Astaxanthin helps increase immunity, growth performance, and antioxidant capacity in puffer fish under high temperature stress.
The present study was conducted to investigate the effects of astaxanthin on growth performance, biochemical parameters, ROS production and immune-related gene expressions of pufferfish (Takifugu obscurus) under heat stress. height. Experimental basic diets supplemented with astaxanthin at a rate of 0 (control), 20, 40, 80, 160 and 320 mg kg-1 were fed to fish for 8 weeks. Results showed that fish fed diets containing 80, 160 and 320 mg/kg astaxanthin significantly improved weight gain and specific growth rate. Furthermore, fish fed moderate dietary astaxanthin increased plasma alkaline phosphatase activity and decreased plasma aspartate aminotransferase and alanine aminotransferase activities. After the feeding trial, fish were subjected to high temperature stress for 48 h. The results showed that astaxanthin could prevent ROS production caused by high temperature stress. Meanwhile, compared with the control group, the astaxanthin group had increased levels of SOD, CAT and HSP70 mRNA under high temperature stress. These results suggest that a basal diet supplemented with 80–320 mg kg−1 astaxanthin can enhance growth, nonspecific immune response and antioxidant defense system as well as improve against high temperature stress in puffer fish.
In aquaculture, aquatic animals often encounter environmental pollutants, high water temperatures, bacterial and viral invasion, and human intervention. All of these adverse environmental factors can induce stress responses in fish (Barton 2002). Water temperature is an important environmental factor in aquaculture, affecting the survival and development of organisms. Any change in water temperature can affect survival, physiological functions, and immune defenses in teleost fish (Bowden 2008; Lee et al. 2014).
In addition, increased temperature increases fish growth by increasing metabolic activities within a certain range, while too high a temperature can lead to physiological dysfunction, increasing the possibility of infection. infections and even death (Verma et al. 2007). Previous studies have shown that high temperature challenge can induce oxidative stress in aquatic organisms (Cheng et al. 2015). Stress-induced ROS are playing a pivotal role in the innate immune defense system (Luo et al. 2014).
To combat oxidative stress and keep the cell’s redox state in balance, antioxidant enzymes and endogenous antioxidants are considered the first cellular defenders against oxidative stress. However, excessive ROS levels induced by stress can damage important biomolecules, such as DNA, proteins, and lipids, and lead to subsequent decline in physiological function (Liu et al. 2014).
In aquaculture, to reduce the adverse effects of excessive stress, many efforts have been made to reduce stress and enhance immunity in aquatic animals using various nutrients. in the diet. Nutritional needs are one of the most important factors in the survival, growth and immunity of economic fish (Bricknell
and Dalmo 2005; Nayak 2010). Astaxanthin is a nutrient involved in the immune system. It is a keto oxycarotenoid widely distributed in crustaceans, salmon meat and seaweed. Astaxanthin has important biological functions, such as antioxidant activity, immune response regulation, and disease resistance (Martin et al. 1999; Chew et al. 2011). Its antioxidant activity is reported to be higher than that of β-carotene, lutein and α-tocopherol (Naguib 2000).
A previous study also reported that astaxanthin can improve the cell-mediated and humoral immune response of mammals (Park et al. 2011). Therefore, astaxanthin is used to improve growth, reproductive performance, and immune system in fish and shellfish (Li et al. 2014;
Jagruthi et al. 2014). The pufferfish (Takifugu obscurus), widely distributed in the Sea of Japan, the East China Sea, and the Yellow Sea, is a commercially important anadromous fish. Wild pufferfish stocks have plummeted due to water pollution and overfishing. In recent years, it has become one of the newest farmed fish species in South China due to its large body size, fast growth rate and high market value. However, farmed puffer fish suffer from serious disease problems due to drugs and stress. Especially in summer, high water temperatures often last for long periods of time, increasing ROS values and the oxidative stress response in fish to the highest level, causing significant economic losses. Therefore, we studied the effects of astaxanthin on growth performance and biochemical parameters after fish culture. In the next experiment, fish fed different types of astaxanthin were challenged at high temperatures, followed by measurement of physiological responses and immune-related gene expression. Our results will provide insight into the physiological responses and molecular mechanisms underlying the protective effects of astaxanthin against high temperature stress in puffer fish.
Materials and methods
Experimental diet
The formula and approximate composition of the basal diet are presented in Table 1. Ingredients were purchased from the Institute of Animal Science, Guangdong Institute of Agricultural Sciences (Guangdong, China). The basal diet was supplemented with six levels of astaxanthin (0, 20, 40, 80, 160 and 320 m kg-1 diet) at the expense of a small amount of cellulose. A basal diet without astaxanthin supplementation was used as a control diet. All ingredients were ground into fine powder through a 60 mm mesh. They are thoroughly mixed until homogeneous in a Hobart-style mixer. Then, lipid and water are added and mixed thoroughly. Each mixture was pelleted (2 mm diameter) using a laboratory pellet press (Institute of Chemical Engineering, South China University of Technology, Guangzhou, China). After air drying, all samples were sealed in plastic bags and stored frozen (-20 °C).
Experimental animals
Puffer fish were taken from a fish farm in Panyu (Guangdong, China). Before starting the experiment, experimental fish were acclimatized to the environment for 2 weeks under laboratory conditions. All fish were fed a basal diet throughout the experiment. At the beginning of the experiment, 540 fish with an average weight of 8.3 ± 0.08 g (mean ± SD) were weighed and randomly allocated into 18 recirculating water tanks (500-L) with 30 individuals each tank. Each experimental diet was randomly assigned to three tanks. Each tank was supplied with a continuous water flow (3 L/min) and continuous aeration through an air stone to maintain dissolved oxygen at or near saturation. Fish were fed twice daily (08:00 and 17:00 h) at a rate of 4-6% of wet body weight. The feeding trial lasted for 8 weeks. Water quality parameters are monitored twice weekly using water quality analysis. During the test, water temperature ranged from 25 to 28 °C, pH was 7.5–7.8, dissolved oxygen was not less than 6.0 mg L-1, and ammonia nitrogen was less than 0.05 mg L-1.
Sample collection
At the end of the rearing experiment, fish were fasted for 24 hours before sampling. Total mass, body weight, body length, organ weight and liver weight were determined to allow analysis of growth indices such as weight gain rate (WGR), feed conversion ratio (FCR), specific growth rate (SGR), body condition coefficient. (CF), liver index (HSI) and visceral index (VSI). Six fish from each tank were anesthetized with diluted MS-222 solution (tricaine methanesulfonate, Sigma,
USA) at a concentration of 100 mg/L, then blood was collected from the tail vein using a 2 mL medical syringe. Blood was separated by centrifugation, and the supernatant was removed and stored at −80°C for subsequent analysis. Additionally, livers were frozen in liquid nitrogen and stored at −80°C for later analysis.
Thermal stress experiment
At the end of the rearing experiment, fish of similar size were sampled from each tank with a water temperature of 25°C and transferred to a smaller tank with a water temperature of 34°C to avoid heat stress. Water temperature was controlled by the Artificial Climate Department (Ningbo, China). After exposure for 24 and 48 h, six fish from each group were randomly sampled and dissected after anesthesia with MS-222. Blood and liver samples were collected to assay ROS production and gene expression.
Measurement of blood biochemistry
Blood biochemical parameters were determined according to the method described by Kikuchi et al. (1994). Alkaline phosphatase (ALP), cholesterol (CHOL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), high density protein (HDL) and low density protein (LDL) levels were measured. automatic. Beckman Cx-4 biochemical analyzer (Beckman Coulter, USA) using kits purchased from Shanghai Junshi Biotech Co., Ltd. All test kits are specifically designed for fish detection.
ROS production
To monitor ROS levels, we used the cell permeation probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma). A 200 µL volume of blood cell suspension was diluted with anticoagulant solution to achieve a final concentration of 1 × 10^6 cells/mL. DCFH-DA was placed at a final concentration of 10 μM for 30 min in the dark at room temperature. The fluorescence of the cell suspension was then analyzed using a flow cytometer (Becton–Dickinson FACSCalibur).
Two scattered lights
parameters (forward scatter and side scatter) of the flow cytometer were used to define the gate that excluded debris and aggregates from all fluorescence analyses. Typically, 10,000 cells are analyzed for two fluorescence signals. ROS production was expressed as mean fluorescence of DCF.
Real-time PCR
Total RNA was isolated from liver tissue using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions, then dissolved in DEPC-treated water. The amount of isolated RNA was then determined by measuring its absorbance at 260 and 280 nm using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, USA) and its integrity was tested using the electrophoresis on a 1.2% agarose gel. Single-stranded complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The cDNA samples were then stored at −80°C for later analysis. Gene specific primers were designed using Primer Premier 5 (Premier Biosoft International, Palo Alto, CA, USA) according to published puffer fish messenger RNA (mRNA) (Table 2). Real-time PCR was amplified in an ABI 7500 real-time PCR machine (Applied Biosystems, USA) using SYBR Premix Ex Taq (TaKaRa, Dalian, China) according to the manufacturer’s recommendations. The reaction mixture is 20 μL, containing 2 μL of cDNA sample, 0.4 μL of ROX, 10 μL of 2× SYBR Premix Ex Taq, 0.4 μL of each of 10 mM forward and reverse primers, and 6.8 μL of dH2O . Real-time PCR conditions were as follows: 94 °C for 10 min; then 45 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s; followed by 10 minutes at 72°C. All samples were run in triplicate and each assay was repeated three times. After the end of the program, threshold cycle (Ct) values were taken from each sample. Relative gene expression levels were evaluated using the 2−ΔΔCT method (Livak and Schmittgen 2001).
Statistical analysis
All data are expressed as means ± standard deviations. Significant differences were assessed by one-way ANOVA followed by Duncan’s multiple range tests. Statistical analysis was performed using SPSS 19.0 software (SPSS, Chicago, IL, USA). P values <0.05 were considered statistically significant.
RESULT
Effects of astaxanthin on the development of puffer fish
The effects of astaxanthin on growth performance of pufferfish are shown in Table 3. Survival rate (SR) did not differ between experimental groups. Compared with the control group, fish fed diets containing 80, 160, and 320 mg/kg astaxanthin significantly improved weight gain and specific growth rate. However, no significant changes were observed in FCR, CF, VSI and HSI between the different feeding groups.
Effects of astaxanthin on plasma biochemical parameters of puffer fish
The effects of astaxanthin on plasma biochemical parameters of puffer fish are shown in Table 4. Compared with the control group, significantly higher ALP activity was observed in fish fed a diet with 40 320 mgkg-1 astaxanthin. Plasma AST activity of fish fed 320 mg/kg astaxanthin was significantly lower than that of the other groups. Plasma ALT activity was significantly lower in fish diets containing 20–320 mg/kg astaxanthin than in fish fed the control diet (P < 0.05). There were no significant differences in CHOL, TG, HDL and LDL between diets.
Effect of astaxanthin on the relative levels of antioxidant enzyme genes in pufferfish under high temperature stress As shown in Figure 1, the expression levels of Mn-SOD mRNA in all groups tended to increase increased during heat stress. Before stress, the transcript levels of Mn-SOD in diets fed fish with 40–320 mg/kg astaxanthin were significantly higher than in the other groups. After heat stress, the expression level of Mn-SOD mRNA in diets fed fish with 160 and 320 mg/kg astaxanthin was significantly higher at 24 and 48 h compared to the control group.
Figure 1. Effect of different dietary astaxanthin levels on the relative expression level of Mn-SOD of puffer fish under high temperature stress. Note that data are expressed as mean ± SD (n = 6). Small diverse letters represent significant differences (P < 0.05) in different groups at the same time point in the Duncan test. Significant differences (P < 0.05) between values obtained before and after stress are marked with asterisks in t tests
Meanwhile, the Mn-SOD mRNA expression level in fish fed diets with 40 mg/kg astaxanthin was significantly higher at 48 hours after heat stress compared to the control group. The expression levels of CAT mRNA in all groups also appeared to increase (Figure 2). Before stress, CAT mRNA expression levels were higher in diets fed to fish with 40, 80, 160, and 320 mg/kg astaxanthin than in the other groups. After stress, the expression level of CAT mRNA in diets fed fish with 40, 80, 160 and 320 mg/kg astaxanthin was significantly higher than the control at 24 and 48 h.
Figure 2 Effect of different levels of astaxanthin in the diet on the relative CAT expression level of pufferfish under high temperature stress. Note that data are expressed as mean ± SD (n = 6). Small diverse letters represent significant differences (P < 0.05) in different groups at the same time point in the Duncan test. Significant differences (P < 0.05) between values obtained before and after stress are marked with asterisks in t tests
Meanwhile, the expression level of CAT mRNA in diets fed fish with 20 mg/kg astaxanthin was significantly higher than the control at 48 hours. Effect of astaxanthin on relative HSP70 levels in puffer fish under high temperature stress. The effects of astaxanthin on relative HSP70 levels in pufferfish are shown in Figure 3. Before stress, the treatment groups showed no effect on relative HSP70 levels compared with the control group. Compared with the level before heat stress, the relative level of HSP70 was significantly increased in all groups at 24 and 48 h after heat stress. Furthermore, the expression level of HSP70 mRNA in diets fed fish with 80, 160 and 320 mg kg−1 astaxanthin was significantly higher than the control at 24 and 48 h after heat stress.
Figure 3. Effect of different dietary astaxanthin levels on the relative expression level of HSP70 in pufferfish under high temperature stress. Note that data are expressed as mean ± SD (n = 6). Small diverse letters represent significant differences (P < 0.05) in different groups at the same time point in the Duncan test. Significant differences (P < 0.05) between values obtained before and after stress are marked with asterisks in t tests
The effect of astaxanthin on ROS production in puffer fish is shown in Figure 4. ROS production in all groups also increased under heat stress. Before stress, there was no significant difference in ROS production among all groups. After stress, compared with the control group, ROS production in fish fed diets containing 160 and 320 mg kg−1 astaxanthin was significantly reduced at 24 and 48 h. Furthermore, ROS production in fish fed diets containing 20, 40, and 80 mg kg−1 astaxanthin decreased significantly after 48 h, while differences between other treatments were not significant.
Discussions
In the present study, fish fed diets containing 80–320 mg kg−1 astaxanthin improved weight gain and specific growth rate, suggesting that dietary astaxanthin has beneficial effects. for the development of puffer fish. Similarly, Li et al. (2014) stated that dietary supplementation of astaxanthin can
Improve body weight of yellow croaker. Jagruthi et al. (2014) reported that dietary astaxanthin supplementation (25, 50 and 100 mg kg−1) significantly increased the growth rate of common carp. Luu et al. (2016) reported that dietary supplementation of astaxanthin can also enhance nutrient utilization and ultimately help improve the growth of yellow catfish. However, some studies indicate that astaxanthin has no effect on weight gain and specific growth rate of fish (Mansour et al. 2006; Sawanboonchun et al. 2008). These differences may be related to different species, fish size, and feeding behavior.
Blood parameters can be used to evaluate the health, physiological status and nutritional status of fish. Previous research has shown that dietary astaxanthin supplementation can improve fish blood health (Rehulka 2000). ALT and AS are common aminotransferases in fish mitochondria and they can be released into the plasma following tissue damage and dysfunction (Ozaki1978). Luu et al. (2016) found that AST and ALT activities were significantly reduced in yellow catfish fed 0.08% astaxanthin compared to control fish. In our study, plasma AST and ALT activities decreased significantly when astaxanthin was supplemented at appropriate doses, indicating that astaxanthin has the potential to improve fish health.
ALP has been reported to be involved in immune defense mechanisms and correlate with immune competence. Furthermore, ALP can enhance the recognition and phagocytosis of pathogens by changing the surface structure of pathogens and enhancing disease resistance. In Wuchang seabream, ALP activity has been shown to decrease under heat stress (Ming et al. 2012).
In the present study, ALP activity was significantly higher in diets fed to fish with 40–320 mg kg−1 astaxanthin. These results suggest that astaxanthin can increase serum ALP levels and counteract the effects of ambient stressors. As is known, temperature stress is one of the most serious threats to aquaculture, leading to impaired immune defenses and disease resistance.
Previous studies have shown that oxidative stress is an important mechanism of high temperature stress, leading to changes in physiological functions (Cheng et al. 2015). ROS caused by environmental stress can trigger cytokine expression, leading to ROS accumulation and cell damage (Luo et al.2014). However, under environmental stress, the balance between ROS production and antioxidant defense mechanisms is disturbed.
Meanwhile, endogenous antioxidant enzymes can be activated to reduce environmentally induced ROS, which depends on the affordances of the antioxidant system. In the present study, ROS production was significantly higher in all groups at 48 h after high temperature stress. However, ROS production was significantly lower in all astaxanthin treatments compared to the control group at 48 h after high temperature stress. These results indicate that the harmful effects of high temperature stress on pufferfish can be mitigated with astaxanthin.
This beneficial effect of astaxanthin as an antioxidant is attributed to its ability to directly scavenge ROS (Naguib 2000). The antioxidant defense system is considered the first cellular defense mechanism against oxidative stress. The antioxidant capacity of fish includes enzymatic and non-enzymatic antioxidant activities. Furthermore, antioxidant defense in fish partly depends on nutritional factors (Zhou et al. 2014). SOD and CAT can scavenge excessive free radicals, playing an important role in immune defense against ambient stressors, including chemical pollution and pathogenic infections (Lortz et al. al. 2000).
SOD is part of the important antioxidant enzyme system, which can convert intracellular free oxygen radicals (O2-) into hydrogen peroxide (H2O2) and molecular oxygen (O2). In addition, SOD also plays an important role in enhancing the immune function of phagocytic cells and the entire body. Previous studies have shown that astaxanthin improved the antioxidant capacity of aquatic animals and prevented the adverse effects of stress (Liu et al. 2016). In our study, the expression levels of SOD mRNA in all groups tended to increase at high temperature for 48 hours. The increase in SOD activity appears to be an adaptive response to the increase in ROS generation due to high temperature stress. Furthermore, both before and after stress, the expression level of SOD mRNA was higher in the astaxanthin group than in the control group. . These results indicate that diet supplemented with astaxanthin can improve the expression level of SOD mRNA to reduce ROS production induced by high temperature stress.
It is known that CAT can decompose hydrogen peroxide into water and molecular oxygen. Thus, CAT activity can reflect antioxidant capacity and is associated with health status. In the present study, both before and after stress, the expression level of CAT mRNA was higher in the astaxanthin group than in the control group. Luu et al. (2016) reported that astaxanthin has good singlet oxygen quenching properties containing unsaturated fatty acids to quench free radicals and can act as a systemic antioxidant. In our study, fish fed a basal diet supplemented with astaxanthin enhanced activation of the antioxidant enzyme system and inhibited ROS production induced by high temperature stress. Therefore, current research indicates that astaxanthin supplementation may enhance antioxidant capacity, leading to greater resistance to stress. As a stress response protein, HSP70 may be involved in stress defense, intracellular trafficking, anticytosis, antigen processing, and immune responses (Pelham 1986). Previous studies have shown that high temperature stress can lead to the induction of heat shock proteins involved in cellular responses (Cheng et al. 2015). Increased production of HSP70 in the body may enhance cellular resistance to environmental stress Fish Physiol Biochem (Pelham 1986). In this experiment, the HSP70 mRNA expression levels of all groups increased significantly under heat stress. An increase in HSP70 levels may be important for protection from cellular damage associated with high temperature stress (Pörtner 2002). Furthermore, many studies have shown that various nutrients in the diet can enhance HSP70 expression in fish (Zhou et al. 2014). In our study, the expression level of HSP70 mRNA in diets fed fish with 80, 160 and 320 mg/kg astaxanthin was significantly higher than the control at 24 and 48 h after heat stress. These results indicate that a basal diet supplemented with moderate dietary astaxanthin can increase the expression level of HSP70 mRNA to enhance the cytoprotective effect.
CONCLUDE
Growth performance of pufferfish was significantly affected by dietary astaxanthin supplementation. Supplementing astaxanthin in the diet can improve fish blood health. Furthermore, dietary astaxanthin supplementation can increase hepatic SOD, CAT and HSP70 gene expression and suppress high temperature-induced ROS production. Combined, our results suggest that dietary supplementation with 80–320 mg kg−1 astaxanthin can increase growth performance, immunity and antioxidant capacity and enhance resistance against High temperature stress in puffer fish.
