198.4 ± 11.7
a
145 ± 22.1
229 ± 10.8
c
580 ± 18.8
cd
302 ± 23.4
cd c
241 ± 28.4
250 ± 34.0
544 ± 16.5c
584 ± 11.2
cd
c
293 ± 10.9
c
257 ± 21.7
590 ± 22.3
cd
c
600 ± 29.7d 314 ± 14.7d
294 ± 5.8
c
257 ± 21.9c
558 ± 15.8c
564 ± 20.6c
249.0 ± 33.5
Hepatopancreas GPx
438 ± 16.9a
481 ± 21.8b
554 ± 18.0c
GST GR
a
133 ± 7.36 92.2 ± 7.65a
b
162 ± 7.87 136 ± 10.5b
b
168 ± 10.0 151 ± 15.0c
204 ± 10.5 170 ± 11.5d
192 ± 4.37 173 ± 10.2d
GPx
382 ± 21.0a
413 ± 19.5b
499 ± 17.8c
500 ± 15.4c
517 ± 16.3
GST
a
203 ± 7.61
b
b
213 ± 9.61
b
97.6 ± 7.52
139 ± 7.91
b
171 ± 16.1
169 ± 14.4
c
GPx
405 ± 29.1a
463 ± 24.7b
557 ± 20.1c
547 ± 17.5c
GST
a
97.1 ± 7.63
109 ± 10.7
GR
49.4 ± 10.0a
109 ± 11.1b
cd
541 ± 16.0c c
cd
207 ± 11.0d 171 ± 16.0d
cd
536 ± 19.5d
199 ± 8.46 168 ± 7.43d
Intestine
GR
139 ± 15.6
a
204 ± 8.24
c
cd
b
525 ± 16.6
b
207 ± 12.1b
179 ± 10.5
c
181 ± 10.7
178 ± 6.67c
545 ± 12.8c
545 ± 12.6c
568 ± 23.3c
206 ± 10.5
c
199 ± 8.23
Muscle ab
bcd
bcd
115 ± 11.8
122 ± 10.3
148 ± 10.9c
138 ± 10.3c
bc
cd
114 ± 8.82
123 ± 6.23
139 ± 10.1c
150 ± 15.3c
129 ± 11.3d 149 ± 20.9c
Values are mean ± SD of three groups of fish, with 5 fish in each group. Mean values with the different superscripts in the same row are significantly different (P \ 0.05)
described by broken-line model [Y = -5.5517x ? 12.586, R2 = 0.920; Ymax = 7.20 (Fig. 1b)]. Based on the above equation, the optimum thiamin level was estimated to be 0.95 mg kg-1 diet.
Discussion The present research used the same animal trial as our previous study by Huang et al. (2011). Our previous study has estimated thiamin requirement of juvenile Jian carp (1.02 mg kg-1 diet) based on percent weight gain and showed that thiamin promoted growth, protein deposition and enhanced digestive and absorption capacity of fish (Huang et al. 2011). As we known, the function of digestive tract and growth of tissues is partly dependent on the structural integrity of fish (Lushchak et al. 2001; Enesco and Leblond 1962). The high degree of unsaturation x-3 fatty acids in fish cell membrane and muscle predisposes them to oxidative stress (Martinez-Alvarez et al. 2005). A number of studies have shown that ascorbic acid (Lee and Dabrowski 2003), myo-inositol (Jiang et al. 2009b)
and pyridoxine (Hu et al. 2011) could improve antioxidant defence in fish. Study in grouper has showed that thiamin decreased the liver lipid peroxidation (Huang et al. 2007). Therefore, the present study investigated the effects of dietary thiamin on oxidative stress and antioxidant responses in serum, hepatopancreas, intestine and muscle of juvenile Jian carp. Malondialdehyde (MDA) content is known to reflect lipid peroxidation, which can be defined as an important consequence of oxidative deterioration of lipids (Lee and Dabrowski 2003). Meanwhile, protein damage can also be directly induced by reactive oxygen species (ROS) (Nordberg and Arne´r 2001). Protein carbonyl (PC) content is regarded as a marker of protein oxidation (Armenteros et al. 2009). The present study demonstrated that MDA and PC contents in serum, hepatopancreas, intestine and muscle were gradually decreased with increasing dietary thiamin levels up to certain values and then remained constant, which was in agreement with the result in juvenile grouper (Huang et al. 2007). This may attribute that thiamin is a water-soluble vitamin, and fish can
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Fish Physiol Biochem
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Fish Physiol Biochem b Fig. 6 Regression analysis and significance of graded level of
thiamin inhibiting lipid peroxidation and protein oxidation may be that it blocked generation of detrimental intermediary metabolite in fish. Glyoxal promoted lipid peroxidation in hepatocytes of rats (Shangari and O’Brien 2004). In human red blood cells, thiamin decreased the formation of glyoxal through increase the activity of transketolase (Thornalley et al. 2001). Besides, advanced glycation end (AGE) products increased MDA content in vessel wall
dietary thiamin and glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) of Jian carp (Cyprinus carpio var. Jian)
maintain the homeostasis through increasing excretion after feeding an overdose of thiamin (Lall and Tibbetts 2009). However, little information is available about the effects of thiamin on the lipid peroxidation and protein oxidation in fish. The benefit effects of dietary
Table 6 Reduced glutathione (GSH, mg L-1) contents in serum, GSH (mg g-1 protein) contents in hepatopancreas, intestine and muscle of Jian carp (C. carpio var. Jian) fed diet with graded levels of thiamin for 60 days Dietary thiamin levels (mg kg-1 diet) 0.25 Serum Hepatopancreas Intestine Muscle
0.48
0.79
1.06
1.37
1.63
2.65
62.8 ± 2.16a
79.4 ± 4.86b
97.2 ± 2.94c
108 ± 2.96d
110 ± 2.65d
111 ± 2.16d
112 ± 2.62d
a
b
c
d
d
d
27.4 ± 0.42d
d
22.6 ± 0.49d 19.3 ± 0.67c
18.5 ± 0.82
a
15.8 ± 0.77 14.8 ± 1.26a
20.3 ± 0.90
b
18.0 ± 0.56 16.8 ± 0.89b
24.3 ± 0.60
c
19.4 ± 0.71 18.6 ± 0.44c
27.3 ± 0.49
d
22.0 ± 0.64 18.5 ± 0.48c
27.0 ± 0.34
d
22.5 ± 0.41 18.5 ± 0.35c
27.7 ± 0.48
22.7 ± 0.34 18.9 ± 0.26c
Values are mean ± SD of three groups of fish, with 5 fish in each group. Mean values with the different superscripts in the same row are significantly different (P \ 0.05)
Fig. 7 Regression analysis and significance of graded level of dietary thiamin and reduced glutathione content (GSH) of Jian carp (C. carpio var. Jian)
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Fish Physiol Biochem
of bovine (Yan et al. 1994). In human endothelial cells, thiamin in its active form can inhibit AGE formation (Beltramo et al. 2008). Calingasan and Gibson (2000) reported that nitric oxide (NO) lead to protein oxidation in mice brain. In humans, thiamin decreased production of NO in endothelial cells (Hazell and Butterworth 2009). Therefore, the mechanism by which dietary thiamin alleviates oxidative damage of lipid and protein needs further investigation. Based on serum MDA and PC content data, the dietary thiamin requirements of juvenile Jian carp were estimated to be 1.07 and 0.95 mg kg-1 diet, respectively, which were similar to thiamin requirement (1.02 mg kg-1) based on percent weight gain reported by Huang et al. (2011). Oxidative injury of lipids and proteins is mostly induced by cellular ROS in vertebrate animals (Knight 2000). Superoxide anions and hydroxyl radicals are the most potent ROS and can cause a wide range of oxidative damage within the cell (Kohen and Nyska 2002). In our study, with the increasing of dietary thiamin levels, the scavenging ability against the superoxide anion and the hydroxyl radical was improved with increasing thiamin levels up to certain values and then plateaued in serum, hepatopancreas, intestine and muscle, suggesting thiamin promoted the elimination of ROS in fish. Thiamin may act as superoxide anion and hydroxyl radical scavenger directly or indirectly in fish. Firstly, thiamin interacts directly with superoxide and peroxide radicals in vitro (Jung and Kim 2003; Lukienko et al. 2000). The scavenging effects of thiamin are probably related to successive transfer of (2H? ? 2e-) from the NH2 group of the pyrimidine ring to radicals (Lukienko et al. 2000). Secondly, thiamin may scavenge free radicals through inhibiting heme oxygenase-1 activity and AGE products accumulation in fish. Heme oxygenase-1 induces formation of hydroxyl radicals through the Fenton reaction in rat mesangial cells (Sandau et al. 1998). Calingasan et al. (1999) reported that thiamin prevented heme oxygenase-1 accumulation in brain of rat. Besides, AGE products enhanced the generation of reactive oxygen intermediates in vessel wall of bovine (Yan et al. 1994). Thiamin in its active form inhibited AGE formation on bovine serum albumin (Beltramo et al. 2008). Thirdly, thiamin may inhibit glutamate N-methyl-D-aspartate (NMDA)
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receptor activation for free radical production in fish. It has been shown that activation of NMDA receptor in neuronal cultures led to the formation of superoxide radicals (Langlais et al. 1997). In rat, thiamin inhibited glutamate NMDA receptors activation via preventing glutamate transports in brain (Bondy and Lee 1993; Hazell et al. 2001). Fish are susceptible to the attack of reactive ROS and have developed antioxidant defence system, including enzymatic and non-enzymatic defences (Martinez-Alvarez et al. 2005). A major component of the antioxidant system in fish consists of antioxidant enzymes, such as SOD, CAT, GPx and GST (Zhou et al. 2011). SOD can dismutate two O2 into H2O2 and O2 and can protect cells from the damage induced by O2 (Zhou et al. 2012). Data in the present study showed that with dietary thiamin levels up to 0.79, 0.48 and 0.48 mg kg-1, SOD activities were markedly increased in serum, hepatopancreas and intestine, while activity of SOD in muscle was not affected by thiamin. The end product of the dismutation reaction, H2O2, can be removed by enzymes CAT and GPx (Kohen and Nyska 2002). GST is an enzymatic multifamily that is able to detoxify xenobiotics containing reactive electrophilic compounds to facilitate their excretion from cells (Lushchak et al. 2001). The present study also showed that the activities of CAT, GPx and GST were increased with increasing dietary thiamin levels up to certain values and then remained constant in serum, hepatopancreas, intestine and muscle. The results suggested that the increases in the activities of these enzymes, noticed in the certain thiamin levels, were sufficient to prevent the rise of lipid peroxidation and protein oxidation. As we known, few studies have yet investigated the effect of thiamin on antioxidant enzyme activities in fish. Huang et al. (2007) reported that the SOD activity was not affected by thiamin in liver of grouper. This may be attributed that SOD is not a main factor affecting oxidative stress in grouper liver. In mammals, thiamin can affect antioxidant enzyme activities in several ways. Firstly, thiamin may correct GPx activity via blockade of triosephosphate accumulation. In bovine erythrocytes, the activity of GPx was inactivated by methylglyoxal, which is derived from triosephosphates (Park et al. 2003). Shangari et al. (2003) reported that thiamin inhibited methylglyoxal
Fish Physiol Biochem
generation via disposal of excess triosephosphates in rat hepatocytes. Furthermore, in rat liver, insulin administration resulted in an increase in Cu/Zn-SOD and CAT activities (Sindhu et al. 2004). Rathanaswami et al. (1991) reported that thiamin elevated insulin secretion in pancreatic islets of rats. Thirdly, thiamin may increase antioxidant enzyme activities by suppression of the expression of the transcription factor p53. It has been reported that p53 repressed the antioxidant gene transcription by binding to the antioxidant response cis elements (AREs) in mouse Hepa1-6 cells (Faraonio et al. 2006). Yang et al. (2004) reported that thiamin could decrease the p53 expression in retina neurons of rats. Taken together, dietary thiamin stimulation of antioxidant enzyme activities of fish may be partly related to these pathways, which needs further investigation. As non-enzymatic antioxidant, reduced GSH is critical to resistance of oxidative stress in organisms (Vardi et al. 2008). In the present study, dietary supplementation of thiamin improved GSH contents levels up to certain values in serum, hepatopancreas, intestine and muscle. To our knowledge, few studies have been reported on the relationship between dietary thiamin and GSH contents in fish. In rats, thiamin increased reduced GSH contents in hepatocytes (Shangari et al. 2007). On the one hand, thiamin may promote GSH synthesis via elevating glutamate concentration. In rat brain, thiamin deficiency caused impairment of glutamate transport and decreased glutamate uptake (Hazell et al. 2001). Glutamate is a constituent amino acid of GSH and necessary for GSH synthesis in enteron of piglets (Stoll et al. 1998). On the other hand, thiamin may promote reduced GSH regeneration from oxidized GSH (GSSG). In goldfish, GR catalyzes the reduction in the oxidized form of glutathione, at the expense of the NADPH (Lushchak et al. 2001). In our study, GR activities were enhanced by dietary thiamin levels up to certain values and then plateaued in serum, hepatopancreas, intestine and muscle. Correlation analysis showed that GSH content was positively correlated with GR activity in serum (r = ?0.986; P \ 0.01), hepatopancreas (r = ?0.948; P \ 0.01), intestine (r = ?0.929; P \ 0.01) and muscle (r = ?0.986; P \ 0.01), respectively. Meanwhile, thiamin increased the activity transketolase, a rate-limiting enzyme of the pentose phosphate pathway, which plays a major role in the production of NADPH for maintaining GSH levels in rat
(Shangari et al. 2007). Nevertheless, the underlying mechanism needs further study. In conclusion, our results show that dietary thiamin supplementation inhibits lipid peroxidation and protein oxidation in fish. The protective effect of thiamin may be attributed to the enhanced free radical scavenging ability or to improved enzymatic and non-enzymatic antioxidant defences. However, the mechanism of thiamin functioned as an antioxidant in fish needs further investigation. Acknowledgments This research was financially supported by the National Department Public Benefit Research Foundation (Agriculture) of China (201003020) and Science and Technology Support Program of Sichuan Province (2011NZ0071). The authors would like to thank the personnel of these teams for their kind assistance.
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