Astaxanthin added to feed increased growth rate, improved coloration, and increased tolerance to hypoxia and ammonia (NH3) stress in white leg shrimp.
Astaxanthin (AX) is a natural carotenoid commonly used as a dietary supplement in the farming of Pacific white shrimp ( Litopenaeus vannamei ). This study evaluated the effects of chemically synthesized astaxanthin on growth performance, survival, stress tolerance, immune response and pigmentation of fed Pacific white shrimp. diet supplemented with 5 levels of AX in the diet containing 0, 20, 40, 80 and 160 mg/kg−1. The feeding trial was conducted for 8 weeks. Results showed that individuals fed the AX-supplemented diet exhibited better growth performance, had a final body weight (FBW), a gain in body weight (BWG), an increased percentage Specific Growth (SGR) and Myostatin ( MSTN ) showed a dose-dependent increase in astaxanthin. After testing for acute hypoxia and ammonia stress, survival (SR) of shrimp fed with diet 40, 80 and 160 mg/kg was higher than that of the control group.. Diet groups AX improved their antioxidant capacity to prevent oxidative damage, including increased activity of acid phosphatase (ACP), total antioxidant capacity (T-AOC) and decreased activity of superoxide dismutase ( SOD) and catalase (CAT), malondialdehyde (MDA) content compared with the control group. In addition, the gene expression of various antioxidant enzymes in the hepatopancreas of shrimp was significantly higher upregulated such as manganese superoxide dismutase (MnSOD) and glutathione S-transferase (GST). The high expression of hypoxia-inducible factor-1α (HIF-1α) and glutamate dehydrogenase (GDH-β), glutamine synthetase (GS) in the 80 and 160 mg kg -1 groups indicated their effect on glycolysis. , which is very important for the required energy production process. The quadratic regression analysis indicated that the optimal levels for growth and pigmentation were 126.94 mg kg-1 and 138.96 mg kg-1, respectively. In summary, Astaxanthin can enhance growth performance, reduce oxidative stress and ammonia, and improve immunity of Pacific white shrimp.
1 . INTRODUCTION
Litopenaeus vannamei is one of the most economically and commercially important marine crustaceans for aquaculture, characterized by fast growth, tender flesh, good stress tolerance, excellent fertility and high nutritional value. These superior features lead to high market value and growing market demand ( Jin and Du, 2015 , Liang et al., 2008 , Pan et al., 2007 ). However, shrimp are susceptible to various environmental factors due to high density intensive farming to increase aquaculture production , when aquaculture systems are not well designed or poorly managed ( Aklakur , 2018 , Fan et al., 2016, Pan et al., 2007 ). In aquaculture systems, inadequate water exchange and high feed intake will produce excessive ammonia levels and cause low dissolved oxygen (DO) in the water ( Chen et al., 2012 ), causes many physiological dysfunctions in the body such as reactive oxygen species (ROS) causing injury, immunosuppression and histological changes ( Hong et al., 2007 , Harris et al., 2001 ). Long-term exposure to the environment eventually leads to disease outbreaks and death of aquatic animals ( Jiang et al., 2005). Because of these risks to aquaculture operations, improvement of these capabilities can be achieved through dietary supplementation with anti-ammonia and antioxidants, such as vitamin C. , α-tocopherol and carotenoids .
Astaxanthin (3,3′-dihydroxy-β, β’-carotene-4,4′-dione), an orange-red carotene, is commonly used as a dietary supplement, colorant, and product. nutritional products in the food industry, which is widely found in marine animals in the wild ( Ambati et al., 2014 , Mezzomo and Ferreira, 2016 ). To date, a lot of valuable information regarding the function of astaxanthin has been discovered in aquaculture. It is reported to be beneficial for the growth, molting and reproduction of aquatic animals ( Ambati et al., 2014 , Lim et al., 2018 , Higuera-Ciapara et al., 2006 , Wade et al. , 2017b). Astaxanthin (AX) is the main pigment in shrimp, giving shrimp an ideal orange-red color ( Shigeru et al., 1994 ), thereby increasing consumer demand ( Wade et al., 2017a ). Another important role of AX is the cellular antioxidant function in animals, as it contains various types of antioxidants and free radical scavengers, and inhibits lipid peroxidation, which can reduces oxidative stress caused by environmental factors ( Miki, 1991 ). Due to its excellent antioxidant activity , AX is also widely used as an immune system promoter and regulator to enhance the immune response and stress tolerance of shrimp against stress hypoxia (Chien and Shiau, 2005 ), stress salinity and ammonia stress ( Flores et al., 2007 , Pan et al., 2003 ). Previous studies have demonstrated that the antioxidant enzymes catalase , superoxide dismutase , peroxidase and reactants with thiobarburic acid (TBARS) in rat plasma and liver are affected by AX ( Ranga et al., 2010 , Saad et al., 2016 ). Therefore, using AX as a feed additive in crustacean farming to improve organism quality is more beneficial for productivity enhancement, which has been reported in crabs and other shrimp species. However, detailed background information on the AX effect of white leg shrimp Litopenaeus vannamei on stress tolerance is still unknown.
Evaluation of biochemical parameters (enzyme activity and relative gene expression) of shrimp is essential in assessing the effects of dietary astaxanthin supplementation under stress. Total antioxidant capacity (T-AOC) is considered to be a comprehensive indicator of antioxidant status and organism health status ( Griendling and Alexander, 1997 ; Castillo et al., 2006 ). Catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) are enzymes that protect oxidation, whose changes reflect the physiological state of the organism or cell under other environmental conditions each other to a certain extent. Therefore, they can be considered as important physiological indicators to determine whether Pacific white shrimp are under pressure from the external environment ( Li et al., 2021 ). Furthermore, as a direct indicator of AX for physiological and immunological effects, the regulation of gene expression of different metabolic enzymes is closely related to the metabolic intensity of vannamei shrimp. Pacific to stress response, such as antioxidant enzymes (glutamate dehydrogenase-β, GDH-β; glutamine synthetase, GS) and ammonia detoxification (Manganese superoxide dismutase, MnSOD; glutathione S-transferase, GST) et al. ( Mayzaud and Conover, 1988 , Ahn et al., 2019). The relative regulation of gene expression may therefore reflect that different AX diets influence the physiological metabolism of Pacific white shrimp under stress.
To date, there has been little literature exploring the effect of AX on resistance to ammonia and hypoxia stress in vannamei L. vannamei . Whether dietary AX supplementation has a beneficial effect on the survival rate of L. vannamei when exposed to hypoxia and ammonia and the immunoregulatory mechanism remains unclear. clear. Therefore, this study was conducted to determine the effect of AX on growth performance, color, antioxidant capacity and ammonia tolerance of vannamei L. vannamei . Our data contribute to improved understanding of the reactivity relationship between AX and hypoxia and ammonia tolerance of L. vannamei , which can provide a reference for feed formulations of L vannamei.
2 . material and method
2.1 . Experimental diet
A chemically synthesized pigment (Astaplus®10%; Guangzhou Juyuan Bio-chem Co., Ltd., Guangzhou, China), with a free astaxanthin content of 10%, was used as an additional source of AX. Due to the relatively large differences in the amounts of AX and other components (sucrose, starch corn , gelatin, natri octenylsuccinate starch, butylated hydroxytoluene) in this source, the contribution of other components is not considered. . The basic dietary formulation and approximate composition analysis are shown in Table 1 . AX was added to the basal diet at concentrations of 0, 20, 40, 80 and 160 mg kg -1 , respectively. Actual concentrations of AX in the five experimental diets are also shown in Table 1 . All dry ingredients are finely ground, weighed and thoroughly mixed, then lipid components and Astaplus®10% are added to the mixture, and finally, water (40% by weight of dry ingredients) is added and mix thoroughly. Portions with a diameter of 1.2 mm are produced using a single screw extruder . The diets were dried in an electric oven at 40°C until the humidity dropped to <10% and stored in a plastic bag at -20°C until use.
2.2 . Trial of shrimp farming and farming management
The experiment was conducted at the Shenzhen Campus, East Sea Fisheries Research Institute of the Chinese Academy of Fisheries (Shenzhen, China). Immature vannamei L. vannamei was purchased from Shenzhen Lianzhong Global Biotechnology Co., Ltd (Shenzhen, China). Before the feeding experiment, all shrimp were acclimated to the experimental conditions and fed a control diet for one week. after acclimatization, shrimp were starved for 24 h and then a total of 800 healthy shrimp with an initial body weight of approximately 0.95 g were randomly assigned to 20 cylindrical fiberglass tanks (500 L, 4 tanks per diet, 40 fish per tank). All shrimp were fed the test diet three times daily at 7:00, 12:00 and 18:00 at 6% body weight per day for eight weeks. Uneaten food and fecal waste are removed by siphon daily. Mortality rates are monitored and recorded daily. Feed allocation was adjusted based on observations of food intake. Shrimp are maintained in a flow system with filtered seawater. During the experiment, the sea water quality parameters were kept as follows: pH from 7.9 to 8.0; temperature, 26–30 °C; salinity 30 ‰; ammonia-N, 0.05–0.07 mg L−1; DO, 5.8–6.7 mg L −1 . The experiment uses the natural light-dark cycle.
2.3 . sample preparation
At the end of the feeding trial, shrimp were starved for 24 h and then all live shrimp in each tank were weighed and counted to determine final body weight and survival. Five shrimp from each tank were randomly collected and stored at -20°C for AX concentration and approximate body analysis. A hemolymph sample was collected from the ventral cavity of six shrimp in each tank. Then, the hemolymph samples were mixed gently in an RNase-free Eppendorf tube, and then kept at 4 °C overnight. It was then centrifuged at 8000 g for 10 min at 4 ̊C to obtain serum. Then, the hepatopancreas and muscle of shrimp were dissected and frozen immediately in liquid nitrogen and stored at -80°C for further determination of gene expression and enzymatic activities. Three shrimp were randomly sampled from each tank for color measurement.
2.4 . stress experiment
After an 8-week feeding trial, experiments on hypoxia and ammonia stress were performed, respectively. A total of 200 individuals in the 5 untreated diet groups were the control group, while the remaining shrimp were treated with hypoxia and ammonia stress. At the end of the challenge trial, blood and hepatopancreas samples were removed from the live shrimp for antioxidant and immunological testing.
2.4.1 . Hypoxia stress test
A total of 150 individuals in five dietary groups were collected for the hypoxia stress experiment. Thirty shrimp per diet were transferred into three plastic bags (three replicates, 10 individuals randomly assigned to each replicate) previously filled with 10 L of fresh seawater without addition or oxygen aeration ( Lu et al., 2019 ). Each plastic bag was tightly tied and survival was recorded every hour up to the mean time to lethality. Finally, the stress test lasted for a period of 6 hours. Dissolved oxygen was measured at baseline and after 6 h of observation. Shrimp were carefully monitored and survival was recorded every hour up to the mean time to lethality. The water temperature, pH and salinity in the bag were kept at 26–30 °C, 7.9–8.0 and 30‰, respectively.
2.4.2 . Ammonia stress test
Similarly, a total of 150 individuals in five dietary groups were collected for the ammonia stress experiment. Thirty shrimp per diet (three replicates, 10 individuals randomly assigned to each replicate) underwent an ammonia stress experiment. Ammonia test solution was prepared by adding ammonium chloride (NH 4 Cl) to each tank. Referring to previous studies, a high concentration ammonia (∼10 mg L -1 ) was designed for the experiment ( Lu et al., 2019 ). Finally, a concentration of 12.85 mg L −1 NH 3-N was determined according to the National Standard of the People’s Republic of China (GB3097–1997) based on salinity of 30‰, pH 8.0, and temperature. degree 26℃ at that time. During stress testing, shrimp survival was carefully monitored and recorded every hour up to the mean death time. The ammonia stress test lasted for a period of 6.5 h.
2.5 . Approximate composition, AX and color analysis
Moisture, crude protein, crude ash and crude lipids of the experimental diet and whole body were determined according to the AOAC procedure ( AOAC, 1995 ). Moisture content was determined by drying in an oven at 105°C for 24 h; Crude protein content was analyzed by Kjeldahl method after acid digestion (1030-auto-analyzer; Tecator, Hoganas, Sweden); Raw ash was determined after burning in a muffle furnace at 550°C to constant mass; Crude lipids were measured via petroleum ether extraction using Soxtec System HT (Soxtec System HT6; Tecator).
Quantitative analysis of free AX content in test diets and shrimp tissues was performed by high performance liquid chromatography (HPLC) according to the method of Wade et al. (2005) , with minor modifications. AX was extracted from minced shrimp and fed with 50 ml of acetone. The acetone extract was then filtered through a sand glass funnel, and the solid residue was extracted twice with 30 ml of acetone in a 40 ̊C water bath for further extraction until the entire extract was essentially pigment-free. get AX extract completely free. The solvents were transferred to a round-bottomed flask and dried by rotating vacuum evaporation at 45°C. The sample solution was obtained by dissolving the residue with 20 ml of methanol until HPLC analysis. HPLC separation was performed on a Shimadzu VP-ODS column (150 L × 4.6: particle size 5 µm). Free AX was determined at 470 nm. The mobile phase, with a flow rate of 0.8 ml min -1 , is as follows: methanol (A), dichloromethane (B), acetonitrile (C), H 2 O (D) (A: B: C: D = 85 :5:5:5). The injection volume was 20 μl. The concentration of free AX was calculated using the peak areas according to the AX standard of known concentrations.
Twelve surviving shrimp from each diet were photographed. Then, all of them were placed in a boiling water bath (>95 °C) for 3 min. After boiling for one minute, the cooked color is evaluated digitally and subjectively scored; The color of raw and cooked shrimp was quantified by color parameters (L* = luminance, a* = redness, b* = yellowness) using a colorimeter (Minolta Chroma Meter CR400, Konica Minolta Sensing). Inc., Osaka, Japan). The value of L* ranges from 0 for pure black to 100 for pure white, a* representing the red-green scale and b* representing the blue-yellow scale. Considering that the colors are different in different parts of the shrimp, the colors of the three parts of the shrimp (head, body and tail) are read by a colorimeter.
2.6 . Testing of antioxidant and immune related parameters
Hepatopancreas was homogenized (1:9) by adding cold saline and the homogenates were centrifuged at 3500 g for 10 min at 4°C, and cold hepatopancreatic supernatant was used. to determine enzyme activity. The total protein content in the supernatant was determined using the BCA Protein Assay Kit (Q045–3, Jiancheng Institute of Biological Engineering, Nanjing, China). The activities of superoxide dismutase (SOD), catalase (CAT), acid phosphatase (ACP), total antioxidant capacity (T-AOC) and malondialdehyde (MDA) in serum and hepatopancreas were measured according to the guidelines of the kit. commercial instruments (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). The SOD activity was determined by the xanthine oxidase (hydroxylamine) method. CAT catalyzes H 2O2, and this reaction can be quickly stopped when ammonium is added. The remaining H2O2 is combined with ammonium molybdate to give a pale yellow compound. The CAT activity was analyzed by determining the difference in absorbance at 405 nm. ACP can catalyze phenyl phosphate to free phenol and phosphoric acid. Under alkaline conditions, phenol can react with 4-aminoantipyrin to give the red quinone derivative. Therefore, the ACP activity was analyzed according to the red tint change at 520 nm. Fe 3+ reduction method was applied to detect T-AOC. The thiobarbituric acid (TBA) method was used to determine the MDA content.
2.7 . Quantitative real-time PCR analysis
Total RNA was extracted using the RNeasy ® Mini Kit (QIAGEN cat. nos. 74104, Germany). RNA was quantified by spectroscopic analysis and quality was assessed using agarose gel electrophoresis at 1%. First-strand cDNA synthesis by reverse transcription was performed based on the manufacturer’s instructions for the PrimeScript™ RT Reagent Kit (TaKaRa cat. nos. RR047A, Japan). Real-time PCR for target genes was performed using a GoTaq® qPCR Master Mix (Promega cat. nos. A6002, USA) and quantified on a LightCycler 480 (Roche Applied Science, based in Switzerland) . Elongation factor 1α (EF1α) was used as a reference gene. The sequences of the evaluated genes were obtained from NCBI GenBank. The specific primer sequences of each gene were designed using Primer Premier 5.0 software ( Table 2 ). RT-PCR consisted of a denaturation step at 95°C for 4 min, followed by 40 cycles of amplification at 95°C for 5 s, incubation at 60°C for 15 s, and extension at 72°C for 15 s. . The relative expression levels of the genes were calculated based on the equation of 2 −ΔΔCT( Livak and Schmittgen, 2001 ). Each data is made into three independent copies.
2.8 . Statistical analysis
One-way ANOVAs were used to test the significance of the overall treatment effects on growth performance, pigmentation, astaxanthin in vivo of farmed shrimp over 8 days of the weekend with varying degrees, and Survival and astaxanthin in vivo of shrimp used in hypoxia and ammonia stress experiments. test, followed by Duncan’s multiple range test (DMRT). Quadratic regression was used to estimate optimal dietary AX levels. All statistics were performed using the SPSS package (version 19.0) and all data are presented as mean with standard error (SE). The difference between treatments was considered significant at the 0.05 level.
3 . result
3.1 . Growth performance and body composition
Growth performance, especially body weight gain and specific growth rate, is an important indicator of shrimp production and efficiency. The final body weight (FBW) of shrimp fed the AX80 and AX160 diets was significantly higher than that of the shrimp fed the AX0 diet (P < 0.05) ( Table 3 ). Similarly, the body weight gain (BWG) and specific growth rate (SGR) of shrimp fed the AX160 diet were significantly higher than those of the shrimp fed the AX0 diet (P < 0.05). . There were no significant differences in shrimp survival (SR) and feed efficiency (FE) between all treatments (P > 0.05).
Approximate whole body compositions of shrimp among all dietary treatments fed diets supplemented with different levels of AX are presented in Table 4 . There were no significant differences in total body moisture , crude protein, ash and crude lipids between all dietary treatments (P > 0.05). The expression level of Myostatin (MSTN) in the muscle of shrimp fed the AX160 diet was higher than that of the shrimp fed the AX0 diet (P < 0.05), which is consistent with the BWG results (Figure 1 ) .
3.2 . Astaxanthin content and shrimp color
The results showed that the concentrations of determined AX in the diet were similar to those of the supplement ( Table 5 ). Furthermore, the AX content in shrimp tissues increased with increasing AX levels. For hepatopancreas , there was a significant accumulation of free AX in shrimp fed with the AX40, AX80 and AX160 diets (P < 0.05). The free AX content in the cortex and muscle showed similar trends with hepatopancreas. The results showed that AX was deposited mainly in the hepatopancreas, followed by the cortex and muscle.
All the dietary groups had marked differences in the coloration of raw and cooked shrimp ( Figure 2 ). Body color changed from light green to light red and light brown starting in the AX20 group, while shrimp fed the astaxanthin diet (AX20, AX40, AX80 and AX160) were redder after cooking and dark in color. gradually as the AX content increases.
Figure 2 . Color of cooked and uncooked L. vannamei shrimp fed 5 levels of astaxanthin (AX) in the diet for 8 weeks. AX levels in the five experimental diets are referenced in Table 1 .
Tricolor parameters (L*, a*, and b*) were used to determine the colorimetric effect of Ax on the head, body, and tail of the experimental shrimp. After treatment, there was no significant change in luminance (L*) values in all dietary groups ( Table 6 ). After cooking, shrimp obtained greater values for brightness (L*), redness (a*) and yellowness (b*) than uncooked shrimp. In addition, an increased trend in red (a*) and yellowness (b*) was observed with the higher AX diet. The values for red color (a*) and yellowness (b*) were much greater in shrimp fed the AX supplemented diet than in shrimp fed the control diet (P < 0.05).
3.3 . Survival rates after hypoxia and ammonia stress
Under hypoxic stress, the hourly SR of shrimp fed the AX40, AX80 and AX160 diets changed less than that of the shrimp fed the AX0 diet over time and the change was minimal. was found in shrimp fed the AX160 diet (Figure 3a) . At the end of the hypoxia stress period, the final SR of shrimp fed the AX40, AX80 and AX160 diets was significantly higher than that of the shrimp fed the AX0 diet (P < 0.05) ( Fig. 3 c). Similar results were observed under ammonia-induced stress ( Fig. 3 b, d).
Figure 3 . Hourly and final survival of L. vannamei fed five diets: under hypoxia (a) and ammonia stress (b); after hypoxia (c) and ammonia stress (d). Data are for mean values based on three independent determinations. Values are means, with their standard errors (three replicates n = 3). Bars with different letters are significantly different ( P <0.05). Astaxanthin (AX) levels in the five experimental diets are referenced in Table 1
3.4 . Antioxidant enzyme activity
Antioxidant indices in the hepatopancreas of shrimp fed with different experimental diets are shown in Table 7 . At the beginning of the experiment, dissolved oxygen was measured as 6.72 mg L -1 , while dissolved oxygen at the end was measured again as 0.9 mg L -1. Before acute stress, no significant differences were found in the SOD and CAT activities between all dietary treatments (P > 0.05), however , the activity of these two enzymes decreased with increasing amount of AX addition. T-AOC increased with increasing amount of AX addition and MDA content decreased significantly in AX40, AX80 and AX160 groups (P < 0.05). After stress due to hypoxia and ammonia, the T-AOC, SOD and CAT activities of shrimp fed with the AX160 diet were significantly higher than those of shrimp fed with the control diet (P < 0.05). The lowest MDA concentration was observed in the AX160 treatment (P < 0.05). The activities of ACP in serum and hepatopancreas after hypoxia and ammonia stress are shown in Figure 4. After hypoxic stress, treatment with AX160 has high serum and hepatopancreatic ACP activity significantly more than AX0 treatment (P < 0.05). After ammonia stress, no significant difference was found in serum ACP activity between all treatments (P > 0.05), while ACP activity was highest in hepatopancreas occurred in shrimp fed the AX160 diet (P < 0.05).
3.5 . Relative expression of antioxidative and stress-related genes in shrimp after acute stress
The effect of dietary AX on the mRNA expression profiles of antioxidant- and stress-related genes in the hepatopancreas of shrimp after acute stress was tested. After hypoxic stress, shrimp fed the AX160 diet had significantly higher MnSOD, GST , and HSP70 expression levels than shrimp fed the control diet (P < 0.05) ( Figure 5)) . Expression levels of HIF-1α were significantly higher in shrimp fed the AX80 and AX160 diets than in shrimp fed the control diet (P < 0.05). After acute ammonia stress, the expression levels of MnSOD and GS in shrimp fed the AX160 diet were significantly up-regulated compared with shrimp fed the AX0 diet (P < 0.05). . Expression levels of GST and GDH-β were significantly higher in shrimp fed the AX80 and AX160 diets than in shrimp fed the control diet. Furthermore, the expression levels of HSP70 mRNA in shrimp fed the AX40 and AX160 diets were significantly down-regulated (P < 0.05).
3.6 . Optimal dietary astaxanthin levels
Quadratic regression was used to estimate optimal dietary AX levels in relation to growth performance ( Figure 6 a) and skin pigmentation ( Figure 6 b). The breakpoint at 126.94 mg kg -1 of the AX diet was estimated using the regression equation y = −0.0047 x 2 + 1.1932 x + 623.39, R 2 = 0, 9975 on body weight gain measurement. Quadratic regression at 138.96 mg kg− 1 AX diet was estimated using the regression equation y = −0.3616x 2 + 5.0249x – 0.7578, R² = 0.8819 on measurement of the redness (a*) of cooked shrimp body.
4 . Discussions
4.1 . Effect of dietary astaxanthin on growth performance
The better growth performance in shrimp fed the astaxanthin diet is probably due to the important regulatory role of AX in aquatic animal intermediate metabolism, which may contribute to the utilization of nutrients. nutrients and ultimately improve growth performance (Wang et al., 2018, Wade et al., 2017a ). Niu et al. (2012) reported a similar result that the BWG and SGR of black tiger shrimp Penaeus fed 0.1% and 0.2% AX were higher than those of the control. Small and associates. (1997) found that AX can shorten the molting cycle of shrimp by increasing ecdysteroid levels, which is beneficial for improving growth. A previous study of AX-supplemented crab diets showed similar results that dietary AX had a positive effect on growth performance ( Amar et al., 2001 , Segner et al., 1989 ) ( Figure 5 c, d).
Notably, our study showed that the growth performance of whiteleg shrimp L. vannamei fed 160 mg/kg -1 AX diet was significantly improved (P < 0.05), this may be related to the increased expression of MSTN in muscle. Myostatin has a negative regulatory effect on muscle growth and differentiation by simultaneously inhibiting protein synthesis in mammals ( Hadjipavlou et al., 2008 ). However, MSTN plays the opposite role in shrimp and its expression level positively regulates growth ( Santis et al., 2011 , Suo et al., 2017). Thus, the difference in MSTN expression better explains the higher growth performance observed in shrimp fed with astaxanthin. However, several studies indicate that growth performance of aquatic animals is not affected by dietary AX. Chien and Shiau (2005) found that synthetic AX could not significantly improve the growth performance of M. japonicas . Similar results were observed in Portunus trituberculatus ( Han et al., 2018 ). The different effects of AX on growth performance of aquatic animals can be attributed to differences in experimental species, animal health, aquaculture systems, and feeding methods. Further studies are needed to elucidate the actual underlying mechanism of AX in aquatic animals.
4.2 . Effect of astaxanthin on colorcoloration
Crustaceans are generally incapable of synthesizing carotenoids de novo ( Velu et al., 2003 ), it seems that dietary astaxanthin supplements compensate for insufficient carotenoids accumulated during intensive culture. In the present study, shrimp fed with the AX diet had a darker orange-red color than wild shrimp ( Barclay et al., 2006 , Boonyaratpalin et al., 2001 ), indicating a positive effect of AX on with the color of shrimp. Color parameters (L*, a* and b*) are one of the important criteria to evaluate the color of crustaceans ( Smith et al., 1992). Although the body L* values of shrimp were not affected by the different diets, the a* and b* values of shrimp fed the astaxanthin-supplemented diet were significantly higher than those of the control. In addition, red parameters (a*) that were significantly different (P < 0.05) from control increased with increasing amount of AX addition, supporting our results for whole-body Ax concentrations . In this study, the free AX content in the liver, cortex and muscle increased with the increase in dietary AX concentration and the pigmenting efficiency of hepatopancreas was higher than that of the cortex and muscle in all. The treatments. Similar results were obtained in black tiger shrimp P. monodon and shrimp Kuruma ( Niu et al., 2012 , Chien and Jeng, 1992). Tissue AX content directly affects the coloration of crustaceans ( Supamattaya et al., 2005 ), and the total carotene content in the hepatopancreas of shrimp is higher than in shell and muscle ( Yanar et al., 2012 ). . Overall, these results suggest that the white leg shrimp L. vannamei can make good use of dietary AX to improve the red color of the body.
4.3 . Effect of astaxanthin on antioxidant capacity of shrimp after stress
The generation of reactive oxygen species (ROS) during stress testing can lead to severe oxidative damage ( Mascio et al., 1991 ). It is widely accepted that ROS-induced damage in organisms can be limited by the action of enzymatic antioxidants ( Sowmya and Sachindra, 2012 ). Previously, the antioxidant system consisting of SOD and CAT was considered as a defense mechanism in the body of crustaceans, helping to eliminate excessive ROS, prevent oxidative stress , and protect the organism against harmful effects. environmental stressors ( Marklund, 1974 ). T-AOC is considered as a comprehensive indicator of antioxidant status and organism health status ( Castillo et al., 2006). In the present study, lethal stress test was used to assess health status by measuring stress tolerance of experimental shrimp. The results showed that the AX supplement group performed better than the control group at LT 50. First, a significant difference (P < 0.05) was found between control and treatment with regard to survival. Second, compared with control shrimp, shrimp fed AX still showed higher antioxidant enzyme activity, when astaxanthin diet increased T-AOC, although SOD and CAT activities decreased in the hepatopancreas. These results are in agreement with the results on Zhang et. al. (2013) and Wu et al. (2017). We speculate that the activity of antioxidant enzymes in the hepatopancreas is significantly reduced with increased oxidative stress exposure, leading to impaired cell biology involved and that AX may initiate other responses. to modulate the antioxidant defense system, ultimately increasing T-AOC.
AX can regulate the expression of shrimp antioxidant enzyme genes and improve resistance to hypoxia and ammonia stress. For example, MnSOD catalyzes O 2- into hydrogen peroxide (H 2 O 2 ) and molecular oxygen. The superoxide generated by ROS is mainly removed by MnSOD in the mitochondrial matrix. The RT-PCR results in this study indicate that AX can enhance endogenous defense by upregulating the expression of antioxidant enzyme genes including MnSOD and GST genes at the mRNA level, thus providing protection against oxidative stress. Truong and associates. (2013) obtained similar results in white leg shrimp Litopenaeus vannamei. These results suggest that AX may exert its effects through influencing the activity of antioxidant enzymes and/or the expression of antioxidant enzyme genes. MDA is one of the end products of lipid peroxidation in tissues and has strong biotoxicity ( Li et al., 2013 ). In our study, both before and after stress, the MDA content showed the opposite trend. These results suggest that AX can inhibit lipid peroxidation and enhance the antioxidant capacity of L. vannamei , similar to previous studies ( Li et al., 2014 ), including Natural AX ( Xie et al., 2018 , Long et al., 2017 ).
4.4 . Effects of AX on immunity and resistance to stress
Due to the lack of an adaptive immune system and mainly relying on the innate immune system, crustaceans are more vulnerable to a number of external factors ( Wang and Wang, 2013 ). For aquaculture, it is extremely important to minimize adverse conditions that can cause greater stress and weaken the host organism. Chien and Shiau (2005) found P. black tiger shrimp fed a high level of AX diet increased SR after low DO challenge. In the present study, SR enhancement in shrimp under high ammonia and low oxygen stress suggests that AX diet can increase shrimp resistance against environmental stressors. As an important phosphatase, ACP is involved in various metabolic processes, so it can be used to assess the resistance of an organism to external stressors ( Xue and Renault). , 2000 ). Several previous studies have found that animals fed an immunostimulant-containing diet can increase serum ACP activity ( Chang et al., 2018 , Deng et al., 2015). Similarly, significantly higher ACP activity in AX160 indicates that appropriate dietary AX can effectively enhance the function of the crustacean immune system. Furthermore, HSP70, as an important component of the cellular machinery to assess the immune function of crustaceans, can be used as a stress-responsive protein to participate in stress defense, anti-apoptotic and immune responses (Franzellitti and Fabbri, 2005 ) . Upregulation of HSP70 in the hepatopancreas due to exposure to hypoxia and ammonia stress was observed ( Franzellitti and Fabbri, 2005 ). In this study, the expression level of HSP70gen was down-regulated in the AX-supplemented groups compared with the control group under acute stress. These results indicate that dietary astaxanthin may partially alleviate the negative effects caused by acute stress. In Penaeus black tiger shrimp , Niu et al. (2014) reported that the expression level of HSP70 mRNA in hypoxia was significantly higher than in normal condition.
HIF-1α is a heterologous transcription factor, which can regulate dozens of genes involved in oxygen transporters and make animals better survive in hypoxia conditions ( Hardy et al., 2012 ). After hypoxic stress, HIF-1α expression levels were higher in shrimp fed the astaxanthin-supplemented diet than in shrimp fed the control diet. Karnakhov et al. (1977) reported that carotene-conjugated double bond chains have good electron acceptor and donor properties, allowing them to attach oxygen molecules. When the organism is in an hypoxic state, the oxygen in the carotene intracellular accumulation can alleviate the hypoxia and make the aquatic organisms more resistant to hypoxia ( Oshima et al., 1993). . Truong and associates. (2013) also showed that AX can partially attenuate the oxidative stress response of L. vannamei under hypoxia. Thus, this result also confirms that AX can participate in the oxygen metabolism of animal cells and provide oxygen to the cells. GDH-β and glutamine GS are key enzymes in maintaining normal ammonia nitrogen levels in shrimp under stress ( Mayzaud and Conover, 1988 ), maintaining ammonia balance through deamination reactions (DeBerardinis) et al., 2007). GDH degrades ammonia to produce glutamate acid and then GS catalyzes glutamate and excess ammonia to synthesize glutamine ( Cooper, 2012 , Silvia et al., 2004). Stress can induce expression levels of GDH and GS genes in the hepatopancreas of vannamei L. vannamei ( Liu et al., 2012 ). Several studies have also demonstrated that the expression levels of GDH and GS were increased after ammonia exposure, indicating that L. vannamei can induce the expression of GDH-β and GS genes under pressure force of ammonia, thereby reducing the ammonia content. In this study, the expression levels of GDH-β and GS in hepatopancreas were significantly increased, which suggests that dietary AX can enhance the ammonia detoxification capacity of vannameiL. vannamei by catalyzing the conversion of ammonia. In summary, changes in expression levels of HSP70, HIF-1α, GDH-β and GS may contribute to our understanding of the metabolic response mechanism of vannamei L. vannamei under stress. hypoxia and ammonia.
5 . CONCLUSION
In summary, our results demonstrated that dietary astaxanthin supplementation could achieve better growth and color performance. Furthermore, dietary astaxanthin supplementation can increase antioxidant capacity and regulate the expression of genes involved in immune response and stress tolerance of L. vannamei . Notably, shrimp groups fed 80 and 160 mg/kg astaxanthin made adaptive changes occurring in the highest antioxidant levels in the hepatopancreas with resistance to stress, providing valuable information value and theoretical basis for the application of AX in aquaculture.
Xiaopin Zhao a 1, Gongpei Wang a c 1, Xuange Liu a, Dingli Guo a, Xiaoli Chen a, Shuang Liu a, Sheng Bi a, Han Lai a, Jimei Zhu b, Dan Ye c, Haifang Wang b, Guifeng Li a