NatuRose Natural Astaxanthin ( Haematococcus algae … · NatuRose Natural Astaxanthin ( Haematococcus algae meal) as a ... laws against this industry are in place to protect the

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NatuRose Natural Astaxanthin (Haematococcus algae meal) as a Carotenoid and Vitamin Source for Salmonids

Introduction NatuRose algae meal is a safe natural source of concentrated astaxanthin derived from a

strain of the microalgae, Haematococcus pluvialis. The majority of the NatuRose carotenoid fraction is astaxanthin, with about 15% of the remainder 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. It has been successfully utilized for pigmenting shrimp (P. monodon, P japonicus), rainbow trout, Coho, Atlantic salmon, poultry eggs, Koi, sea bream (Tai), yellowtail, and ornamental fish (marine and fresh water). For a full description of NatuRose natural astaxanthin, refer to NatuRose Technical Bulletin #050. NatuRose Haematococcus algae has been approved as a feed additive for salmonids by the Canadian Food Inspection Agency (CFIA 990535) and US Food and Drug Administration approval for salmonid feeds was achieved in August 2000 (21 CRF 73.185). Similar registrations are in progress in the European Community.

Salmon Farming Wild catches of salmon have been declining over the past 10 years and have probably reached a maximum sustainable level. Conversely, salmon and trout farming have increased substantially during this period. The world production of farmed salmon in 1998 (Atlantic and Pacific) was estimated at over 800,000 metric tons of which Norway accounted for about 46% of the total. These figures are expected to reach one million metric tons by the year 2000 and 1.2-1.5 million tons by 2005. Although Norway has been a leading producer of farmed salmon for decades, its share of the total production is narrowing and is expected to produce 600,000 tons by 2005. Chile entered into salmon farming later but is growing fast as a producer. Ideal conditions in Southern Chile will allow for significant expansion to about 500,000 tons in the coming years (Fish Farming International, April 1998). Scotland has increased its production considerably in recent years but will near its potential of 100,000 tons by 2005, and Ireland is expected to produce 40,000 tons. Farmed salmon in Europe, excluding Norway, is not expected to exceed 200,000 tons per year. The United States has only limited potential as a producer due to high costs, and Canada has capacity for only 50,000 tons annually. Although Alaska has considerable potential for raising captured salmon, laws against this industry are in place to protect the salmon fishing operations. Wild landed salmon are not expected to increase and would be expected to fluctuate at about 800,000 tons annually, thus the total supply of salmon by 2010 is predicted to be at or in excess of 2 million tons (Hempel, 1997, Fish Farming International, April 1998). Just 30 years ago salmon was an expensive delicacy only available in certain parts of the world during the season. Advances in production technologies have led to increased production levels and declining prices for the last three years. Thus, salmon is now considered a commodity that is available worldwide without seasonal fluctuation. The worldwide CIF prices are expected to level off at $3 per kg in the year 2002 which would make profitable production difficult in the

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UK and USA, but both Chile and Norway would be able to thrive. The three major consumer markets for salmon are the USA, Europe, and Japan. While Japan is by far the largest single market, much of its consumption is wild caught now but this trend is reversing toward farmed salmon. The majority of salmon currently consumed in Europe is farmed. Although salmon is almost unknown in China and many other Asian countries, these regions represent the largest future markets. Imports into China have increased 500% in 1997 from the previous year with Norway conducting major marketing campaigns into the country (Hempel, 1997). The continued growth of salmonid farming has created an enormous demand for pigments. The flesh color of salmonids is due to the absorption and deposition of dietary oxygenated carotenoids, primarily astaxanthin. Salmonids are unable to synthesize astaxanthin de novo, only plants, bacteria, algae, and fungi are capable of this. Therefore, carotenoid pigments must be supplied in their artificial aquaculture diet (Steven, D.M. 1948;Goodwin 1984). In the natural marine environment, astaxanthin is biosynthesized in the food chain within microalgae or phytoplankton as the primary production level. The microalgae are consumed by zooplankton, insects or crustaceans such as krill which accumulate astaxanthin, and in turn are ingested by salmonids (Kitahara 1984 and Foss et al., 1987). Function of Carotenoids Carotenoids are a group of over 700 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. 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. Astaxanthin was first characterized and termed in 1938 from an extract of the lobster, Homarus astacus. The pigment in Haematococcus was called “haematochrom” until 1944 when Tisher correctly identified the principal carotenoid as astaxanthin. The astaxanthin molecule, as shown in Figure 1, has two asymmetric carbons located at the 3 and 3’ positions of the benzenoid rings on either end of the molecule. Different enantiomers of the molecule result from the exact way that the hydroxyl groups (-OH) are attached to the carbon atoms at these centers of asymmetry. When the hydroxyl group is attached so that it projects above the plane of the molecule it is said to be in the R configuration. Conversely, when the hydroxyl group is attached to project below the plane of the molecule it is said to be in the S configuration. Thus the three possible enantiomers are designated 3R, 3’R, 3S, 3’S and 3R, 3’S (meso). Free astaxanthin and its mono- and diesters from Haematococcus have optically pure (3S,3'S)-chirality (Grung et al., 1992 and Renstrom et al., 1981). Wild rainbow trout caught in alpine lakes of Austria are intensely pigmented with astaxanthin. A quantitative and qualitative analysis of the carotenoids in the skin and flesh reveals that only the 3S, 3S’ isomer of astaxanthin found in these fishes. Since

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salmonids are unable to epimerize the 3-hydroxy groups, it was concluded that their dietary carotenoid was also 3S, 3'S-astaxanthin, contained in the freshwater crustaceans of their diet (Schiedt et al., 1986, Storebakken et al., 1985). This is consistent with the food chain studies in various lakes by Storebakken et al., (1984) where the same chiral composition of astaxanthin was found in the crustaceans as in the fishes Salvelinus alpinus and Salmo trutta. Another study revealed that fish caught from the wild in Scotland, Ireland and Norway contained greater than 80% 3S, 3’S astaxanthin in the flesh (Schiedt, 1981). HPLC separation of astaxanthin isomers has been used to identify the eggs of escaped salmon since wild fish contain about 80% 3S, 3’S astaxanthin and farmed fish contain 35% or less (Lura, 1991;Turujman et al., 1997). Figure 1-Astaxanthin Isomers

The use of carotenoids as pigments in aquaculture is well documented. An extensive body of data emphasizes the vital role of carotenoids in the physiology and overall health and concludes

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that carotenoids are essential nutrients that should be included in all aquatic diets at a minimum level of 10 ppm (Craik, 1985; Torrissen, 1990; Grung et al. 1993). It appears the broader roles of astaxanthin include functions as an antioxidant and provitamin A, enhancing immune response, reproduction, growth, maturation and photoprotection of eggs. For example, supplementation of the diet with astaxanthin decreases the period of shrimp postlarval development by inducing molting hormones (Petit, 1993). Additionally, growth rates are less with P. japonicus fed a carotenoid-free diet compared to groups fed astaxanthin-supplemented diets. It has also been demonstrated that carotenoids supplied in the diet to fry of Atlantic salmon have an increased growth rate. The mobilization of carotenoids in fish and their transport from the flesh to the skin and ovaries during maturation indicates a role as a fertilization hormone, photoprotective element, and stress protection from elevated temperatures or ammonia.

Interestingly, a recent groundbreaking study in Norway by Christiansen and his colleagues demonstrated that Atlantic salmon fry have a definitive requirement for astaxanthin in their diet for growth and survival. Fish fed diets with astaxanthin below 5.3 ppm were found to have marginal growth, those fed levels above 5.3 ppm had significantly higher lipid levels accompanied by lower moisture levels. When fry were fed astaxanthin concentrations below 1 ppm, survival rates plummeted. More than 50% of the fry fed diets with less than 1.0 ppm astaxanthin died during the experimental period, survival of those groups receiving higher concentrations had survival rates greater than 90%. Thus, Atlantic salmon have the distinction as being the first salmonid species for which astaxanthin has been shown to be an essential vitamin, with minimum levels being about 5.1 ppm. However, higher astaxanthin levels of 13.7 ppm in the feed continued to improve the fish lipid levels another 20% to a plateau point and gave better overall performance than the 5.3 ppm feeds. The results also strongly suggested a provitamin A function for astaxanthin over the same fry-feeding period (Christiansen et al., 1995).

In a continuation of the prior study, Atlantic salmon juveniles with a mean weight of 1.75 g. were supplied with feeds with various levels of astaxanthin (0, 5.3, 36 and 190 ppm) for 10 weeks to study the effects on growth, survival and vitamin A content. It was found that the juvenile groups that had no astaxanthin supplied in the diet actually lost weight over the experimental period, while those with 5.3 ppm had a reduced SGR (specific growth rate) and BWI (body weight increase) compared to the higher doses. Groups fed the higher concentrations of 36 or 190 ppm of astaxanthin had the highest SGR, BWI, and survival rates. The lipid content was also significantly higher in those groups fed astaxanthin at 36 and 190 ppm, while lower levels of astaxanthin resulted in less lipid and protein but a concomitant increase in moisture and ash content. The flesh astaxanthin and vitamin A content was found to be dose dependent. The results corroborate other studies that astaxanthin does indeed function as a provitamin A source for juvenile Atlantic salmon with which body stores increase with dosage. It appears that the dietary needs for astaxanthin increases with the growth stage. Whereas the minimum levels for fry appear to be about 5.3 ppm, juveniles require a higher concentration as demonstrated by the feeds containing 36 ppm. It is apparent that astaxanthin has a specific role in salmonid fry and juveniles linked to vitamin A metabolism and perhaps other functions. Stored astaxanthin from the flesh and skin apparently cannot be used as a vitamin A source, it must be converted at the point of uptake within the intestinal mucosa (Christiansen et al, 1996). Other studies have

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definitively linked a role for astaxanthin in the regulation or metabolism of vitamin E (α-tocopherol), vitamin A and retinol (Christiansen 1994 and 1995b). Carotenoids can also be characterized by their capacity to interact with the chemically reactive species of oxygen, singlet oxygen. Thus, a further role of carotenoids may be in the protection of lipid tissues from peroxidation, since cold water fish such as salmonids have a high level of polyunsaturated fats in their membranes (Tacon, A., 1981; Craik, J. 1985; Torrissen, O.J., 1984; Burton, G.W., 1984). Astaxanthin is approximately 10 times stronger than other carotenoids, including β-carotene, in terms of antioxidant activity and 100 times greater than vitamin E (alpha-tocopherol). Astaxanthin also showed strong activity as an inhibitor of lipid peroxidation mediated by active forms of oxygen and has been proposed as the “super vitamin E” (Miki, 1991; Ranby and Rabek 1978). Mobilization of Carotenoids About 90% of the carotenoids found in the fish tissue are located in the flesh, but significant amounts can also be found in the skin and ovaries of maturing fish. Hydroxy-carotenoids in the skin are present mainly as esters (Hata and Hata, 1975; Kitahara, 1983). Although the majority of the carotenoid fraction of crustaceans is esterified astaxanthin, only the free form of astaxanthin is present in the flesh of salmonids (Kitahara, 1984; Foss et al., 1987). Dietary astaxanthin esters are hydrolyzed in the intestine by an nonspecific lipase rapidly and absorbed into the serum through the intestinal tract lumen as the free form, and subsequently deposited into the flesh (Torrissen 1979; Schiedt 1981). Carotenoids are most likely emulsified in the mixed micelle together with bile and other lipid components, prolipase and lipase (Leger, 1985). Ingested astaxanthin begins to appear in the serum 3 hours after feeding, and then rapidly increases. Transport of carotenoids across the intestinal wall results in the carotenoids being packaged into high density lipoproteins and very high density lipoproteins (HDL and VHDL), which enable the delivery of lipid soluble molecules such as carotenoids through the bloodstream (Kitahara, 1983; Schiedt et al. , 1985; Nakamura et al., 1985; Ando S., 1986a,b). Deposition of carotenoids in salmonid muscle is dependent on the specific binding of the pigment liposomes to muscle cells. Liposomes can vary in size and density depending on the life cycle of the fish, some transporting carotenoids from the intestine to the liver and another class transports from the liver to the tissues. In parallel, the pigmentation process changes throughout the life cycle. Smaller juvenile fish tend to deposit most of the accumulated carotenoid into the skin as an esterified form, whereas older fish deposit pigment into the flesh in the free form (Mori et al., 1989; Arai et al., 1987; Meyers, 1992; Greene and Selivonchick 1987; Nickell and Bromage, 1997). During maturation, new classes of lipoprotein predominates, vitellogenin and lipovitellin, which transports carotenoids from the muscles to the liver where they are processed and directed to the developing ovaries (Kitahara, 1983; Schiedt et al. , 1985; Nakamura et al.,1985; Ando S., et al., 1986; Ando S., 1986). During this process astaxanthin is transported to the ovaries from the flesh or the gastrointestinal tract by vitellogenin, the female-specific serum protein. Thus, significant amounts of carotenoids are deposited in the eggs of salmonids from mobilization of

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flesh astaxanthin. Transport of astaxanthin to sperm has also been observed in rainbow trout (Czeczuga, 1975; Ando 1986a,b). Isomerization or oxygenation of carotenoids has been shown not to occur at any step. The rates and detailed mechanisms of these absorption, transport, and deposition processes are not well understood but appears to be species specific and varies considerably with diets, genetics, environmental conditions, and even between individuals irrespective of their sex. (Mori et al., 1989; Arai et al., 1987; Greene and Selivonchick 1987; Nickell and Bromage, 1997; Foss et al., 1984). The liver appears to be the major metabolic and excretory organ for carotenoids. It is the site of vitamin A storage and transformation, synthesis of lipoproteins and transformation between esterified carotenoids and their free form. The carotenoids in the skin are localized in the xanthophores while the non-carotenoid pigments are localized in the erythrophores, astaxanthin is retained in the flesh in the non-esterified form (Goodwin, 1984). Carotenoids bind to a hydrophobic pocket on the surface of actomyosin with one of the ionone rings. The hydroxyl and keto groups contribute to the further stabilization of the complex by weak hydrogen bonding. Carotenoids are lost from the flesh and skin due to insufficient dietary intake, metabolic degradation and excretion. It is estimated that the retention rate of carotenoids in salmonids ranges from 3-18% (Storebakken 1992; Henmi, 1989). Source of Carotenoids The predominant source of carotenoids for farmed salmonids has been synthetic astaxanthin (Carophyll Pink) which has been used for pigmentation for the last 20 years, although it just received U.S. Food and Drug Administration approval in 1996 (21 CFR 73.35). Synthetic canthaxanthin (Carophyll Red) was approved by the U.S. FDA in 1969 for poultry (21 CFR 73.75) and in 1998 for use in salmonid feeds and has also been widely used as a pigment source. Natural sources of astaxanthin for commercially raised salmonids have utilized processed crustacean wastes from krill, shrimp, crab and crawfish, or the fermentative yeast Phaffia rhodozyma. Studies have demonstrated that there is practically no difference between krill astaxanthin ester and synthetic free astaxanthin in their absorption and deposition by Coho salmon. Nor is there a significant variation among optical isomers of astaxanthin in the deposition efficiency in the flesh of Coho salmon fed diets containing krill astaxanthin esters (Mori et al., 1989; Arai et al., 1987). However, crustacean waste products (oils and meals) generally contain less than 1000 ppm of astaxanthin, which necessitates exceedingly high inclusion rates (5-10%) into feeds for efficient pigmentation. Furthermore, crustacean sources contain high amounts of moisture, ash, fluoride and chitin that limits the percentage of these products that can be included in salmonid feeds. Another natural source of astaxanthin has been derived from Phaffia rhodozyma, however the concentrations are typically 0.5% (5,000 ppm) which requires adding a large amount to feeds for sufficient pigmentation leading to higher ash contents. Description of NatuRose With the growing movement in organic or “green” salmon and trout farming, a problem has arisen in the certification process. Nearly all of the obstacles have been overcome concerning

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natural feed ingredients, the use of antibiotics and medications, and the environmental issues, however a concentrated source of natural carotenoid pigments has not been available. Without dietary carotenoids the flesh of organically farmed fish has been pale-gray which significantly lowers market value as the consumer expects a natural pinkish-orange color. Synthetics such as Carophyll Pink and Carophyll Red are manufactured by a complex chemical process and are not certifiable as a natural pigment. In 1997, a natural astaxanthin product from Haematococcus algae meal (NatuRose ) was developed which provides a high concentration of carotenoid pigment for the feeds of farm raised fish. The carotenoid fraction of Haematococcus algae meal contains about 70% monoesters of astaxanthin, 10% diesters of astaxanthin, 5% free astaxanthin, and the remainder consisting of β-carotene, canthaxanthin, lutein and other carotenoids. Results of numerous trials with Haematococcus algae meal in salmon and trout demonstrate equivalent or higher pigmentation to synthetic astaxanthin by color card and Minolta colorimeter. HPLC analysis of the flesh generally reveals a 10-15% lower astaxanthin content but the incorporated canthaxanthin and lutein fill this gap and give a more natural and evenly distributed color. Due to the protective cell wall, astaxanthin from Haematococcus is not readily bioavailable when whole cells are added to feeds. The NatuRose production process includes a technique which “cracks” greater than 95% of the cells to enable maximum bioavailability. NatuRose is formulated to contain 1.5 % astaxanthin and is available worldwide for pigmentation of salmonids, sea bream, yellowtail as well as and crustaceans and broodstock.

Full approval in Japan has been received as a pigment in feeds and food and registration for salmonids in Canada has been approved (CFIA 990535). US Food and Drug Administration approval was achieved in August 2000 (21 CFR 73.185) and similar registrations are in progress in the European Community. Although there is no tolerance limitations due to safety, an amount of 25-100 parts per million of carotenoids in the final feed, or 1.67 to 6.67 kg of NatuRose per metric ton of feed, is sufficient to give the desired pigmentation in various salmonid species. A specific proposed use level of NatuRose Haematococcus algae meal in the finished feed is not suggested as numerous variables affect pigmentation. Some of the factors that affect the rate at which astaxanthin is deposited in muscle tissue include the salmonid species, growth stage, feed composition, salinity, and water temperature. Additionally, fish farmers have their own pigmentation regime that may change with season or growth stage. Therefore, it is recommended that NatuRose be used in accordance with Good Manufacturing Practices (GMP) and levels that will enhance the pinkish orange color of salmonid flesh to an acceptable level. Full details about NatuRose can be obtained from NatuRose Technical Bulletin #050. Pigmentation of Trout with NatuRose. One of the first published reports studied the effect of supplementing trout diets with Haematococcus algae meal during a 100 day feeding trial. Intact and homogenized (cracked) Haematococcus algae meal was compared to Carophyll Pink and a control without pigment. Mean total carotenoid and astaxanthin levels were measured in trout flesh and skin. The report demonstrated the necessity of cracked cells for bioavailability of the pigment, as intact algal

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spores were poorly utilized. The disruption of the homogenized spores was not complete (60%) and may have reduced their bioavailability in the “broken” ration. No significant differences were seen in trout growth, or mortalities. The carotenoid levels and ester distribution measured in both flesh and skin were consistent with wild and farmed trout. The study established that Haematococcus algae meal caused significant astaxanthin deposition in trout flesh and skin, as well as visual enhancement of the flesh coloration, and concludes that it is a safe and effective pigment source for rainbow trout (T.R. Sommer et al., 1991). Another published trial utilized non-homogenized Haematococcus cells in a 4 week feeding trial with rainbow trout to compare the pigmentation of muscle tissue with synthetic carotenoids. Rainbow trout were fed algae up to 6% of the diet without any major effect on growth or mortalities. The report reiterated the importance of comminution, or “cracking” of algal cells to increase bioavailability of the carotenoids. Although the algal cells were not cracked in this study it established that the astaxanthin and canthaxanthin, which represented 85% of total carotenoids, caused significant pigment deposition in the trout muscle. The final muscle carotenoid was 6.2 mg/kg, which is considered acceptable for market. The authors concluded that Haematococcus algae meal is a safe and effective source of pigment for rainbow trout (Choubert and Heinrich, 1993). Dr. Ron Hardy conducted one recent study at the Hagerman Fish Culture Experiment Station in 1997. Rainbow trout (average weight 200 g.) were selected at random in groups of ten fish, and placed into twenty fiberglass tanks, each supplied with untreated spring water. The fish were fed twice per day and six days per week at approximately 0.75% total body weight. This feeding regime was instituted for a period of 12 weeks. Five diets were produced by compression pelleting with a basal composition of 44% protein and 14% lipid, similar to commercial trout feeds. The control diet contained no pigment source, the other test diets consisted of 25 ppm or 50 ppm astaxanthin from Carophyll Pink, and 25 or 50 ppm astaxanthin from NatuRose. A randomized design was used to assign diets to replicate tanks of fish. From the initial population of fish, and at 28-day intervals, ten fish per treatment were sacrificed and three judges using a color card assessed the pigmentation of the fillets. Each reader scored the color of the dorsal and mid-section of each fillet and the scores were averaged. Tristimulus color measurements (L*a*b) were also made on each fillet with a Minolta Colorimeter. The carotenoid concentration in the fillet samples was quantified by spectrophotometric and HPLC analysis after extraction with acetone. Differences in fillet color among dietary treatment groups were evident at the end of the study. Fish fed the control feed without astaxanthin had white, unpigmented fillets and Roche color scores below 11, which was the lowest grade on the cards. Roche color scores of fillets from fish fed the diet containing 50 ppm astaxanthin from NatuRose averaged 15.0, similar to scores of fillets from fish fed synthetic astaxanthin (15.2). Carotenoid retention, feed conversion ratios, and weight gains were calculated with no significant differences among the dietary treatment groups. No mortalities or disease was observed during the course of the trial. A follow-up trial is in progress at Hagerman Experimental Station and will be reported in the March 1999 update of this bulletin.

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In another study by a major Chilean salmon company (here called, “BS”), pens with unpigmented kamloop trout with an initial average weights of 200 grams were fed dietary astaxanthin (ax) from either NatuRose or synthetic Carophyll Pink. The pen that received NatuRose contained 12 thousand fishes, the pen that received the synthetic astaxanthin had 17 thousand. In each of the adjacent pens, the usual Growers pigmentation strategy was used concerning dose in the different growth stages, and in the quantity and periodicity of feed supplied. Commercial extruded feeds with 47% protein and 30% fat, and a target level of 50 ppm of astaxanthin were used. Pellets of 3mm, 4, 5 and 7 mm were produced on commercial extruders as fish increased in size. Both feeds were manufactured on the same day using identical conditions. Samples were taken of the ingredients after final mixing, at the outlet of the extruder, and after drying. These samples were analyzed for astaxanthin content by HPLC, the results are described in Table 1.

Table 1- Astaxanthin (ppm) in feed during manufacture of NatuRose at BS. Run # Mixed ingredients At outlet of

extruder Final product, after

adding oil % loss

1 58.8 48.7 45.2 23.1 2 58.8 49.9 46.9 20.2

Similar studies made at other Chilean feed manufacturing plants with NatuRose had losses

ranging from 6-23% depending on the particular extruder conditions. Comparative losses in the preparation of feeds containing synthetic astaxanthin during the BS trial averaged 17.2%.

Differential feeding was initiated on Jan. 21,1998 and represents day 0. Six fish were sampled at random approximately every 30 days (Feb. 18, March 20, April 24, May 28, July 14 and August 25) and analyzed for pigmentation by color card, Minolta colorimeter, astaxanthin content by HPLC, as well as weight gain and other characteristics. The sampled fish were kept in black plastic bags under ice and color card and Minolta measurements were performed in the processing plant of BS within one hour after which fish were caught. Sub-samples were frozen and sent to Fundacion Chile for HPLC and spectrophotometric analysis. Fillets from the August 25 sampling were placed in a freezer for stability testing at the end of 90 days.

Unfortunately, most of the Chilean salmon growing centers suffered outbreaks of Rickettsia (SRS) during the winter of 1998. The center where the experiment was conducted required the use of flumequine-medicated feed between June 20 and July 8, which deteriorated the salmon performance in both pens. Evolution of pigmentation as measured by color card is shown in Figure 2.

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Figure 2

Pigmentation increased progressively in both pens, though a stalled period is noticeable at the mid-point during the Rickettsia outbreak. At harvest, both groups reached an excellent color, as can be seen in photographs (not included here). The independent panel of judges noted that the NatuRose fish had a more natural color compared to synthetically colored fish, better uniformity of pigmentation and less variability among individual fish. Color values for both groups were corraborated with Minolta measurements as well. The laboratory stated that the more natural color and uniformity may be due to the esterified form of astaxanthin contained in NatuRose which is similar to the one ingested by wild salmonids (krill), and consequently more physiologically compatible.

Astaxanthin accumulation in flesh from an average of 6 fish was measured by HPLC and shown in Figure 3 .

Color Card Scores of Kamloop Trout10

11.1

0

13.5

0

15.0

0

15.1

7 16.2

1

16.2

8

10

11.7

5 12.6

0

15.0

0

15.2

5 16.3

3

16.3

9

9

10

11

12

13

14

15

16

17

0 30 60 90 120 180 210Days

Col

or C

ard

Scor

e

NatuRose

Synthetic

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Figure 3 The group receiving NatuRose lagged slightly behind the synthetic-fed group as the Rickettsia

outbreak was apparently more severe in this group. However, a smaller coefficient of variability indicated a higher uniformity between the NatuRose samples at the endpoint.

Table 2 shows the development of biomass as determined by the inventory and average body weight of fishes, routinely performed monthly by the Grower.

Table 2- Increment of biomass.

Synthetic astaxanthin NatuRose # fish Ave. body

wt. (kg) Biomass

Ton Biomass

incr. (ton) # fish Ave. body

wt. (kg) Biomass

Ton Biomass

incr. (ton) 0 Days 16,771 0.15 2.52 11,878 0.14 1.72

30 Days 16,557 0.20 3.31 0.80 11,702 0.20 2.31 0.58 60 Days 16,292 0.35 5.70 2.39 11,557 0.38 4.40 2.10 90 days 15,852 0.68 10.80 5.09 11,079 0.77 8.49 4.08

120 Days 15,411 1.33 20.47 9.67 10,601 1.51 16.02 7.53 150 Days 12,954 1.70 22.05 1.58 8,984 1.90 17.07 1.05 180 Days 12,448 2.00 24.90 2.85 8,769 2.17 19.03 1.96 210 Days 11,962 2.40 28.71 3.81 8,559 2.61 22.34 3.31

*Treatment with flumequine at 150 days for 13 days.

Astaxanthin by HPLC: Kamloop Trout0.

1

3.40

8.21 9.

47

15.7

2

15.1

4

0.1

5.96

9.27

13.4

5 16.3

7

15.7

7

1.85 2.16

02468

10121416

0 30 60 90 120 180 210Days

Ast

axan

thin

(pp

m)

NatuRose

Synthetic

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The deteriorated performance of fish during the flumequine treatment is apparent. At 60, 90 and 210 days of feeding, tissue slices of kidney, liver, brain, gonads and muscle were preserved under appropriate conditions and sent to Fish Pathology Associates Laboratory (Guelph, Canada) for histopathological examination. The report did not reveal any abnormalities.

On the basis of the figures from the previous table, the following calculations of the efficiency of retention of both pigments can be made in Table 3. NatuRose had 92% of the retention efficiency compared to synthetic astaxanthin.

Table 3- Retention efficiency Synthetic ax NatuRose ax Day 30 Day 210 Day 30 Day 210

Biomass, (ton) 3.31 28.71 2.31 22.34 Astaxanthin In muscle, (ppm) 2.16 15.77 1.85 15.14

Initial biomass, (g.) 7.15 452.74 4.26 338.21 Incr. In biomass, (g.) 445.59 333.95

Supply Feed,(ton) 37.729 30.663 Astaxanthin in feed, (g.) 3018 2453

Retention efficiency, % 14.76 13.61

In 1998 a study was conducted at the University of Plymouth (Plymouth, UK Fish Nutrition Unit) to study trout pigmentation with NatuRose compared with that of synthetic astaxanthin. The trial allowed evaluation of three dietary treatments and a control, each in quadruplicate. A total of 300 rainbow trout with a mean weight of 100 grams were randomly assigned to 12 tanks, which were identical with respect to temperature, water quality parameters and volumes. The experimental diets were formulated to simulate commercial feeds for salmonids and were supplemented with the different sources of astaxanthin as the major pigmenting carotenoid. Diet 1 (control) contained 50 ppm synthetic astaxanthin, diet 2 contained 50 ppm astaxanthin from NatuRose, and diet 3 contained 70 ppm astaxanthin from NatuRose. During the acclimation period (weeks 0-4), the fish were fed to satiation using a non-pigmented expanded feed (Standard Expanded, Trouw Aquaculture, Wincham, Cheshire, UK). Twenty-four fish were then sampled at week 4, the time zero acclimation period, and sub-sampling of 36 fish each was done at weeks, 8, 12 and 16. During the experimental period of weeks 4-16, the feed ration was calculated based on chart levels to ensure good growth but minimal feed waste. Analyses were performed on fish fillets by Roche color card and HPLC of extracts. Other parameters such as weight, feed conversion rate (FCR), and specific growth rate (SGR) were calculated.

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Table 4 Mean weight of fish (g), measure biweekly to assess growth. Week 50 ppm synthetic 50 ppm NatuRose 70 ppm NatuRose

4 99.31 99.43 102.03 6 139.55 139.46 142.05 8 178.05 178.81 182.12

10 212.65 208.76 212.65 12 242.32 238.61 243.68 14 280.26 274.72 283.38 16 326.87 315.06 322.00

SGR 1.42 1.37 1.36 FCR 1.02 0.97 0.97

The results of the flesh deposition of the rainbow trout showed that there was a significant visual pink coloration after week 8. The pigmentation trend continued through the end of the trial for each diet. At week 16 representing the final sampling, the Color Score Units (CSU) were 27.92, 28.59 and 29.43 for the three experimental diets respectively. Although subjective, the diet of 50 ppm NatuRose elicited higher color scores than the synthetic astaxanthin. The 70 ppm NatuRose diet had significantly higher color scores than either the 50 ppm synthetic or 50 ppm NatuRose diets. Table 5: Roche color score for unskinned fillets, using SalmoFan, +/- standard deviation

Week 50 ppm synthetic 50 ppm NatuRose 70 ppm NatuRose 4 N/A N/A N/A 8 23.42 +/- 0.22 22.67 +/- 0.09 24.72+/- 0.35

12 27.19 +/- 0.17 27.45 +/- 0.25 27.92 +/- 0.92 16 27.92 +/- 0.19 28.59 +/- 0.12 29.43 +/- 0.13

The flesh astaxanthin extracted from each of the samples paralleled the visual scores. At week 4 (0 time), there was no astaxanthin detected in the flesh sample. The week 8 sampling showed significant deposition in the flesh of each group and the trend continued through the end of the trial. At week 16 representing the final sampling, the flesh astaxanthin concentrations were 3.26 +/- 0.57, 3.61 +/- 1.28 and 3.81 +/- 0.30 for each of the diets respectively. Notably, the diet 3 group with 70 ppm astaxanthin from NatuRose attained the same average flesh pigmentation at only 12 weeks (3.23 ppm) as that of the diet 1 group (50 ppm synthetic astaxanthin) after 16 weeks.

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Table 6: Flesh astaxanthin levels with test diets (mg/kg) Week Diet 1: 50 ppm synthetic Diet 2: 50 ppm NatuRose Diet 3: 70 ppm NatuRose

4 Not detected Not detected Not detected 8 2.33 +/- 0.38 1.92 +/- 0.36 2.54 +/- 0.45 12 2.83 +/- 0.29 2.91 +/- 0.25 3.23 +/- 0.92 16 3.26 +/- 0.57 3.61 +/- 1.28 3.81 +/- 0.30

Figure 4 There were no apparent health problems with any of the groups at the conclusion of the study, nor evidence for internal lesions or abnormalities. The investigators concluded that NatuRose was an effective substitute for synthetic astaxanthin and recommended the natural algal source as a source of pigmentation for trout and salmon.

Pigmentation of Salmon with NatuRose A study conducted by a major aquaculture research center in Norway compared synthetic astaxanthin and Haematococcus algae meal as carotenoid sources in extruded feeds for penned Atlantic salmon. The study measured the pigmentation of the fillet and the absorption rates after a 13-month period. At the start of the trial, 500 salmon of average weight of 400 g. were marked with Floy tags and transferred to the two experimental cages. The sea cages were 2.75 x 5.5 x 6 m deep, and were conveyed to larger cages at the mid-point of the trial. The feeds were made in several batches to minimize storage losses of the astaxanthin, and formulated to give proximate chemical compositions, only differing in pigment sources. Each month a total of 10 fish were sampled from each cage for determination of astaxanthin concentration in dorsal muscle close to the head of the fillet. The astaxanthin concentration in the fillet was analyzed by standard HPLC methods after acetone extraction, flesh

Flesh astaxanthin levels with tes t die ts

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

4 8 12 16We e k

Fle

sh a

stax

anth

in

(mg/

kg)

50 ppm s ynthetic:Diet 150 ppm NatuRose:Diet 270 ppm NatuRose:Diet 3

15

color in L* a* b* values were analyzed by Minolta colorimeter. The “L” value represents the lightness/darkness factor, “a” value represents the red chromaticity, and the “b” value represents the yellow chromaticity. The color determination was based on an average of 3 samples from the dorsal muscle taken from the anterior, posterior and mid-section of the fish. Color scores of the two groups were similar at the conclusion of the trial. After 11 months, the diets resulted in a corresponding mean pigment level of 3.3 mg/kg and 3.0 mg/kg for the synthetic astaxanthin and Haematococcus algae meal feeds, respectively. Although the Haematococcus algae cells used in this study were not fully cracked and bioavailable, the study concluded that Haematococcus algae meal was an effective alternative pigment source for Atlantic salmon. A trial conducted by a multinational feed manufacturer studied the pigmentation qualities of Haematococcus algae meal at two dietary inclusion levels. Three tanks were utilized for the trial, each measuring 2.4 meters with depths of 0.6 meters. Tanks were stocked with 31 salmon of mean initial weight of 650 grams. The quantity of feed was uplifted every week to allow for weight increase, the trial continued for five months before final analysis. The average astaxanthin in the flesh of salmon fed diets containing Haematococcus algae meal was 3.42 ppm as measured by HPLC and it was concluded that it would be capable of pigmenting salmon to an acceptable color when used in diets at 75 ppm. No toxic effects or neoplasia were reported and mortalities during the study ranged from 3-6%. A major Canadian feed manufacturer tested the pigmentation capacity of Carophyll Red, Carophyll Pink, and Haematococcus algae meal in salmon. Coho smolts of approximately 45 grams were placed into two raceways for the duration of the trial. The pigments were added in a pelleted salmon feed at 50 ppm and the fish were fed to satiation seven days per week for five months. Ten fish were sampled bimonthly for weight and flesh color from each section. Analysis indicated that each of the three pigment sources gave similar visual pigmentation deposition. There were no significant differences in color intensity at the end of the trial. The rate of color development was also similar for all three groups. Flesh color was significantly correlated with the fish weight and time the fish had been fed the diets containing the specific pigments. Haematococcus algae meal was shown to be capable of pigmenting salmon to an acceptable color for market. A 1997 study conducted by an international Chilean salmon company (“MH”) compared the pigmentation potential of synthetic astaxanthin with NatuRose. Four pens with duplicates of Atlantic salmon were utilized for the feeding experiments during the 4-month trial. A total of 300 fish in each pen had of average initial weights of 2.6 kgs. Target levels of 75 ppm astaxanthin from either Carophyll Pink or NatuRose were incorporated into the diets, new feed batches were made every 40 days for both groups. An initial sample of 30 fish was taken, and thereafter samples were taken at 35, 106 and 113 days for analysis. NatuRose efficiency was 83% that of synthetic astaxanthin when measured in mg of astaxanthin/kg fish by HPLC analysis. However, the color card scores revealed that there were no significant differences in pigmentation, which might be attributed to the incorporation of other carotenoids from NatuRose such as canthaxanthin (Figure 5 & Table 7). The colors of the fillets and steaks from the NatuRose-fed fish were more consistent, better distributed and had a more “natural orange appearance” as

16

judged by an independent panel. There were no significant differences in specific growth rate, feed conversion rate, or mortalities between the NatuRose and synthetic astaxanthin groups. Figure 5

Table 7-Pigmentation of fillets in Roche Color Units

Synthetic asta NatuRose

Time Score S.D. Score S.D. 30 days 13.98 2.09 13.89 1.26 60 days 14.94 2.61 14.63 3.10 90 days 14.80 2.22 14.84 1.30 120 days 14.23 1.54 14.93 2.24

The storage and stability tests of NatuRose revealed a 97.2% average recovery during the premix process and 94% stability after feed extrusion. A shelf-life stability of the feeds showed that over 90% of the original pigment was retained after 90 days at ambient conditions (Figure 6).

Color Card Scores of Atlantic Salmon

13.4

0

13.8

9 14.6

3

14.8

4

14.9

3

13.4

0 13.9

8 14.9

4

14.8

0

14.2

3

9.00

10.00

11.00

12.00

13.00

14.00

15.00

0 30 60 90 120Days

Col

or C

ard

Scor

e

NatuRose

Synthetic

17

Figure 6-NatuRose Stability in Feeds Under Warehouse Conditions

Further feed stability tests demonstrated over 90% stability of pigments after 15 days up to 35 C (Figure 7). Figure 7-NatuRose Stability in Feeds at Various Temperatures

Stability of NatuRose in Chilean salmon feedWarehouse Conditions

50

60

70

80

90

100

0 10 20 30 40 50 60 70 80 90 100 110 120Days

% S

tabi

lity

NatuRose Stability in Feeds

50

60

70

80

90

100

110

0 2 4 6 8 10 12 14 16Days

% S

tabi

lity

5°

20°

35°

18

In a second study by a major Chilean salmon company (here called, “UM”), two adjoining

net pens were used which contained approximately 18,000 Atlantic salmon each. The fish were of similar origin and prior management with an average initial weight of 1.7 kg. Fish had received an 80 ppm feed containing synthetic astaxanthin (Carophyll Pink) since August 1997, having reached the pigmentation levels indicated in Table 8. Table 8- Pigmentation of fish at the initiation of trial

Roche color card Ax (ppm) HPLC Filets 13.77 (0.38) 3.56 (14.13) Steaks 3.35 (4.29)

*Values between parenthesis indicate coefficient of variability.

The extruded feeds for the trial were normal commercial formulations that were manufactured with 43% protein and 30% fat on 2 different dates. Fish received identical feeds, only differing in the pigment source; one contained NatuRose and the other contained the synthetic astaxanthin, Carophyll Pink. In both pens, the pigmentation strategy normally used by the Grower was exercised concerning dose in the different growth stages, and in the quantity and periodicity of feed supplied.

On March 4, 1998, 5 mm pellets with a target level of 70 ppm of astaxanthin were prepared and on April 21st, 7 mm pellets with a target of 80 ppm astaxanthin were produced. Trial feeds were manufactured in sequence on the same dates with the same ingredients other than the astaxanthin source. Differential feeding was initiated on March 11th, and was considered Day 0 for this trial.

During feed fabrication by extrusion, samples were taken and the losses in astaxanthin content were calculated. Since the losses in the first batches were higher than expected, the amount added in the second batch was uplifted accordingly as shown in Table 9. Table 9- Contents of astaxanthin (ppm), during fabrication.

Pigment Target ax ppm Content in ingreds. Final ax % loss March 4 NatuRose

Synthetic 70 70

80 80

52.5 65

34.3 18.7

April 21 NatuRose Synthetic

80 80

110 100

90.8 82.9

17.4 17.1

After the March 4th run, the specific conditions were investigated and altered to increase stability of astaxanthin from NatuRose. Losses in the April manufacturing were similar for both pigments and considerably less than in the March run for NatuRose. The March 4th feeds were supplied until April 25th, after which date the fish received the second batch of feeds produced on

19

April 21st. The initial sampling were followed by samplings conducted on April 13th, May 8th and July 10th after which fish were harvested.

During samplings, six fishes from each pen were grabbed at random, in which the following observations were made: pigmentation in filets and steaks measured with the Roche score card and Minolta colorimeter, astaxanthin content by HPLC and total carotenoids measured by spectrophotometry. Roche and Minolta measurements were performed in the Processing Plant, within one hour after which fishes were caught; samples were kept in black plastic bags, under ice.

Again, most of the Chilean salmon farms suffered outbreaks of Rickettsia (SRS) during the winter growing period of 1998. The center where this study was conducted also suffered an outbreak that required the use of medicated feed between April 24 and May 13 that deteriorated the salmon performance in both pens. Evolution of pigmentation during the experiment is shown in the Table 10.

Table 10- Roche color card and carotenoid content. Feb.19 Day 0 Color card: 13.77 (0.38) HPLC 3.56 (14.13) Color Card HPLC Total carotenoids NatuRose Synthetic NatuRose Synthetic NatuRose Synthetic

April 13 15.28 (1.2) 15.20 (2.7) 5.50 (32.0) 6.62 (88.2) May 8 14.50 (2.8) 14.08 (2.4) 4.15 (17.8) 4.65 (22.6) 5.40 (40.5) 5.60 (17.4) July 10 14.80 (3.0) 15.20 (3.8) 4.70 (8.8) 5.51 (10.3) 5.47 (39.4) 6.30 (107)

*Values between parentheses indicate coefficient of variability

It is worth noting that, in general, values obtained with NatuRose had a smaller coefficient of variability. This indicates better uniformity in the color of the salmon possibly attributable to the more “natural” form of astaxanthin contained in NatuRose, similar to the one ingested by wild salmonids, and consequently more physiologically compatible.

As can be seen, color and carotenoid content of fish increased during the period in which fishes were not affected by disease, with respect to levels observed at the initiation. This trend was broken during the infection phase and reverted in the last period. Similar deterioration in body weight gain can be observed in the Table 11, which shows the evolution of the biomass as determined by the inventory and average body weight of fishes performed monthly by the growers.

Table 11- Biomass increment.

Synthetic astaxanthin NatuRose astaxanthin # fish Avg. body

wt. (kg) Biomass Ton.

Biomass incr. (ton)

# fish Avg. body wt. (kg)

Biomass Ton.

Biomass incr. (ton)

Jan. 22 17,882 1.03 18.33 17,842 1.19 21.30 March 20 17,667 1.69 29.80 11.48 17,625 1.83 32.27 10.97

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April 24 17,432 1.89 33.00 3.19 17,412 2.23 38.81 6.4 May 22 16,922 2.14 36.15 3.15 16,836 2.42 40.79 1.98 June 23 16,600 2.72 45.14 8.99 16,452 3.01 49.44 8.64

Treatment with antibiotics: April 24-May 13

On the basis of the figures from the previous table, the following calculations of the

efficiency of retention of both pigments can be made in Table 12.

Table 12- Retention efficiency. Synthetic ax NatuRose March 11 April 13 March 11 April 13 Fish Biomass, ton. 27.99 32.18 30.54 34.64 Astaxanthin In muscle, (ppm) 3.56 6.62 3.56 5.50 Asta in biomass, (g.) 99.65 213.01 108.72 190.54 Incr. in asta biomass, (g.) 113.36 81.82 Supply Feed, (ton) 8.175 30.663 Astaxanthin in feed, ( g) 542.00 368.81 Retention efficiency, % 20.92 22.19

Calculations of the previous tables are based on the “ideal” period, with no diseases, no

stress caused by fish harvesting or other manipulations, with rapid body gain and efficient feed conversion. Henmi (1989) and Hardy (1992), indicate retention figures near 18% as typical.

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Choubert G. and O. Heinrich. 1993. Carotenoid pigments of the green alga Haematococcus pluvialis: assay on rainbow trout, Oncorhynchus mykiss, pigmentation in comparison with synthetic astaxanthin and canthaxanthin. Aquaculture 112:217-226. Christiansen R., O. Lie and O.J. Torrissen. 1994. Effect of astaxanthin and vitamin A on growth and survival during first feeding of Atlantic salmon, Salmo salar. Aquacult. Fish. Management. 25:903-914. Christiansen R., O. Lie, and O.J. Torrissen. 1995. Growth and survival of Atlantic salmon, Salmo salar L., fed different dietary levels of astaxanthin. First-feeding fry. Aquaculture Nutrition. 1:189-198. Christiansen R., J. Glette, O. Lie, O.J. Torrissen and R. Waagbo. 1995b. Antioxidant status and immunity in Atlantic salmon, Salmo salar L., fed semi-purified diets with and without astaxanthin supplementation. J. Fish Diseases 18:317-328. Christiansen R., O. Lie, and O.J. Torrissen. 1994. Effect of astaxanthin and vitamin A on growth and survival during first feeding of Atlantic salmon, Salmo salar L. Aquaculture and Fisheries Management 25:903-914. Christiansen R., and O.J. Torrissen. 1996. Growth and survival of Atlantic salmon, Salmo salar L. fed different dietary levels of astaxanthin. Juveniles. Aquaculture Nutrition. 2:55-62. Craik J.C. 1985. Egg quality and egg pigment content in salmonid fishes. Aquaculture 47:61-88.. Czeczuga B. 1975. Carotenoids in fish. IV. Salmonidae and Thumallidae from Polish waters. Hydrobiologia 46:223-239. D’Abramo Louis R. 1997. Crustacean Nutrition. Advances in World Aquaculture, Volume 6. World Aquaculture Society. Louisiana State University. Baton Rouge, LA. Foss P., B. Renstrom, S. Liaaen-Jensen, E. Austreng, and K. Streiff. 1984. Carotenoids in diets of rainbow trout for salmonids. I. Pigmentation of rainbow trout with the individual optical isomers of astaxanthin in comparison with canthaxanthin. Aquaculture 41: 213-226. Foss P., Renstrom B., and S. Liaaen-Jensen. 1987a. Natural Occurrence of enatiomeric and meso astaxanthin in crustaceans including zooplankton. Comp. Biochem. Physiol. 86B:313-314. Foss P., B. Renstrom, and S. Liaaen-Jensen. 1987b. Carotenoids in diets for salmonids. V. Pigmentation of rainbow trout and sea trout with astaxanthin and astaxanthin dipalmitate in comparison with canthaxanthin. Aquaculture 65:293-305. Goodwin. T.W. and M. Jamikorn. 1954. Studies in carotenogenesis. II. Carotenoid synthesis in the alga Haematococcus pluvialis. Biochem. J. 57: 376-381. Goodwin T.W. 1984. In: The Biochemistry of the Carotenoids. Volume II. Tunicates and Fish. Chapter 8. pp 122-153. Chapman and Hall, London. Greene D. and D. Selivonchick. 1987. Lipids metabolism in fish. Prog. Lipid Res. 26:53-85.

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Grung M., F.M.L. D’Souza, M. Borowitzka, and S. Liaaen-Jensen. 1992. Algal carotenoids 51. Secondary carotenoids 2. Haematococcus pluvialis aplanospores as a source of (3S, 3'S)-astaxanthin esters. J. Appl. Phycol. 4: 165-171. Grung M., Y.S. Svendsen, and S. Liaaen-Jensen. 1993. The carotenoids of eggs of wild and farmed cod. Comp. Biochem. Physiol. 106B:237-242. Hansen L.P. and P. Pethon. 1985. The food of Atlantic salmon, Salmo salar, caught by long-line in Northern Norwegian waters. J. Fish Bio. 26:553-562. Hardy, R.W. and R. J. Roberts. 1998. Atlantic Salmon-Species Profile. International Aqua Feed. Number 2, 21-23. Hata M. and M. Hata. 1975. Carotenoid pigments in rainbow trout Salmo gairneri irideus. Tohuku J. Agric. Res. 26(1):35-40. Hempel E. December 1997. Seafood International. pp 29-31. Henmi H., M. Hata and M. Hata. 1989. Astaxanthin and/or canthaxanthin-actomyosin complex in salmon muscle. Nippon Suisan Gakkasishi. 55:1583-1589. Kitahara T. 1983. Behavior of carotenoids in the chum salmon (Oncorhynchus keta) during anadromous migration. Comp. Biochem. Physiol. 76B:97-101. Kitahara T. 1984. Carotenoids in the Pacific salmon during the marine period. Comp. Biochem. Physiol. 78B:859-862. Kvalheim B. and G. Knutsen. 1985. Pigmentation of salmon with astaxanthin from microalgae. Norsk. Fiskeoppdrett 10(3): 4 (abstract). Leger C. 1985. Digestion, absorption and transport of lipids. In: C.B. Cowey, A.M. mackie and J.C. Bell (editors). Nutrition and Feeding of Fish. Academic press, New York, N.Y. pp 299-331. Lura H. and H. Saegrov. 1991. A method of separating offspring from farmed and wild Atlantic salmon (Salmo salar) based on different ratios of optical isomers of astaxanthin. Can. J. Fish. Aquat. Sci. 48:429-433. Maoka T., M. Katsuyama, N. Kaneko, and T. Matsuno. 1985. Stereochemical investigation of carotenoids in the antarctic krill Euphausia superba. Bull. Jap. Soc. Sci. Fish. 51:1671-1673. Meyers S.P. and C. Huei-Mei. 1992. Astaxanthin and its role in fish culture. Proc. of Freshwater Fish Culture. No. 3, 153-165. Miki W. 1991. Biological functions and activities of animal carotenoids. Pure and Applied Chem. 63:141-146. Mori T., K. Makabe, K. Yamaguichi, S. Konosu, and S. Arai. 1989. Comparison between krill astaxanthin diester and synthesized free astaxanthin supplemented to diets in their absorption and deposition by juvenile salmon (Oncorhynchus kisutch). Comp. Biochem. Physiol. 93B:255-258.

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Nakamura K., M. Hata, and M. Hata. 1985. A study on astaxanthin in Salmon Oncorhynchus keta serum. Bull Jpn. Soc. Sci. Fish. 51(6):979-983. Nickell D. and N. Bromage. 1997. Problems of Pigmentation. Fish farmer. Jan/Feb:48-51. No H.K. and T. Storebakken. 1991. Color stability or rainbow trout fillets during frozen storage. J. Food Sci. 56(4):969-972 and 984. Petit H. 1993. Effet de la supplementation en carotenoides de la nourriture sur le developpement lavaire et postlarvaire de Penaeus japonicus (Bate, 1988). Crustacace Decapode, Peneida. These de Doctorat de I’Universite de Montpelier, France. Ranby B. and J.F. Rabek, editors. 1978. Singlet Oxygen. Wiley, Chichester, England Renstrom B., G. Borch, O. Skulberg, and S. Liaaen-Jensen. 1981. Optical purity of (3S,3'S)-astaxanthin from Haematococcus pluvialis. Phytochem. 20(11): 2561-2564. Renstrom B. and S. Liaaen-Jensen. 1981. Fatty acid composition of some esterified carotenols. Comp. Biochem. Physiol. B., Comp. Biochem. 69: 625-627. Ronneberg H., B. Renstrom, K. Aereskjold, and S. Liaaen-Jensen. 1980. Natural occurrence of enantiomeric and meso-astaxanthin 1. Lobster eggs (Homarus gammarus) Helv. Chim. ACTA 63:711-715. Schiedt K., F.J. Leunberger, and M. Vecchi.1981. 44. Natural occurrence of enantiomeric and meso-astaxanthin. 5. Ex wild salmon (Salmo salar and Oncorhynchus). Helv. Chim. ACTA 64:449-457. Schiedt K., F. J. Leunberger, M.Vecchi, and E. Glinz. 1985. Absorption, retention and metabolic transformations of carotenoids in rainbow trout, salmon and chicken. Pure Appl. Chem. 57(5):685-692. Schiedt K., M. Vecchi, M., and E. Glinz. 1986. Astaxanthin and its metabolites in wild rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 83B:9-12. Sommer T.R., W.T. Potts, and N.M. Morrissy. 1991. Utilization of microalgal astaxanthin by rainbow trout (Oncorhynchus mykiss). Aquacult. 94: 79-88. Steven D.M. 1948. Studies on animal carotenoids. I. Carotenoids of the brown trout (Salmo trutta Linn.) J. Exp. Biol. 25:369. Storebakken T., P. Foss, T. Asgaard, and S. Liaaen-Jensen. August 1984. Carotenoids in food chain studies-Optical isomer composition of astaxanthin in crustaceans and fish from two sub-alpine lakes. In 7th International Symposium on Carotenoids. 27-31. Abstract P31. Storebakken T., P. Foss, E. Austreng, and S. Liaaen-Jensen. 1985. Carotenoids in diets for salmonids. II. Epimerization studies with astaxanthin in Atlantic salmon. Aquaculture 44:259-269. Storebakken T. and H.K. No. 1992. Pigmentation of rainbow trout. Aquaculture. 100:209-229. Tacon A.G. 1981. Speculative review of possible carotenoid function in fish. Prog. Fish. Cult. 43(4):205-208.

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Takashi M., M. Katsuyama, N. Kaneko, and T. Matsuno. 1985. Stereo investigation of carotenoids in the antarctic krill Euphausia superba. Bull. Japn. Soc. Sci. Fish. 51(10):1671-1673. Torrissen O.J. and O.R. Braekkan. 1979 The utilization of astaxanthin forms by rainbow trout (Salmo gairdneri) Proceedings of the World Symposium on Finfish Nutrition and Fishfeed Technology. Hamburg 20-23 June 1978. Vol II Heenemann. Berlin, pp 377-382. Torrissen O.J. 1984. Pigmentation of salmonids-effect of carotenoids in eggs and start-feeding diets on survival and growth rate. Aquaculture 43:185-193. Torrissen O.J., R. W. Hardy, and K.D. Shearer. 1989. Pigmentation of salmonids-carotenoid deposition and metabolism. Aquatic Sciences 1(2):209-225. Torrissen O.J. 1989. Biological activities of carotenoids in fish. Proc. Third Int. Symp. On Feeding and Nutr. In Fish. (August/September) 387-399. Torrissen O.J. 1990. Biological activities of carotenoids in fishes. The current status of fish nutrients in aquaculture. pp. 387-399. In, Proceedings of the third international symposium on feeding and nutrition in fish. Eds. M. Takeda and T. Watanabe. Tokyo University of Fisheries, Tokyo, Japan. Turujman S., W. Wamer, R.R. Wei, and R. H. Albert. 1997. Rapid liquid chromatographic method to distinguish wild salmon from aquacultured salmon fed synthetic astaxanthin. Journal of AOAC International. 80:622-632. Vecchi M., V. Muduna, and E. Glinz. 1987. HPLC separation and determination of astacene, semiastacene, and other keto-carotenoids. J. High Res. Chrom. And Chrom. Commun. 10:348-351. Wan P.J., F. Zhang, and R.J. Hron. 1995. Extraction, composition, and stability of pigments from crawfish shell waste. In, Nutrition and Utilization Technology in Aquaculture (Eds: Lim C.E. and D.J. Sessa) Chapter 19, pp. 255-268. AOCS Press. Yuan, J., Gong, X., and F. Chen. 1996. Separation and identification of astaxanthin esters and chlorophylls in Haematococcus lacustris by HPLC. Biotechnology Techniques. 10(9):655-660. NatuRose Technical Bulletin #055 Revision Date: March 28, 2001 Contact: Dr. R. Todd Lorenz Cyanotech Corporation Phone: 808-326-1353 FAX: 808-329-3597 Email: [email protected] www.cyanotech.com R. Todd Lorenz 1999