NatuRose: Natural Astaxanthin as a Pigment Source for Ornamental Fish and Animals
Introduction
NatuRose™ algae meal is a safe natural source of astaxanthin derived from a unique strain of the microalgae Haematococcus pluvialis. The majority of the NatuRose carotenoid fraction is astaxanthin, with about 15% of the remaining fraction consisting of canthaxanthin, lutein, and beta-carotene. NatuRose is spray-dried and formulated into a fine dark red powder and is currently used worldwide as a coloration and nutrition source for numerous species of animals. It has been successfully used for pigmenting shrimp (P. monodon, P. japonicus), rainbow trout, coho, Atlantic salmon, poultry eggs, and sea bream. NatuRose natural astaxanthin has also been extensively tested and has demonstrated exceptional pigmentation of koi and tropicals (marine and fresh water).
Carotenoids
Carotenoids are a group of over 600 natural lipid-soluble pigments that are primarily produced within phytoplankton, algae, and plants. These pigments are responsible for the broad variety of colors in nature; most notable are the brilliant yellow, orange, and red colors of fruits, leaves, and aquatic animals. Among all of the numerous classes of natural colors, the carotenoids are the most widespread and structurally diverse pigmenting agents. Although plants, algae, and some fungal and bacterial species synthesize carotenoids, animals cannot produce them de novo. Carotenoids are absorbed in animal diets, sometimes transformed into other carotenoids, and incorporated into various tissues. For example, flamingos ingest algae containing high levels of beta-carotene and convert this yellow carotenoid into canthaxanthin and astaxanthin before depositing it into the feathers and tissues as red plumage. Some fish species such as koi and various crustaceans (P. japonicus and P. monodon) have the enzymatic mechanisms to convert carotenoids into other forms such as astaxanthin. The crustacean Taliepus subsists on seaweeds and kelp and converts the beta-carotene and xanthophylls to astaxanthin, which is then deposited into the exoskeleton.
The color of various carotenoids is related to the number of alternating double-bond pairs in the long polyene chain of the molecule, known as the chromophore (Figure 1). Specifically, light energy is absorbed by the carotenoid polyene system between 400-700 nm, and is converted into vibrational energy and heat, each carotenoid having a unique resonance in this regard. The carrot root contains predominantly (-carotene, which consists of 9 double-bond pairs within the polyene chain, and confers a yellow to orange color. The carotenoid (-carotene is composed of 10 alternating double-bond pairs and confers a deeper orange color, whereas the red color of ripe tomatoes and the flesh of watermelons is conferred by lycopene, which consists of 11 alternating double-bonds in the polyene chain. Although the polyene structure of astaxanthin is composed of 9 double-bonds similar to (-carotene, the keto and hydroxyl groups of the terminal ring structures contribute to the perceived color through absorption resonance.
Carotenoproteins and Carotenolipoproteins
In combination with proteins and lipids, carotenoids also give rise to the wide range of blue, green, purple, and brownish colors of marine life. Complexes of carotenoids and proteins, known as carotenoproteins and carotenolipoproteins, dominate in the exoskeleton of crustaceans, coelenterata, and many other marine species. Although astaxanthin itself appears as a red pigment, when it is complexed with various proteins the light absorbance shifts, which results in green, yellow, blue, and brown colors. This is demonstrated by the appearance of the red color when crustaceans are cooked, which is the result of the denaturation of the proteins and release of the astaxanthin chromophore. The American lobster, Homarus americanus, displays an abundance of green colors but also with shades of black, red and blue, which is almost entirely astaxanthin complexed with protein and chitin. Astaxanthin at over 90% purity has been identified from the blue goose-barnacle within the inner and outer body tissues as well as the ripe eggs. The green astaxanthin protein of lobsters, ovoverdin, persists unchanged until shortly before the eggs hatch, whereby the protein moiety is then released, liberating astaxanthin.
Ubiquity of Astaxanthin in the Marine Environment
In the marine environment phytoplankton are the primary production level of carotenoids. The ocean's phytoplankton constitute the worlds most prolific biochemical factory, estimated at over 40 billion metric tons of matter by dry weight annually. A conservative estimate of only 0.01% of this quantity represents the carotenoid content of this biomass, which is equivalent to several hundred millions tons of these pigments produced each year. Phytoplankton are ingested by zooplankton or crustaceans such as krill, and in turn become prey for salmon or trout which deposit the characteristic red carotenoid, astaxanthin, into their flesh. Ornamental fish obtain carotenoids from feeding upon algae, coral, or prey that has accumulated these pigments.
The copepod and euphausid microcrustaceans are by far the most abundant of all marine animals and act as the primary food for a wide variety of larger animals. Astaxanthin and tunaxanthin, either as a complexed or non-complexed form, appear to be the most abundant carotenoids found in all marine animals. The sponge Axinella crista-galla contains predominately astaxanthin as a non-complexed form and displays a radiant red color. Interestingly, the blue colors of Velella and Porpita jellyfish are due to astaxanthin carotenoprotein complexes, which shift the absorbed light pattern to appear bluish. When this blue protein material is denatured by heating or treatment with alcohol, the blue color disappears and the red astaxanthin fraction is liberated. Evidence now indicates that either free or conjugated carotenoids are associated with every branch of the coelenterate phylum, though sometimes is not apparent in white species until red eggs are produced. Carotenoids are not confined to the soft tissue parts, as astaxanthin is found firmly bonded to the calcareous skeleton in hydrocoral species, gorgonians, and horny corals such as the sea fan. The purple hydrocoral of Southern California, Allopora californica, owes its prevailing blue color to a complex of astaxanthin, protein, and calcium (Ronneberg et al., 1978). The crimson-colored skeleton of Distichopora violacea, the pinkish-red skeleton of D. coccinea, and the orange skeleton of D. nitida all contain various complexes of pure astaxanthin. The major species of Stylaster coral, including S. rosea, the pinkish-orange S. elegans, and S. sanguineus contain nearly pure astaxanthin bonded in the skeleton. The bright red integument as well as the pyloric caeca digestive apparatus of many asteroids is primarily astaxanthin. These include the sand star, Astropecten californicus, the webbed star Patiria miniata, the red-tipped spines of Astrometis sertulifera, and the large colorful predators Pisaster ochraceus and P. giganteus. The purple pigmented slug, Flabellinopsis, contains astaxanthin exclusively, which is traceable to its principle prey, the hydroid Eudendrium ramosum. Similar examples are found in numerous species of serpent stars, sea urchins, sea cucumbers, and molluscs.
Role of Carotenoids in Marine Animals
The luminous carotenoid colors of tropical fish are not only keys for species identification and mating signals, they have significant physiological roles as well. The use of carotenoids as pigments in aquaculture is well documented, and it appears their broader functions include a role as an antioxidant, provitamin A activity, immune enhancement, hormone, reproduction, growth, maturation, and photoprotection. The sand-burrowing shrimp, Emerita analoga, feeds upon algae and detritus within the sand layers. It has been found that the seasonal astaxanthin levels in the carapace and eggs parallels the exposure to sunlight, indicating that the carotenoids serve to protect external proteins and eggs from ultraviolet exposure. An extensive body of data stresses the vital role of carotenoids in the physiology and overall health and concludes that carotenoids are essential nutrients that are required in all aquatic diets. Beta-carotene is converted to vitamin A, which is required for the biochemical processes involved in vision. Furthermore, vitamin A plays an important role in the growth, development, and integrity of mucous surfaces. However, the majority of research concerning astaxanthin and other carotenoids has been aimed at its role in photoprotection and as an antioxidant in quenching of oxygen radicals.
Some species only asimilate astaxanthin and not other carotenoids. The brightly colored anemone, Metridium, with its plumed tentacles and extensible column, is one of the more spectacular forms of this organism. It too varies in shades of brown, yellow, orange, and red, but contains predominately astaxanthin esters in the somatic and ovarian tissues. Large adult females are raised in segregated aquariums simulating natural conditions and fed chowdered white fish muscle supplemented with various carotenoids. Ova samples were taken by syringe through a hollow needle from the animal's enteron and analyzed at various times. When these animals were fed a supplement of beta-carotene or zeaxanthin as the sole carotenoid, they absorbed the pigment through their digestive tubules but failed to assimilate it into developing ova or tissue, which resulted in colorless ova. However, when astaxanthin was administered into the diets, the carotenoid was esterified and transported to the ripening ova, which then pigmented red. The astaxanthin then acts as a antioxidant and photoprotectant for the developing eggs.
Astaxanthin also comprises the bulk of the carotenoids in salmonids (salmon and trout), crustaceans (shrimp, krill, crawfish), and many coelenterates, echinodermata, and tropical fish. The abundant deposits of astaxanthin in the liver of the ribbon-like oarfish Regalicus glesne and lumpsucker Cyclopterus lumpus, and in the skin, flesh, and eggs of Salmo and Oncorhyncus, are all result from their diet of various small crustaceans. The marine dorado Beryx decadactylus stores astaxanthin in the skin, mouth, gills, and iris of the eyes. The red varieties of the goldfish, Carassius auratus, also stores significant quantities of astaxanthin in the skin, which can be increased by inclusion of carotenoid in an artificial diet. This species oxidizes lutein, zeaxanthin, and beta-carotene to astaxanthin esters and deposits in the skin at a level of about 45% of the total carotenoids, whereas esterified 4-ketolutein comprises about 41%. The California sheep-head Pimelometopan pulchrum is an unusual sequential hermaphrodite, and also has the ability to biochemically convert tunaxanthin and zeaxanthin into esterified astaxanthin before deposition into its deep red skin.
Use of NatuRose in Artificial Diets
Marine animals have a constant source of carotenoids from their diet to maintain their pigmentation. However, captive animals are typically fed artificial diets that are poor or completely lacking in these critical carotenoids. The anemone Actinia equina exists in red, brown and green varieties. Specimens that are denied food or raised from the egg stage with a carotenoid-free diet lack pigmentation. However, when carotenoids are fed back to these depleted animals they regain their natural characteristic colors. The initially red animals recovered their red color and the green and brown forms resumed storage of their respective pigments. These experiments demonstrate not only that pigmentation involves genetic factors, but also that carotenoids have metabolic turnover and must be supplied in the diet to be maintained in the animals.
One of the greatest challenges in the tropical marine ornamental industry is to replicate the accurate natural color of the fish in the captive environment. Numerous operations that have mastered the art and science of propagation have failed to successfully market their fish due to faded colors. Various products have been introduced to alleviate this problem, but none has performed so effectively and consistently as NatuRose natural astaxanthin. On a commercial scale, inclusion rates of as low as 30 ppm astaxanthin from NatuRose is used on a daily basis in over 600 aquariums of fresh and salt water fishes as a supplement to live and flake foods. A very noticeable improvement in color can be seen on most species of Tetras, Cichlids, Gouramis, Goldfish, Koi, and Danios, as well as many other species. Stocks of over 50,000 individual fishes were as healthy and vigorous as ever, if not more so, since the initiation of this large-scale program (Aquarium Center, Inc. of Randallstown Maryland). NatuRose added to feeds of 40-day-old Amphiprion ocellaris caused a dramatic enhancement of color and market value after only 10 days (Reef Propagations Inc.). The largest breeder of marine ornamentals in the world, C-Quest (Puerto Rico), augments their own feeds with NatuRose to enhance pigmentation and fertility.
At Kohala Crawfish Farm (Kapa'au, Hawaii), NatuRose (at 25 ppm astaxanthin) is mixed into Moore Clarke "Nutra Starter Feed" and fed to Red Velvet Swordtails and Neon Green Swordtails. By the fifth feeding, pigmentation is noticeably improved, and by 30 days the color has achieved market quality. Feed not supplemented with NatuRose do not meet market quality standards.
Further feeding trials at Palaau Prawn and Shrimp Company (Kaunakakai, Hawaii) using NatuRose mixed into Rangen Trout Swim-Up feed demonstrated excellent efficacy in Rosy Barbs, Cichlids, gouramis, koi, and Red Velvet Swordtails.
Since NatuRose is a concentrated natural astaxanthin powder, it can be incorporated into any type of food delivery system such as dry flake, crumble, or frozen food. Thus, formulations can be made using the preferred food and application of the grower that has already been tested and accepted. When feeds are to be manufactured, a measured amount of NatuRose is added, similar to using a premix. Reef Propagations Inc. adds NatuRose to a semi-moist granulated food and experiences good adherence and very little wash off upon feeding. Aquarium Center Inc. mixes 1 part of NatuRose to 500 parts of a cooked food preparation (30 ppm astaxanthin), which is a mixture of fresh ingredients including beef liver, whole eggs, wheat germ, and Spirulina powder. Good distribution within the mixture will ensure consistency within the final feed. Depending on the application, NatuRose containing 1.5% astaxanthin (15,000 ppm) is incorporated into feeds at a final concentration of 45-90 ppm (3-6 grams/kg of feed).
As with all carotenoids, astaxanthin is sensitive to light, heat, and oxygen, so precautions should be taken to protect this carotenoid from oxidation. NatuRose is manufactured with antioxidants to enhance the stability of the carotenoids, and is shipped in sealed foil laminate bags or bottles that contain a packet of the oxygen absorber, Ageless. Foil bags or bottles should be kept at room temperature (72°F/22°C) or cooler to allow maximum stability during storage. The unused portion of the foil bag should be sealed and stored under the proper conditions to protect against light, heat, and oxygen. Packets of Ageless can be removed and disposed after opening foil bags. Pelleting of feeds offers the optimal conditions of low heat and oxygen exposure, and losses of astaxanthin are as low as 1-4%. The extrusion process popular with many feed manufacturers is also compatible with NatuRose, and losses generally range from 4-12%. NatuRose is very stable in oils; top-coating (top-dressing) of pellets with a NatuRose/oil mixture has also proven successful.
Fresh feeds are often made by aquaculturists raising tropical fish or crustacean broodstock in a batch fashion, and the unused portion is frozen. NatuRose can be blended directly into these feeds and will offer excellent stability while stored as refrigerated or frozen. Used with the proper storage and handling conditions, NatuRose will yield excellent stability and provide a superior source of natural astaxanthin for koi as well as fresh water and marine ornamentals.
Sources
D'Abramo, Louis R. 1997. Crustacean Nutrition: Advances in World Aquaculture, Volume 6. World Aquaculture Society. Louisiana State University. Baton Rouge, LA.
Fox, D. Biochromy: Natural Coloration of Living Things. 1979. University of California Press, Ltd. London, England.
Ronneberg, H. G. Borch, D. Fox, and S. Liaaen Jensen. 1979. Animal carotenoids 19. "Alloporin, a new carotenoprotein." Comp. Biochem. Physiol.