Effect of a Dunaliella extract on growth performance, health condition, immune response and disease resistance in black tiger shrimp (Penaeus monodon)

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1 Aquaculture 248 (2005) Effect of a Dunaliella extract on growth performance, health condition, immune response and disease resistance in black tiger shrimp (Penaeus monodon) Kidchakan Supamattaya a, *, Suphada Kiriratnikom a, Mali Boonyaratpalin b, Lesley Borowitzka c a Aquatic Animal Health Research Center, Department of Aquatic Science, Faculty of Natural Resources, Prince of Songkla University, HatYai 90110, Thailand b Department of Fisheries, Kaset Klang, Bangkhen, Chatujak, Bangkok 10900, Thailand c Cognis Australia Pty Ltd., Lot 6, Weston Road, Pickering Brook, WA 6076, Australia Abstract The effects of commercially available Dunaliella extract (Algro NaturalR) on growth, immune functions and disease resistance were determined in black tiger shrimp (Penaeus monodon). Trial I was performed in small shrimp (1 2 g body weight). Shrimp fed mg of the Dunaliella extract/kg diet for 8 weeks showed higher weight gain and survival compared to the control. There was no significant difference in total hemocyte count and phenoloxidase activity among treatment. Shrimp fed 300 mg of the extract/kg diet exhibited higher resistance to WSSV infection than other groups and also became more tolerable to the stress (low dissolved oxygen condition). Color intensity of boiled shrimp was highest in groups fed mg of the Dunaliella extract/kg diet. Trial II was performed in juvenile shrimp (12 15 g body weight) fed the same diet in Trial I for 6 weeks. Total hemocyte counts were negatively correlated to the increase in Dunaliella extract in test diet (r = 0.97). Phenoloxidase activity and clearance of pathogenic vibrio from the hemolymph were not significantly different. Total carotenoid and astaxanthin levels were highest in shrimp fed mg of the Dunaliella extract. In conclusion, the Dunaliella extract showed beneficial effects as a shrimp feed supplement. D 2005 Elsevier B.V. All rights reserved. Keywords: Dunaliella extract; Beta-carotene; Black tiger shrimp; Penaeus monodon 1. Introduction * Corresponding author. Tel.: ; fax: address: kidchakan.s@psu.ac.th (K. Supamattaya). In shrimp culture industries, color is one of the major factors, which determines the market price of black tiger shrimp (Penaeus monodon) in the international market. Supplementation of carotenoid pigments into diets has been demonstrated to yield /$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi: /j.aquaculture

2 208 K. Supamattaya et al. / Aquaculture 248 (2005) higher pigments in shrimp. Moreover, carotenoid pigments have positive effect on the immunological and stress response (Latscha, 1991; Estermann, 1994). However, synthetic astaxanthin is still expensive for use in aquaculture. Several attempts have been made to find alternative sources of astaxanthin and other carotenoids such as yeast, Phaffia sp. (Sanderson and Jolly, 1994), and many species of algae (Liao et al., 1993; Sommer et al., 1991; Boonyaratpalin et al., 2000). In black tiger shrimp, the color is provided mainly by astaxanthin as the free or esterified form (Tanaka et al., 1976). Black tiger shrimp are unable to biosynthesize carotenoid de novo but can convert h- carotene and synthetic canthaxanthin in feed to deposit in the body as astaxanthin (Boonyaratpalin et al., 2001). Therefore, price and efficiency are used for determining the sources of pigment use in shrimp feed. Moreover, carotenoids are known to enhance immune function and disease resistance in higher animal (Bendich and Shapiro, 1986; Jyounchi et al., 1993; Thompson et al., 1995)., but little information is available for aquatic animal especially in shrimp. In this study, different levels of carotenoid extract from the green alga, Dunaliella was used in shrimp feed and the effects of this material, at different levels on growth performance, health condition, immune function and disease resistance in black tiger shrimp were evaluated. 2. Materials and methods 2.1. Test animal Trial 1: small shrimp Ten thousand PL-15 P. monodon were grown on commercial shrimp feed in two 5-ton tanks for one and a half months until weight attained g, then graded to the same size and stocked in cm aquaria with a density of 15 shrimp per aquarium Trial 2: juvenile shrimp One thousand healthy shrimp with a weight range of g were purchased from a shrimp farm where no severe disease outbreak has been recorded. One hundred and twenty shrimp were stocked in each 10- ton concrete tank equipped with aeration. Each concrete tank was covered with black plastic blanket to control the temperature in the pond ( C). Five tanks were used in this study. Shrimp were acclimated and were fed with commercial shrimp feed five times daily until satiation for 1 week before start feeding with test diet. Sediment from the pond bottom was clean. Water exchange was done daily with 10% changing rate, and water salinity was kept constant at 1.5% throughout the experiment Test diet Dunaliella extract Dunaliella extract was provided by Cognis Pty Australia. Dunaliella salina was cultured in saline water. After harvesting, the natural h-carotene was concentrated by a physical process without petrochemical solvents. The natural h-carotene concentrate was then spray-dried and concentration of total carotenoids was controlled as 2% w/w Test diet and feed processing Five isonitrogenous test diets were formulated to contain 40% protein and 8% fat. All diet have the same composition except the level of the Dunaliella extract (Algro NaturalR, provided by Cognis Pty Australia) added to achieve 0, 125, 200 and 300 as mg h-carotene/kg diet 1, 2, 3 and 4, respectively. NaCl 0.9% was added to diet 5 as another control group of Dunaliella extract (Algro NaturalR powder contained 0.9% NaCl) (Table 1). Composition of the test diets for trial 1 and 2 are the same except the pellet size. Ingredients were ground, weighed according to each formulation in Table 1, and mixed in bowl mixer for 5 min; then oil was added and mixed well again. After that, 350 ml of freshwater/kg feed was added and mixed well for another 5 min. Then, passed through the meat grinder, the spaghetti-like feed was broken into pellet and dried in an air flow oven at 60 8C for 4 6 h or until the moisture was less than 10%. The dry pellets were kept in two layers of plastic bag in the refrigerator until use Growth performance analysis For the experiment in small shrimp, the stocking rate was 15 shrimp per aquarium ( m) and each treatment contained six replications. Shrimp

3 K. Supamattaya et al. / Aquaculture 248 (2005) Table 1 Compositions of test diet Ingredients g/kg T1 T2 T3 T4 T5 Fish meal Shrimp head meal Squid meal Wheat gluten Soybean meal Wheat flour Rice flour Fish oil Lecithin Cholesterol Vitamin-mix a Choline Vitamin E Vitamin C Mineral mixture b BHT Zeolite Algro NaturalR (2%) Salt (NaCl) a Vitamin mixture (mg/ kg diet): thiamin 22.5; riboflavin 20; nicotinic acid 36.7; Ca pantothenate 24; inositol 98; biotin 0.5; folic acid 1.68; vitamin B ; menadione 13.28; vitamin A 1150 IU; vitamin D IU; BHT 1; PABA 20. b Mineral mixture (g/kg diet) KH 2 PO 4 1; CaHPO 4 1; NaH 2 PO 4 1.5; KCl 0.5. was fed to satiation five times daily for 8 weeks. Data for growth performance were recorded such as growth, weight gain, survival, feed conversion ratio (FCR), and body color (score from color fan). Data recorded was determined every 2 weeks for the 8 weeks experimental period Immune function analysis After 6 weeks culture period of juvenile shrimp in trial 2, body color of test animal was examined by using a color fan. Immune functions and disease resistance were performed in the test group of juvenile shrimp as follows Total hemocyte count Hemolymph from each shrimp was collected by using a 1-ml syringe with 25 G needle from the base of a walking leg. After withdrawal, the hemocytes was immediately diluted with 0.5% trypan blue in 2.6% NaCl and hemocyte was counted using a hemacytometer and calculated as number of blood cell (total hemocytes/mm 3 ) Phenoloxidase activity Twenty shrimp from each experimental group were collected on week 6 for PO activities determination. PO activity from hemocytes was measured by the following methods. Hemolymph was collected from each shrimp using a 1-ml plastic syringe. K-199 medium (Itami et al., 1994) with 3% l-cysteine was used as an anticoagulant. After the blood was withdrawn, the hemocyte was washed three times with K-199 medium. Hemocyte lysate was prepared in a cacodylate buffer ph 7.4 by using a sonicator (Vibra Cellk sonicator and material, USA) at 30 amplitudes for 20 s. Phenoloxidase activity from hemocyte lysate was measured by the modified method of Smith and Soderhall (1983). l-3,4-dihydroxyphenylalanine (l- DOPA) (4 mg/ml in cacodylate buffer) was used as a substrate and enzyme activity was measured at 630 nm. Protein content in hemocyte lysate was measured by using Lowry s method (Lowry et al., 1951). Phenoloxidase activity was defined by the increase of OD /min/mg protein Bacterial clearance ability At the end of the 6 weeks feeding period, 20 shrimp from each experimental group of trial 2 were tested for their ability to remove the foreign particles. The ability of each shrimp to remove bacterial cell from the blood circulation system was measured by the modified method of Martin et al. (1993). A bacterial suspension was prepared from 24 h cultured of Vibrio harveyi. A bacterial suspension of 0.1 ml ( cells/ml) was injected into the tail muscle of each shrimp and were then kept in aquaria equipped with seawater and aeration for 3 h. Hemolymph was collected from each shrimp without an anticoagulant and 30 Al of whole blood (without dilution) was dropped on TCBS agar. A two-fold dilution of whole blood was made using sterile 2.6% NaCl solution. The number of bacteria was counted on the TCBS as above. The bacterial cells in the hemolymph of each shrimp was reported as cfu/ ml. Hemolymph from five of sham-injected shrimp were also performed for the initial number of vibrio in the blood.

4 210 K. Supamattaya et al. / Aquaculture 248 (2005) Table 2 Average weigh (g) of small shrimp fed with experimental diet for 8 weeks Week 0 Week 2 Week 4 Week 6 Week 8 T1 (control) 1.22F F F F F0.59 T2 (125 mg h-carotene) 1.22F F F F F0.38 T3 (200 mg h-carotene) 1.21F F F F F0.18 T4 (300 mg h-carotene) 1.22F F F F F0.73 T5 (0.9% NaCl) 1.22F F F F F0.61 MeanFS.D. from six replications. No significant difference was observed in mean ( P N0.05). Each replication contained 15 shrimp per aquarium Stress tolerance At the end of experiment, in trial 1, 20 shrimp from each treatment were transferred to duplicates of stress test chambers (50-l glass aquaria). Low dissolved oxygen conditions were maintained by stop the water flow through system and aeration, using a plastic sheet overlying on the water surface in each stress test chamber. Dissolved oxygen was linearly decreased to mg/l within 10 h. Dissolved oxygen in the test chamber was measured by an oxygen meter (YSI model 57). The stress condition was carried on only in the daytime for 10 h (7:00 a.m. 5:00 p.m.) per day and then returned to normal condition in the evening. Shrimp was stressed under this condition for 10 consecutive days. Mortality of the shrimp in each group was recorded for 10 days period Disease resistance Virus stock preparation For preparation of viral stock, normal and healthy shrimp weighing about g was injected with white spot syndrome virus (WSSV) and kept in a 250-l tank until they showed signs of the red disease with white spots and positive by PCR (polymerase chain reaction technique). Viral stock used for injection was prepared by withdrawing the hemolymph from the moribund shrimp and diluted to 1:2 with PBS and stored at 80 8C until use. The diluted hemolymph was further diluted at 1:10,000, 1:50,000, 1:100,000 with sterile 1.5% NaCl and 20 shrimp were injected with 0.1 ml of each dilution of viral suspension. Mortality was recorded for days and LD 50 was determined by using probit analysis Challenge procedure At termination of the experiment in small shrimp, the shrimp from each treatment were transferred to duplicates of 150-l glass aquaria with 10 shrimp per aquarium. Stock virus was prepared in sterile 1.5% NaCl and diluted to a certain concentration. Each shrimp was injected intramuscularly on the sixth segment with 0.1 ml of viral suspension at the concentration of LD 50. The negative control group was injected with sterile 1.5% NaCl solution. Shrimp were fed regularly as normal condition with 20% water exchanged daily. Moribund or dead shrimp were removed each day and the cause of mortality examined due to the virus by polymerase chain reaction technique (Takahashi et al., 1996). The mortality was recorded everyday for a period of 15 days. Table 3 Growth performance of small shrimp after feeding with experimental diet for 8 weeks Weight gain (%) Survival rate (%) FCR T1 (control) F41.89 a 88.89F3.44 b 1.98F0.19 T2 (125 mg h-carotene) F29.52 b F0.00 c 1.68F0.14 T3 (200 mg h-carotene) F16.41 ab F0.00 c 1.80F0.09 T4 (300 mg h-carotene) F58.98 b F0.00 c 1.72F0.23 T5 (0.9% NaCl) F44.46 a 86.67F0.00 a 1.85F0.28 MeanFS.D. from six replications. Each replication contained 15 shrimp per aquarium. Means not sharing a common superscript are significantly different ( P b0.05).

5 K. Supamattaya et al. / Aquaculture 248 (2005) Table 4 Color score and visual apparent of small shrimp a in each treatment after boiling for 3 min Color score Visual apparent T1 (control) b20 Light orange T2 (125 mg h-carotene) 26 Orange red T3 (200 mg h-carotene) 27 Orange red T4 (300 mg h-carotene) Orange red T5 (0.9% NaCl) b20 Light orange a Eighteen animals were visualized from each treatment (three animals were sampling from each replication) Carotenoids analysis After 6-week periods of feeding trial in juvenile shrimp (trial 2), 10 shrimp from each treatment were sampled and analyzed for total carotenoids by the method described by Sommer et al. (1991). Thin layer chromatography (TLC) from the method described by Yamada et al. (1990) was used for the analysis of free astaxanthin, astaxanthin mono-ester and astaxanthin di-ester Statistical analysis All data were subjected to analysis with Duncan s new multiple range test (Duncan, 1955). 3. Results 3.1. Trial 1 (small shrimp) Growth performance During 8 weeks of feeding period, there was no significant difference in average weight of the shrimp among each treatment (Table 2). Weight gain, percent survival and feed conversion ratio (FCR) of the shrimp in each treatment are shown in Table 3. Weight gain of shrimp fed diet supplemented with mg h-carotene/kg diet was significantly higher than the shrimp fed control diet (T1) or diet containing 0.9% NaCl (T5) ( P b0.05), as well as the percent survival, which was lowest in the group fed control diet (T1) and the diet supplemented with 0.9% NaCl (T5). However, it was not significantly different in FCR among each treatment Body color The body color of each treatment could be distinguished by the naked eye in week 3 after feeding with test diet. Shrimp in treatments 1 and 5 looked pale blue, while dark blue or dark brown was observed in T2 T4. The color in T4 was more intense than the others. After 8 weeks of feeding trial, color score of boiled shrimp in T3 and T4 was highest and the lowest score was found in T1 and T5 as described in Table Blood parameters and disease resistance There was no significant difference in total hemocyte count and phenoloxidase activity among each treatment as shown in Table 5. The percent survival after challenged with WSSV (1:10 6 ) is shown in Fig. 1. After 15 days of challenged period survival of shrimp fed with 300 mg h-carotene/kg diet was significantly higher than the others (Table 5) Stress tolerance During 9 days of stress period, the survival of shrimp fed diet containing 300 mg h-carotene/kg Table 5 Blood parameters and survival after challenge test of small shrimp fed experimental diet for 8 weeks Hemocytes count y (10 4 cell/mm 3 ) PO activity y (U/min/mg protein) Survival yy (%) T1 (control) 6.83F F F0 a T2 (125 mg h-carotene) 7.22F F F4.71 ab T3 (200 mg h-carotene) 7.30F F F4.71 ab T4 (300 mg h-carotene) 7.64F F F12.47 b T5 (0.9 % NaCl) 8.05F F F8.16 a y MeanFS.D. from 15 shrimp. No significant difference was observed in mean ( P N0.05). yy Survival after 15 days of challenge test with WSSV, meanfs.d. from three replications, means not sharing a common superscript are significantly different ( P b0.05).

6 212 K. Supamattaya et al. / Aquaculture 248 (2005) Table 6 Total hemocyte counts and phenoloxidase activity of juvenile shrimp fed experimental diet for 6 weeks Total hemocyte count (10 4 cell/ mm 3 ) Phenoloxidase activity (U/min/mg protein) T1 (control) 7.04F2.70 bc F T2 (125 mg h-carotene) 5.87F2.95 abc F T3 (200 mg h-carotene) 5.45F1.97 ab F T4 (300 mg h-carotene) 4.91F2.17 a F T5 (0.9% NaCl) 7.91F2.00 c F MeanFS.D. from 20 shrimp. Means within columns not sharing the same superscript are significantly different ( P b0.05). Fig. 1. Survival (%) of shrimp in each treatment after challenged with WSSV for 15 days. diet (T4) was the highest as shown in Fig. 2. Moreover, survival of all groups fed h-carotene supplemented diet were significantly higher than control groups ( P b0.05) Trial 2 (juvenile shrimp) Blood parameters At termination of experiment on week 6, total hemocyte were significantly higher in control and the shrimp fed diet containing 0.9% NaCl ( P b 0.05). Total hemocyte counts were negatively correlated to the increasing of h-carotene in test diet (r = 0.97). Phenoloxidase activity was not significantly different among treatments (Table 6) Body colors and carotenoids analysis The difference in body color of shrimp in each treatment was first observed on week 4, the shrimp fed pigment supplemented diet exhibited more dark-brown color when compared with control and the group fed with 0.9% NaCl (Fig. 3). At the end of feeding trial, color intensity of boiled shrimp in T3 and T4 were higher than T2, while the light-yellow color was observed in control and the group fed with 0.9% NaCl (Fig. 4). Color scores of boiled shrimp fed each experimental diet are shown in Table 7. The carotenoid content Fig. 2. Survival (%) of shrimp in each treatment during 9 days of stress period. Fig. 3. Body color of juvenile shrimp after fed each experimental diet for 6 weeks.

7 K. Supamattaya et al. / Aquaculture 248 (2005) Fig. 4. Color of shrimp fed each experimental diet after boiling for 5 min. of the shrimp is summarized in Table 8. Total carotenoid was highest in the shrimp fed with mg h-carotene/kg diet. However, free astaxanthin was highest in the shrimp fed 300 mg h-carotene/kg diet Bacterial clearance ability At 3 h post injection of V. harveyi suspension, vibrio count in the hemolymph from shrimp fed each experimental diet ranged from cfu/ml (Table 9). However, vibrio counts in hemolymph were not significantly different among treatment. The vibrio counted from sham-injected shrimp ranged 8 12 cfu/ml hemolymph. 4. Discussion It is considered that penaeid shrimp cannot biosynthesize carotenoids from mevalonic acid, but can alter dietary carotenoids by oxidation and deposit them in their tissues. Tanaka et al. (1976) reported on the metabolism of carotenoids in kuruma shrimp and suggested that some of dietary carotenoid pigments such as astaxanthin, h-carotene, isocryptoxanthin, echinenone, canthaxanthin, phoenicoxanthin, zeaxanthin and 4-ketozeaxanthin were converted into astaxanthin in shrimp body. Especially, astaxanthin was the most effective substance for pigmentation in shrimp when compared with h-carotene and canthaxanthin. (Yamada et al., 1990). However, Liao et al. (1993) studied the pigmentation of the black tiger shrimp by feeding the shrimp with diets containing different carotenoid sources, e.g., h-carotene, spirulina, Phaffia yeast and krill oil. A marked increase of carotenoid content in the carapace was observed in the group fed with spirulina supplemented diets and suggested that zeaxanthin, which is the major carotenoid in spirulina, was rapidly converted to astaxanthin. Moreover, 125 mg/kg of synthetic h- carotene or mg/kg h-carotene extracted from D. salina (Betatene) have been reported as a pigment sources in black tiger shrimp and demonstrated that black tiger shrimp has the metabolic ability to converted h-carotene into astaxanthin (Boonyaratpalin et al., 2001). Such finding was correlated with our results, mg/kg of h- carotene from D. salina (Algro NaturalR) showed its high efficiency for pigmentation in black tiger shrimp. From previous reports, dietary carotenoids were converted into astaxanthin and deposited in the shrimp body in free form by the association with protein and exists as carotenoprotein and esterified forms which are predominantly a monoester and di ester of long-chain fatty acids (Foss et al., 1987; Yamada et al., 1990). However, the incorporation of astaxanthin esters in test diets seemed less effective than the free form as reported by Liao et al. (1993) Moreover, Barbosa et al. (1999) reported that astaxanthin in rainbow trout serum was increased with increasing dietary lipids levels. No evidence showed the positive effect of dietary carotenoid on growth and feed efficiency in black tiger shrimp is in agreement with Liao et al. (1993), Table 7 Color score and visual apparent of the juvenile shrimp a fed experimental diet after boiling for 3 min Color score Visual apparent T1 (control) Light orange T2 (125 mg h-carotene) Orange red T3 (200 mg h-carotene) Orange red T4 (300 mg h-carotene) Orange red T5 (0.9% NaCl) Light orange a Eighteen animals were visualized from each treatment (three animals were sampled from each replication).

8 214 K. Supamattaya et al. / Aquaculture 248 (2005) Table 8 Total carotenoid and astaxanthin (mg/kg) content in juvenile shrimp* fed experimental diet for 6 weeks Total carotenoid (ppm) Di ester astaxanthin (ppm) Mono ester astaxanthin (ppm) Free astaxanthin (ppm) T1 (control) 8.29F0.91 a 1.86F0.23 a 0.12F0.16 a 1.29F0.35 a T2 (125 mg h-carotene) 19.43F2.98 b 2.65F0.18 a 1.69F0.23 bc 3.42F0.33 a T3 (200 mg h-carotene) 20.88F2.34 b 6.94F0.86 b 2.94F0.88 c 3.27F1.54 a T4 (300 mg h-carotene) 24.52F1.22 b 6.23F1.45 b 2.71F0.45 c 9.93F1.51 b T5 (0.9% NaCl) 11.63F1.79 a 1.77F0.17 a 0.54F0.33 ab 2.01F0.22 a * Analysis was performed from 3 replicates of 10 animals from each treatment. Means not sharing a common superscript are significantly different ( P b0.05). Chien and Jeng (1992) and Boonyaratpalin et al. (2001). However, the antioxidant activity of carotenoid pigments are involved in many physiological systems in the aquatic animal (Estermann, 1994; Hunter, 2000). Amar et al. (2001) studied the effect of carotenoids and vitamin A, C and E supplemented diets on bio-defense mechanisms in rainbow trout and found that serum complement activity in both beta-carotene and astaxanthin vitamin-supplement fed groups were significantly higher than control fish. Phagocytic activity was also high in the vitamin-supplemented astaxanthin and beta-carotene fed groups compared with control. Moreover, the vitamin-supplemented astaxanthin and h-carotene fed groups also exhibited better nonspecific cytotoxicity for the peripheral blood lymphocytes. Thus, among the carotenoids studied, h-carotene and astaxanthin elevated humoral factors such as serum complement and lysozyme activity, as well as cellular factors such as phagocytosis and nonspecific cytotoxicity. However, the concentration of h-carotene used in our study did not affect the immune parameters but Table 9 Total bacteria count in hemolymph after injected with suspension of V. harveyi for 3 h Total vibrio count (10 4 cfu/ml) T1 (control) 2.15F1.62 T2 (125 mg h-carotene) 2.88F2.81 T3 (200 mg h-carotene) 3.44F2.58 T4 (300 mg h-carotene) 3.90F2.52 T5 (0.9% NaCl) 2.55F1.48 Vibrio counted from sham-injected shrimp was 8 12 cfu/ml hemolymph. MeanFS.D. from 20 shrimp. No significant difference was observed in mean ( P N0.05). enhanced the resistance to white spot syndrome virus (WSSV). In addition, Thompson et al. (1995), who reported that the immunomodulatory effects of vitamin A and/or astaxanthin in rainbow trout were small, only serum antiprotease activity was increased by dietary vitamin A (18 mg/kg diet) or astaxanthin (100 mg/kg diet). Astaxanthin may serve as an intracellular oxygen supply for shrimp, allowing survival under the hypoxic conditions in the pond bottom (Chien and Jeng, 1992). Chien et al. (1999) reported that black tiger shrimp fed diet containing 360 mg synthetic astaxanthin/kg diet had better survival when exposed to oxygen depletion stress. This finding agreed with our study and concluded that deposited h-carotene from Dunaliella extract may constitute an intracellular reserve of oxygen in black tiger shrimp. From our results, the increase of dietary h-carotene reduced the total hemocytes of juvenile black tiger shrimp, while the phenoloxidase activity was not different from others, such phenomenon was not found in small shrimp. It is hypothesized that the large shrimp has a difference in physiological function from smaller shrimp. h-carotene may act as an antioxidant in the hemolymph of juvenile shrimp and reduce hemocyte production. Similar results have been reported by Nakano et al. (1995) who found that the activity of serum glutamic oxaloacetic transaminase (GOT) was lower in rainbow trout fed a diet containing red yeast (Phaffia yeast) or synthetic astaxanthin when compared with those of fish fed control diet. Moreover, the decline in HSI (hepatosomatic indices) has demonstrated that dietary astaxanthin has the potential to improve the function of the liver. Additionally, Rehulka (2000) reported that rainbow trout fed diet containing astaxanthin had significantly lower levels of red blood cells count, hematocrit,

9 K. Supamattaya et al. / Aquaculture 248 (2005) hemoglobin, triacylglycerol and Ca 2+ in the plasma. This finding shows that dietary carotenoids influenced blood components. Several reports have demonstrated a positive effect of microbial products that improve the pigmentation in many aquatic animals (Gentles and Haard, 1991; Sommer et al., 1991; Sanderson and Jolly, 1994). However, Tangeras and Slinde (1994) suggested that the thick cell wall in Phaffia rhodozyma restricted pigment availability, because of the lack of enzyme able to degrade the cell wall components in digestive tract of salmonid fish. In the application of algae Haematocuccus pluvialis in rainbow trout, Sommer et al. (1991) reported that the astaxanthin level in the fish body was higher in fish fed cell wall disrupted algal cells when compared with intact cell supplemented diet. Moreover, a high supplementation of dried algal cells caused retardation of growth in black tiger shrimp (Liao et al., 1993) and striped jack (Watanabe et al., 1990). Thus, supplementation of concentrated carotenoids, which extracted from microbial cells, are more practical than the incorporation of whole cells in feedstuffs. References Amar, E.C., Kiron, V., Satoh, S., Watanabe, T., Influence of various dietary synthetic carotenoids on bio-defence mechanisms in rainbow trout, Oncorhynchus mykiss (Walbaum). Aquacult. Res. 32, Barbosa, M.J., Morais, R., Choubert, G., Effect of carotenoid source and dietary lipid content on blood astaxanthin concentration in rainbow trout (Oncorhynchus mykiss). Aquaculture 176, Bendich, A., Shapiro, S.S., Effect of h-carotene and canthaxanthin on the immune responses of the rat. J. Nutr. 116, Boonyaratpalin, M., Supamattaya, K., Borisuth, C., The immune system in black tiger shrimp, Penaeus monodon Fabricius: VIII. 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