Astaxanthin protects the hepatopancreas against oxidative agents in white shrimp
The present study investigated the effects of dietary astaxanthin (AX) on growth performance, antioxidant parameters, and recovery of hepatopancreatic damage in Pacific white shrimp ( Litopenaeus vannamei). To evaluate the hepatopancreatoprotective function of Astaxanthin in shrimp, we compared the effects of five diets under oxidized fish oil conditions with different levels of astaxanthin over a 50-day trial period. The diets were formulated as follows: (i) OFO (oxidized fish oil); (ii) OFO/AX150 (oxidized fish oil + AX150 mg/kg); (iii) OFO/AX250 (oxidized fish oil + AX250 mg/kg); (iv) OFO/AX450 (oxidized fish oil + AX450 mg/kg); and (v) control group (fresh fish oil). The results showed that oxidized fish oil with a peroxide value (POV) of 275.2 meq/kg significantly reduced the final body weight of white shrimp ( P > 0.05) and caused some changes clear histopathological changes in the hepatopancreas. Growth performance was significantly higher in shrimp fed the OFO/AX450 diet than in shrimp fed the OFO diet ( p < 0.05). However, there was no significant difference when comparing the OFO/AX450 diet with the control diet containing fresh fish oil ( p > 0.05). Furthermore, shrimp on the OFO/AX450 diet showed significant improvement in hepatopancreatic health and reduced malondialdehyde (MDA) compared to shrimp on the OFO diet ( p < 0.05). Dietary astaxanthin improved the antioxidant capacity of L. vannamei by increasing lymphatic catalase (CAT) activity. The acute salinity change test showed that the survival rate of shrimp on the OFO/AX450 diet was higher than that on the OFO diet (p < 0.05), suggesting that AX may contribute to enhancing antibacterial resistance. bear stress. In summary, our data show that AX has a dose-dependent protective effect against oxidative damage caused by oxidized fish oil in shrimp.
1. INTRODUCE
The marine carotenoid astaxanthin (AX) is naturally found in a variety of aquatic organisms, such as microalgae, crustaceans (crabs, lobsters, and shrimps), and fish (salmon) [ 1 , 2 ]. AX has a hydrophobic polyunsaturated structure at both ends of the conjugated olefin structure, which facilitates its precise localization in cell membranes and circulating lipoproteins. In fact, AX exhibits a powerful antioxidant function as a powerful scavenger of oxygen free radicals, to reduce oxidative stress and lipid peroxidation [ 3 ]. Astaxanthin is well known as a powerful antioxidant that has been reported to surpass β-carotene or lutein and even α-tocopherol [ 4 ]. In crustaceans, several studies have demonstrated that dietary astaxanthin can increase total antioxidant capacity (TOC), improve growth performance [5, 6, 7]. survival [ 8 ] and enhance resistance to various environmental stresses, including salinity stress [ 9 ], hypoxic stress [ 10 ] and high temperature stress [ 11 ]. Therefore, dietary astaxanthin, which is a superior antioxidant, can be used to improve growth performance and enhance stress tolerance of marine species.
Lipid peroxidation is the redox breakdown of polyunsaturated fatty acids (PUFA) through a free radical chain reaction. Lipid peroxide can damage lipid-rich cell membranes of organisms [ 12 ]. Fish oil, which is rich in PUFAs, is necessary to maintain the fluidity of the cell membranes of aquatic animals. However, PUFAs are susceptible to peroxidation to form toxic lipid hydroperoxides, the main oxidation product, through a process involving free radicals. Unstable lipid hydroperoxides can readily decompose to fatty acid alkoxy [ 13 ] or decompose to release a series of secondary oxidation products, such as aldehydes, ketones, alcohols and carboxylic acids [ 14 ]. These oxidation by-products are the main source of unpleasant flavors and odors in decomposing marine organisms, due to the destruction of cellular biofilms and deterioration of animal health [ 15 , 16 ] . Previous studies have shown that the use of oxygenated oils in fish diets can delay development or survival by modulating hemoglobin concentration and glycolytic activity, lipid peroxidation [ 17 , 18 , 19 , 20 ] and α-tocopherol deposition [ 21 ]. In contrast, our understanding of the effects of lipid oxidation on crustaceans is limited. Koshio et al. (1994) reported that oxidized oil can lead to reduced growth, reduced postlarvae size, and poor health in Penaeus japonicas [ 22 ]. Laohabanjong et al. (2009) showed that fishmeal lipid oxidation can lead to abnormalities in erythrocyte infiltration, tubular epithelial atrophy, and nodule formation in black tiger shrimp Penaeus monodon [ 23 ]. Wang et al. (2015) found that moderate and high levels of oil oxidation can reduce the survival rate and foraging efficiency of juvenile Chinese crabs Eriocheir sinensis [ 24 ]. Although oxidized fish oil has negative effects on crustaceans, only a few studies have explored how to reduce the adverse effects of lipid peroxidation through dietary management.
Pacific white shrimp ( Litopenaeus vannamei ) is the most commonly farmed crustacean in South China. In recent years, environmental degradation has seriously affected shrimp farming, especially in the subtropical regions of Guangdong province, China, which has a humid climate and high temperatures all year round [ 25 , 26 ]. Commercially, animal feed or rations are often stored in paper bags, making lipids susceptible to oxidation. Several studies have shown that oxidative damage in aquatic animals caused by oxidized lipids can be prevented by exogenous antioxidants, such as vitamin E or α-tocopherol supplementation [ 21, 24 , 27 ]. In this study, the potential benefits and protective effects of dietary Astaxanthin in white shrimp L. vannamei were evaluated in shrimp fed with oxygenated fish oil (OFO) for a period of 50 days. The chronic effects of dietary OFO and the role of dietary AX, as a possible chemoprotective agent against OFO, have been observed. The objectives of this study were (1) to evaluate the long-term effects of dietary OFO on growth performance and hepatopancreatic injury of L.vannamei and (2) to determine the benefits of Astaxanthin in dietary supplementation. diet in preventing or ameliorating the effects of dietary OFO.
2. Result
2.1. Growth performance and feed utilization
The growth performance and survival rate of shrimp are shown in Table 1 . After a 50-day feeding trial, the final body weight (FBW) of shrimp fed the OFO + AX450 diet was significantly higher than that of shrimp fed the OFO diet ( p < 0.05). However, there was no significant difference between shrimp fed the OFO + AX450 diet and the control fresh fish oil diet ( p > 0.05). The lowest weight gain (WG), specific growth rate (SGR) and survival were found in shrimp fed the OFO diet, although no significant differences were found in WG, SGR and survival among other dietary groups ( p > 0.05). Similarly, a higher feed conversion ratio (FCR) was found in shrimp fed the OFO + AX150 diet than in shrimp fed the OFO diet, and no significant differences were found. among other dietary groups ( p > 0.05).
Table 1. Growth performance and feed utilization of young L. vannamei shrimp, fed different diets in a 50-day feeding trial.
2.2. Concentrated Astaxanthin
The effect of the experimental diets on Astaxanthin concentration in shrimp shells (% dry weight) is presented in Figure 1. We observed increasing levels of Astaxanthin in the peel as we increased dietary Astaxanthin levels. Shrimp fed the OFO diet showed significantly lower shell AX levels than shrimp fed the OFO + AX250 and OFO + AX450 diets ( p < 0.05). We observed no significant difference between shrimp fed with control fresh fish oil and the OFO + AX150 diet ( p > 0.05).
Figure 1. Astaxanthin content in L. vannamei shells fed different diets in a feeding trial.
2.3. Shrimp survival rate after acute salinity change test
Shrimp survival after a 5-h acute salinity stress test in all diet groups is presented in Figure 2 . The lowest survival rate was found in shrimp fed the OFO diet and was significantly lower than that of shrimp fed the OFO + AX450 diet ( p < 0.05). No significant differences were observed in shrimp fed with control fresh fish oil, OFO + AX150 and OFO + AX250 diets ( p > 0.05).
Figure 2. Survival rate (%) of L.vannamei fed control diet and test diet after acute salinity change for 5 hours.
2.4. Hepatopancreatic and hemolytic immune parameters
The hemolytic and hepatopancreatic immune parameters of shrimp are shown in Table 2 . High levels of hemolytic and hepatopancreatic malondialdehyde (MDA) in shrimp were induced by the OFO diet, thus the hemolytic MDA content of shrimp was significantly higher than that of the control group ( p < 0.05). The lowest hemolytic catalase (CAT) activity was found in shrimp fed the OFO diet, and CAT activity showed an increasing trend with increasing dietary AX.
Table 2. Some lymphatic and hepatopancreatic parameters of young L. vannamei shrimp fed with different diets.
2.5. Muscle fatty acid composition
The fatty acid composition of shrimp muscle is presented in Table 3 . The unsaturated fatty acid content (monounsaturated and polyunsaturated fatty acids) in shrimp muscle fed the OFO diet was significantly lower than that of shrimp fed the OFO + AX250 diet ( p < 0 .05). In shrimp fed the OFO diet, the levels of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and omega-3 (n -3) polyunsaturated fatty acids (PUFA) were lowest, while n -6 PUFA is the highest. .
Table 3. Fatty acid composition in muscle of white shrimp L. vannamei (% weight)
2.6. Hepatopancreatic histology
We observed lesions in the hepatopancreas of shrimp fed the OFO diet. The hepatopancreatic duct epithelial cells were vacuolated and some were broken. A melanotic appearance of epithelial cells and a significant reduction of B cells were evident ( Figure 3 b). Dietary AX supplementation significantly reduced hepatopancreatic tissue damage caused by oxidized fish oil, thus observing similar or even better histopathological picture compared to the control group ( Figure 3 a). Furthermore, increased distance between adjacent ducts and the abnormal lumen was observed in the control group ( Figure 3 a). Concomitant AX treatment preserved the normal electron microscopic appearance of the hepatopancreas, producing a histology similar to or even better than that observed in the control group ( Figure 3 c–e )
Figure 3. Hepatopancreas from experimental diets treated with L. vannamei . ( a ) Hepatopancreas of shrimp fed on contol diet for 50 days ( b ) Hepatopancreas of shrimp fed on oxidized fish oil diet for 50 days. Bar = 50 μm. The hepatopancreatic duct epithelial cells were vacuolated and some were broken. Melanization of epithelial cells has appeared. ( c ) Hepatopancreas from shrimp fed with oxygenated fish oil + 150 mg/kg AX diet for 50 days. Bar = 50 μm. Cross-section of the middle region of the renal tubule shows that the tubules are well-arranged and appear as a star shape within the tubular lumen. Different types of cells can be observed and the number of these cells is more than the control group and OFO group. ( d ) Hepatopancreas from shrimp fed with oxygenated fish oil + 250 mg/kg AX diet for 50 days. Bar = 50 μm. ( e ) Hepatopancreas from shrimp fed with oxygenated fish oil + 450 mg/kg AX diet for 50 days. Bar = 50 μm. Both ( e ) and ( f ) show that the renal tubules often appear as star shapes within the lumen. Different cell types can also be observed. ALU, abnormal lumen; BL, basal lamina; REc, ruptured epithelial cell; Mel, melanocytes; *, star shape of the cell lumen; *, star shape of the heart.
TEM analysis demonstrated that in the hepatopancreas of shrimp fed the OFO and OFO/AX450 diets for 50 days, the rough endoplasmic reticulum in many B cells underwent marked swelling and vesicles and showed large vacuoles, reduced inner mitochondrial envelopes, disappearance of cristae in severe cases, and blurred RER structures ( Figure 4 a,b). Dietary supplementation of OFO/AX450 reduced OFO-induced hepatopancreatic injury ( Figure 4 c,d).
Figure 4. Hepatopancreas from test diet treated with Litopenaeus vannamei . a – d TEM ( a ) and ( b ). Hepatopancreas from shrimp fed an OFO diet for 50 days. Hepatocytes showed moderate edema, intact cell membranes, moderate swelling, and vacuolization of organelles in the cytoplasm, more vacuoles in the cell, and reduced local electron density. Mitochondria (M) show mild swelling, most of the mitochondrial matrix is slightly weakened, internal cristae are reduced, and cristae disappear in severe cases; Faint RER structure. Glycogen (GL) is abundant. Autophagy (AP) is abundant. Lipid droplets (LDs) are present individually. There are many lysosomes (L) and secondary lysosomes (SL). ( c ) and ( d ) Hepatopancreas from shrimp fed the OFO/AX450 diet for 56 days. Hepatocytes showed mild edema, cell membranes were intact, intracellular organelles were slightly swollen, and there were some vacuoles. autophagy (AP), mitochondria (M), large vacuoles (V), nucleoli (Nu), nucleus (N), Glycogen (GL), lysosomes (L), secondary lysosomes (SL), rough endoplasmic reticulum (RER).
3. DISCUSS
The present study investigated whether Astaxanthin supplements provide protection against the chronic effects of dietary OFO on growth performance and hepatotoxicity in Pacific white shrimp. OFO diets are associated with reduced nutrient digestibility [ 28 ] and low nutritional value of feed ingredients, by destroying PUFA content and other essential food components [ 29 ]. Several studies have demonstrated that feeding oxidized fats does not reduce the growth of several different fish species [ 12, 30, 31 ]. In contrast, OFO-induced growth reduction was also observed in several aquatic animals, including Labeo rohita fingerlings [ 32 ], Pagrus major [ 33 ], and M. salmoide [ 34 ]. Yang et al. (2015) demonstrated a significant growth reduction in young L. vannamei shrimp fed an OFO diet (peroxide value (POV): 234.84 meq/kg) [ 35 ]. Reduced weight gain in animals fed oxidized lipids in the diet may be due to altered appetite, leading to poor growth performance [ 21 , 24 , 27 ].
Dietary astaxanthin, as a powerful antioxidant, can enhance nutrient utilization and ultimately improve growth. In addition, AX can increase stress tolerance and plays an important role in the intermediary metabolism of aquatic animals [36, 37, 38]. In this study, adequate AX supplementation in the OFO diet significantly improved the growth performance of L. vannamei . Astaxanthin functions similarly to Vitamin E [ 39 ] and Selenium (Se) [ 40 ] in protecting lipid oxidation, thereby maximizing nutrient availability in the diet of L. vannamei [ 6 , 10 ].
The thiobarbituric acid reactive substances (TBARS) test is widely used as a single assay to measure the MDA of lipid peroxidation byproducts. In this study, we show that hemolytic MDA levels are significantly increased in shrimp fed an OFO diet, suggesting that OFO induces an oxidative stress state. The AX diet eliminated lipid peroxidation levels, as MDA levels decreased in shrimp lymph when AX was included in the OFO diet. Similar to our study, other researchers have documented increased TBARS levels in serum and liver due to dietary oil oxidation [ 21 , 24 , 27 ]. CAT is one of the main antioxidant enzymes responsible for scavenging reactive oxygen species (ROS) and serves as a protective mechanism to avoid tissue damage caused by free radicals and phagocytosis. [ 5 ]. We show that CAT activity in hemolytic disease of shrimp fed the OFO diet is significantly reduced. This result indicates that, under the OFO diet, the ability of shrimp to scavenge free radicals is significantly reduced. As a powerful biological antioxidant, moderate concentrations of Astaxanthin in the diet can inhibit MDA accumulation and thereby reduce the observed CAT activity. Previous studies have demonstrated that Astaxanthin has potent free O quenching activity and that dietary Astaxanthin can reduce oxidative stress [6, 10]. Our results suggest that the AX diet can partially alleviate the oxidative stress induced by the OFO diet in L. vannamei , which may help reduce the damage caused by reactive oxygen species .
Previous studies have shown that dietary Astaxanthin can enhance stress tolerance in aquatic animals. Pan et al. (2003) and Chien and Shiau (2005) showed that P. monodon fed on an AX diet had increased survival following thermal, osmotic, ammonia, and low dissolved oxygen (DO) challenges [ 41 , 42 ]. Chien and Shiau (2005) reported that under low DO conditions, M. Japonicus fed on algae or synthetic AX diets had longer survival times than the control group [ 41 ]. In an acute salinity change test, we showed that shrimp fed the OFO diet had a lower survival rate than the control group, while shrimp fed the AX supplement had a higher survival rate. compared to the OFO group. These results indicate that AX supplementation can improve poor tolerance to salt stress in shrimp fed with OFO.
It is well known that OFO can cause pathological changes in the hepatopancreas of terrestrial and aquatic organisms. Chen et al. (2012) suggested that increased oxidative stress in M. salmoides fed with oxidized lipids may be responsible for the observed pathological changes [ 43 ]. The hepatopancreas is an important digestive organ that has several functions, including absorption, digestion, storage, excretion, and detoxification in crustaceans [ 44 , 45 ]. The hepatopancreas is mainly composed of branching ducts and different types of epithelial cells lining the ducts. The hepatopancreas of crustaceans is sensitive to dietary contaminants and is often used to monitor the effects of various toxins [ 46 ]. In this study, histological examination of shrimp fed the OFO diet revealed degenerative features in the renal tubules and epithelial cells of the hepatopancreas, but the addition of Astaxanthin to the diet reduced these changes. this histopathological change ( Figure 3 ). From TEM analysis, we found that the rough endoplasmic reticulum in B cells was clearly swollen and blistered. These results suggest that shrimp may benefit from the AX diet by ameliorating any pathological changes related to oxidized lipids.
According to the National Research Council (2011) [ 47 ], diets containing oxidized lipids can be considered essential fatty acid deficiencies. In the present study, intramuscular fatty acid and intrathecal AX composition were altered by dietary supplementation of OFO and AX. Muscle UFA reached its highest value in the OFO + AX250 diet and was significantly higher than in the OFO diet. The current Astaxanthin content was reduced from 1.24 mg/kg in the control diet to 0.72 mg/kg in the OFO diet, which may indicate that shrimp consuming AX supplements may be resistant to oxidative stress. Correlation analysis showed that hepatopancreatic MDA not only had a highly significant positive correlation with UFA but also had a significant negative correlation with survival rate in the feeding test or after the acute salinity change test. . This may suggest that mortality and stress tolerance are related to lipid peroxidation in the hepatopancreas. Survival rate after acute salinity change test was significantly positively correlated with shell AX content, suggesting a direct or indirect relationship between AX and stress tolerance. Astaxanthin concentrations stored in shrimp increased with increasing dietary AX levels. Furthermore, Astaxanthin may reduce tissue lipid peroxidation, and reduced lipid peroxidation products may help protect muscle UFA content from the adverse effects of oxidation. Therefore, the present study hypothesizes that Astaxanthin may play an important role in detoxifying peroxidation caused by oxidized fish oil in the diet.
4. Materials and methods
4.1. Prepare for the diet
Vannamei shrimp were fed with five isonitrogenous and isolipidic diets. Diets were formulated with varying levels of oxidized fish oil and/or astaxanthin (AX) supplement (Lucantin @Pink10%; BASF SE, Ludwigshafen, Germany). The dietary conditions were as follows: (i) OFO (oxidized fish oil); (ii) OFO/AX150 (oxidized fish oil + AX150 mg/kg); (iii) OFO/AX250 (oxidized fish oil + AX250 mg/kg); (iv) OFO/AX450 (oxidized fish oil + AX450 mg/kg); and (v) control group (fresh fish oil) ( Table 4 ). The peroxide value of oxidized fish oil is 275.2 meq/kg. Each diet was fed four groups of shrimp. Briefly, all powder ingredients are weighed accurately, mixed thoroughly, and then lipids and water are added. The cold-extruded pellets (1.2 mm diameter) were air-dried to a moisture content of approximately 10%. The dried pellets were placed in vacuum-packed bags and stored at −20°C until use. A total of 40 g of each diet was sampled for biochemical analysis [ 48 ].
Table 4. Composition and nutritional levels of the experimental diets.
Oxidized fish oil was prepared as follows: (1) fish oil was oxidized by heating at 70°C under strong aeration conditions; (2) After 36 hours, the peroxide value (POV) was monitored every 8 hours until a high oxidation level (275.2 meq/kg) was reached. Peroxide value is determined according to AOAC. Briefly, 5 g of oil, 0.5 mL of saturated KI solution, and 30 mL of a solvent mixture consisting of acetic acid and chloroform (3:2) were combined. Titration was performed with 0.1 mol/L Na2S2O3, using 1% starch indicator. In the same way, we repeat this with the reagent blank test. Then we calculate and analyze the measurement results. Calculation: X = [( V − V 0) × N × 0.1269]/m ( X : Peroxide value of the sample, %. V : Volume of sodium thiosulfate solution consumed by the sample, mL. V 0 : Blank sample volume consumption of sodium thiosulfate solution, mL.N : Molar concentration of sodium thiosulfate standard solution, mol/L.) [ 33 ].
4.2. Shrimp and experimental conditions
Young L. vannamei shrimp was supplied by Evergreen South Ocean Tech Co. Ltd, Zhan-jiang, China. Before the experiment, shrimp were acclimatized to their new environment by placing them in the tank for two weeks, fed with a commercial diet (Guangdong Evergreen Group, Zhan-jiang, China) and provided with seawater. circulating filter with aeration. Shrimp (initial weight 0.53 g) were then randomly assigned to 20 fiberglass tanks (300 L, 0.6 m2 bottom, 4 tanks per diet, 30 shrimp per tank). All groups were hand-fed four times a day (at 7:00, 12:00, 17:00, and 21:00) by hand, with a total food intake of approximately 9% of body weight. To maintain proper culture conditions, all uneaten food and feces were evacuated throughout the 50-day experimental period.
During the experiment, the temperature ranged from 27 to 30 °C, salinity about 27‰ –30‰, pH 7.7–8.0, nitrogen – ammonia not more than 0.05 mg/L and dissolved oxygen not less than 6.5 mg/L.
4.3. Sample collection
All shrimp were fasted for 24 hours before sampling. During sampling, shrimp were individually weighed and collected for further analysis. For each tank, 5 shrimp were randomly collected for analysis of shell Astaxanthin and muscle fatty acid (FA) content, while 6 shrimp were randomly collected for blood and hepatopancreas sampling. Hematoma was sampled from the abdominal cavity or around the ventricles using a 1 mL syringe. Hemolysate samples were kept refrigerated for 24 h at 4°C and then centrifuged for 10 min (4°C, 8000 rpm). The supernatant was used to analyze enzyme activity and malondialdehyde (MDA) content. Six hepatopancreas samples were immediately placed into liquid nitrogen and stored at −80 °C. Before enzyme activity analysis, the hepatopancreas of each shrimp was individually weighed (0.5 g) and homogenized. in 10× (w/v) phosphate buffer (0.1 mol L -1 , pH 6.4) on ice. The homogenate was centrifuged (6000 rpm, 10 min) at 4°C and an aliquot of the supernatant was used to determine hepatic MDA.
4.4. Histopathological research
Hepatopancreas samples from three shrimp (per tank) were fixed in 4% neutral formalin and then embedded in paraffin. Tissue sections (8 μm thickness) of the hepatopancreas were stained with hematoxylin and eosin (H&E) stain and the slides were finally examined under a light microscope for histopathological lesions.
For TEM microscopy, specimens were fixed in 2.5% glutaraldehyde with 0.1 M phosphate buffer and postfixed in 1% OsO 4 . Specimens were embedded in Spurr’s medium epoxy resin after dehydration in graded acetone (Polysciences Ltd., Warrington, PA, USA). A Leica UCT ultrasound machine was used to cut ultrathin sections, and then the ultrathin sections were stained with lead citrate and a saturated solution of uranylacetate in 50% ethanol. Ultrathin sections were then screened with a TEM (FEI Tecnai G2 20, Netherlands) at 150 kV and images were acquired with a Megaview III camera (SIS GmbH), equipped with AnalySIS software.
4.5. Fatty acid composition
Total lipids were extracted with chloroform: methanol (2:1, v/v). Capillary gas chromatography (GC) was used to determine fatty acid composition. The HP6890 (FID detector; Agilent Technologies, Palo Alto, CA, USA) and SPTM-2380 column (30 m × 0.25 mm × 0.20 µm) were used on the GC. Separation is performed with nitrogen as the carrier gas. The column temperature was increased from 140 to 240 °C at 4 °C min -1 , held at 140 °C for 5 min, and at 240 °C for 10 min, with the detector at 260 °C. Use a split injector (50:1) at 260°C. Each fatty acid was determined by retention time according to chromatographic standards (Sigma, Northampton, UK). Peak areas were determined using Varian software (Varian, Inc., Palo Alto, CA, USA). The concentration of each fatty acid was expressed as a percentage of the total fatty acids.
4.6. Astaxanthin composition of the peel
According to the method of Chien and Shiau (2005) [ 41 ], the shells of 10 shrimp in each tank were dissected, freeze-dried, minced and placed in a 50 mL polypropylene centrifuge tube. Acetone solvent (20 mL) was added to each tube to homogenize the mixture (Polytron PT-MR-3000; PT. Hartono IstanaTeknologi, Indonesia) at 12700× g for 1 min and then centrifuged (Hitachi 18 PR-52; Hitachi Ltd ., Tokyo, Japan) at 12700× g for 5 min. The pellet was resuspended and centrifuged with an additional 20 mL of acetone, until the acetone extract was clear. The pooled acetone extract was transferred to a 250mL separating funnel divided with 30mL of n-hexane and washed three times with 10% NaCl to remove residual acetone. The extract was then reduced to 10 mL using a rotary evaporator. The extract was then filtered through a 0.2 Am Millipore filter and stored in three 2 mL brown vials. AX content was determined using high-performance liquid chromatography (Agilent 1200; Agilent Technologies, Waldbronn, Germany). Standards of chromatographically pure Astaxanthin were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). HPLC conditions were adjusted according to the previous method of Yuan et al. (1996) [ 49 ]. Chromatographic peaks were identified by comparing retention times with known standards.
4.7. Enzyme activity and lipid peroxidation assays
Lipid peroxidation (LPO) was assessed by measuring thiobarbituric acid reactive substances (TBARS) in the acid heating reaction, as previously described by Esterbauer and Cheeseman (1990) [ 50 ]. Determination of LPO content in shrimp was performed using an MDA detection kit (A003-1, Jian-Cheng Institute of Biological Engineering, Nan jing, China). Results are expressed as nmol MDA equivalent per milligram of hepatopancreatic protein and mmol MDA equivalent per liter of hemolymph.
Catalase (CAT) activity was determined using an assay kit (Jian-Cheng Institute of Biotechnology, Nan jing, China). Tissue CAT activity was measured spectrophotometrically at 405 using a SpectraMax M5 microplate reader (Molecular Devices, Minneapolis, MN, USA). One unit of CAT activity is defined as the amount of enzyme that catalyzes the decomposition of 1.0 μmol H 2 O 2 per minute. Results were expressed as U/L of hemolymph [ 48 ].
iNOS activity was determined using the iNOS assay kit (Jiancheng Institute of Bioengineering, Nanjing, China). Briefly, 10% brain homogenate (30 μL) or serum (15 μL) was mixed with the working solution provided in the kit and after incubation at 37°C for 15 min, the reaction was completed. Finish by adding the finishing solution provided in the kit. kits. Absorbance at 530 nm was recorded on a Hitachi U-2010 spectrophotometer (Tokyo, Japan), and iNOS activity was calculated according to the reference of a standard curve. The amount of NO produced in the samples was determined by measuring the absorbance at 550 nm.
4.8. Acute salinity change experiment
A total of 10 shrimp in each tank were randomly selected for the acute salinity change experiment. The test involved instantly reducing salinity from 29% to 10% by adding dechlorinated fresh water. Shrimp were carefully monitored and mortality recorded throughout the entire 5-hour experiment.
4.9. Calculation and statistical analysis
The parameters are calculated as follows:
Percent weight gain (WG, %) = 100 × ( Wt − Wi )/ Wi
Specific growth rate (SGR, % day −1 ) = 100 × (Ln Wt − Ln Wi )/ t
Feed conversion ratio (FCR) = feed consumed (g, dry weight)/weight gain (g, wet weight)
Survival rate (%) = 100 × (final number of shrimp)/(initial number of shrimp)
where Wt is the final body weight (g), Wi is the initial body weight (g), and t is the experimental period in days.
Analyzes of all data included testing for homogeneity, and if similar variances were observed, a one-way ANOVA was performed to determine the main effect of dietary manipulation. When significant differences (P 0.05) emerged after one-way ANOVA, group means were further compared using Duncan’s multiple range test. On the other hand, if the data do not have similar variance, the non-parametric Kruskal–Wallis test is applied, followed by pairwise multiple comparisons if the results of the Kruskal–Wallis test show a significant difference. significantly ( P 0.05). Correlation analyzes were also used to determine relationships between biochemical parameters. All data were analyzed using SPSS 19.0 software (SPSS, Chicago, IL, USA) and results are presented as means ± SEM.
5. Conclusion
Overall, the present study demonstrated that dietary oxidized fish oil (POV: 275.2 meq/kg) can induce obvious histopathological changes and oxidative stress in L. vannamei shrimp. . vannamei . Furthermore, dietary Astaxanthin can ameliorate these effects by increasing CAT activity and reducing MDA accumulation in shrimp plasma. Shrimp fed an Astaxanthin diet were better able to sustain an acute salinity stress tolerance test. Further studies are needed to verify the potential mechanism of AX in alleviating oxidative stress and hepatotoxicity in shrimp and other crustaceans.
by Yingying Yu 1,2,3,Yang Liu 2,Peng Yin 1,Weiwen Zhou 1,Lixia Tian 1,Yongjian Liu 1,Donghui Xu 3 andJin Niu 1,*ORCID
1
State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, School of Life Science, Sun Yat-sen University, Guangzhou 510275, China
2
Guangdong Key Laboratory of Animal Molecular Design and Precision Breeding, School of Life Science and Engineering, Foshan University, Foshan 528225, Guangdong, China
3
Laboratory of Traditional Chinese Medicine and Marine Drugs, Department of Biochemistry, Traditional Chinese Medicine and Marine Drugs, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China