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Effects of astaxanthin on antioxidant capacity of golden pompano (Trachinotus ovatus) in vivo and in vitro

Effects of astaxanthin on antioxidant capacity of golden pompano (Trachinotus ovatus) in vivo and... The objective of this research was to study the effect of astaxanthin (AST) on growth performance and antioxidant capacity in golden pompano (Trachinotus ovatus) both in vivo and in vitro. In the in vivo study, two diets were formulated with or without astaxanthin supplementation (D1 and D2; 0 and 200 mg/kg) to feed fish for 6 weeks. In the in vitro study, cells from hepatopancreas of golden pompano were isolated and four treatments with or without astaxanthin and H O supplementation were applied (control group: without both astaxanthin and H O 2 2 2 2 treated; H O group: just with H O treated; H O + AST group: with both astaxanthin and H O treated; AST group: 2 2 2 2 2 2 2 2 just with AST treated). Results of the in vivo study showed that weight gain (WG) and special growth rate (SGR) significantly increased with astaxanthin supplemented (P < 0.05). Feed conversion ratio (FCR) of fish fed D2 diet was significantly lower than that of fish fed D1 diet (P < 0.05). Hepatic total antioxidant capacity (T-AOC) and the reduced glutathione (GSH) of golden pompano fed D2 diet were significant higher than those of fish fed D1 diet (P < 0.05). Superoxide dismutase (SOD) was significantly declined as astaxanthin was supplemented (P < 0.05). Results of the in vitro study showed that the cell viability of H O group was 52.37% compared to the control 2 2 group, and it was significantly elevated to 84.18% by astaxanthin supplementation (H O + AST group) (P < 0.05). 2 2 The total antioxidant capacity (T-AOC) and the reduced glutathione (GSH) of cell were significant decreased by oxidative stress from H O (P < 0.05), but it could be raised by astaxanthin supplementation (H O vs H O + AST), 2 2 2 2 2 2 and the malondialdehyde (MDA) was significant higher in H O group (P < 0.05) and astaxanthin supplementation 2 2 could alleviate the cells from lipid peroxidation injury. In conclusion, dietary astaxanthin supplementation can improve the growth performance of golden pompano. Moreover, astaxanthin can improve the golden pompano hepatic antioxidant capacity both in vivo and in vitro study by eliminating the reactive oxygen species. Keywords: Golden pompano, Astaxanthin, Growth performance, Antioxidant capacity Background normal physiological conditions, the excessive ROS can be Reactive oxygen species (ROS) are oxidative products, pro- removed by internal antioxidants and anti-oxidative sys- duced continuously in the course of normal aerobic cellular tems (Chen et al. 2015), including counter balance such as metabolism and respiratory burst (Chew 1995), which par- enzymes (like superoxide dismutase, catalase, and glutathi- ticipate in a variety of biological processes, including nor- one peroxidase), functionalized large molecules (albumin, mal cell growth, induction and maintenance of the ferritin, and ceruloplasmin) and small molecules (ascorbic transformed state, programmed cell death, and cellular sen- acid, α-tocopherol, β-carotene, and uric acid) (Martinez-Al- escence (Finkel 2003). However, ROS can, in turn, damage varez et al. 2005). The dietary antioxidants most widely healthy cells if they are not eliminated (Chew 1995). Under used include vitamin E, vitamin C, carotenoids, flavanoids, zinc, and selenium (Chew and Park 2004). Among those, * Correspondence: gzniujin2003@163.com; 1657864417@qq.com carotenoids reach the highest plasma and tissue concentra- Equal contributors tions, despite their lower intake (Olmedilla et al. 2007). Institute of Aquatic Economic Animals, School of Life Science, Sun Yat-sen Carotenoids, more than 600 known types, can be classi- University, NO.135 at Xingang Xi Road, Haizhu District, Guangzhou 510275, Guangdong Province, China fied into two categories, xanthophyll and carotenes. Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xie et al. Fisheries and Aquatic Sciences (2017) 20:6 Page 2 of 8 Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′dione, Pink, 10% astaxanthin, DSM Nutritional Products AST) is a xanthophyll carotenoid which is found in many France SAS) (Table 1). The method of diet preparation microorganisms and marine animals, such as shrimp, was the same as described by Niu et al. ( 2015). Briefly, crayfish, crustaceans, salmon, trout, krill, microalgae as all dry ingredients were finely ground, weighed, mixed well as yeast. Its molecule consists of 40 carbon atoms, di- manually for 5 min, and then transferred to a Hobart vided into a central portion containing 22 carbon atoms mixer (A-200 T Mixer Bench Model unit; Resell Food linked with 13 conjugated double bonds and two terminal Equipment Ltd., Ottawa, ON, Canada) for another benzene rings containing hydroxyl and ketone groups, giv- 15 min mixing. During the mixing, 6 N NaOH was ing rise to the higher polar structure of AST compared added to establish a pH level of 7–7.5. The pH of the with other carotenoids (Britton 1995).The antioxidant ac- diet was obtained by homogenizing a 5-g portion of the tivity of astaxanthin was found to be approximately 10 diet with 50 mL of distilled water with a glass-electrode times stronger than β-carotene (Shimidzu et al. 1996). pH meter on the supernatant (Robinson et al. 1981). Except for its antioxidant capacity, AST is also recog- Soya lecithin was added to pre-weighed fish oil and nized to have growth performance and survival rate pro- mixed until homogenous. The oil mix was then added to moting in Atlantic salmon (Salmo salar) (Christiansen the Hobart mixer slowly while mixing was still continu- and Torrissen 1996) and red porgy (Pagrus pagrus)(Kali- ing. All ingredients were mixed for another 10 min. nowski et al. 2011), skins coloration enhancing in large Then, distilled water (about 30–35%, v/w) was added to yellow croaker (Larimichthys croceus) (Yi et al. 2014) and the mixture to form dough. Dough of even consistency Atlantic salmon(Baker et al. 2002), anti-lipid peroxidation was passed through a pelletizer with a 2.5-mm-diameter (Leite et al. 2010), and immune response reinforcing in Astronotus ocellatus (Alishahi et al. 2015) properties. Table 1 Ingredients and proximate composition of the two Golden pompano (Trachinotus ovatus)belongs to family experimental diets (%) carangidae, genus Trachinotus. It is a warm-water species Ingredients Diet 1 Diet 2 (25–32 °C) and a carnivorous fish that preys mainly on zoo- Fish meal 32.00 32.00 plankton, small crustaceans, shellfish, and small fish (Liu Soybean meal 30.00 30.00 and Chen 2009). T. ovatusis is widely distributed in China, Wheat flour 20.00 19.80 Japan, Australia, and other countries (Huo-sheng 2006). Krill meal 2.00 2.00 Pompano is considered one of the most desirable food fishes, and it commands a significantly higher price than Fish oil 8.00 8.00 many other marine and freshwater species (Tutman et al. Soya lecithin 2.00 2.00 2004). Recently, pompano is widely farmed owing to its Monocalcium phosphate 2.00 2.00 high price in the market and resilience to salinity and Pre-vitamin 1.00 1.00 temperature ranges (Tutman et al. 2004), and annual out- Pre-mineral 1.00 1.00 put was over 100,000 tons. The suitable dietary protein and Choline 0.50 0.50 lipid levels for golden pompano are 46.0 and 6.5% (Wang et al. 2013). The optimum carbohydrate level for juvenile Vc 0.50 0.50 golden pompano could be 11.2–16.8% of the diet (Zhou DL-Met 0.40 0.40 et al. 2015). The optimal requirements of methionine (Niu Lys-HCL (78%) 0.60 0.60 et al. 2013), arginine (Lin et al. 2015), and n-6 long-chain AST 0 0.20 polyunsaturated fatty acid arachidonic acid (ARA) (Qi et al. Sum 100 100 2016) for golden pompano have been determined as well. Nutrient levels However, fewer studies were conducted to investi- gate the effects of antioxidants on golden pompano. Moisture 9.00 7.54 To date,theeffectsofAST on variouskinds of Crude protein 40.64 40.55 fishes are mainly reported in vivo and rarely found Crude fat 10.71 10.90 in vitro. We used in vivo and in vitro models to Ash 15.21 15.24 study and compare the effect of astaxanthin on anti- a −1 Pre-vitamin (mg or g kg diet): thiamin, 25 mg; riboflavin,45 mg; pyridoxine oxidant ability of golden pompano, respectively. HCl, 20 mg; vitamin B12, 0.1 mg; vitamin K3,10 mg; inositol, 800 mg; pantothenic acid, 60 mg; niacin acid, 200 mg; folic acid, 20 mg; biotin, 1.20 mg; retinal acetate, 32 mg; cholecalciferol, 5 mg; α-tocopherol, 120 mg; Methods ascorbic acid, 2000 mg; choline chloride, 2500 mg; ethoxyquin 150 mg; and Diet preparation and dietary treatments wheat middling, 14.012 g (Niu et al. 2013) b −1 . Pre-mineral (mg or g kg diet): NaF, 2 mg; KI, 0.8 mg; CoC 6H O (1%), In this study, two isonitrogenous and isoenergetic semi- l2 2 . . . . 50 mg; CuSO 5H O, 10 mg; FeSO H O, 80 mg; ZnSO H O, 50 mg; MnSO H O, 4 2 4 2 4 2 4 2 purified diets were formulated supplementing with or . . 60 mg; MgSO 7H O, 1200 mg; Ca(H PO ) H O, 3000 mg; NaCl, 100 mg; and 4 2 2 4 2 2 without astaxanthin (D1: 0%; D2: 0.2%; CAROPHYLL zoelite, 15.447 g (Niu et al. 2013) Xie et al. Fisheries and Aquatic Sciences (2017) 20:6 Page 3 of 8 die (Institute of Chemical Engineering, South China was sterilized by alcohol, and its abdomen was dissected University of Technology, Guangzhou, China). The diets with sterile instruments from the anus toward the head. were dried until the moisture was reduced to <10%. The Liver tissue was excised and rinsed three times with phos- dry pellets were placed in plastic bags and stored 20 °C phate buffer solution. The liver tissue was then minced until fed. into pieces of 1 mm and transferred to a 15 mL tube to which a solution of 0.25% trypsin (1:20 w/v; Sigma) was Animal rearing and experimental procedures added. The mixture was trypsinized on a thermostatic The feeding trial was conducted at an experimental sta- water bath to obtain the cell suspension, which was tion of South China Sea Fisheries Research Institute of shaken every 5 min. Then, the mixture was filtered Chinese Academy of Fishery Sciences (Sanya, Hainan). through a 100-mesh sieve. The cell suspensions were Prior to the start of the trial, juvenile T. ovatus were ac- pooled and centrifuged at 1000 rpm for 10 min, and the climated to a commercial diet for 2 weeks and were fed cell pellet was washed and resuspended in a culture twice daily to apparent satiation. At the beginning of the medium. The cell number was counted using a haemocyt- feeding trial, the fish were starved for 24 h, weighed, and ometer, and cell viability was estimated immediately fol- then fish with similar size (initial body weight 23.65 ± lowing isolation using the trypan blue exclusion assay. 0.10 g) were randomly allotted to 8 sea cages (1.0 m × 1.0 m × 1.5 m; four cages per diet treatment); each cage Cell culture and treatments was stocked with 30 fish. Each experimental diet was A final cell density of hepatocytes was adjusted to 2 × 10 −1 randomly assigned to four cages. The feeding frequency cells mL in L-15 culture medium (Jinuo Co, Hangzhou, was once daily at 8:00 and lasted for 6 weeks. To prevent China) supplemented with 2 mM L-glutamine (Sigma) the waste of pellets, fish were slowly hand-fed to sati- and 10% foetal bovine serum (Gibco). Cells were seeded ation based on visual observation of their feeding behav- into 12-well culture plates with 500 μLcellsuspension per ior. Feed consumption was recorded for each cage every well. Cells were cultured in a humidified atmosphere at day. Water quality parameters were monitored daily. 28 °C. Once seeded, cells were allowed to attach to culture plates for 24 h. At 24 h, 50% of the culture medium Sample collection (250 μL) was removed and replaced with fresh medium. At the end of the feeding trial, fish were starved for 24 h Then PBS, 100 mM H O , 1000 ng/mL astaxanthin dis- 2 2 and then weighed and counted the total number. Ten solved in dimethyl sulfoxide (DMSO) (final concentration fish from each cage were randomly collected for sam- 0.01%), and H O plus astaxanthin were added in the 2 2 pling: four for analysis of whole-body composition and wells. Every treatment was replicated in three wells. Cell six were anesthetized to obtain weights of individual viability was evaluated by trypan blue exclusion test, and whole body, viscera, and liver. The livers were rapidly re- cells were harvested for antioxidant capacity analysis after moved and frozen in the liquid nitrogen separately for the treatments. As an additional measure of cell viability, analysis of lipid peroxidation and antioxidant status. lactate dehydrogenase (LDH) activity in the extracellular medium (an indicator of membrane leakage (Misra and Biochemical composition analysis Niyogi 2009) was measured. Chemical composition of diets and fish were determined by standard methods (Latimer 2012). Moisture was de- Antioxidant capacity analysis termined by oven drying at 105 °C until a constant Hepatic and cell samples were homogenized in ice-cold weight was obtained. Crude protein content (N × 6.25) phosphate buffer (1:10 dilution) (phosphate buffer; 0.064 M, was determined according to the Kjeldahl method after pH 6.4). The homogenate was then centrifuged for 20 min acid digestion using an Auto Kjeldahl System (1030- (4 °C, 3000 g), and aliquots of the supernatant were used to Autoanalyzer; Tecator, Höganäs, Sweden). Crude lipid quantify hepatic T-AOC, GSH, SOD, and MDA. was determined by the ether extraction method using a The levels of enzyme activity and lipid peroxidation Soxtec extraction System HT (Soxtec System HT6, were measured with commercial ELISA kits (Randox La- Tecator). Ash content was determined after samples boratories Ltd.) in accordance with the instructions of the were placed in a muffle furnace at 550 °C for 4 h. manufacturer. The assays are briefly described as follows: The T-AOC is the representative of enzyme and none- Isolation of liver cells nzyme original antioxidant in the body; these antioxidants 3+ 2+ Golden pompano was purchased from a market in can reduce the ferric ion (Fe )to ferrous ion (Fe ). The Guangzhou, China. Hepatocytes were isolated according latter combines with phenanthroline and produces a to the methods of Wan et al. (2004) with some modifica- stable chelate, which can be measured by spectrophoto- tions. In the procedure, a fish was kept in 0.01% potassium graphy at 520 nm (Xiao et al. 2004). The T-AOC was de- permanganate solution for half an hour, after that, its skin termined in units per milligram of tissue protein. Xie et al. Fisheries and Aquatic Sciences (2017) 20:6 Page 4 of 8 Total superoxide dismutase (SOD) activity was mea- Results sured by using a xanthine oxides (Marklund and Mark- Growth performance in vivo lund 1974). The ratio of autooxidation rates of the Growth, feed utilization, and biometric parameters of ju- samples with or without hepatic homogenate was deter- venile pompano fed different dietary astaxanthin levels are mined at 550 nm. One unit of SOD activity was calculated shown in Table 2. Results showed that final body wet using the amount of superoxide dismutase required to in- weight (FBW), weight gain (WG), and special growth rate hibit the reduction of nitrobluete trazolium by 50%. (SGR) significantly increased with astaxanthin supple- The formation of 5-thio-2-nitrobenzoate (TNB) was mented (P < 0.05). Feed conversion ratio (FCR) of golden followed spectrophotometrically at 412 nm (Vardi et al. pompano fed the diets supplemented with astaxanthin 2008). The amount of GSH in the extract was determined was significantly lower than that of fish fed the control as μmol/mg protein utilizing a commercial GSH as the diet (P < 0.05), while no significant differences were found standard. The results are expressed as μmol/mg protein. in survival rate between the two diet treatments (P >0.05). Lipid-peroxidation levels were determined based on Hepatosomatic somatic indices (HSI), visceral somatic in- the malondialdehyde (MDA) level generated by oxidizing dices (VSI), and condition factor (CF) were significantly fatty acids. In the presence of thiobarbituric acid, malon- decreased in astaxanthin-supplemented diet treatment. dialdehyde started producing colored thiobarbituric- acid-reacting substances (TBARS) that were measured at Growth performance in vitro 532 nm (Buege and Aust 1978). In the in vitro study, the cell viability of H O group was 2 2 Lactate dehydrogenase (LDH) can catalyze lactate into 52.37% compared to the control group (PBS group), and pyruvate, which react with 2,4-dinitrophenylhydrazine it could be significantly elevated to 84.18% with astax- and produce a stable compound, which was measured anthin supplementation (H O + AST group) (P < 0.05) 2 2 by spectrophotography at 450 nm. (Fig. 1). The highest lactate dehydrogenase (LDH) activ- ity was found in H O group, and it was 159.02% com- 2 2 Calculations and statistical analysis pared to the control group, it could be significantly The parameters were calculated as follows: lessened to 122.96% with astaxanthin supplementation (H O + AST group) (P < 0.05) (Fig. 2). Weight gain rateðÞ WG; %¼ 100 2 2 ðÞ final body weight ‐ initial body weight =initial body weight Whole-body composition ‐1 Specific growth rate SGR; % day ¼ 100 Whole-body composition of golden pompano fed different ðÞ Ln final mean weight ‐ Ln initial mean weight = number of days dietary astaxanthin levels are shown in Table 3. There were no significant differences in whole-body composition of fish Feed conversion ratioðÞ FCR ¼ dry diet fed=wet weight gain between the two diet treatments (P >0.05). Survival rateðÞ % ¼ 100 ðÞ final number of fish =ðÞ initial number of fish Table 2 Growth performance and survival of golden pompano fed diets with and without supplementation of astaxanthin Viscerosomatic indexðÞ VSI; % ¼ 100 Diets Diet 1 Diet 2 ðÞ viscera weight; g =ðÞ whole body weight; g −1 (AST mg kg ) 0 200 Hepatosomatic indexðÞ HSI; %¼ 100 IBW (g) 32.72 ± 0.21 32.58 ± 0.06 ðÞ liverweight; g =ðÞ whole body weight; g FBW (g) 63.71 ± 0.66a 67.23 ± 0.22b WG (%) 95.30 ± 0.02a 106.36 ± 0.01b Condition factorðÞ CF; g=cm3¼ 100 −1 SGR (% day ) 1.59 ± 0.02a 1.72 ± 0.01b ðÞ bodyweight; g = body length; cm FCR 1.78 ± 0.02a 1.53 ± 0.02b Data from each treatment were subjected to one-way SR (%) 97.78 ± 0.01 96.67 ± 0.00 analysis of variance (ANOVA). Homogeneity of variance HSI (%) 1.13 ± 0.06a 0.94 ± 0.03b was verified using Bartlett and Levene’s test. When over- VSI (%) 5.97 ± 0.11a 5.42 ± 0.15b all differences were significant, Tukey’s multiple range CF (g/cm3) 3.4 ± 0.06a 3.04 ± 0.05b tests was used to compare the mean values among indi- Values are mean ± SEM of four replicates, and values in the same row with vidual treatments. The level of significant difference was different letters are significant different (P < 0.05). Diet 1 meant golden set at P < 0.05. Statistical analysis was performed using pompano groups fed diets without supplementation of astaxanthin (AST). Diet 2 meant groups with supplementation of astaxanthin the SPSS19.0 (SPSS Inc., Michigan Avenue, Chicago, IL, IBW initial body weight, FBW final body weight, WG weight gain, SGR special USA) for Windows, and the results are presented as growth rate, FCR feed conversion ratio, SR survival rate, HSI hepatosomatic means ± SEM (standard error of the mean). index, VSI viscerosomatic index, CF condition factor Xie et al. Fisheries and Aquatic Sciences (2017) 20:6 Page 5 of 8 antioxidant, can repair the cells from the oxidative stress. The total antioxidant capacity (T-AOC) and the 100 reduced glutathione (GSH) of oxidative stress group (H O ) were the lowest and significantly lower than 2 2 those of the control group (P < 0.05), but it could be sig- nificantly improved by astaxanthin supplementation (H O + AST group) (P < 0.05). The astaxanthin supple- 2 2 mented groups were significant higher than the other ones (P < 0.05). The SOD and MDA showed the highest value in H O group, which were significantly higher 2 2 than those in the control group (P < 0.05), but it also Control H2O2 H2O2 +AST AST could be significantly modified by astaxanthin supple- Fig. 1 The relative cell viability in different groups. Control column mentation (H O + AST group) (P < 0.05). The astax- 2 2 meant treating with neither H O nor AST, H O column with H O 2 2 2 2 2 2 anthin supplemented groups showed the significantly only, H O + AST column with both H O and AST, and AST column 2 2 2 2 higher antioxidant capacity than the other groups (P < with AST only. Data are expressed as mean ± SEM of three replicates; 0.05). values in the column sharing the same superscript letter are not significantly different; however, values in the column with the different superscript letter are significantly different Discussion Growth performance and proximate composition Antioxidant capacity analysis in vivo Carotenoids are reported to improve growth perform- The antioxidant status of juvenile pompano in vivo study ance of fish with the reason that carotenoids may exert a are presented in Table 4. The hepatic total antioxidant cap- positive influence on intermediary metabolism in aquatic acity (T-AOC) and the reduced glutathione (GSH) in fish animals (Segner et al. 1989), which enhance nutrient fed diet supplemented with astaxanthin were significantly utilization, ultimately resulting in improving growth higher than that of fish fed the control diet (P <0.05). On (Amar et al. 2001). The other possible mechanism may the contrary, superoxide dismutase (SOD) declined with be to adjust the intestinal flora breaking down indigest- astaxanthin supplementation significantly (P < 0.05), while ible feed components to extract more nutrients and to hepatic malondialdehyde (MDA) content was not affected stimulate the production of enzymes transporting fats by astaxanthin supplementation (P > 0.05). for growth instead of storage (James et al. 2006). Kali- nowski et al. (2011) believed that astaxanthin could en- Antioxidant capacity analysis in vitro hance lipid utilization in whole fish and liver, providing The antioxidant status of hepatocytes in the vitro study more energy and consequently enhancing growth per- are shown in Table 5. The H O as an oxidizing agent formance. In the present experiment, the growth per- 2 2 can totally damage the healthy cells, and astaxanthin, an formance (FBW, WG, and SGR) and feed utilization of fish fed diet with supplemental astaxanthin were signifi- cantly higher than that of fish fed the control diet. This result was in agreement with those in previous studies on Atlantic salmon (Christiansen and Torrissen 1996), red porgy (Kalinowski et al. 2011), Astronotus ocellatus 120 (Alishahi et al. 2015), and large yellow croaker (Li et al. 2014). However, effect of carotenoids on fish growth is 80 controversial. Many earlier studies have reported that dietary astaxanthin has no significant influence on growth and flesh composition of fish (Tejera et al. 2007; Table 3 Whole-body compositions (% dry weight) of golden Control H2O2 H2O2 +AST AST pompano fed diets with and without supplementation of astaxanthin Fig. 2 Lactate dehydrogenase (LDH) activity in the extracellular medium in different groups. Control column meant treating with Protein Lipid Ash Moist neither H O nor AST, H O column with H O only, H O + AST 2 2 2 2 2 2 2 2 Diet 1 61.77 ± 1.17 26.68 ± 0.17 17.71 ± 0.23 70.19 ± 1.17 column with both H O and AST, and AST column with AST only. 2 2 Diet 2 61.68 ± 1.35 26.33 ± 0.46 17.41 ± 0.94 71.68 ± 1.15 Data are expressed as mean ± SEM of three replicates; values in the column sharing the same superscript letter are not significantly Values are mean ± SEM of four replicates, and values in the same column with different letters are significant different (P < 0.05). Diet 1 meant golden different; however, values in the column with the different superscript pompano groups fed diets without supplementation of astaxanthin (AST). Diet letter are significantly different 2 meant groups with supplementation of astaxanthin Xie et al. Fisheries and Aquatic Sciences (2017) 20:6 Page 6 of 8 Table 4 Hepatic antioxidant statuses of golden pompano fed diets with and without supplementation of astaxanthin T-AOC (U/mg protein) SOD (U/mg protein) GSH (μmol/g protein) MDA (nmol/mg protein) Diet 1 0.11 ± 0.01a 240.87 ± 5.76a 82.44 ± 4.87a 0.41 ± 0.02 Diet 2 0.15 ± 0.01b 214.24 ± 5.71b 118.52 ± 8.93b 0.41 ± 0.06 Values are mean ± SEM of four replicates, and values in the same column with different letters are significant different (P < 0.05). Diet 1 meant golden pompano groups fed diets without supplementation of astaxanthin (AST). Diet 2 meant groups with supplementation of astaxanthin T-AOC total antioxidant capacity, GSH reduced glutathione, SOD superoxide dismutase, MDA malondialdehyde Zhang et al. 2012; Pham et al. 2014; Yi et al. 2014). Kop H O may decrease the total antioxidant capacity, the 2 2 and Durmaz (2008) believed that the effectiveness of ca- supplementation of astaxanthin can repair it to the same rotenoids in terms of deposition and physiological func- level with the control group. tion is species-specific in fish and not all fish species The stress response might increase free radical con- possess the same pathways for the metabolism of carot- tents, which may result in the increase of the lipid per- enoids. The mechanisms related to these findings have oxidation content and lipid peroxidation injury (Liu not yet been clearly elucidated. Our latest research re- et al. 2010). Malondialdehyde (MDA) is a product of sults showed that the dietary astaxanthin can increase lipid peroxidation, through crosslinking with the nucleo- the apparent digestibility coefficient of the diet and fur- philic groups of proteins, nucleic acids, and amino phos- ther promote the expression of insulin-like growth fac- pholipids, accumulation of MDA leads to cell toxicity, tors (IGFs); moreover, as members of the family of accelerating the damage of cells and tissues (Buege and transforming growth factors β, myostatin is affected by Aust 1978). The antioxidants and antioxidant enzyme dietary astaxanthin (unpublished data). system can play a significant role in resisting lipid oxide damage (Liu et al. 2010). Carotenoids may serve as an Antioxidant capacity analysis antioxidant in systems containing unsaturated fatty acids H O is a strong oxidizer, produced in cell metabolism, to quench free radicals (Mansour et al. 2006). The re- 2 2 but the excessive dose may be cytotoxic. As is shown, sults showed that the MDA were not significant different cell viability was sharply decreased with H O supple- when no stress appeared in the present in vivo study. 2 2 mented and the increased LDH leakage into the extra- However, once the cells suffered from oxidative stress in cellular media by H O indicated the occurrence of the present in vitro study, the MDA was increased and 2 2 oxidative stress membrane damage in our present the cell viability was decreased, but supplemented astax- in vitro study. Cellular antioxidative defense mechanisms anthin could totally decrease the MDA value and save can intercept the ROS both enzymatically and non- cells from the stress. Increased T-AOC and decreased enzymatically. Total antioxidant capacity (T-AOC) is an MDA in the in vitro study reflected that supplemented overall indicator of the antioxidant status of an individ- astaxanthin in media can be totally conducive to elimin- ual, representing the level of enzyme and nonenzyme ate the reactive oxygen species and protect the hepato- original antioxidantin of the body (Xiao et al. 2004). As cytes of golden pompano from free radicals. The MDA the value increases, the antioxidant defense against free in (H O + AST) group was lower than that in H O 2 2 2 2 radical reaction and reactive oxygen intermediates in- group, which indicated that AST can alleviate the lipid creases (Chien et al. 2003). In both of the in vivo and oxide damage. in vitro study, the T-AOC in the liver of the fish and in Superoxide dismutase (SOD), a cytosolic enzyme that the hepatocytes supplemented with astaxanthin were is specific for scavenging superoxide radicals, is the first higher, meaning that astaxanthin can improve the anti- enzymes to respond against oxygen radicals and import- oxidant status whether in vivo or in vitro. Although ant endogenous antioxidants for protection against Table 5 The antioxidant statuses of hepatocytes treated with or without astaxanthin and H O supplementation 2 2 T-AOC SOD GSH (μmol/g protein) MDA (nmol/mg protein) (U/mg protein) (U/mg protein) Control 0.35 ± 0.01b 2682.76 ± 127.04b 17.81 ± 0.83b 0.13 ± 0.01a H O 0.22 ± 0.02a 3264.92 ± 76.26c 5.92 ± 0.91a 0.40 ± 0.01c 2 2 H O + AST 0.37 ± 0.01bc 2726.34 ± 74.17b 28.24 ± 1.11c 0.23 ± 0.01bc 2 2 AST 0.41 ± 0.01c 2312.19 ± 69.94a 136.51 ± 4.11d 0.12 ± 0.01a Values are mean ± SEM of three replicates, and values in the same column with different letters are significant different (P < 0.05). Control meant golden pompano groups fed diets without supplementation of astaxanthin (AST). Control groups meant treating with neither H O nor AST, H O groups meant treating with H O 2 2 2 2 2 2 only, H O + AST groups meant treating with both H O and AST, and AST groups meant treating with AST only 2 2 2 2 T-AOC total antioxidant capacity, GSH reduced glutathione, SOD superoxide dismutase, MDA malondialdehyde Xie et al. Fisheries and Aquatic Sciences (2017) 20:6 Page 7 of 8 oxidative stress (Winston and Di Giulio 1991). Lygren Funding The design of the study and collection, analysis, interpretation of data, and et al. showed that high levels of dietary fat-soluble anti- writing the manuscript were supported by the Project of Science and oxidants, such as astaxanthin and vitamin E, there was a Technology of Guangdong Province (2013B090500110, 2013B090600045), the reduced need for endogenous antioxidant enzymes, such Central Institutes of Public Welfare Projects (2014A08YQ02), and the Special Project of Marine Fishery Science and Technology of Guangdong Province as total SOD (Lygren et al. 1999). The higher the SOD (A201601C11). value, the more superoxide radicals need to be reacted (Qingming et al. 2010). It was found that the activities of Authors’ contributions JN, JW, and YW designed the study. JJX wrote the article. JJX, QQL, and XC liver SOD were significantly decreased by dietary astax- performed the experiment. JN and JJX analyzed and interpreted the data. All anthin supplementation in olive flounder (Paralichthys authors have read, commented upon, and approved the final article. olivaceus) (Pham et al. 2014); large yellow croaker (Pseu- Competing interests dosciaena crocea) (Li et al. 2014) and rainbow trout (On- The authors declare that they have no competing interests. corhynchus mykiss) (Zhang et al. 2012). In this present study, SOD was significant lower in vivo and vitro study Consent for publication Not applicable both supplemented with astaxanthin, implying that astaxanthin can eliminate reactive oxygen species to Ethics approval and consent to participate avoid the cells and tissues to produce more SOD. Once All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals in Sun Yat-sen Univer- suffering from oxidative stress, the cells may produce sity, Guangzhou, China, and conformed to the National Institutes of Health much more endogenous SOD, as is shown in the study, Guide for Care and Use of Laboratory Animals (publication no. 85–23, revised to protect the body or cells from being hurt. 1985). Glutathione (GSH), ubiquitous non-enzymatic antioxi- dants in the cells, is known to play an important role in Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in the scavenging of free radicals and thus protect the im- published maps and institutional affiliations. portant cellular macromolecules and organelles from oxidative damage (Misra and Niyogi 2009). Its role in Author details South China Sea Fisheries Research Institute, Chinese Academy of Fishery the detoxification of ROS is important (Mallikarjuna Sciences, Guangzhou 510300, China. School of Life Science and Technology, et al. 2009). When suffered from oxidative stress, GSH 3 Shanghai Ocean University, Shanghai 201306, China. Institute of Aquatic was significantly lower in the present in vitro study. One Economic Animals, School of Life Science, Sun Yat-sen University, NO.135 at Xingang Xi Road, Haizhu District, Guangzhou 510275, Guangdong Province, mechanism for oxidative stress induced GSH depletion China. may involve enhanced utilization of GSH for the detoxi- fication of free radicals and other oxidants produced as a Received: 25 January 2017 Accepted: 18 April 2017 result of H O exposure (Shaw 1989). Vogt suggested 2 2 that the increase of lipid peroxidation was not apparent Reference until after GSH levels had been depleted (Vogt and Alishahi M, Karamifar M, Mesbah M. Effects of astaxanthin and Dunaliella salina on skin carotenoids, growth performance and immune response of Richie 2007). Astaxanthin can improve the GSH content Astronotus ocellatus. Aquac Int. 2015;23(5):1239–48. in both our in vivo and in vitro study. Amar E, Kiron V, Satoh S, Watanabe T. Influence of various dietary synthetic carotenoids on bio‐defence mechanisms in rainbow trout, Oncorhynchus mykiss (Walbaum). Aquacult Res. 2001;32(s1):162–73. Conclusions Baker R, Pfeiffer A-M, Schöner F-J, Smith-Lemmon L. Pigmenting efficacy of In conclusion, dietary astaxanthin supplementation can astaxanthin and canthaxanthin in fresh-water reared Atlantic salmon, Salmo improve the growth performance of golden pompano. salar. Anim Feed Sci Technol. 2002;99(1):97–106. Britton G. Structure and properties of carotenoids in relation to function. FASEB J. 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Qingming Y, Xianhui P, Weibao K, Hong Y, Yidan S, Li Z, Yanan Z, Yuling Y, Lan D, � Our selector tool helps you to find the most relevant journal Guoan L. Antioxidant activities of malt extract from barley (Hordeum vulgare L.) � We provide round the clock customer support toward various oxidative stress in vitro and in vivo. Food Chem. 2010;118(1):84–9. � Convenient online submission Robinson EH, Wilson RP, Poe WE. Arginine requirement and apparent absence of a lysine-arginine antagonist in fingerling channel catfish. J Nutr. 1981;111(1):46–52. � Thorough peer review Segner H, Arend P, Von Poeppinghausen K, Schmidt H. The effect of feeding � Inclusion in PubMed and all major indexing services astaxanthin to Oreochromis niloticus and Colisa labiosa on the histology of � Maximum visibility for your research the liver. Aquaculture. 1989;79(1–4):381–90. Shaw S. Lipid peroxidation, iron mobilization and radical generation induced by Submit your manuscript at alcohol. 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Effects of astaxanthin on antioxidant capacity of golden pompano (Trachinotus ovatus) in vivo and in vitro

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Springer Journals
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Copyright © 2017 by The Author(s)
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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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2234-1757
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10.1186/s41240-017-0052-1
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Abstract

The objective of this research was to study the effect of astaxanthin (AST) on growth performance and antioxidant capacity in golden pompano (Trachinotus ovatus) both in vivo and in vitro. In the in vivo study, two diets were formulated with or without astaxanthin supplementation (D1 and D2; 0 and 200 mg/kg) to feed fish for 6 weeks. In the in vitro study, cells from hepatopancreas of golden pompano were isolated and four treatments with or without astaxanthin and H O supplementation were applied (control group: without both astaxanthin and H O 2 2 2 2 treated; H O group: just with H O treated; H O + AST group: with both astaxanthin and H O treated; AST group: 2 2 2 2 2 2 2 2 just with AST treated). Results of the in vivo study showed that weight gain (WG) and special growth rate (SGR) significantly increased with astaxanthin supplemented (P < 0.05). Feed conversion ratio (FCR) of fish fed D2 diet was significantly lower than that of fish fed D1 diet (P < 0.05). Hepatic total antioxidant capacity (T-AOC) and the reduced glutathione (GSH) of golden pompano fed D2 diet were significant higher than those of fish fed D1 diet (P < 0.05). Superoxide dismutase (SOD) was significantly declined as astaxanthin was supplemented (P < 0.05). Results of the in vitro study showed that the cell viability of H O group was 52.37% compared to the control 2 2 group, and it was significantly elevated to 84.18% by astaxanthin supplementation (H O + AST group) (P < 0.05). 2 2 The total antioxidant capacity (T-AOC) and the reduced glutathione (GSH) of cell were significant decreased by oxidative stress from H O (P < 0.05), but it could be raised by astaxanthin supplementation (H O vs H O + AST), 2 2 2 2 2 2 and the malondialdehyde (MDA) was significant higher in H O group (P < 0.05) and astaxanthin supplementation 2 2 could alleviate the cells from lipid peroxidation injury. In conclusion, dietary astaxanthin supplementation can improve the growth performance of golden pompano. Moreover, astaxanthin can improve the golden pompano hepatic antioxidant capacity both in vivo and in vitro study by eliminating the reactive oxygen species. Keywords: Golden pompano, Astaxanthin, Growth performance, Antioxidant capacity Background normal physiological conditions, the excessive ROS can be Reactive oxygen species (ROS) are oxidative products, pro- removed by internal antioxidants and anti-oxidative sys- duced continuously in the course of normal aerobic cellular tems (Chen et al. 2015), including counter balance such as metabolism and respiratory burst (Chew 1995), which par- enzymes (like superoxide dismutase, catalase, and glutathi- ticipate in a variety of biological processes, including nor- one peroxidase), functionalized large molecules (albumin, mal cell growth, induction and maintenance of the ferritin, and ceruloplasmin) and small molecules (ascorbic transformed state, programmed cell death, and cellular sen- acid, α-tocopherol, β-carotene, and uric acid) (Martinez-Al- escence (Finkel 2003). However, ROS can, in turn, damage varez et al. 2005). The dietary antioxidants most widely healthy cells if they are not eliminated (Chew 1995). Under used include vitamin E, vitamin C, carotenoids, flavanoids, zinc, and selenium (Chew and Park 2004). Among those, * Correspondence: gzniujin2003@163.com; 1657864417@qq.com carotenoids reach the highest plasma and tissue concentra- Equal contributors tions, despite their lower intake (Olmedilla et al. 2007). Institute of Aquatic Economic Animals, School of Life Science, Sun Yat-sen Carotenoids, more than 600 known types, can be classi- University, NO.135 at Xingang Xi Road, Haizhu District, Guangzhou 510275, Guangdong Province, China fied into two categories, xanthophyll and carotenes. Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xie et al. Fisheries and Aquatic Sciences (2017) 20:6 Page 2 of 8 Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′dione, Pink, 10% astaxanthin, DSM Nutritional Products AST) is a xanthophyll carotenoid which is found in many France SAS) (Table 1). The method of diet preparation microorganisms and marine animals, such as shrimp, was the same as described by Niu et al. ( 2015). Briefly, crayfish, crustaceans, salmon, trout, krill, microalgae as all dry ingredients were finely ground, weighed, mixed well as yeast. Its molecule consists of 40 carbon atoms, di- manually for 5 min, and then transferred to a Hobart vided into a central portion containing 22 carbon atoms mixer (A-200 T Mixer Bench Model unit; Resell Food linked with 13 conjugated double bonds and two terminal Equipment Ltd., Ottawa, ON, Canada) for another benzene rings containing hydroxyl and ketone groups, giv- 15 min mixing. During the mixing, 6 N NaOH was ing rise to the higher polar structure of AST compared added to establish a pH level of 7–7.5. The pH of the with other carotenoids (Britton 1995).The antioxidant ac- diet was obtained by homogenizing a 5-g portion of the tivity of astaxanthin was found to be approximately 10 diet with 50 mL of distilled water with a glass-electrode times stronger than β-carotene (Shimidzu et al. 1996). pH meter on the supernatant (Robinson et al. 1981). Except for its antioxidant capacity, AST is also recog- Soya lecithin was added to pre-weighed fish oil and nized to have growth performance and survival rate pro- mixed until homogenous. The oil mix was then added to moting in Atlantic salmon (Salmo salar) (Christiansen the Hobart mixer slowly while mixing was still continu- and Torrissen 1996) and red porgy (Pagrus pagrus)(Kali- ing. All ingredients were mixed for another 10 min. nowski et al. 2011), skins coloration enhancing in large Then, distilled water (about 30–35%, v/w) was added to yellow croaker (Larimichthys croceus) (Yi et al. 2014) and the mixture to form dough. Dough of even consistency Atlantic salmon(Baker et al. 2002), anti-lipid peroxidation was passed through a pelletizer with a 2.5-mm-diameter (Leite et al. 2010), and immune response reinforcing in Astronotus ocellatus (Alishahi et al. 2015) properties. Table 1 Ingredients and proximate composition of the two Golden pompano (Trachinotus ovatus)belongs to family experimental diets (%) carangidae, genus Trachinotus. It is a warm-water species Ingredients Diet 1 Diet 2 (25–32 °C) and a carnivorous fish that preys mainly on zoo- Fish meal 32.00 32.00 plankton, small crustaceans, shellfish, and small fish (Liu Soybean meal 30.00 30.00 and Chen 2009). T. ovatusis is widely distributed in China, Wheat flour 20.00 19.80 Japan, Australia, and other countries (Huo-sheng 2006). Krill meal 2.00 2.00 Pompano is considered one of the most desirable food fishes, and it commands a significantly higher price than Fish oil 8.00 8.00 many other marine and freshwater species (Tutman et al. Soya lecithin 2.00 2.00 2004). Recently, pompano is widely farmed owing to its Monocalcium phosphate 2.00 2.00 high price in the market and resilience to salinity and Pre-vitamin 1.00 1.00 temperature ranges (Tutman et al. 2004), and annual out- Pre-mineral 1.00 1.00 put was over 100,000 tons. The suitable dietary protein and Choline 0.50 0.50 lipid levels for golden pompano are 46.0 and 6.5% (Wang et al. 2013). The optimum carbohydrate level for juvenile Vc 0.50 0.50 golden pompano could be 11.2–16.8% of the diet (Zhou DL-Met 0.40 0.40 et al. 2015). The optimal requirements of methionine (Niu Lys-HCL (78%) 0.60 0.60 et al. 2013), arginine (Lin et al. 2015), and n-6 long-chain AST 0 0.20 polyunsaturated fatty acid arachidonic acid (ARA) (Qi et al. Sum 100 100 2016) for golden pompano have been determined as well. Nutrient levels However, fewer studies were conducted to investi- gate the effects of antioxidants on golden pompano. Moisture 9.00 7.54 To date,theeffectsofAST on variouskinds of Crude protein 40.64 40.55 fishes are mainly reported in vivo and rarely found Crude fat 10.71 10.90 in vitro. We used in vivo and in vitro models to Ash 15.21 15.24 study and compare the effect of astaxanthin on anti- a −1 Pre-vitamin (mg or g kg diet): thiamin, 25 mg; riboflavin,45 mg; pyridoxine oxidant ability of golden pompano, respectively. HCl, 20 mg; vitamin B12, 0.1 mg; vitamin K3,10 mg; inositol, 800 mg; pantothenic acid, 60 mg; niacin acid, 200 mg; folic acid, 20 mg; biotin, 1.20 mg; retinal acetate, 32 mg; cholecalciferol, 5 mg; α-tocopherol, 120 mg; Methods ascorbic acid, 2000 mg; choline chloride, 2500 mg; ethoxyquin 150 mg; and Diet preparation and dietary treatments wheat middling, 14.012 g (Niu et al. 2013) b −1 . Pre-mineral (mg or g kg diet): NaF, 2 mg; KI, 0.8 mg; CoC 6H O (1%), In this study, two isonitrogenous and isoenergetic semi- l2 2 . . . . 50 mg; CuSO 5H O, 10 mg; FeSO H O, 80 mg; ZnSO H O, 50 mg; MnSO H O, 4 2 4 2 4 2 4 2 purified diets were formulated supplementing with or . . 60 mg; MgSO 7H O, 1200 mg; Ca(H PO ) H O, 3000 mg; NaCl, 100 mg; and 4 2 2 4 2 2 without astaxanthin (D1: 0%; D2: 0.2%; CAROPHYLL zoelite, 15.447 g (Niu et al. 2013) Xie et al. Fisheries and Aquatic Sciences (2017) 20:6 Page 3 of 8 die (Institute of Chemical Engineering, South China was sterilized by alcohol, and its abdomen was dissected University of Technology, Guangzhou, China). The diets with sterile instruments from the anus toward the head. were dried until the moisture was reduced to <10%. The Liver tissue was excised and rinsed three times with phos- dry pellets were placed in plastic bags and stored 20 °C phate buffer solution. The liver tissue was then minced until fed. into pieces of 1 mm and transferred to a 15 mL tube to which a solution of 0.25% trypsin (1:20 w/v; Sigma) was Animal rearing and experimental procedures added. The mixture was trypsinized on a thermostatic The feeding trial was conducted at an experimental sta- water bath to obtain the cell suspension, which was tion of South China Sea Fisheries Research Institute of shaken every 5 min. Then, the mixture was filtered Chinese Academy of Fishery Sciences (Sanya, Hainan). through a 100-mesh sieve. The cell suspensions were Prior to the start of the trial, juvenile T. ovatus were ac- pooled and centrifuged at 1000 rpm for 10 min, and the climated to a commercial diet for 2 weeks and were fed cell pellet was washed and resuspended in a culture twice daily to apparent satiation. At the beginning of the medium. The cell number was counted using a haemocyt- feeding trial, the fish were starved for 24 h, weighed, and ometer, and cell viability was estimated immediately fol- then fish with similar size (initial body weight 23.65 ± lowing isolation using the trypan blue exclusion assay. 0.10 g) were randomly allotted to 8 sea cages (1.0 m × 1.0 m × 1.5 m; four cages per diet treatment); each cage Cell culture and treatments was stocked with 30 fish. Each experimental diet was A final cell density of hepatocytes was adjusted to 2 × 10 −1 randomly assigned to four cages. The feeding frequency cells mL in L-15 culture medium (Jinuo Co, Hangzhou, was once daily at 8:00 and lasted for 6 weeks. To prevent China) supplemented with 2 mM L-glutamine (Sigma) the waste of pellets, fish were slowly hand-fed to sati- and 10% foetal bovine serum (Gibco). Cells were seeded ation based on visual observation of their feeding behav- into 12-well culture plates with 500 μLcellsuspension per ior. Feed consumption was recorded for each cage every well. Cells were cultured in a humidified atmosphere at day. Water quality parameters were monitored daily. 28 °C. Once seeded, cells were allowed to attach to culture plates for 24 h. At 24 h, 50% of the culture medium Sample collection (250 μL) was removed and replaced with fresh medium. At the end of the feeding trial, fish were starved for 24 h Then PBS, 100 mM H O , 1000 ng/mL astaxanthin dis- 2 2 and then weighed and counted the total number. Ten solved in dimethyl sulfoxide (DMSO) (final concentration fish from each cage were randomly collected for sam- 0.01%), and H O plus astaxanthin were added in the 2 2 pling: four for analysis of whole-body composition and wells. Every treatment was replicated in three wells. Cell six were anesthetized to obtain weights of individual viability was evaluated by trypan blue exclusion test, and whole body, viscera, and liver. The livers were rapidly re- cells were harvested for antioxidant capacity analysis after moved and frozen in the liquid nitrogen separately for the treatments. As an additional measure of cell viability, analysis of lipid peroxidation and antioxidant status. lactate dehydrogenase (LDH) activity in the extracellular medium (an indicator of membrane leakage (Misra and Biochemical composition analysis Niyogi 2009) was measured. Chemical composition of diets and fish were determined by standard methods (Latimer 2012). Moisture was de- Antioxidant capacity analysis termined by oven drying at 105 °C until a constant Hepatic and cell samples were homogenized in ice-cold weight was obtained. Crude protein content (N × 6.25) phosphate buffer (1:10 dilution) (phosphate buffer; 0.064 M, was determined according to the Kjeldahl method after pH 6.4). The homogenate was then centrifuged for 20 min acid digestion using an Auto Kjeldahl System (1030- (4 °C, 3000 g), and aliquots of the supernatant were used to Autoanalyzer; Tecator, Höganäs, Sweden). Crude lipid quantify hepatic T-AOC, GSH, SOD, and MDA. was determined by the ether extraction method using a The levels of enzyme activity and lipid peroxidation Soxtec extraction System HT (Soxtec System HT6, were measured with commercial ELISA kits (Randox La- Tecator). Ash content was determined after samples boratories Ltd.) in accordance with the instructions of the were placed in a muffle furnace at 550 °C for 4 h. manufacturer. The assays are briefly described as follows: The T-AOC is the representative of enzyme and none- Isolation of liver cells nzyme original antioxidant in the body; these antioxidants 3+ 2+ Golden pompano was purchased from a market in can reduce the ferric ion (Fe )to ferrous ion (Fe ). The Guangzhou, China. Hepatocytes were isolated according latter combines with phenanthroline and produces a to the methods of Wan et al. (2004) with some modifica- stable chelate, which can be measured by spectrophoto- tions. In the procedure, a fish was kept in 0.01% potassium graphy at 520 nm (Xiao et al. 2004). The T-AOC was de- permanganate solution for half an hour, after that, its skin termined in units per milligram of tissue protein. Xie et al. Fisheries and Aquatic Sciences (2017) 20:6 Page 4 of 8 Total superoxide dismutase (SOD) activity was mea- Results sured by using a xanthine oxides (Marklund and Mark- Growth performance in vivo lund 1974). The ratio of autooxidation rates of the Growth, feed utilization, and biometric parameters of ju- samples with or without hepatic homogenate was deter- venile pompano fed different dietary astaxanthin levels are mined at 550 nm. One unit of SOD activity was calculated shown in Table 2. Results showed that final body wet using the amount of superoxide dismutase required to in- weight (FBW), weight gain (WG), and special growth rate hibit the reduction of nitrobluete trazolium by 50%. (SGR) significantly increased with astaxanthin supple- The formation of 5-thio-2-nitrobenzoate (TNB) was mented (P < 0.05). Feed conversion ratio (FCR) of golden followed spectrophotometrically at 412 nm (Vardi et al. pompano fed the diets supplemented with astaxanthin 2008). The amount of GSH in the extract was determined was significantly lower than that of fish fed the control as μmol/mg protein utilizing a commercial GSH as the diet (P < 0.05), while no significant differences were found standard. The results are expressed as μmol/mg protein. in survival rate between the two diet treatments (P >0.05). Lipid-peroxidation levels were determined based on Hepatosomatic somatic indices (HSI), visceral somatic in- the malondialdehyde (MDA) level generated by oxidizing dices (VSI), and condition factor (CF) were significantly fatty acids. In the presence of thiobarbituric acid, malon- decreased in astaxanthin-supplemented diet treatment. dialdehyde started producing colored thiobarbituric- acid-reacting substances (TBARS) that were measured at Growth performance in vitro 532 nm (Buege and Aust 1978). In the in vitro study, the cell viability of H O group was 2 2 Lactate dehydrogenase (LDH) can catalyze lactate into 52.37% compared to the control group (PBS group), and pyruvate, which react with 2,4-dinitrophenylhydrazine it could be significantly elevated to 84.18% with astax- and produce a stable compound, which was measured anthin supplementation (H O + AST group) (P < 0.05) 2 2 by spectrophotography at 450 nm. (Fig. 1). The highest lactate dehydrogenase (LDH) activ- ity was found in H O group, and it was 159.02% com- 2 2 Calculations and statistical analysis pared to the control group, it could be significantly The parameters were calculated as follows: lessened to 122.96% with astaxanthin supplementation (H O + AST group) (P < 0.05) (Fig. 2). Weight gain rateðÞ WG; %¼ 100 2 2 ðÞ final body weight ‐ initial body weight =initial body weight Whole-body composition ‐1 Specific growth rate SGR; % day ¼ 100 Whole-body composition of golden pompano fed different ðÞ Ln final mean weight ‐ Ln initial mean weight = number of days dietary astaxanthin levels are shown in Table 3. There were no significant differences in whole-body composition of fish Feed conversion ratioðÞ FCR ¼ dry diet fed=wet weight gain between the two diet treatments (P >0.05). Survival rateðÞ % ¼ 100 ðÞ final number of fish =ðÞ initial number of fish Table 2 Growth performance and survival of golden pompano fed diets with and without supplementation of astaxanthin Viscerosomatic indexðÞ VSI; % ¼ 100 Diets Diet 1 Diet 2 ðÞ viscera weight; g =ðÞ whole body weight; g −1 (AST mg kg ) 0 200 Hepatosomatic indexðÞ HSI; %¼ 100 IBW (g) 32.72 ± 0.21 32.58 ± 0.06 ðÞ liverweight; g =ðÞ whole body weight; g FBW (g) 63.71 ± 0.66a 67.23 ± 0.22b WG (%) 95.30 ± 0.02a 106.36 ± 0.01b Condition factorðÞ CF; g=cm3¼ 100 −1 SGR (% day ) 1.59 ± 0.02a 1.72 ± 0.01b ðÞ bodyweight; g = body length; cm FCR 1.78 ± 0.02a 1.53 ± 0.02b Data from each treatment were subjected to one-way SR (%) 97.78 ± 0.01 96.67 ± 0.00 analysis of variance (ANOVA). Homogeneity of variance HSI (%) 1.13 ± 0.06a 0.94 ± 0.03b was verified using Bartlett and Levene’s test. When over- VSI (%) 5.97 ± 0.11a 5.42 ± 0.15b all differences were significant, Tukey’s multiple range CF (g/cm3) 3.4 ± 0.06a 3.04 ± 0.05b tests was used to compare the mean values among indi- Values are mean ± SEM of four replicates, and values in the same row with vidual treatments. The level of significant difference was different letters are significant different (P < 0.05). Diet 1 meant golden set at P < 0.05. Statistical analysis was performed using pompano groups fed diets without supplementation of astaxanthin (AST). Diet 2 meant groups with supplementation of astaxanthin the SPSS19.0 (SPSS Inc., Michigan Avenue, Chicago, IL, IBW initial body weight, FBW final body weight, WG weight gain, SGR special USA) for Windows, and the results are presented as growth rate, FCR feed conversion ratio, SR survival rate, HSI hepatosomatic means ± SEM (standard error of the mean). index, VSI viscerosomatic index, CF condition factor Xie et al. Fisheries and Aquatic Sciences (2017) 20:6 Page 5 of 8 antioxidant, can repair the cells from the oxidative stress. The total antioxidant capacity (T-AOC) and the 100 reduced glutathione (GSH) of oxidative stress group (H O ) were the lowest and significantly lower than 2 2 those of the control group (P < 0.05), but it could be sig- nificantly improved by astaxanthin supplementation (H O + AST group) (P < 0.05). The astaxanthin supple- 2 2 mented groups were significant higher than the other ones (P < 0.05). The SOD and MDA showed the highest value in H O group, which were significantly higher 2 2 than those in the control group (P < 0.05), but it also Control H2O2 H2O2 +AST AST could be significantly modified by astaxanthin supple- Fig. 1 The relative cell viability in different groups. Control column mentation (H O + AST group) (P < 0.05). The astax- 2 2 meant treating with neither H O nor AST, H O column with H O 2 2 2 2 2 2 anthin supplemented groups showed the significantly only, H O + AST column with both H O and AST, and AST column 2 2 2 2 higher antioxidant capacity than the other groups (P < with AST only. Data are expressed as mean ± SEM of three replicates; 0.05). values in the column sharing the same superscript letter are not significantly different; however, values in the column with the different superscript letter are significantly different Discussion Growth performance and proximate composition Antioxidant capacity analysis in vivo Carotenoids are reported to improve growth perform- The antioxidant status of juvenile pompano in vivo study ance of fish with the reason that carotenoids may exert a are presented in Table 4. The hepatic total antioxidant cap- positive influence on intermediary metabolism in aquatic acity (T-AOC) and the reduced glutathione (GSH) in fish animals (Segner et al. 1989), which enhance nutrient fed diet supplemented with astaxanthin were significantly utilization, ultimately resulting in improving growth higher than that of fish fed the control diet (P <0.05). On (Amar et al. 2001). The other possible mechanism may the contrary, superoxide dismutase (SOD) declined with be to adjust the intestinal flora breaking down indigest- astaxanthin supplementation significantly (P < 0.05), while ible feed components to extract more nutrients and to hepatic malondialdehyde (MDA) content was not affected stimulate the production of enzymes transporting fats by astaxanthin supplementation (P > 0.05). for growth instead of storage (James et al. 2006). Kali- nowski et al. (2011) believed that astaxanthin could en- Antioxidant capacity analysis in vitro hance lipid utilization in whole fish and liver, providing The antioxidant status of hepatocytes in the vitro study more energy and consequently enhancing growth per- are shown in Table 5. The H O as an oxidizing agent formance. In the present experiment, the growth per- 2 2 can totally damage the healthy cells, and astaxanthin, an formance (FBW, WG, and SGR) and feed utilization of fish fed diet with supplemental astaxanthin were signifi- cantly higher than that of fish fed the control diet. This result was in agreement with those in previous studies on Atlantic salmon (Christiansen and Torrissen 1996), red porgy (Kalinowski et al. 2011), Astronotus ocellatus 120 (Alishahi et al. 2015), and large yellow croaker (Li et al. 2014). However, effect of carotenoids on fish growth is 80 controversial. Many earlier studies have reported that dietary astaxanthin has no significant influence on growth and flesh composition of fish (Tejera et al. 2007; Table 3 Whole-body compositions (% dry weight) of golden Control H2O2 H2O2 +AST AST pompano fed diets with and without supplementation of astaxanthin Fig. 2 Lactate dehydrogenase (LDH) activity in the extracellular medium in different groups. Control column meant treating with Protein Lipid Ash Moist neither H O nor AST, H O column with H O only, H O + AST 2 2 2 2 2 2 2 2 Diet 1 61.77 ± 1.17 26.68 ± 0.17 17.71 ± 0.23 70.19 ± 1.17 column with both H O and AST, and AST column with AST only. 2 2 Diet 2 61.68 ± 1.35 26.33 ± 0.46 17.41 ± 0.94 71.68 ± 1.15 Data are expressed as mean ± SEM of three replicates; values in the column sharing the same superscript letter are not significantly Values are mean ± SEM of four replicates, and values in the same column with different letters are significant different (P < 0.05). Diet 1 meant golden different; however, values in the column with the different superscript pompano groups fed diets without supplementation of astaxanthin (AST). Diet letter are significantly different 2 meant groups with supplementation of astaxanthin Xie et al. Fisheries and Aquatic Sciences (2017) 20:6 Page 6 of 8 Table 4 Hepatic antioxidant statuses of golden pompano fed diets with and without supplementation of astaxanthin T-AOC (U/mg protein) SOD (U/mg protein) GSH (μmol/g protein) MDA (nmol/mg protein) Diet 1 0.11 ± 0.01a 240.87 ± 5.76a 82.44 ± 4.87a 0.41 ± 0.02 Diet 2 0.15 ± 0.01b 214.24 ± 5.71b 118.52 ± 8.93b 0.41 ± 0.06 Values are mean ± SEM of four replicates, and values in the same column with different letters are significant different (P < 0.05). Diet 1 meant golden pompano groups fed diets without supplementation of astaxanthin (AST). Diet 2 meant groups with supplementation of astaxanthin T-AOC total antioxidant capacity, GSH reduced glutathione, SOD superoxide dismutase, MDA malondialdehyde Zhang et al. 2012; Pham et al. 2014; Yi et al. 2014). Kop H O may decrease the total antioxidant capacity, the 2 2 and Durmaz (2008) believed that the effectiveness of ca- supplementation of astaxanthin can repair it to the same rotenoids in terms of deposition and physiological func- level with the control group. tion is species-specific in fish and not all fish species The stress response might increase free radical con- possess the same pathways for the metabolism of carot- tents, which may result in the increase of the lipid per- enoids. The mechanisms related to these findings have oxidation content and lipid peroxidation injury (Liu not yet been clearly elucidated. Our latest research re- et al. 2010). Malondialdehyde (MDA) is a product of sults showed that the dietary astaxanthin can increase lipid peroxidation, through crosslinking with the nucleo- the apparent digestibility coefficient of the diet and fur- philic groups of proteins, nucleic acids, and amino phos- ther promote the expression of insulin-like growth fac- pholipids, accumulation of MDA leads to cell toxicity, tors (IGFs); moreover, as members of the family of accelerating the damage of cells and tissues (Buege and transforming growth factors β, myostatin is affected by Aust 1978). The antioxidants and antioxidant enzyme dietary astaxanthin (unpublished data). system can play a significant role in resisting lipid oxide damage (Liu et al. 2010). Carotenoids may serve as an Antioxidant capacity analysis antioxidant in systems containing unsaturated fatty acids H O is a strong oxidizer, produced in cell metabolism, to quench free radicals (Mansour et al. 2006). The re- 2 2 but the excessive dose may be cytotoxic. As is shown, sults showed that the MDA were not significant different cell viability was sharply decreased with H O supple- when no stress appeared in the present in vivo study. 2 2 mented and the increased LDH leakage into the extra- However, once the cells suffered from oxidative stress in cellular media by H O indicated the occurrence of the present in vitro study, the MDA was increased and 2 2 oxidative stress membrane damage in our present the cell viability was decreased, but supplemented astax- in vitro study. Cellular antioxidative defense mechanisms anthin could totally decrease the MDA value and save can intercept the ROS both enzymatically and non- cells from the stress. Increased T-AOC and decreased enzymatically. Total antioxidant capacity (T-AOC) is an MDA in the in vitro study reflected that supplemented overall indicator of the antioxidant status of an individ- astaxanthin in media can be totally conducive to elimin- ual, representing the level of enzyme and nonenzyme ate the reactive oxygen species and protect the hepato- original antioxidantin of the body (Xiao et al. 2004). As cytes of golden pompano from free radicals. The MDA the value increases, the antioxidant defense against free in (H O + AST) group was lower than that in H O 2 2 2 2 radical reaction and reactive oxygen intermediates in- group, which indicated that AST can alleviate the lipid creases (Chien et al. 2003). In both of the in vivo and oxide damage. in vitro study, the T-AOC in the liver of the fish and in Superoxide dismutase (SOD), a cytosolic enzyme that the hepatocytes supplemented with astaxanthin were is specific for scavenging superoxide radicals, is the first higher, meaning that astaxanthin can improve the anti- enzymes to respond against oxygen radicals and import- oxidant status whether in vivo or in vitro. Although ant endogenous antioxidants for protection against Table 5 The antioxidant statuses of hepatocytes treated with or without astaxanthin and H O supplementation 2 2 T-AOC SOD GSH (μmol/g protein) MDA (nmol/mg protein) (U/mg protein) (U/mg protein) Control 0.35 ± 0.01b 2682.76 ± 127.04b 17.81 ± 0.83b 0.13 ± 0.01a H O 0.22 ± 0.02a 3264.92 ± 76.26c 5.92 ± 0.91a 0.40 ± 0.01c 2 2 H O + AST 0.37 ± 0.01bc 2726.34 ± 74.17b 28.24 ± 1.11c 0.23 ± 0.01bc 2 2 AST 0.41 ± 0.01c 2312.19 ± 69.94a 136.51 ± 4.11d 0.12 ± 0.01a Values are mean ± SEM of three replicates, and values in the same column with different letters are significant different (P < 0.05). Control meant golden pompano groups fed diets without supplementation of astaxanthin (AST). Control groups meant treating with neither H O nor AST, H O groups meant treating with H O 2 2 2 2 2 2 only, H O + AST groups meant treating with both H O and AST, and AST groups meant treating with AST only 2 2 2 2 T-AOC total antioxidant capacity, GSH reduced glutathione, SOD superoxide dismutase, MDA malondialdehyde Xie et al. Fisheries and Aquatic Sciences (2017) 20:6 Page 7 of 8 oxidative stress (Winston and Di Giulio 1991). Lygren Funding The design of the study and collection, analysis, interpretation of data, and et al. showed that high levels of dietary fat-soluble anti- writing the manuscript were supported by the Project of Science and oxidants, such as astaxanthin and vitamin E, there was a Technology of Guangdong Province (2013B090500110, 2013B090600045), the reduced need for endogenous antioxidant enzymes, such Central Institutes of Public Welfare Projects (2014A08YQ02), and the Special Project of Marine Fishery Science and Technology of Guangdong Province as total SOD (Lygren et al. 1999). The higher the SOD (A201601C11). value, the more superoxide radicals need to be reacted (Qingming et al. 2010). It was found that the activities of Authors’ contributions JN, JW, and YW designed the study. JJX wrote the article. JJX, QQL, and XC liver SOD were significantly decreased by dietary astax- performed the experiment. JN and JJX analyzed and interpreted the data. All anthin supplementation in olive flounder (Paralichthys authors have read, commented upon, and approved the final article. olivaceus) (Pham et al. 2014); large yellow croaker (Pseu- Competing interests dosciaena crocea) (Li et al. 2014) and rainbow trout (On- The authors declare that they have no competing interests. corhynchus mykiss) (Zhang et al. 2012). In this present study, SOD was significant lower in vivo and vitro study Consent for publication Not applicable both supplemented with astaxanthin, implying that astaxanthin can eliminate reactive oxygen species to Ethics approval and consent to participate avoid the cells and tissues to produce more SOD. Once All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals in Sun Yat-sen Univer- suffering from oxidative stress, the cells may produce sity, Guangzhou, China, and conformed to the National Institutes of Health much more endogenous SOD, as is shown in the study, Guide for Care and Use of Laboratory Animals (publication no. 85–23, revised to protect the body or cells from being hurt. 1985). Glutathione (GSH), ubiquitous non-enzymatic antioxi- dants in the cells, is known to play an important role in Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in the scavenging of free radicals and thus protect the im- published maps and institutional affiliations. portant cellular macromolecules and organelles from oxidative damage (Misra and Niyogi 2009). Its role in Author details South China Sea Fisheries Research Institute, Chinese Academy of Fishery the detoxification of ROS is important (Mallikarjuna Sciences, Guangzhou 510300, China. School of Life Science and Technology, et al. 2009). When suffered from oxidative stress, GSH 3 Shanghai Ocean University, Shanghai 201306, China. Institute of Aquatic was significantly lower in the present in vitro study. One Economic Animals, School of Life Science, Sun Yat-sen University, NO.135 at Xingang Xi Road, Haizhu District, Guangzhou 510275, Guangdong Province, mechanism for oxidative stress induced GSH depletion China. may involve enhanced utilization of GSH for the detoxi- fication of free radicals and other oxidants produced as a Received: 25 January 2017 Accepted: 18 April 2017 result of H O exposure (Shaw 1989). Vogt suggested 2 2 that the increase of lipid peroxidation was not apparent Reference until after GSH levels had been depleted (Vogt and Alishahi M, Karamifar M, Mesbah M. 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