Background

Reactive oxygen species (ROS) are oxidative products, produced continuously in the course of normal aerobic cellular metabolism and respiratory burst (Chew 1995), which participate in a variety of biological processes, including normal cell growth, induction and maintenance of the transformed state, programmed cell death, and cellular senescence (Finkel 2003). However, ROS can, in turn, damage healthy cells if they are not eliminated (Chew 1995). Under normal physiological conditions, the excessive ROS can be removed by internal antioxidants and anti-oxidative systems (Chen et al. 2015), including counter balance such as enzymes (like superoxide dismutase, catalase, and glutathione peroxidase), functionalized large molecules (albumin, ferritin, and ceruloplasmin) and small molecules (ascorbic acid, α-tocopherol, β-carotene, and uric acid) (Martinez-Alvarez et al. 2005). The dietary antioxidants most widely used include vitamin E, vitamin C, carotenoids, flavanoids, zinc, and selenium (Chew and Park 2004). Among those, carotenoids reach the highest plasma and tissue concentrations, despite their lower intake (Olmedilla et al. 2007).

Carotenoids, more than 600 known types, can be classified into two categories, xanthophyll and carotenes. Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′dione, AST) is a xanthophyll carotenoid which is found in many microorganisms and marine animals, such as shrimp, crayfish, crustaceans, salmon, trout, krill, microalgae as well as yeast. Its molecule consists of 40 carbon atoms, divided into a central portion containing 22 carbon atoms linked with 13 conjugated double bonds and two terminal benzene rings containing hydroxyl and ketone groups, giving rise to the higher polar structure of AST compared with other carotenoids (Britton 1995).The antioxidant activity of astaxanthin was found to be approximately 10 times stronger than β-carotene (Shimidzu et al. 1996).

Except for its antioxidant capacity, AST is also recognized to have growth performance and survival rate promoting in Atlantic salmon (Salmo salar) (Christiansen and Torrissen 1996) and red porgy (Pagrus pagrus) (Kalinowski et al. 2011), skins coloration enhancing in large yellow croaker (Larimichthys croceus) (Yi et al. 2014) and Atlantic salmon(Baker et al. 2002), anti-lipid peroxidation (Leite et al. 2010), and immune response reinforcing in Astronotus ocellatus (Alishahi et al. 2015) properties.

Golden pompano (Trachinotus ovatus) belongs to family carangidae, genus Trachinotus. It is a warm-water species (25–32 °C) and a carnivorous fish that preys mainly on zooplankton, small crustaceans, shellfish, and small fish (Liu and Chen 2009). T. ovatusis is widely distributed in China, Japan, Australia, and other countries (Huo-sheng 2006). Pompano is considered one of the most desirable food fishes, and it commands a significantly higher price than many other marine and freshwater species (Tutman et al. 2004). Recently, pompano is widely farmed owing to its high price in the market and resilience to salinity and temperature ranges (Tutman et al. 2004), and annual output was over 100,000 tons. The suitable dietary protein and lipid levels for golden pompano are 46.0 and 6.5% (Wang et al. 2013). The optimum carbohydrate level for juvenile golden pompano could be 11.2–16.8% of the diet (Zhou et al. 2015). The optimal requirements of methionine (Niu et al. 2013), arginine (Lin et al. 2015), and n-6 long-chain polyunsaturated fatty acid arachidonic acid (ARA) (Qi et al. 2016) for golden pompano have been determined as well.

However, fewer studies were conducted to investigate the effects of antioxidants on golden pompano. To date, the effects of AST on various kinds of fishes are mainly reported in vivo and rarely found in vitro. We used in vivo and in vitro models to study and compare the effect of astaxanthin on antioxidant ability of golden pompano, respectively.

Methods

Diet preparation and dietary treatments

In this study, two isonitrogenous and isoenergetic semi-purified diets were formulated supplementing with or without astaxanthin (D1: 0%; D2: 0.2%; CAROPHYLL Pink, 10% astaxanthin, DSM Nutritional Products France SAS) (Table 1). The method of diet preparation was the same as described by Niu et al. ( 2015). Briefly, all dry ingredients were finely ground, weighed, mixed manually for 5 min, and then transferred to a Hobart mixer (A-200 T Mixer Bench Model unit; Resell Food Equipment Ltd., Ottawa, ON, Canada) for another 15 min mixing. During the mixing, 6 N NaOH was added to establish a pH level of 7–7.5. The pH of the diet was obtained by homogenizing a 5-g portion of the diet with 50 mL of distilled water with a glass-electrode pH meter on the supernatant (Robinson et al. 1981). Soya lecithin was added to pre-weighed fish oil and mixed until homogenous. The oil mix was then added to the Hobart mixer slowly while mixing was still continuing. All ingredients were mixed for another 10 min. Then, distilled water (about 30–35%, v/w) was added to the mixture to form dough. Dough of even consistency was passed through a pelletizer with a 2.5-mm-diameter die (Institute of Chemical Engineering, South China University of Technology, Guangzhou, China). The diets were dried until the moisture was reduced to <10%. The dry pellets were placed in plastic bags and stored 20 °C until fed.

Table 1 Ingredients and proximate composition of the two experimental diets (%)
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Animal rearing and experimental procedures

The feeding trial was conducted at an experimental station of South China Sea Fisheries Research Institute of Chinese Academy of Fishery Sciences (Sanya, Hainan). Prior to the start of the trial, juvenile T. ovatus were acclimated to a commercial diet for 2 weeks and were fed twice daily to apparent satiation. At the beginning of the feeding trial, the fish were starved for 24 h, weighed, and then fish with similar size (initial body weight 23.65 ± 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 was stocked with 30 fish. Each experimental diet was randomly assigned to four cages. The feeding frequency was once daily at 8:00 and lasted for 6 weeks. To prevent the waste of pellets, fish were slowly hand-fed to satiation based on visual observation of their feeding behavior. Feed consumption was recorded for each cage every day. Water quality parameters were monitored daily.

Sample collection

At the end of the feeding trial, fish were starved for 24 h and then weighed and counted the total number. Ten fish from each cage were randomly collected for sampling: four for analysis of whole-body composition and six were anesthetized to obtain weights of individual whole body, viscera, and liver. The livers were rapidly removed and frozen in the liquid nitrogen separately for analysis of lipid peroxidation and antioxidant status.

Biochemical composition analysis

Chemical composition of diets and fish were determined by standard methods (Latimer 2012). Moisture was determined by oven drying at 105 °C until a constant weight was obtained. Crude protein content (N × 6.25) was determined according to the Kjeldahl method after acid digestion using an Auto Kjeldahl System (1030-Autoanalyzer; Tecator, Höganäs, Sweden). Crude lipid was determined by the ether extraction method using a Soxtec extraction System HT (Soxtec System HT6, Tecator). Ash content was determined after samples were placed in a muffle furnace at 550 °C for 4 h.

Isolation of liver cells

Golden pompano was purchased from a market in Guangzhou, China. Hepatocytes were isolated according to the methods of Wan et al. (2004) with some modifications. In the procedure, a fish was kept in 0.01% potassium permanganate solution for half an hour, after that, its skin was sterilized by alcohol, and its abdomen was dissected with sterile instruments from the anus toward the head. Liver tissue was excised and rinsed three times with phosphate buffer solution. The liver tissue was then minced into pieces of 1 mm3 and transferred to a 15 mL tube to which a solution of 0.25% trypsin (1:20 w/v; Sigma) was added. The mixture was trypsinized on a thermostatic water bath to obtain the cell suspension, which was shaken every 5 min. Then, the mixture was filtered through a 100-mesh sieve. The cell suspensions were pooled and centrifuged at 1000 rpm for 10 min, and the cell pellet was washed and resuspended in a culture medium. The cell number was counted using a haemocytometer, and cell viability was estimated immediately following isolation using the trypan blue exclusion assay.

Cell culture and treatments

A final cell density of hepatocytes was adjusted to 2 × 106 cells mL−1 in L-15 culture medium (Jinuo Co, Hangzhou, China) supplemented with 2 mM L-glutamine (Sigma) and 10% foetal bovine serum (Gibco). Cells were seeded into 12-well culture plates with 500 μL cell suspension per well. Cells were cultured in a humidified atmosphere at 28 °C. Once seeded, cells were allowed to attach to culture plates for 24 h. At 24 h, 50% of the culture medium (250 μL) was removed and replaced with fresh medium. Then PBS, 100 mM H2O2, 1000 ng/mL astaxanthin dissolved in dimethyl sulfoxide (DMSO) (final concentration 0.01%), and H2O2 plus astaxanthin were added in the wells. Every treatment was replicated in three wells. Cell viability was evaluated by trypan blue exclusion test, and cells were harvested for antioxidant capacity analysis after the treatments. As an additional measure of cell viability, lactate dehydrogenase (LDH) activity in the extracellular medium (an indicator of membrane leakage (Misra and Niyogi 2009) was measured.

Antioxidant capacity analysis

Hepatic and cell samples were homogenized in ice-cold phosphate buffer (1:10 dilution) (phosphate buffer; 0.064 M, pH 6.4). The homogenate was then centrifuged for 20 min (4 °C, 3000 g), and aliquots of the supernatant were used to quantify hepatic T-AOC, GSH, SOD, and MDA.

The levels of enzyme activity and lipid peroxidation were measured with commercial ELISA kits (Randox Laboratories Ltd.) in accordance with the instructions of the manufacturer. The assays are briefly described as follows: The T-AOC is the representative of enzyme and nonenzyme original antioxidant in the body; these antioxidants can reduce the ferric ion (Fe3+) to ferrous ion (Fe2+). The latter combines with phenanthroline and produces a stable chelate, which can be measured by spectrophotography at 520 nm (Xiao et al. 2004). The T-AOC was determined in units per milligram of tissue protein.

Total superoxide dismutase (SOD) activity was measured by using a xanthine oxides (Marklund and Marklund 1974). The ratio of autooxidation rates of the samples with or without hepatic homogenate was determined at 550 nm. One unit of SOD activity was calculated using the amount of superoxide dismutase required to inhibit the reduction of nitrobluete trazolium by 50%.

The formation of 5-thio-2-nitrobenzoate (TNB) was followed spectrophotometrically at 412 nm (Vardi et al. 2008). The amount of GSH in the extract was determined as μmol/mg protein utilizing a commercial GSH as the standard. The results are expressed as μmol/mg protein.

Lipid-peroxidation levels were determined based on the malondialdehyde (MDA) level generated by oxidizing fatty acids. In the presence of thiobarbituric acid, malondialdehyde started producing colored thiobarbituric-acid-reacting substances (TBARS) that were measured at 532 nm (Buege and Aust 1978).

Lactate dehydrogenase (LDH) can catalyze lactate into pyruvate, which react with 2,4-dinitrophenylhydrazine and produce a stable compound, which was measured by spectrophotography at 450 nm.

Calculations and statistical analysis

The parameters were calculated as follows:

$$ \mathrm{Weight}\ \mathrm{gain}\ \mathrm{rate}\ \left(\mathrm{WG},\ \%\right) = 100 \times \left(\mathrm{final}\ \mathrm{body}\ \mathrm{weight}\ \hbox{-}\ \mathrm{initial}\ \mathrm{body}\ \mathrm{weight}\right)/\mathrm{initial}\ \mathrm{body}\ \mathrm{weight} $$
$$ \mathrm{Specific}\ \mathrm{growth}\ \mathrm{rate}\ \left(\mathrm{SGR},\ \%\ \mathrm{da}{\mathrm{y}}^{\hbox{-} 1}\right) = 100 \times \left(\mathrm{Ln}\ \mathrm{final}\ \mathrm{mean}\ \mathrm{weight}\ \hbox{-}\ \mathrm{Ln}\ \mathrm{initial}\ \mathrm{mean}\ \mathrm{weight}\right)/\ \mathrm{number}\ \mathrm{of}\ \mathrm{da}\mathrm{ys} $$
$$ \mathrm{Feed}\ \mathrm{conversion}\ \mathrm{ratio}\ \left(\mathrm{FCR}\right) = \mathrm{dry}\ \mathrm{diet}\ \mathrm{fed}/\mathrm{wet}\ \mathrm{weight}\ \mathrm{gain} $$
$$ \mathrm{Survival}\ \mathrm{rate}\ \left(\%\right) = 100 \times \left(\mathrm{final}\ \mathrm{number}\ \mathrm{of}\ \mathrm{fish}\right)/\left(\mathrm{initial}\ \mathrm{number}\ \mathrm{of}\ \mathrm{fish}\right) $$
$$ \mathrm{Viscerosomatic}\ \mathrm{index}\ \left(\mathrm{VSI},\ \%\right) = 100 \times \left(\mathrm{viscera}\ \mathrm{weight},\ \mathrm{g}\right)/\left(\mathrm{whole}\ \mathrm{body}\ \mathrm{weight},\ \mathrm{g}\right) $$
$$ \mathrm{Hepatosomatic}\ \mathrm{index}\ \left(\mathrm{HSI},\ \%\right) = 100 \times \left(\mathrm{liverweight},\ \mathrm{g}\right)\ /\left(\mathrm{whole}\ \mathrm{body}\ \mathrm{weight},\ \mathrm{g}\right) $$
$$ \mathrm{Condition}\ \mathrm{factor}\ \left(\mathrm{CF},\ \mathrm{g}/\mathrm{cm}3\right) = 100 \times \left(\mathrm{body}\mathrm{weight},\ \mathrm{g}\right)/\left(\mathrm{body}\ \mathrm{length},\ \mathrm{c}{\mathrm{m}}^3\right) $$

Data from each treatment were subjected to one-way analysis of variance (ANOVA). Homogeneity of variance was verified using Bartlett and Levene’s test. When overall differences were significant, Tukey’s multiple range tests was used to compare the mean values among individual treatments. The level of significant difference was set at P < 0.05. Statistical analysis was performed using the SPSS19.0 (SPSS Inc., Michigan Avenue, Chicago, IL, USA) for Windows, and the results are presented as means ± SEM (standard error of the mean).

Results

Growth performance in vivo

Growth, feed utilization, and biometric parameters of juvenile pompano fed different dietary astaxanthin levels are shown in Table 2. Results showed that final body wet weight (FBW), weight gain (WG), and special growth rate (SGR) significantly increased with astaxanthin supplemented (P < 0.05). Feed conversion ratio (FCR) of golden pompano fed the diets supplemented with astaxanthin was significantly lower than that of fish fed the control diet (P < 0.05), while no significant differences were found in survival rate between the two diet treatments (P > 0.05). Hepatosomatic somatic indices (HSI), visceral somatic indices (VSI), and condition factor (CF) were significantly decreased in astaxanthin-supplemented diet treatment.

Table 2 Growth performance and survival of golden pompano fed diets with and without supplementation of astaxanthin
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Growth performance in vitro

In the in vitro study, the cell viability of H2O2 group was 52.37% compared to the control group (PBS group), and it could be significantly elevated to 84.18% with astaxanthin supplementation (H2O2 + AST group) (P < 0.05) (Fig. 1). The highest lactate dehydrogenase (LDH) activity was found in H2O2 group, and it was 159.02% compared to the control group, it could be significantly lessened to 122.96% with astaxanthin supplementation (H2O2 + AST group) (P < 0.05) (Fig. 2).

Fig. 1
figure 1

The relative cell viability in different groups. Control column meant treating with neither H2O2 nor AST, H2O2 column with H2O2 only, H2O2 + AST column with both H2O2 and AST, and AST column with AST only. Data are expressed as mean ± SEM of three replicates; 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

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Fig. 2
figure 2

Lactate dehydrogenase (LDH) activity in the extracellular medium in different groups. Control column meant treating with neither H2O2 nor AST, H2O2 column with H2O2 only, H2O2 + AST column with both H2O2 and AST, and AST column with AST only. Data are expressed as mean ± SEM of three replicates; 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

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Whole-body composition

Whole-body composition of golden pompano fed different dietary astaxanthin levels are shown in Table 3. There were no significant differences in whole-body composition of fish between the two diet treatments (P > 0.05).

Table 3 Whole-body compositions (% dry weight) of golden pompano fed diets with and without supplementation of astaxanthin
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Antioxidant capacity analysis in vivo

The antioxidant status of juvenile pompano in vivo study are presented in Table 4. The hepatic total antioxidant capacity (T-AOC) and the reduced glutathione (GSH) in fish fed diet supplemented with astaxanthin were significantly higher than that of fish fed the control diet (P < 0.05). On the contrary, superoxide dismutase (SOD) declined with astaxanthin supplementation significantly (P < 0.05), while hepatic malondialdehyde (MDA) content was not affected by astaxanthin supplementation (P > 0.05).

Table 4 Hepatic antioxidant statuses of golden pompano fed diets with and without supplementation of astaxanthin
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Antioxidant capacity analysis in vitro

The antioxidant status of hepatocytes in the vitro study are shown in Table 5. The H2O2 as an oxidizing agent can totally damage the healthy cells, and astaxanthin, an antioxidant, can repair the cells from the oxidative stress. The total antioxidant capacity (T-AOC) and the reduced glutathione (GSH) of oxidative stress group (H2O2) were the lowest and significantly lower than those of the control group (P < 0.05), but it could be significantly improved by astaxanthin supplementation (H2O2 + AST group) (P < 0.05). The astaxanthin supplemented groups were significant higher than the other ones (P < 0.05). The SOD and MDA showed the highest value in H2O2 group, which were significantly higher than those in the control group (P < 0.05), but it also could be significantly modified by astaxanthin supplementation (H2O2 + AST group) (P < 0.05). The astaxanthin supplemented groups showed the significantly higher antioxidant capacity than the other groups (P < 0.05).

Table 5 The antioxidant statuses of hepatocytes treated with or without astaxanthin and H2O2 supplementation
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Discussion

Growth performance and proximate composition

Carotenoids are reported to improve growth performance of fish with the reason that carotenoids may exert a positive influence on intermediary metabolism in aquatic animals (Segner et al. 1989), which enhance nutrient utilization, ultimately resulting in improving growth (Amar et al. 2001). The other possible mechanism may be to adjust the intestinal flora breaking down indigestible feed components to extract more nutrients and to stimulate the production of enzymes transporting fats for growth instead of storage (James et al. 2006). Kalinowski et al. (2011) believed that astaxanthin could enhance lipid utilization in whole fish and liver, providing more energy and consequently enhancing growth performance. In the present experiment, the growth performance (FBW, WG, and SGR) and feed utilization of fish fed diet with supplemental astaxanthin were significantly 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 (Alishahi et al. 2015), and large yellow croaker (Li et al. 2014). However, effect of carotenoids on fish growth is controversial. Many earlier studies have reported that dietary astaxanthin has no significant influence on growth and flesh composition of fish (Tejera et al. 2007; Zhang et al. 2012; Pham et al. 2014; Yi et al. 2014). Kop and Durmaz (2008) believed that the effectiveness of carotenoids in terms of deposition and physiological function is species-specific in fish and not all fish species possess the same pathways for the metabolism of carotenoids. The mechanisms related to these findings have not yet been clearly elucidated. Our latest research results showed that the dietary astaxanthin can increase the apparent digestibility coefficient of the diet and further promote the expression of insulin-like growth factors (IGFs); moreover, as members of the family of transforming growth factors β, myostatin is affected by dietary astaxanthin (unpublished data).

Antioxidant capacity analysis

H2O2 is a strong oxidizer, produced in cell metabolism, but the excessive dose may be cytotoxic. As is shown, cell viability was sharply decreased with H2O2 supplemented and the increased LDH leakage into the extracellular media by H2O2 indicated the occurrence of oxidative stress membrane damage in our present in vitro study. Cellular antioxidative defense mechanisms can intercept the ROS both enzymatically and non-enzymatically. Total antioxidant capacity (T-AOC) is an overall indicator of the antioxidant status of an individual, representing the level of enzyme and nonenzyme original antioxidantin of the body (Xiao et al. 2004). As the value increases, the antioxidant defense against free radical reaction and reactive oxygen intermediates increases (Chien et al. 2003). In both of the in vivo and in vitro study, the T-AOC in the liver of the fish and in the hepatocytes supplemented with astaxanthin were higher, meaning that astaxanthin can improve the antioxidant status whether in vivo or in vitro. Although H2O2 may decrease the total antioxidant capacity, the supplementation of astaxanthin can repair it to the same level with the control group.

The stress response might increase free radical contents, which may result in the increase of the lipid peroxidation content and lipid peroxidation injury (Liu et al. 2010). Malondialdehyde (MDA) is a product of lipid peroxidation, through crosslinking with the nucleophilic groups of proteins, nucleic acids, and amino phospholipids, accumulation of MDA leads to cell toxicity, accelerating the damage of cells and tissues (Buege and Aust 1978). The antioxidants and antioxidant enzyme system can play a significant role in resisting lipid oxide damage (Liu et al. 2010). Carotenoids may serve as an antioxidant in systems containing unsaturated fatty acids to quench free radicals (Mansour et al. 2006). The results showed that the MDA were not significant different when no stress appeared in the present in vivo study. However, once the cells suffered from oxidative stress in the present in vitro study, the MDA was increased and the cell viability was decreased, but supplemented astaxanthin could totally decrease the MDA value and save cells from the stress. Increased T-AOC and decreased MDA in the in vitro study reflected that supplemented astaxanthin in media can be totally conducive to eliminate the reactive oxygen species and protect the hepatocytes of golden pompano from free radicals. The MDA in (H2O2 + AST) group was lower than that in H2O2 group, which indicated that AST can alleviate the lipid oxide damage.

Superoxide dismutase (SOD), a cytosolic enzyme that is specific for scavenging superoxide radicals, is the first enzymes to respond against oxygen radicals and important endogenous antioxidants for protection against oxidative stress (Winston and Di Giulio 1991). Lygren et al. showed that high levels of dietary fat-soluble antioxidants, such as astaxanthin and vitamin E, there was a reduced need for endogenous antioxidant enzymes, such as total SOD (Lygren et al. 1999). The higher the SOD value, the more superoxide radicals need to be reacted (Qingming et al. 2010). It was found that the activities of liver SOD were significantly decreased by dietary astaxanthin supplementation in olive flounder (Paralichthys olivaceus) (Pham et al. 2014); large yellow croaker (Pseudosciaena crocea) (Li et al. 2014) and rainbow trout (Oncorhynchus mykiss) (Zhang et al. 2012). In this present study, SOD was significant lower in vivo and vitro study both supplemented with astaxanthin, implying that astaxanthin can eliminate reactive oxygen species to avoid the cells and tissues to produce more SOD. Once suffering from oxidative stress, the cells may produce much more endogenous SOD, as is shown in the study, to protect the body or cells from being hurt.

Glutathione (GSH), ubiquitous non-enzymatic antioxidants in the cells, is known to play an important role in the scavenging of free radicals and thus protect the important cellular macromolecules and organelles from oxidative damage (Misra and Niyogi 2009). Its role in the detoxification of ROS is important (Mallikarjuna et al. 2009). When suffered from oxidative stress, GSH was significantly lower in the present in vitro study. One mechanism for oxidative stress induced GSH depletion may involve enhanced utilization of GSH for the detoxification of free radicals and other oxidants produced as a result of H2O2 exposure (Shaw 1989). Vogt suggested that the increase of lipid peroxidation was not apparent until after GSH levels had been depleted (Vogt and Richie 2007). Astaxanthin can improve the GSH content in both our in vivo and in vitro study.

Conclusions

In conclusion, dietary astaxanthin supplementation can improve the growth performance of golden pompano. Moreover, astaxanthin can improve their hepatic antioxidant capacity in both vivo and vitro study by eliminating the reactive oxygen species.