The Uses of Astaxanthin in Aquaculture

Astaxanthin is a naturally occurring red carotenoid pigment found primarily in crustaceans (e.g., crabs, crayfish, lobsters and krill), salmon, and other marine life and microorganisms, including microalgae [1–3]. Fish, like other animals, lack the ability to synthesize astaxanthin de novo and must obtain it from food. Wild fish obtain astaxanthin from prey, while farmed fish obtain it from feed. Natural astaxanthin has anti-inflammatory, antioxidant, coloring, immunity-enhancing, anti-atherosclerotic, anti-aging and other effects, and is widely used in the functional food, dietary supplement, beverage, cosmetics and health care industries [4-6].

In recent years, astaxanthin has not only been used in human health products, but also as a feed additive in animal farming. The use of astaxanthin in aquaculture can improve animal immunity, reduce mortality, and reduce the abuse of antibiotics [7]. In addition, astaxanthin plays a key role in the pigmentation of some aquatic animals, such as rainbow trout and ornamental fish [8]. This paper reviews the sources, physical and chemical properties, and applications of astaxanthin in aquaculture.

1 Sources of astaxanthin

The main sources of astaxanthin are simple microorganisms, especially algae, yeasts, fungi and bacteria. Astaxanthin cannot be synthesized biochemically by animals, but can accumulate in animal tissues by eating organisms containing astaxanthin. Natural astaxanthin extracts usually contain other types of carotenoids (β-carotene, canthaxanthin and lutein) that are associated with biological activity. Different microorganisms and marine animals, including microalgae, yeast, the muscles of wild and farmed salmon, trout, krill and crayfish, complex plants, and some birds are natural sources of astaxanthin. Commercial astaxanthin is usually chemically synthesized or produced by Xanthophyllomyces dendrorhous or Haematococcus Pluvialis [9-11]. The astaxanthin content in wild salmon meat is 3–38 mg·kg-1, while the astaxanthin content in rainbow trout meat is 12–25 mg·kg-1 [12]. Therefore, fillets from wild and farmed salmon can be used as a dietary source of astaxanthin.

Driven by market demand and ecological benefits, the production of astaxanthin from microalgae and the practical application of astaxanthin in aquaculture have become research hotspots in recent years. Researchers have studied the isolation of astaxanthin-rich microalgae strains and the mechanism by which algae cells synthesize astaxanthin. Currently, Haematococcus pluvialis, Chlorella zofingiensis and Scenedesmus obliquus have been identified as algal strains with the potential to produce natural astaxanthin (see Table 1). Astaxanthin or astaxanthin-rich microalgae have been widely used as feed additives for salmon, shrimp and ornamental fish to improve animal immunity, prevent the abuse of antibiotics and increase pigmentation. The use of algal astaxanthin to raise fish has been considered a viable way to achieve sustainable development in the aquaculture industry.

2 Physical and chemical properties of astaxanthin

Astaxanthin is a carotenoid with the molecular formula C40H52O4 (relative molecular mass 596.84), density 1.071 g·mL-1, melting point 216 °C, and boiling point 774 °C. Astaxanthin has poor solubility in water and is easily soluble in organic solvents. Its solubility in dichloromethane, chloroform, dimethyl sulfoxide and acetone is 30 mg·mL-1, 10 mg·mL-1, 0.5 mg·mL-1 and 0.2 mg·mL-1, respectively [20].

The molecular structure of astaxanthin is shown in Figure 1A. It is composed of a conjugated double bond in the middle consisting of four isoprene units, and an α-hydroxycanthaxanthin hexameric ring structure at both ends. C-3 and C-3 ' have chiral centers. Since a chiral center can have two conformations, the two chiral carbon atoms C-3 and C-3' of astaxanthin can exist in the R or S form, so astaxanthin has three isomers: (3S, 3S'), (3R, 3R') and (3R, 3S') (see Figures 1B~D), where (3S, 3S ') and (3R, 3R ') are enantiomers, each pair of enantiomers having opposite optical rotation, which causes plane polarized light to rotate left or right, while the (3R, 3S ') isomer has no optical rotation.

Astaxanthin exists in two states: free and esterified. Synthetic astaxanthin is mainly in the free state (3S, 3R'), while natural astaxanthin is mainly in the esterified state (3S, 3S') and (3R, 3R'). Astaxanthin produced by Rhodopseudomonas palustris is mainly the cis (3R, 3R') diester [21], and the astaxanthin contained in Haematococcus pluvialis is mainly the all-trans (3S, 3S') monoester [22]. As a member of the fat-soluble carotenoid family, the special structure of astaxanthin may lead to the following disadvantages in application:

(1) Hydrophobicity. Astaxanthin is an oil-loving substance with poor solubility in non-polar solvents. Astaxanthin has two hydroxyl groups at each end, and each hydroxyl group can interact with a fatty acid to form an ester. The esterified astaxanthin is more hydrophobic than free astaxanthin [23].

(2) Instability: Due to the structure of the unsaturated conjugated double bond, astaxanthin monomers are extremely unstable. During processing and storage, they are prone to degradation and discoloration due to changes in light, temperature and oxygen content, which causes them to lose their original biological activity and results in poor quality and color of the final product.

3 Application of astaxanthin in aquaculture

For a long time, astaxanthin has been added to fish feed as a natural pigment for the cultivation of salmon, bream and some ornamental fish. In recent years, more and more studies have found that astaxanthin has a positive effect on the weight gain and immune response of aquatic animals, and the application of astaxanthin has been expanded to crustaceans and some economic fish species. Carotenoids are closely related to a large number of physiological functions in plants and animals. The color of most aquatic animals and animal tissues is attributed to the presence of various carotenoids [24-25]. As the main carotenoid in aquatic animal tissues, astaxanthin has the functions of pigmentation, anti-oxidation and anti-stress.

3.1 Effect of astaxanthin on the coloring function of aquatic animals

Pigmentation is an important process in the farming of salmon, trout, seabream, ornamental fish and crustaceans, and consumers prefer pink salmon with a high astaxanthin content. In addition, the market value of ornamental fish depends partly on their skin color or muscle color. The color of aquatic animals plays an important role in consumer acceptance, perceived quality and price. Farmed crustaceans with light yellow shells generally have a lower market price than those with red or dark orange shells. In most cases, fish cannot synthesize pigments in their bodies, but can absorb exogenous pigments. Therefore, adding astaxanthin to the feed can improve the coloring ability of fish and crustaceans and the concentration of astaxanthin in their tissues [26-27].

Salmon, trout and sea bream are widely distributed in the Pacific and Atlantic Oceans, and are rich in protein, lipids, natural pigments and minerals, as well as other essential nutrients for the human body. In the market, the pigmentation in the skin and muscles of salmon, trout and sea bream to a certain extent determines their quality. However, in the wild, the decline of fishery resources has severely limited the market supply of these high value-added fish. In order to provide humans with high-quality aquatic products, the aquaculture of salmon, trout and sea bream has developed rapidly, increasing the market demand for fish feed.

Sigurgisladottir et al. [28] added 88.6 mg·kg-1 astaxanthin to Atlantic salmon feed. The results showed that the astaxanthin content in the salmon muscle of the experimental group was 2 .0~2.5 μg·kg-1 , while the control group had 0.5~0.8 μg·kg-1 , indicating that the addition of astaxanthin increased the pigmentation in the salmon muscle. In rainbow trout farming, the content of astaxanthin in the fish feed was increased from 0 to 27.6 mg·kg-1, resulting in an increase in the total carotenoid content and muscle chroma value in the muscles. With an astaxanthin content of 27.6 mg·kg-1 to 46.1 mg·kg-1, there was no significant increase in total carotenoid content and chroma value [29]. The main reason for this phenomenon may be that excessive astaxanthin cannot be fully absorbed by aquatic animals.

Zhang Chunyan et al. [30] added 1.0 g·kg-1 synthetic astaxanthin (Ast group) and 4.4 g·kg-1 Haematococcus pluvialis extract (HE group, astaxanthin content 100 mg·kg-1) to rainbow trout feed, and the control group was the basal diet. After feeding for 6 weeks, it was found that the muscle redness and yellow were significantly higher than those in the control group. A. Kurnia et al. [31] showed that the astaxanthin content in the tissues of bream fed with synthetic and natural astaxanthin reached 7.76 mg·kg-1 and 12.7 mg·kg-1 , respectively. C. Georges et al. [32] fed rainbow trout with synthetic astaxanthin (AST group) and natural astaxanthin derived from Haematococcus pluvialis (ALG group), and the results showed that the plasma and muscle astaxanthin concentrations, muscle color saturation (C*), redness (a*) and yellowness (b*) of the AST group rainbow trout were higher than those of the ALG group, indicating that astaxanthin from different sources has different effects on the pigmentation of different aquatic animals.

Jiang J. F. et al. [33] added astaxanthin of different sources and concentrations (20, 50, 75 and 100 mg·kg-1) to the feed of red sea strawberries (Pseudochromis fridma⁃ nii) and conducted a 42-day feeding trial to study the effect of astaxanthin on the coloration of red sea strawberries. The results proved that the brightness of the fish in the groups fed 75 mg·kg-1 and 100 mg·kg-1 natural astaxanthin and 100 mg·kg-1 synthetic astaxanthin was significantly lower than that of the control group, and the skin color saturation was significantly higher than that of the control group, and the hue of all groups (except the 25 mg·kg-1 natural astaxanthin group) was significantly different from that of the control group.

N. A. Bell et al. [34] added astaxanthin-rich and protein-rich lobster powder to the feed of goldfish (Carassius auratus), and found that the brightness, redness and yellowness of the goldfish increased. Wang Junhui et al. [35] studied the effect of different astaxanthin addition levels on the body color of koi carp (Cyprinus carpio L.), and found that the addition of astaxanthin had no effect on the brightness of the body color, and that the redness (a*) and yellowness (b*) of the body color tended to increase first and then decrease with the increase of astaxanthin addition. In addition to affecting the pigmentation of ornamental fish, the addition of astaxanthin to the feed can also affect the color-based behavior of ornamental fish. E. Lewis et al. [36] found that the addition of astaxanthin to the feed of red rose fish (Puntius titteya) can reduce their mirror attack behavior and mate selection behavior.

Many species of crustaceans such as hermit crabs, red king crabs, crayfish, clawed lobsters, spiny lobsters and grass shrimp will lose or not develop pigmentation if they do not consume enough carotenoids [37-41]. Pigmentation in banana shrimp has been shown to be inherited [42], suggesting that there may be a genetic basis for the retention of carotenoid pigments. Long X. W. et al. [43] found that when adult Chinese mitten crabs (Eriocheir sinensis) were fed a diet containing Haematococcus pluvialis powder rich in natural astaxanthin, the redness (a*) of the ovaries and shells of the Chinese mitten crabs increased significantly with the increase in the amount of Haematococcus pluvialis powder in the diet, while the brightness (L*) and yellowness (b*) showed a downward trend. This result is consistent with Han T Haematococcus pluvialis powder was added to the diet, the redness (a*) of the ovaries and carapaces of the Chinese mitten crab increased significantly, while the brightness (L*) and yellowness (b*) showed a downward trend. This result is consistent with the research results of Han T. et al. [44] on the swimming crab (Portunus trituberculatus).

3.2 Effect of astaxanthin on the antioxidant function of aquatic animals

Reactive oxygen species (ROS) are a natural product of aerobic metabolism in living organisms and are a type of free radical necessary for survival. Reactive oxygen species play an important role in regulating certain cellular activities, apoptosis, the immune system, gene transcription, etc., but they are highly harmful to cells. To deal with the constantly generated reactive oxygen species, the body relies on an antioxidant defence system that includes both enzymatic and non-enzymatic antioxidants. Enzymatic antioxidants include peroxidase (POX), superoxide dismutase (SOD) and catalase (CAT); non-enzymatic antioxidants include vitamin E, vitamin C, trace elements and carotenoids carotenoids. ROS is highly reactive towards lipids, proteins and nucleotides. The reaction of ROS with polyunsaturated fatty acids (PUFA) leads to lipid peroxidation. This reaction produces a new ROS, which continues to react with new PUFA, leading to a cycle of oxidative reactions. Aquatic animals are rich in n-3 PUFA and are extremely vulnerable to attacks by oxygen and other free radicals.

In recent years, astaxanthin, an effective antioxidant found in certain marine organisms, has attracted increasing attention from researchers [45-46]. Because astaxanthin contains conjugated double bonds, hydroxyl groups and keto groups in its molecular structure, it can react with oxygen free radicals to scavenge free radicals and exert an antioxidant effect. Astaxanthin has been shown to have strong antioxidant activity, 10 times that of carotenoids (such as canthaxanthin, β-carotene and lutein), 65 times that of vitamin C and 100 times that of vitamin E [47]. The antioxidant activity of astaxanthin is manifested in its ability to scavenge singlet oxygen, superoxide and hydroxyl radicals, as well as other reactive oxygen species (ROS), reactive nitrogen species (RNS) and inhibit lipid peroxidation [48]. Astaxanthin reacts with fat to form astaxanthin esters, which can effectively inhibit the chemical reaction between intracellular oxygen free radicals and unsaturated fatty acids, thereby reducing oxidative damage to DNA and inflammatory responses, improving the body's immune response, and easily penetrating biological membranes in a way that minimizes damage to the membrane, thereby protecting the membrane and fatty acids from lipid peroxidation.

O. Z. Barim et al. [49] added vitamin E (VE), vitamin C (VC), vitamin A (VA), astaxanthin (AST) and β-carotene (βC) to the diet of Danube crayfish (Astacus leptodactylus). During the experiment, The number and size of eggs were measured during the experiment, and the contents of VE, VC, VA, AST, βC and malondialdehyde (MDA) in the tissues were determined. The results showed that the number and size of eggs in the VE and AST groups were the best in each group. In the AST group, the MDA content in the liver, ovary, gill and muscle tissues was the lowest, indicating that the astaxanthin group had the resistance was the highest. Yi X. et al. [50] showed that adding astaxanthin to the diet of yellow croaker could increase the activity of SOD and GSH-Px in the yellow croaker liver, thereby increasing the overall antioxidant level of the yellow croaker body.

P. Ma S. L. et al. [51] studied the effect of adding astaxanthin to the diet of Hali otus discus hannai) added to the diet of astaxanthin on its antioxidant capacity, and found that compared with the control group, the astaxanthin group of Haliotis discus hannai serum SOD and CAT content increased significantly, while the MDA content decreased significantly. SOD and CAT reflect the body's ability to compensate for free radicals, and the higher the content, the higher the antioxidant capacity.

3.3 Effect of astaxanthin on the stress resistance and disease resistance of aquatic animals

High-density aquaculture operations often expose animals to various physical stresses. These stresses come from factors such as grading, transportation, handling, vaccination, and crowding, which cause animals to be in a state of high stress and immunosuppression, which may disrupt the dynamic balance between aquatic animals and their surroundings, thereby triggering a stress response. Excessive stress leads to physiological dysfunction, reduced growth rate, immunosuppression, susceptibility to disease invasion, and even death in aquatic animals [52-53]. Therefore, it is important to alleviate the adverse conditions caused by stress in aquaculture research.

An early study showed that a daily intake of a diet containing 230–810 mg·kg-1 of astaxanthin for 4 weeks could improve the tolerance of Penaeus monodon to a high salt environment [54] . Y. H. Chien et al. [55] noted that a diet supplemented with 360 mg·kg-1 astaxanthin for 1 week could induce tolerance to low dissolved oxygen levels in P. monodon larvae. After being tested with different stress factors, Penaeus monodon fed an astaxanthin-containing diet (80 mg·kg-1) for 8 weeks showed higher antioxidant defense capacity (lower SOD levels) and better liver function (lower AST and ALT), as well as improved resistance to hyperosmotic and thermal stress.

Similarly, when fed a diet containing 71.5 mg·kg-1 of astaxanthin for 8 weeks, Penaeus monodon exhibited an astonishing antioxidant status and resistance to ammonia stress at different levels (0.02, 0.2, 2, 20 mg·L-1) [56].

Lower SOD, AST and ALT indicate that various antioxidant enzymes in the tissues of P. monodon are consumed after biological stress, enhancing antioxidant capacity and liver function. The above studies show that astaxanthin is a very important nutrient for P. vannamei under physiological stress caused by external pressure. K. Supamattaya et al. [57] fed P. vannamei with a feed containing 200–300 mg·kg-1 of Dunaliella salina extract, could tolerate low oxygen environments of 0.8–1 mg·L-1 and significantly resist viral white spot syndrome (WSSV). It was also found that the tissue of Penaeus monodon fed with Dunaliella salina extract had a higher astaxanthin content, indicating that they could quickly convert β-carotene into astaxanthin.

Similar results were found in a large number of studies on Litopenaeus vannamei and other crustaceans. Zhang J. et al. [58] found that Litopenaeus vannamei (with a dietary supplement of 125–150 mg·kg -1 astaxanthin, in addition to upregulating the expression levels of hypoxia-inducible factor (HIF-1α), cytosolic manganese superoxide dismutase (cMnSOD) and CAT mRNA, also increased total antioxidant capacity and tolerance to hypoxic stress (0.8 mg·L-1). In a related study, Niu J. et al. [59] demonstrated that feeding a combined diet of astaxanthin (100 mg·kg-1) and cholesterol (1%) enhanced the mRNA gene expression of HIF-1α factor and heat shock protein HSP70 in the Vanamei shrimp, which improved its tolerance during a 36 h simulated in vivo transport.

Wang H. et al. [60] further found that after being fed 80 mg·kg-1 astaxanthin for 4 weeks, Penaeus vannamei had high resistance to white spot syndrome virus (WSSV), which was related to the improvement of hemolymph immune indicators such as phagocytic activity, total blood cell count, serum phenoloxidase activity, serum anti-superoxide radical activity, serum lysozyme activity, and serum antibacterial activity. After 10 weeks of feeding on a diet containing different doses of astaxanthin (50–150 mg·kg-1), Japanese shrimp (Macrobrachium nipponense) can tolerate various physical and chemical stresses, such as anoxic supply (0.5 mg·L-1), ammonia stress (0.75 mg·L-1) and cold stress (0 ℃) [61]. Jiang X. D. et al. [62] added 30–120 mg·kg-1    of Haematococcus pluvialis powder, and found that compared with the control group (without adding Haematococcus pluvialis powder), the mortality of each added group was greatly reduced when experiencing ammonia stress. It was also found that the expression of antioxidant enzyme genes in each group with added Haematococcus pluvialis was significantly enhanced, which may be related to its low mortality.

Astaxanthin also has a beneficial effect on fish. Liu F. et al. [53] studied the effect of dietary astaxanthin intake on the stress resistance of Pelteobagrus fulvidraco. When fed at a dose of 80 mg·kg-1 for 60 days, it can increase the content of liver HSP70, liver SOD, and serum total protein (TP) and tolerance to acute crowding stress. Subsequent tests of attack by Proteus mirabilis showed that astaxanthin can significantly reduce the mortality of Pelteobagrus fulvidraco under crowding stress. dose for 60 days, it can increase the content of liver HSP70, liver SOD, serum total protein (TP), and tolerance to acute crowding stress. The subsequent attack test by Proteus mirabilis showed that the resistance of the astaxanthin group was significantly enhanced. Li M. Y. et al. [63] studied the effects of astaxanthin supplementation on lipopolysaccharide-induced oxidative stress and immune response in Channa argus, and found that astaxanthin can increase the expression of heat shock proteins HSP70 and HSP90 and glucocorticoid receptor genes.

Another study also found that astaxanthin inhibits the production of pro-inflammatory cytokines by inhibiting the NF-κB and MAPKs signaling pathways [64]. Xie J. J. et al. [65] added Haematococcus pluvialis powder to the diet of golden pomfret (Trachinotus ovatus) with Haematococcus pluvialis powder for 8 weeks, and then subjected to an acute hypoxia stress test (1.2 mg·L-1). It was found that the group supplemented with Haematococcus pluvialis powder alleviated the inflammatory response by activating the Nef2-ARE signaling pathway to antagonize the NF-κB signaling pathway.

4 Summary

Astaxanthin is one of the most important carotenoids in nature and has attracted the attention of animal nutritionists due to its wide range of effects on the health of aquatic animals. The addition of astaxanthin to the diet of aquatic animals not only improves the body color of the animals, but also has strong antioxidant, anti-stress and anti-disease functions, and protects the cells and tissues of aquatic animals from stress and oxidative damage. With the large-scale and intensive development of China's aquaculture industry, the application of astaxanthin will become more and more widespread.

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