How do phospholipids affect astaxanthin deposition in the Japanese marsh shrimp, Penaeus vannamei?

 The body color of crustaceans depends on their carotenoid content [ 1]. However, due to the lack of relevant enzymes, crustaceans cannot synthesize carotenoids from scratch, but they can deposit or convert carotenoids from food directly into their own carotenoids [2]. Astaxanthin is the most important carotenoid in crustacean body coloration. It binds to proteins to form blue astaxanthin proteins, which give many crustaceans their dark cyan blue body color. Under high temperature conditions, the proteins are denatured and the astaxanthin in the chelate dissociates to reveal its own red color [3]. Body color is an important indicator of the commercial quality of crustaceans.

In captivity, the few effective color sources and unstable composition of compound feeds have led to the fact that the body color of crustaceans is much lower than that of the pure natural wild species [4]. These deficiencies can be compensated to a certain extent by adding specific effective color sources to the compound feeds [5]. However, astaxanthin is expensive, and the cost of adding astaxanthin accounts for 10%~20% of the cost of feed ingredients [ 6], and increasing the deposition rate of astaxanthin in the animal body is a potential way to reduce the cost.

 

As a fat-soluble pigment, astaxanthin requires the presence of lipids for digestion, absorption and transport. Phospholipids are good surfactants because of their polar and nonpolar amphiphilic properties; they are easily emulsified and have the ability to emulsify fats [7]. It has been shown that the addition of phospholipids can improve the absorption of astaxanthin in the intestine and help astaxanthin deposition in the body [8].

 

The structure of astaxanthin contains a long unsaturated conjugate system with unstable electron orbitals in the outer layer, which is highly efficient in scavenging free radicals in cells [9]. The free astaxanthin scavenging capacity is 500 times higher than that of vitamin E. When astaxanthin scavenges reactive oxygen radicals in vivo, it is reduced to the corresponding ketone and other reduction products. Therefore, factors that induce oxidative stress may reduce astaxanthin deposition. Phospholipids, on the other hand, can increase the activity of antioxidant enzymes and thus reduce the production of reactive oxygen radicals [10]. Therefore, they may have synergistic effects in the antioxidant process, which may be another potential way that phospholipids favor astaxanthin deposition.

 

Japanese marsh shrimp (Macrobrachium nipponense), also known as river shrimp, prawns, belonging to the family Macrobrachium, marsh shrimp genus, is an important species in China's aquaculture, only in 2016, the output reached 272,600 t [11]. The color of the carapace of Japanese marsh shrimp is also an important basis for evaluating its commercial quality. Therefore, the present study was conducted to investigate the effects of phospholipids on astaxanthin deposition and their interactions during oxidative stress, to provide theoretical support for the effective utilization of astaxanthin in feeds, and to provide a reference for revealing the antioxidant regulatory mechanism of the Japanese marsh shrimp.

 

1 Materials and Methods

1.1 Test feed

Fish meal, cottonseed meal, soybean meal, rapeseed meal and peanut meal were used as the main protein sources, while fish oil, soybean oil and phospholipids were used as the main fat sources. The phospholipid levels were set at 0, 20 and 40 g/kg, and the levels of astaxanthin were set at 0 and 4 g/kg respectively. Six groups were designed according to the levels of phospholipids and astaxanthin added to the feed: control group without phospholipids and astaxanthin (CON group), group with 20 and 40 g/kg phospholipids (PL1 and PL2 groups), group with 4 g/kg astaxanthin only (AX group), group with 20 g/kg phospholipids + 4 g/kg astaxanthin and group with 40 g/kg phospholipids + 4 g/kg astaxanthin (AXPL1 and AXPL2 groups). The composition and nutrient levels of the diets are shown in Table 1. The solid ingredients were mixed and pulverized through an 80-mesh sieve, and the micro-ingredients such as vitamins and minerals were mixed by the stepwise expansion method. Fish oil, soybean oil and phospholipids were added and mixed with water to homogenize the mixture, and then the feed was pressed by a twin-screw extruder (LY Twin-screw Extruder, Changzhou Jiafa Pelleting and Drying Equipment Co., Ltd.) into feeds with a particle size of 1.0 mm, air-dried at ambient temperature, and then stored in a sealed container at -20 ℃ for spare use.

 

1.2 Experimental design and culture management

The experiment was conducted at the graduate workstation of Suzhou Yangcheng Lake National Modern Agricultural Demonstration Zone Development Co. The Japanese marsh shrimp was purchased from a local shrimp nursery. The shrimp were reared in indoor cement ponds for 15 d, during which time they were fed with the control diet. Before grouping, the shrimp were fasted for 24 h. The 2,160 shrimp with an average body weight of (1.00±0.00) g were randomly divided into 6 groups of 3 replicates of 120 shrimp each. The shrimp were randomly assigned to 18 round plastic tanks with a volume of 400 L (water capacity 300 L), each with 120 tails, and 3 tanks were fed with each diet. The tanks were equipped with pots of Eriocalycium and PVC mesh as attachments for the shrimp. The tanks were fed once a day at 07:30 and once a day at 18:30. Dead shrimp were collected and weighed during the culture period. The water used for aquaculture was Yangcheng Lake water that had been settled for at least 48 hours. During the test period, the water temperature was 16-20 ℃, pH was 7.40-8.20, ammonia concentration was below 0.05 mg/L, and the dissolved oxygen concentration was kept at 6.40-7.30 mg/L by continuous oxygenation, and the water was sucked out once every 3 days and changed by 1/3.

After 4 weeks of fasting for 24 h, the total weight of shrimps in each tank was weighed and counted, 20 test shrimps were randomly taken from each tank to prepare the analytical samples directly, and another 20 shrimps were randomly taken for the ammonia stress test: the ammonia concentration of the water body was adjusted to ( 32.25±0.42) mg/L with ammonium chloride (as measured by Nano-colorimetry [12]), injected into a 300 L aquarium, and then the shrimps were transferred into it, and then the samples were sampled and analyzed after 24 h of stress.

 

1.3 Sample Preparation and Analytical Methods

1 .3.1 Sample Preparation

The shrimp were dissected on an ice tray, and the hepatopancreas tissue was rinsed with 4 ℃ pre-cooled deionized water, fixed in 4% formaldehyde solution, and stored in the refrigerator at 4 ℃ for histomorphometric observation. The rest of the liver tissue was rinsed with 4 ℃ pre-cooled deionized water, put into EP tubes, wrapped in tin foil and snap-frozen in liquid nitrogen, and stored in the refrigerator at -80 ℃.

 

1 .3.2 Determination of feed nutrient levels

The moisture content was determined by low-temperature freeze-drying method (LJB18 type freeze-dryer, Beijing Sihuan Scientific Instrument Factory Co., Ltd.), the crude protein content was determined by Kjeldahl nitrogen determination method (GB 5009.5-2010), and the crude fat content was determined by Soxhlet extraction method (GB/T 14772-2008).

 

1 .3.3 Hepatopancreas astaxanthin extraction and assay

Astaxanthin extraction and determination methods refer to Wang et al. 13] method, weighing 0.20 g of tissue samples in a mortar grinding, 30 mL acetone non-destructive transfer into a 50 mL centrifuge tube centrifugation, repeat the extraction 3 times, and merge the supernatant in the dispenser funnel. To the separatory funnel to add a certain proportion of n-hexane, sodium chloride (NaCl) and double-distilled water mixture, mix thoroughly, let stand, and then in a 50 mL volumetric flask to determine the volume. The astaxanthin content of the prepared sample was determined by high performance liquid chromatography (HPLC). The column was SUPELCOSILTM LC-18-DB HPLC Column: 250 mm×4.6 mm, the mobile phase was methanol:acetonitrile = 9∶1, the flow rate was 1 mL/min, the detection wavelength was 475 nm, the column temperature was room temperature, and the injection volume was 20 μL. The standardized curve of astaxanthin was y = 76,369x + 162.29 (R2 = 0.99).

 

1 .3.4 Determination of hepatopancreatic total antioxidant capacity (T⁃AOC) and total superoxide dismutase (T⁃SOD) activity

Weigh 0.10 g of hepatopancreatic tissue, add 9 times the volume of 0.86% pre-cooled saline, make homogenate with a high-speed homogenizer (S10 high-speed homogenizer, Shanghai Huyan Industrial Co., Ltd.), centrifugate at 2,500 r/min for 10 min, and then measure the T ⁃AOC and T ⁃SOD activities in the supernatant with the kit (Nanjing Jianjian Institute of Biological Engineering).

U/mg prot is the number of units of enzyme activity per milligram of protein.

 

1 .3.5 Hepatopancreatic superoxide dismutase (SOD) mRNA expression assay

Hepatopancreatic tissue samples stored at -80 ℃ were ground in liquid nitrogen, and total RNA was extracted according to the instruction manual of Trizol. The RNA was digested with DNase Ⅰ, and the concentration of the obtained RNA was detected by ultraviolet spectrophotometer and 1% agarose gel electrophoresis, then the first strand of cDNA was synthesized according to the instructions of PrimeScriptTM RT 1st strand cDNA Synthe⁃ sis Kit, and was stored at -20 ℃ for spare use. The first strand of cDNA was synthesized according to the instructions of PrimeScriptTM RT 1st strand cDNA Synthe⁃ sis Kit. The 20 μL reaction system consisted of 2 μL of diluted cDNA sample, 6 μL of sterilized distilled water, 10 μL of SYBR Premix Ex Taq, and 1 μL each of upstream and downstream primers at 8 μmol/L. The analyzing instrument was a Line Gene 9600 Fluorescence Quantitative PCR Instrument. The reaction program was as follows: pre-denaturation at 95 ℃ for 20 s, 95 ℃ for 15 s, 60 ℃ for 2 s, with a total of 40 cycles; melting section at 95 ℃ for 15 s, 60 ℃ for 60 s, and 95 ℃ for 15 s. The melting curves were plotted by decreasing from 95 ℃ to 60 ℃ (4 ℃/s) for each reaction. The results were analyzed by the 2-ΔΔCt method [ 15].

 

1 .3.6 Hepatopancreatic morphology

Formaldehyde-fixed hepatopancreatic tissue samples were dehydrated, transparent, dipped in wax, embedded, and sectioned at a thickness of 5 μm, and stained with hematoxylin-eosin (HE) according to conventional tissue sectioning procedures. The slices were observed and photographed with a microimaging system (AXoskop microscope, Carl Zeiss, Germany; color video camera, JVCKY-F30, Japan).

 

1.4 Data analysis

Data were statistically analyzed by SPSS 22.0 software, and differences between groups were analyzed by one⁃way ANOVA and three⁃way ANOVA (three factors: phospholipids, astaxanthin, and ammonia stress), and Duncan's method for multiple comparisons was used when the differences were significant. Data were expressed as mean ± standard deviation (n = 3). Differences between the groups before and after ammonia stress were examined by t-test of independent samples. p < 0.05 was considered significant.

 

2 Results

2.1 Astaxanthin content in hepatopancreas

As shown in Fig. 1, before ammonia stress (non-stressed group), astaxanthin content in the hepatopancreas of the PL1, PL2 and AX groups was increased compared with that of the CON group, but the difference was not significant (P > 0.05); and astaxanthin content in the hepatopancreas of the AXPL1 and AXPL2 groups was significantly increased (P < 0.05). After ammonia stress (stress group), the astaxanthin content in the hepatopancreas of AX, AXPL1 and AX⁃PL2 groups was significantly higher than that of the CON group (P < 0.05), and the AXPL2 group was significantly higher than that of the AX group (P < 0.05). Meanwhile, astaxanthin content in the hepatopancreas of AX, AXPL1 and AXPL2 groups after ammonia stress was significantly higher than that before ammonia stress (P<0.05). As shown in Table 3, the three-way ANOVA showed that phospholipids, astaxanthin and ammonia stress significantly increased astaxanthin content in the hepatopancreas (P<0.05), and there was a significant two-way and three-way interaction among the three factors (P<0.05).

 

2.2 Hepatopancreatic antioxidant indices

As shown in Figures 2 and 3, before ammonia stress, hepatopancreatic T ⁃AOC increased in all experimental groups compared with the CON group, among which PL1 and AX groups were significantly higher than the CON group (P<0.05); hepatopancreatic T ⁃SOD activity increased in all experimental groups compared with the CON group, but there was no significant difference (P>0.05). After ammonia stress, hepatopancreatic T⁃AOC was significantly increased in all experimental groups compared with the CON group (P<0.05), and hepatopancreatic T⁃SOD activity was significantly increased in the PL2 and AXPL2 groups (P<0.05). Meanwhile, hepatopancreatic T ⁃AOC and T ⁃SOD activities decreased in all groups after ammonia stress, hepatopancreatic T ⁃AOC in all groups after ammonia stress (except PL2 group) was significantly lower than that before ammonia stress (P<0.05), and hepatopancreatic T ⁃SOD activity in CON group after ammonia stress was significantly lower than that before ammonia stress (P<0.05). As shown in Table 3, the results of three-way ANOVA showed that hepatopancreatic T ⁃AOC and T ⁃SOD activities were significantly increased by phospholipids (P<0.05), hepatopancreatic T ⁃AOC and T ⁃SOD activities were decreased by astaxanthin (P>0.05), and hepatopancreatic T ⁃AOC and T ⁃SOD activities were significantly decreased by ammonia duress (P<0.05). The two-by-two and three-by-three interactions of hepatopancreatic T ⁃AOC among the three factors were significant (P<0.05).

 

2.3 Hepatopancreatic SOD mRNA expression

As shown in Figure 4, before ammonia stress, hepatopancreatic SOD mRNA expression was significantly up-regulated in the PL1 and PL2 groups compared with the CON group (P<0.05), and there was no significant change in hepatopancreatic SOD mRNA expression in the AX group (P>0.05); hepatopancreatic SOD mRNA expression was significantly down-regulated in the AXPL1 and AXPL2 groups (P<0.05) and was significantly lower than that of the PL1, PL2 and AX groups (P<0.05). The expression of hepatopancreatic SOD mRNA in AXPL1 and AXPL2 groups was significantly down-regulated (P<0.05), and significantly lower than that in PL1, PL2 and AX groups (P<0.05). After ammonia stress, hepatopancreatic SOD mRNA expression was significantly up-regulated in the PL2 group compared with that in the CON group (P<0.05), while there was no significant change in the hepatopancreatic SOD mRNA expression in the PL1, AX and AXPL1 groups (P>0.05), and the hepatopancreatic SOD mRNA expression in the AXPL2 group was significantly down-regulated (P<0.05). Meanwhile, the hepatopancreatic SOD mRNA expression was down-regulated in the CON, PL1 and PL2 groups (P>0.05) and up-regulated in the AX, AXPL1 and AXPL2 groups (P>0.05) after ammonia stress compared with that before ammonia stress. As shown in Table 3, three-way ANOVA showed that the effects of phospholipid and ammonia stress on hepatopancreatic SOD mRNA expression were not significant (P>0.05), and astaxanthin significantly down-regulated hepatopancreatic SOD mRNA expression (P<0.05). The effects of astaxanthin and ammonia stress, the interaction between astaxanthin and ammonia stress, and the interaction between astaxanthin and ammonia stress were significant (P<0.05).

 

2.4 Hepatopancreatic morphology and structure

Photomicrographs of pre-stress hepatopancreatic tissue sections of the Japanese swamp shrimp (Litopenaeus vannamei) fed on different diets are shown in Fig. 5. The hepatopancreatic B-cell numbers were relatively higher in PL1, PL2, AX, AXPL1 and AXPL2 groups compared with the CON group.

 

3 Discussion

The content of astaxanthin in the hepatopancreas is strongly influenced by the feed [ 16] . The addition of astaxanthin alone can increase the content of astaxanthin in the hepatopancreas, and when phospholipids are present, the accumulation of astaxanthin in the hepatopancreas is further promoted. Astaxanthin is not water-soluble, so its intestinal absorption is slow and its effective utilization rate is low [17]. Phospholipids are amphiphilic, and their polar heads have high affinity with the hydroxyl groups in astaxanthin molecules[18] , which can form mixed micelles with astaxanthin and emulsify astaxanthin[19] , thus contributing to the solubilization of astaxanthin, improving the intestinal absorption ability, and thus increasing the content of astaxanthin in the hepatopancreas. The results of this study suggest that when astaxanthin is added to the feed, sufficient amount of phospholipids should be added at the same time, so as to improve the utilization of astaxanthin.

Aquatic animals produce a certain amount of free radicals during normal growth and metabolism, and more free radicals are produced under stress conditions [20]. Accumulation of free radicals in the body can damage DNA and lead to apoptosis, thus reducing the growth performance, disease resistance and survival rate of aquatic animals [21]. Free radical scavenging defense systems exist in the body, including enzymatic (SOD, etc.) and non-enzymatic (astaxanthin, etc.) antioxidant systems. Phospholipids have been found to stimulate the transcription and translation of the antioxidant enzyme system, affecting the expression level of the SOD gene and the activity of antioxidant enzymes, whereas astaxanthin has a direct antioxidant function[22] and is able to scavenge free radicals efficiently in the cell[9] , thus decreasing the reliance on enzyme-promoted antioxidants, lowering the activity of hepatopancreatic SOD[23] , and down-regulating the expression of hepatopancreatic SOD mRNA[24] . In this experiment, phospholipids significantly increased T ⁃SOD activity and T ⁃AOC, while astaxanthin significantly down-regulated hepatopancreatic SOD mR ⁃ NA expression and decreased hepatopancreatic T ⁃SOD activity and T ⁃AOC, which is consistent with the above reports.

 

Under mild stress conditions, aquatic animals scavenge overproduced free radicals by increasing T ⁃SOD activity and T ⁃AOC [25]. However, the antioxidant enzymes decreased under strong stress conditions [26]. In the present study, the hepatopancreatic T ⁃SOD activity and T ⁃AOC in the CON group were significantly lower than those before ammonia stress, suggesting that the ammonia concentration in the present experiment was a strong stress to the Japanese marsh shrimp, exceeding its normal physiological regulation and damaging the antioxidant system [27]. The addition of phospholipids and astaxanthin alone or in combination significantly increased hepatopancreatic T ⁃ AOC (except PL2 group) and hepatopancreatic T ⁃ SOD activity, indicating that the addition of phospholipids and astaxanthin can help to improve the scavenging ability of hepatopancreas and reduce the oxidative stress damage.

 

It is noteworthy that in the present study, astaxanthin content in the hepatopancreas of AX, AXPL1, and AXPL2 groups after ammonia stress was significantly higher than that before ammonia stress. This may imply that the non-enzymatic antioxidant system represented by astaxanthin plays an important role in the oxidative stress of Litopenaeus vannamei and may even be the preferred antioxidant pathway in Litopenaeus vannamei under stress [28]. After ammonia stress, the hepatopancreas of the AXPL2 group showed the greatest increase in astaxanthin content, which was significantly higher than that of the AX group, suggesting that higher phospholipid levels may facilitate endogenous astaxanthin utilization by the Japanese marsh shrimp (Litopenaeus vannamei). In the CON, PL1 and PL2 groups, where no astaxanthin was added to the diet, no significant increase in hepatopancreatic astaxanthin content was observed after stress, probably due to the low accumulation of astaxanthin in the body and insufficient astaxanthin to be utilized.

According to Zhao Weihong et al [29], B cells are hepatopancreatic cells with nutrient absorption function. Oxidative damage and toxicity can reduce the number of B cells in the hepatopancreas [30]. Laboratory conditions are characterized by a high number of stressors, such as light and water change management, and the fact that the shrimp is subjected to a certain level of environmental stress during normal rearing. Compared with the CON group, the number of hepatopancreatic B cells increased in both phospholipids and astaxanthin alone and in combination, which is presumably related to the protective effects of phospholipids and astaxanthin against oxidative damage in the hepatopancreas.

 

4 CONCLUSIONS

In conclusion, the addition of phospholipids to the diet is beneficial to the deposition of exogenous astaxanthin and the call of endogenous astaxanthin by the Japanese marsh shrimp (Litopenaeus vannamei), and the deposition of astaxanthin in the body can reduce the hepatopancreatic T ⁃AOC and T ⁃SOD activities of the Japanese marsh shrimp (Litopenaeus vannamei) and protect the morphology of the hepatopancreas.

 

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