Astaxanthin: a powerful antioxidant used in aquaculture for coloration with aquatic animal health implications

Authors

• Shaylee Martling 1a, Jill M. Voorhees 1a, Maggie J. Erlenbusch 1a, Isabel Nachtigal 1a,* and Michael E. Barnes 1a South Dakota Game, Fish and Parks 1, McNenny State Fish Hatchery, 19619 Trout Loop, Spearfish, South Dakota, 57783, USA

Abstract

Astaxanthin is a xanthophyll with unique properties that make it a potent antioxidant and photoreceptor. It is synthesized in lower trophic level organisms, such as microalgae, yeast, and some other microbes. It is also synthetically manufactured. The use of astaxanthin for pigmentation in aquaculture is well documented, as are the numerous benefits for humans from the consumption of astaxanthin. However, little research has been conducted on its potential health benefits to aquatic species. Astaxanthin has recently been identified as a semi-essential nutrient for some common aquaculture species, such as crustaceans and salmonids, but its effectiveness as a health supplement in aquatic species is unclear. This review aims to summarize the varied current uses of astaxanthin in aquaculture, as well as the potential effects of astaxanthin on the aquatic animal species which receive it.

Introduction

Aquaculture has become a major producer of food for human consumption. In 2020, 56% of the seafood eaten by people was grown; 44% was obtained via wild capture [1]. In addition, aquaculture accounted for 83% of the freshwater aquatic animals consumed, with both freshwater and saltwater production split approximately equally between aqua-culture and the harvest of wild stocks [1]. Feed ingredients that maximize growth, maintain health, and improve the appearance of aquatic animals are essential to the continued growth of aquaculture. One important ingredient is astaxanthin, which is a xanthophyll, an oxidized form of β-carotene naturally synthesized by lower trophic level organisms such as microalgae, yeast, and some microbes [2-4]. It is also synthetically manufactured [5,6]. Astaxanthin has historically and widely been used in aquaculture as a pigment to color fish flesh [7-10]. However, fish and shellfish grown in aquaculture cannot synthesize astaxanthin de novo [2,11,12]. It must be included in their diets [13,14].

While considerable initial research has examined the astaxanthin dosages and feeding durations required to produce the desired coloration in cultured aquatic animals, additional benefits have begun to appear. Astaxanthin is a potent antioxidant [11,15], with likely positive effects on the immune function and overall health of fish and shellfish, as well as their survival during hatchery rearing [2,16]. Improvements in the growth of fish and crustaceans receiving dietary astaxanthin have also been observed, although these results are somewhat inconsistent [17]. Astaxanthin also appears to positively impact reproduction and subsequent egg survival in some fish species [18].

This review article describes astaxanthin from an aquaculture perspective. The chemical properties and characteristics of astaxanthin are described first, followed by a discussion of natural and artificial astaxanthin sources. A review of the effects of astaxanthin on oxidative stress and immune responses occurs next, followed by a review of those studies examining astaxanthin effects on aquatic animal growth and reproduction. Lastly, the use of astaxanthin as a pigment source for fish and shellfish is reviewed, including the dosages, feeding durations, and retention times.

Chemical Formula and Properties

Carotenoids are a group of naturally occurring pigments [11]. There are two classes of carotenoids, xanthophylls and carotenes, whose chemical formulas differ. Xanthophylls are distinguishable by the presence of oxygen in addition to a double-bonded polyene carbon chain, whereas carotenes do not have oxygen. Astaxanthin (3,3’-dihydroxy-β, β’carotene-4, 4’-dione) is a xanthophyll [19-21]. It contains hydroxyl and keto moieties on either side of the ionone ring at either end of the polyene chain which give the molecule both lipophilic and hydrophilic properties. It is generally naturally occurring in esterified forms, with many different isomers [2,19-24]. Esterified astaxanthin is found primarily in the skin of fish [25,26], however, most, if not all, astaxanthin found in the muscle of salmonid fish is unesterified or free [27-29].

Unlike crustaceans, fish and other higher trophic level animals are unable to synthesize astaxanthin and must acquire it through food [2,11,12]. For example, salmonid fish cannot epimerize 3-hydroxy groups, but 3S,3’S astaxanthin isomer is in the muscle tissue, indicating that astaxanthin must have a dietary source [30,31]. Higuera-Ciapara et al. [14] reported that salmonids obtain astaxanthin from zooplankton, which in turn accumulate astaxanthin primarily by ingesting Spirulina and Haematococcus algae (green microalgae). Storebakken et al. [32] isolated the chiral isomer of astaxanthin in crustaceans consumed by wild salmonids.

Sources

Natural astaxanthin is produced mainly at the primary trophic level by higher plants, microalgae, bacteria, and some fungi. Examples of astaxanthin-producing species include some microalgae (Haematococcuslacustriss, Chromochloriszofingiensis, Scenedesmus obliquus), red yeast (Phaffiarhodozyma), and many other algal species [33].

Astaxanthin was first discovered in lobster (Homerus gammarus) in 1938 and was extracted from Haematococcusalgae in 1944. Naturally sourced astaxanthin has traditionally been from crustacean by-products [2,3]. Recently, extraction of astaxanthin is possible from red yeast, [34-36] and microalgae [6,18,22,23,37-39] which are emerging as a sustainable natural sources [4].

Astaxanthin is harvested from microalgae in one of two ways. The first process involves two steps. Microalgae are cultured to produce biomass, and then the microalgae are exposed to ultra-violet light or stressed. The stress can be applied by using chemicals, temperature, or lack of nutrients. The second method uses a one-step process where the microalgae are grown at a low level of stress for simultaneous biomass and astaxanthin production. The microalgae respond to the stressful environmental conditions by becoming dormant and forming cysts full of astaxanthin as protection against oxidative stress. The microalgal cysts are surrounded by a tough sporopollenin or algaenan cell wall which requires either mechanical or chemical processes for astaxanthin extraction. Extraction makes up about 20-to-30% of the production cost of astaxanthin [4,19,40]. The two-step process is the most widely used and is likely the most efficient process because the production of the algal biomass is not ideal under any stress conditions [41]. Synthetic astaxanthin is commonly used [8,15,36,39,42-49] and is much less expensive to produce than natural forms [5,6]. Approximate costs for synthetic astaxanthin range from $1,000-to-$2,000 USD per kg, while natural sources are approximately $7,000 USD per kg [40,50].

Oxidative Stress and Organism Health

Mitochondrial metabolic activity constantly produces free radicals, reactive oxygen, and nitrogen species that can cause oxidative damage to proteins and genetic material. While a small amount of reactive oxygen species is necessary for cell signaling and homeostasis, an over-abundance is known to contribute to genomic mutations and oxidative stress, such as the irreversible modification of a number of biologically-important molecules such as proteins and lipids [15,16,38,49]. Carotenoids protect against chronic stress by preventing lipid peroxidation and reducing oxidative stress, thereby reducing the inflammatory response [3,7,30]. Part of the initial stress response of an organism is mild inflammation, which involves the generation of oxidants. While this immediate oxidative response is necessary for fighting infectious agents, it can be damaging if it becomes chronic. Carotenoids help prevent chronic inflammation because they are potent antioxidants [11]. Astaxanthin is a multifaceted molecule with 100 to 500 times the potency of other carotenoids and antioxidant vitamins [15,48]. Its unique polar structure allows it to embed in cell membranes, providing protection against lipid peroxidation inside the cell membrane and allowing it to scavenge free radicals outside the cell membrane. Because of these unique properties, it is highly anti-carcinogenic, anti-diabetic, anti-ageing, anti-inflammatory, anti-tumor, anti-bacterial, ultra-violet light protective, cardio-protective, ocular-protective, neuro-protective, hepato-protective, and gastro-protective, with positive effects on athletic performance, fertility, immune response, and disease resistance in humans [2,3,16,20,21,38,51-55].

Although astaxanthin is a well-known pigment in fish and crustaceans, relatively few studies have investigated the potential health benefits of astaxanthin to these organisms [2,38,53]. Just as in mammals, astaxanthin is likely important for various functions other than coloration, such as immune function, antioxidant capacity, and reproductive performance [56-58]. Although focusing on astaxanthin use for pigmentation, Pham et al. [6] did investigate its antioxidant properties. More recent studies have focused on the health effects of astaxanthin on cultured crustaceans [15,37,39,59,60]. Yu et al. [60] reported that in Pacific white shrimp (Litopenaeusvannamei), astaxanthin supplementation was associated with increased survival and hepatopancreatic health. In juvenile red king crab (Paralithodes camtschaticus) in Alaska, astaxanthin supplementation enhanced survival [37]. Wang et al. [47] found that dietary astaxanthin increased immune response and tolerance against freshwater shock stress in kuruma shrimp (Marsupenaeus japonicus). Adult Chinese mitten crab (Eriocheir sinensis) showed a marked decrease in antioxidant enzyme activity [59]. These results are in contrast to some aquatic species as well as mammalian studies, which observed increased antioxidant enzyme activity [3,51,52]. Long et al. [39] studied green microalgae powder in Chinese mitten crabs and found no significant difference in hepatosomatic index between treatments. However, this index is a relatively crude indicator of antioxidant activity, and the old age of the crabs may have negatively influenced the results [39]. Lastly, astaxanthin mitigates the oxidative stress caused by microplastics in fish, but this occurs at the expense of skin pigmentation [61].

Growth

The impact of astaxanthin on crustacean growth is uncertain, likely because of species-specific nutritional differences, differences in study durations, and differences in diet compositions among the studies. Wu et al. [59], Long et al. [39], Wang et al. [15], and Ma et al. [62] found no significant effect on the growth. However, Daly et al. [37], Zhang et al. [56], and Wang et al. [49] both reported improved growth with the use of astaxanthin. These studies either used juvenile crabs or crabs that molted during the experiment. Wang et al. [49] used two levels of astaxanthin and three levels of vitamin E in kuruma shrimp and found the treatment with high levels of astaxanthin and medium levels of vitamin E outperformed the other treatments. Zhang et al. [56] found Pacific white shrimp had similar growth and survival as controls when fed only 25 mg/kg astaxanthin when stressed with low oxygen levels. However, there was increased survival with fish fed 75-125 mg/kg astaxanthin.

Just as with crustaceans, the effect of astaxanthin on fish growth is also uncertain. Similar to the studies involving invertebrates, the studies evaluating astaxanthin in fish are not uniform. Not only are the astaxanthin effects likely influenced by species-specific nutritional differences, the studies also have different study durations and use diets with different ingredients, many of which could potentially influence astaxanthin absorption or utilization. Some studies have shown a positive relationship between astaxanthin and fish growth [18,43,47,48,63,64] while others have found no relationship [6,37,48,56,65]. Palma et al. [48] found increased egg quality and juvenile growth and survival when astaxanthin was fed to parental females in long snout seahorses (Hippocampus guttulatus). Hansen et al. [47] found female spawning age Atlantic cod (Gadus morhua) to have increased egg production and efficiency, with higher fertilization success, egg survival, and larval growth when fed a diet with astaxanthin included. Feeding astaxanthin for six weeks improved the growth of red tilapia (Oreochromis spp.), and also improved skin coloration [66].

Reproduction

Little research has been conducted on the effects of astaxanthin consumption on aquatic animal reproduction. In salmonids, studies examining the possible relationship between astaxanthin consumption and reproductive success have produced mixed results. Christiansen and Torrissen [67] reported no significant effects on egg fertilization or survival when Atlantic salmon (Salmo salar) broodstock diets were supplemented with synthetic astaxanthin. Choubert et al. [68] also did not observe any relationship among astaxanthin and several reproduction parameters in rainbow trout (Oncorhynchus mykiss). In contrast, Ahmadi et al. [17] found a positive correlation between synthetic astaxanthin and fertilization, eyed-egg percentage, and percent hatch in rainbow trout, and suggested that astaxanthin supplementation of brood stock diets are necessary for optimal reproductive performance in rainbow trout. Sawanboonchun et al. [45] and Hansen et al. [47] found an increase in egg quality and larval production in Atlantic cod.

Pigmentation

Carotenoids are one of four main pigment groups (melanins, purines, pteridiums, and carotenoids) that produce yellow, red, and orange pigments in fish and crustaceans [69]. Carotenoids in the skin of fish are deposited in xanthophores and erythrophores. Astaxanthin is generally the most efficiently absorbed carotenoid pigment, although this may vary by species [69].

Astaxanthin is most widely known for its role in the pigmentation of salmonid muscle [14,70]. Increased pigmentation in food fish increases market demand and customer satisfaction [2,10,38,53,71]. Astaxanthin is also an important pigment for crustaceans [2,6,38], because coloration is also a key component of customer satisfaction and market demand [39,59].

Tissue Integration

Astaxanthin cannot be synthesized de novo by salmonids and therefore must be ingested as part of their diet [14]. Once ingested, the food undergoes enzymatic digestion and then enters the intestine where any astaxanthin esters are hydrolyzed by lipases. They are then absorbed into the blood serum through the intestinal lumen as the free form of astaxanthin and deposited in the muscle tissue [72-74]. Most salmonids fed supplemental astaxanthins receive it in the synthetic free form because it is more readily absorbed than the naturally occurring esterified forms; the degree of esterification influences absorption [23,24].

Once ingested and dependent on temperature, astaxanthin typically begins to appear in blood serum three hours after feeding with levels increasing rapidly from that point. When astaxanthin is conveyed across the lumen wall it enters the blood stream where it is transported in high density lipoproteins and very high-density lipid proteins [26,31,75-77]. Once astaxanthin-containing lipoproteins reach muscle tissue, attachment to the cells is dependent upon specific binding sites. Astaxanthin binds to actomyosin using one ionone ring. Depending upon the developmental stage of the fish, lipoproteins carrying astaxanthin can vary in size and density, with high density lipoproteins dominating in early life stages and very low density lipoproteins increasing dramatically with age. Some transport pigment from the intestine to the liver and others transport pigment from the liver to other tissues. Astaxanthin distribution and deposition changes throughout the salmonid life cycle, with younger fish depositing more esterified form in their skin and maturing fish depositing more free form in their muscle tissue [9,78-81]. With the onset of sexual maturity astaxanthin, originally obtained from the diet, begins transference from the flesh to reproductive organs and eggs.

At all fish ages, the esterified form of astaxanthin is more likely to be deposited in the skin with the free form being deposited in the muscle tissue [82]. Once consumed, astaxanthin deposition is dependent on several factors including the rate of absorption, transport, metabolism, and excretion [29,83]. Considerable research has focused on the effects of these variables on pigmentation in the muscle of food fish such as rainbow trout [9,35], Atlantic salmon [36], coho salmon (Oncorhynchus kisutch) [8,84], Australian snapper (Pagrus auratus) [46], and red porgy (Pagruspagrus) [61]. Iwamoto et al. [84] suggested that pigmentation may be mostly determined by genetics. In addition, March and MacMillan [44] also concluded that the genetics has a large influence on astaxanthin absorption and deposition in Atlantic salmon. Micah et al. [85] documented 4,250 differently expressed genes affecting numerous metabolic and physiological pathways in blood parrotfish (Viejamelanurus x Amphilopuscitrinellus) fed astaxanthin,

Feed composition contributes to astaxanthin deposition efficiency. Increased dietary fat concentrations in rainbow trout increases astaxanthin digestibility, transport [86], absorption [87] and retention efficiency [88]. If higher lipid levels lead to changes in fish growth or feed conversion ratios, dietary astaxanthin must be adjusted to obtain desired pigment levels [89]. Lipid type and quality play an important role in the absorption of carotenoids and flesh pigmentation. Atlantic salmon fed diets containing animal fats had lower levels of astaxanthin in their muscle tissue than those receiving fish oil [90]. Compared to more-highly-saturated fats, polyunsaturated fatty acids increase astaxanthin retention in the muscles of salmonids in diets with high levels of vitamin E [91].

The source and type (astaxanthin or canthaxanthin) of carotenoids also influences pigmentation. Pham et al. [6] fed juvenile olive flounder (Paralichthysolivaceus) either synthetic astaxanthin, green algae extract, whole green algae, or paprika extract to assess the effects of each treatment on flesh color. Both paprika and whole green algae had a significantly better effect on the flesh pigmentation than the other treatments [6]. Teimouri and Amirkolaie [68] investigated feeding synthetic astaxanthin and canthaxanthin to an aquarium species. After supplementation with five different astaxanthin or canthaxanthin concentrations, carotenoid concentrations and coloration parameters were consistently higher in fish fed astaxanthin than in those fed canthaxanthin. Red porgy fed astaxanthin from shrimp meal also had significantly better coloration than those fed either a control diet or one with canthaxanthin [63]. March and MacMillan [44] looked at the effects of feeding different astaxanthin concentrations on carotenoid absorption and deposition in rainbow trout, Chinook salmon (Oncorhynchus tschawytscha), and Atlantic salmon. They found rainbow trout had the highest astaxanthin concentration in muscle tissue and the most visible pigmentation, chinook salmon and rainbow trout were equally variable in pigmentation, and Atlantic salmon had the lowest muscle astaxanthin concentrations and the lowest visible pigmentation.

The coloration of ornamental fish can be safely enhanced using astaxanthin [92]. Song et al. [93] observed improved skin pigmentation in discus fish (Symphysodon spp.) receiving at least 200 mg/kg of dietary astaxanthin for four weeks. The external coloration of goldfish (Carassius auratus) was also improved with relatively low levels of dietary astaxanthin [85] while considerable higher levels were used with blood parrotfish to achieve changes in skin coloration [94]. Astaxanthin Both natural and synthetic sources of astaxanthin improved the coloration of orchid dottyback (Pseudochromisfridmani) with natural source astaxanthin deemed a more effective colorant [95]. Clown anemonefish (Amphiprionocellaris) skin pigmentation was positively related to the dietary astaxanthin concentrations and the duration of feeding astaxanthin-containing diets [96]. In spinecheek anemonefish (Premnasbiaculeatus), external coloration was achieved after feeding 214 mg/l astaxanthin for 115 days [97]. Supplemental astaxanthin improved the external orange-red coloration of red zebra cichlid (Maylandiaesterae) [98], while differing levels of carotenoids in commercial diets influenced the external color of goldfish (Carassius auratus) [99].

Astaxanthin is extremely sensitive to light, heat, moisture, and oxygen exposure and can be damaged during feed manufacturing [100,101]. Storage in sealed dark packaging at cold temperatures and even vacuum-packaging is recommended. Decreased efficiency of pigmentation could be caused by any milling processes or storage practices of feed that contain astaxanthin, which would lead to premature decomposition [101,102].

Dosages and Retention

There is an inverse relationship between dietary astaxanthin dose and deposition rate in the flesh of salmonids. Bjerkeng et al. [103] reported pigment concentration in muscle directly increased with increasing dietary doses of astaxanthin. However, increasing dietary astaxanthin reduced retention rates. Feeding lower doses of astaxanthin over an extended period produces the best pigment retention [8]. In rainbow trout, astaxanthin inclusion levels of 50-to-70 mg/kg astaxanthin appear to be optimal [44,103,104]. March and MacMillan [44] reported the highest levels of rainbow trout pigmentation were achieved at 27 weeks with 40 mg/kg of dietary astaxanthin or 22 weeks with 70 or 100 mg/kg astaxanthin. Storebakken and No [69] stated that little extra flesh pigmentation can be gained in rainbow trout at dietary astaxanthin levels higher than 50-to-60 mg/kg.

There is very little information on retention duration after the cessation of feeding astaxanthin. However, astaxanthin levels do not decrease even after several months of starvation in fish [28,105,106]. Brown et al. [107] reported no decrease in muscle coloration in rainbow trout after the elimination of dietary astaxanthin. A compilation of astaxanthin studies in fish and invertebrates are provided in Tables 1 and 2, respectively.

Conclusion

The carotenoid astaxanthin is a potent antioxidant available from both natural and synthesized sources. It has documented benefits to mammalian health that have yet to be fully investigated and described for aquatic animals. Its underlying physiological mechanisms of action, which have been researched in mammals, also need to be further detailed for fish and crustaceans. Study results are likely influenced by species and genetic differences in the ability to absorb and utilize astaxanthin, as well as the source of astaxanthin used. The stability of astaxanthin also likely influences study results. In its most bioavailable form, astaxanthin is the least stable, and even in the more stable forms it is highly susceptible to oxidation. Feed manufacturing, shipment, and storage could be exerting a substantial influence on astaxanthin potency.

Sustainable astaxanthin sourced from microalgae is promising if production and processing can be streamlined. With recirculating aquaculture systems expanding rap-idly in commercial aquaculture, the use of astaxanthin has tremendous potential. It could provide a buffer against various stressors inherent to fish and shellfish rearing, potentially improving growth, and decreasing the likelihood of catastrophic disease outbreaks.

Acknowledgments

Thanks to Alexis Gerber for her assistance with manuscript formatting.

Conflicts of interest

The authors declare no conflict of interest.

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