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Optimal dietary levels of lecithin and cholesterol for black tiger prawn Penaeus monodon larvae and postlarvae
Aquaculture 167 Ž1998. 273–281
Optimal dietary levels of lecithin and cholesterol for black tiger prawn Penaeus monodon larvae and postlarvae Chalee Paibulkichakul a , Somkiat Piyatiratitivorakul a,b,) , Prasat Kittakoop c , Voranop Viyakarn a , Arlo W. Fast d , Piamsak Menasveta a,b,c a
Department of Marine Science, Faculty of Science, Chulalongkorn UniÕersity, Bangkok 10330, Thailand b Aquatic Resources Research Institute, Chulalongkorn UniÕersity, Bangkok 10330, Thailand c Marine Biotechnology Research Unit, National Genetic Engineering and Biotechnology Center, Chulalongkorn UniÕersity, Bangkok 10330, Thailand d Hawaii Institute of Marine Biology, UniÕersity of Hawaii, Kaneohe, HI 96744, USA Accepted 13 July 1998
Abstract The effect of lecithin and cholesterol on growth and survival of larval and postlarval Penaeus monodon was evaluated using semi-purified diets containing four levels of lecithin Ž0.0, 0.5, 1.0 and 1.5%. and three levels of cholesterol Ž0.0, 0.5 and 1.0%.. Three early stages Žzoeal, mysid and postlarval. of P. monodon were fed the experimental diets. Growth and survival of shrimp fed diets containing 1.0 and 1.5% lecithin were not significantly different Ž P ) 0.05. but these groups had significantly greater growth and survival than those fed 0.0 and 0.5% lecithin diets. Shrimp fed diets containing 1.0% cholesterol had significantly greater Ž P - 0.05. growth and survival than that of shrimp fed diets containing 0.0 and 0.5% cholesterol. There was no interaction between lecithin and cholesterol on growth and survival of P. monodon. During a low salinity stress test, PL-15 shrimp fed diets containing 1.0% cholesterol had significantly greater Ž P - 0.05. tolerance to low salinity exposure. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Lecithin; Cholesterol; Larval feed; Salinity stress test; Penaeus monodon; Black tiger prawn; Marine shrimp
)
Corresponding author. Tel.: q66 2 2188161 fax: q66 2 2544259; e-mail: [email protected]
0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 3 2 7 - 5
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C. Paibulkichakul et al.r Aquaculture 167 (1998) 273–281
1. Introduction Currently, shrimp larviculture primarily depends on live food organisms such as micro-algae, rotifers, copepods and Artemia sp., all of which are often seasonally available. Furthermore, the quality of live feed is not consistent and declines after harvest and storage. A high quality artificial feed that can serve as a replacement for live feeds is needed. An essential step in the development of artificial feed for larval shrimp is defining nutrient requirements. Lecithin and cholesterol are two such nutrients. Phospholipids can be synthesized by shrimp, but the rate of synthesis is too slow to satisfy metabolic requirements of young crustaceans ŽD’Abramo et al., 1981; Kanazawa, 1983; Kanazawa et al., 1985.. All crustaceans, including marine shrimp, are incapable of synthesizing steroid ring compounds ŽAkiyama et al., 1992.. Lecithin plays important roles in lipid and carbohydrate metabolism in the liver of fish, and as an emulsifying agent in biological systems associated with transportation and absorption of fatty acids within the body. Lecithin is an essential component of biomembrane systems in all eukaryotic cells. Cholesterol is an essential precursor of bile acids, steroid hormones, molting hormones, vitamin D 3 and prostaglandins ŽSteffens, 1989; Tacon, 1990; Akiyama et al., 1992.. Appropriate phospholipid and cholesterol supplementation is known to promote growth and survival of many marine and freshwater crustaceans ŽCastell et al., 1975; D’Abramo et al., 1981, 1984, 1985; Kanazawa et al., 1985; Briggs et al., 1988; Chen and Jenn, 1991.. Most studies of dietary lecithin and cholesterol requirements have focused on juvenile and larger shrimp rather than early life stages of marine shrimp. Therefore, our present study was designed to determine optimal dietary levels of lecithin and cholesterol for growth, survival and low salinity tolerance of postlarval Penaeus monodon.
2. Materials and methods P. monodon eggs were obtained from the Andaman Sea broodstock and were hatched at the Aquaculture Laboratory, Department of Marine Science, Chulalongkorn University in Bangkok, Thailand. After hatching, shrimp larvae were cultured in a static water hatchery system as described by Wilkenfeld et al. Ž1983.. This system had a 50% water exchange every two days. Salinity was maintained at 30‰, temperature at 28 " 18C, pH at 7.75 " 0.25, dissolved oxygen at 6.5–7.7 mgrl, and ammonia concentration at - 0.5 mgrl. Twelve hours of dim light, and 12 h of darkness were provided. Two larval stages Žzoeal and mysid. and postlarvae were used to evaluate the effects of dietary lecithin and cholesterol on growth and survival, while 15 day old postlarvae ŽPL-15. were used in the low salinity stress tests. The experiment was 4 = 3 factorials in a completely randomized design with three replicates per dietary treatment combination. Diets included combinations of four levels of lecithin Ž0.0, 0.5, 1.0 and 1.5%. and three levels of cholesterol Ž0.0, 0.5 and 1.0%.. Diets were semi-purified with mean protein and lipid levels of 50 and 8%, respectively. These diets were produced using methods modified from Kanazawa et al. Ž1985. and
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Table 1 Composition of the test diets before addition of lecithin or cholesterol Ingredients
Dry matter Ž%.
Casein Dextrin Semi-refined fish oil Mineral mixturea Vitamin mixtureb Lecithinc Cholesterold Carrageenan a-cellulose
55.0 15.5 8.0 8.0 4.0 0–1.5 0–1.0 5.0 2.0–4.5
a
Mineral mixture 100 g contains: K 2 HPO4 2.0 g, Ca 3 ŽPO4 . 2 2.720 g, MgSO4 .7H 2 O 3.041g, NaH 2 PO4 .2H 2 O 0.790 g. b Vitamin mixture 100 g contains: r-aminobenzoic acid, 10.0 mg; biotin, 0.40 mg; inositol, 400.0 mg; nicotinic acid, 40.0 mg; Ca-pantothenate, 60.0 mg; pyridoxine-HCl, 12.0 mg; riboflavin, 8.0 mg; thiamin-HCl, 4.0 mg; menadione, 4.0 mg; a-tocopherol, 20.0 mg; cyanocobalamine, 0.08 mg; calciferol, 1.20 mg; folic acid, 0.80 mg; choline chloride, 120.0 mg; ascorbic acid, 20 mg; astaxanthin, 10 mg. c Soy lecithin, feed grade. d Ninety five percent cholesterol, laboratory grade, Sigma.
ingredient composition is presented in Table 1. All diets were ground and sieve sorted into three sizes; - 53 m for the zoeal diet, 53–125 m for the mysid diet, and ) 125–500 m for the postlarval diet. All diets were prepared at a temperature - 408C and stored in dark containers at y208C until used. In preparation for the zoeal trials, stage VI nauplii were separated into 4-l, cylindrical rearing units at a density of 100 naupliirl. The trial was terminated after larvae reached mysis stage I. For the mysid and postlarval trials, mysis stage I and PL-1 were prepared the same as in the zoeal trial, but densities were 30 and 10 individualsrl, respectively. During each trial, shrimp were fed four times daily Ž0800, 1200, 1600 and 2000 h.. Each morning Ž0900–1000 h. 10 larvae from each replicate of the zoeal and mysid trials were randomly sampled for growth determination and then returned. Growth index was calculated using the method of Kanazawa et al. Ž1985.. The total length of postlarvae was determined 15 days after metamorphosis Žat PL-15.. During the low salinity stress tests, ten PL-15 from each dietary feed group were placed in 500 ml of dilute seawater Ž2‰.. Shrimp mortalities were observed every 5 min for 2 h. Data were analyzed using a probit analysis to determine median tolerance time for each treatment group. 3. Results There was no significant Ž P ) 0.05. interaction between dietary lecithin and cholesterol on growth and survival of larval and postlarval P. monodon. Therefore, the effects of lecithin and cholesterol on growth and survival are discussed separately. Mean zoeal growth rate was not significantly different Ž P ) 0.05. for the four levels of dietary lecithin ŽFig. 1.. For the mysid stage, the growth rate of shrimp fed the diet
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Fig. 1. Effects of lecithin and cholesterol on growth of P. monodon zoeal, mysid and postlarval stages. Mean values are shown at the top of each bar. Means with the same superscript are not significantly different.
containing 1.5% lecithin was significantly greater Ž P - 0.05. than that of shrimp fed diets containing 0.0 and 0.5% lecithin. Postlarval shrimp fed diets containing 0.5, 1.0 and 1.5% lecithin had a growth rate that was significantly greater Ž P - 0.05. than that of postlarvae fed a diet containing no lecithin.
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The growth rates of larval and postlarval P. monodon fed diets containing each level of cholesterol were significantly different Ž P - 0.05. ŽFig. 1.. In all cases, the highest dietary cholesterol content Ž1.0%. always resulted in the greatest growth rate.
Fig. 2. Effects of lecithin and cholesterol on survival of P. monodon zoeal, mysid and postlarval stages. Mean values are shown at the top of each bar. Means with the same superscript are not significantly different.
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Fig. 3. Effects of lecithin and cholesterol on survival of P. monodon PL-15 exposed to low salinity Ž2‰. stress. Median tolerance times ŽTL 50 . in minutes are shown at the top of each bar. Values with the same superscript are not significantly different.
The effects of lecithin and cholesterol on survival of larval and postlarval P. monodon followed essentially the same patterns as those for growth rate ŽFig. 2.. Each larval stage fed diets containing 1.0 and 1.5% lecithin had significantly greater Ž P - 0.05. survival than that for 0.0 and 0.5% lecithin containing diets. Shrimp fed diets containing 1.0% cholesterol had the greatest survival, significantly greater than that of shrimp fed a diet containing no cholesterol. As found in survival and growth responses, there was no significant Ž P ) 0.05. interaction between lecithin and cholesterol content of diets on PL-15 tolerance of P. monodon to low salinity stress tests ŽFig. 3.. Survival of P. monodon fed diets containing different levels of lecithin was not significantly different Ž P ) 0.05.. However, regression analysis indicates that shrimp fed diets containing higher levels of lecithin survived significantly Ž r s 0.96; P - 0.05. longer than shrimp fed low concentrations of lecithin when exposed to low salinity. Survival of PL-15 shrimp fed diets containing 1.0% of cholesterol was significantly greater Ž P - 0.05. than that PL-15 shrimp fed other diets ŽFig. 3.. Regression analysis revealed a significant correlation Ž r s 0.99; P - 0.05. between dietary levels of cholesterol and median tolerance time of shrimp exposed to low salinity. 4. Discussion Our findings of no interactions between dietary lecithin and cholesterol relative to survival, growth and salinity tolerance of larval and postlarval P. monodon agree with published results for other crustaceans, including; Homarus americanus ŽKean et al., 1985., Macrobrachium rosenbergii ŽBriggs et al., 1988., Penaeus penicillatus ŽChen and Jenn, 1991. and juvenile P. monodon ŽChen, 1993.. Although lecithin and cholesterol are essential nutrients, the nutritional functions of these compounds are independent. The dietary phospholipid requirement for P. penicillatus was reported as 1.25% of the total diet ŽChen and Jenn, 1991., and 0.4–2.0% of the total diet for other marine
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shrimp ŽAkiyama et al., 1992.. Reported dietary cholesterol requirements for Penaeus japonicus vary from 0.5% ŽKanazawa et al., 1971. to 2.0% ŽDeshimaru and Kuroki, 1974., and 0.5% or higher for P. penicillatus ŽChen and Jenn, 1991.. In our present study, P. monodon larvae and postlarvae fed a diet containing either 1.0 or 1.5% of lecithin had significantly better growth and survival, whereas 1.0% cholesterol added to the diet significantly increased growth and survival of P. monodon. Greater tolerance to low salinity stress was also associated with higher survival and increased growth of shrimp. Mammals synthesize phospholipids from 1,2-diglycerides with the aid of CDP-choline ŽImai and Sakagami, 1966, cited by Kanazawa et al., 1985.. Larvae of P. japonicus, however, are believed to have a limited ability to biosynthesize phospholipid because of the inefficacy of CDP-choline ŽKanazawa et al., 1985.. Our study indicates that P. monodon larvae are similar to P. japonicus larvae in their ability to biosynthesize phospholipids, based on our observed growth and survival of P. monodon. This indicates that dietary phospholipids such as phosphatidylcholine and phosphatidylinositol improved growth and survival of P. monodon larvae and PL by effects other than enhancement of dietary lipid emulsification in the digestive tract. Previous work has shown that the hemolymph of juveniles of P. japonicus contains high-density lipoproteins rich in phospholipids as the major lipoproteins ŽTeshima and Kanazawa, 1980a., and also that the high-density lipoproteins were involved in the transport of lipids through the hemolymph ŽTeshima and Kanazawa, 1980b.. We, therefore, suspect that phosphatidylcholine and phosphatidylinositol containing high levels of n-3 and n-6 fatty acids serve as the lipid moieties of high-density lipoproteins in P. japonicus larvae. Conklin et al. Ž1980. and D’Abramo et al. Ž1981. demonstrated a positive effect of phospholipid on survival of juvenile Homarus americanus. Kanazawa et al. Ž1985. demonstrated the relative efficacy of different sources of phospholipids in improving growth and survival of P. japonicus. The relative nutritional value of phospholipids on growth and survival of crustaceans seems to vary with the quality of esterified fatty acids, in addition to the kinds of compounds esterified with phosphoric acid at the C-3 position. Studies of the effects of lecithin and cholesterol on juvenile, Macrobrachium rosenbergii ŽBriggs et al., 1988. and lobster H. americanus ŽKean et al., 1985. have found that there is no interaction between lecithin and cholesterol in influencing growth or survival. They used semi-purified diets such as crab protein rather than purified diet such as casein as the primary protein source. D’Abramo et al. Ž1982. indicated that the absence of soy lecithin in purified diets fed to juvenile lobsters caused a significant decrease in the concentration of total cholesterol and phospholipid in the serum, possibly owing to insufficient levels of certain dietary amino acids when casein was the main source of protein. Kanazawa et al. Ž1971. demonstrated that diets containing 0.5% cholesterol significantly improved the growth and survival of P. japonicus. Teshima et al. Ž1983. reported that diets containing 1.0% cholesterol improved growth and survival of P. japonicus larvae. At these cholesterol levels, larval shrimp grew as well as those fed natural diets. Sheen et al. Ž1994. reported that diets containing 0.2–0.8% cholesterol improved growth and survival of shrimp.
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During our trials, we observed greater mortality of zoea during the first three days of this stage, compared with that for the last three days. This higher mortality was associated with incomplete ecdysis and feed residue in the digestive tract. This mortality syndrome has been previously observed and may be related to problems with sterol metabolism ŽConklin et al., 1980; Bowser and Rosemark, 1981.. Additional amounts of dietary cholesterol may overcome this problem. In our study survival, growth and salinity tolerance continued to increase even at the highest cholesterol levels used Ž1.0%. ŽFigs. 1–3.; therefore optimal dietary cholesterol levels may well exceed 1.0%.
Acknowledgements Financial support for this research was provided by the Thailand Research Fund ŽTRF..
References Akiyama, D.M., Dominy, W.G., Lawrence, A.L., 1992. Penaeid shrimp nutrition. In: Fast, A.W., Lester, L.J. ŽEds.., Marine Shrimp Culture: Principles and Practices. Elsevier Science Publishing, New York, pp. 535–568. Bowser, P.R., Rosemark, P., 1981. Mortalities of cultured lobsters, Homarus, associated with a molt–death syndrome. Aquaculture 23, 11–18. Briggs, M.R.P., Jauncey, K., Brown, J.H., 1988. The cholesterol and lecithin requirements of juvenile prawn (Macrobrachium rosenbergii) fed semi-purified diets. Aquaculture 70, 121–129. Castell, J.D., Mason, E.C., Covey, J.F., 1975. Cholesterol requirements of juvenile American lobsters (Homarus americanus). J. Fish. Res. Board Can. 38, 1431–1435. Chen, H.Y., 1993. Requirements of marine shrimp, Penaeus monodon, juvenile for phosphatidylcholine and cholesterol. Aquaculture 109, 165–176. Chen, H.Y., Jenn, J.S., 1991. Combined effects of dietary phosphatidylcholine and cholesterol on the growth, survival and body lipid composition of marine shrimp, Penaeus peniciliatus. Aquaculture 96, 167–178. Conklin, D.E., D’Abramo, L.R., Bordner, C.E., Baum, N.A., 1980. A successful purified diet for the culture of juvenile lobsters: the effect of lecithin. Aquaculture 21, 243–249. D’Abramo, L.R., Bordner, C.E., Conklin, D.E., Baum, N.A., 1981. Essentiality of dietary phosphatidylcholine for the survival of juvenile lobsters. J. Nutr. 111, 425–431. D’Abramo, L.R., Bordner, C.E., Conklin, D.E., 1982. Relationship between dietary phosphatidylcholine and serum cholesterol in the lobster Homarus sp. Mar. Biol. 67, 231–235. D’Abramo, L.R., Bordner, C.E., Conklin, D.E., Baum, N.A., 1984. Sterol requirement of juvenile lobsters, Homarus sp. Aquaculture 42, 13–25. D’Abramo, L.R., Wright, J.S., Wright, K.H., Bordner, C.E., Conklin, D.E., 1985. Sterol requirement of cultured juvenile crayfish, Pacifastacus leniusculus. Aquaculture 49, 245–255. Deshimaru, O., Kuroki, K., 1974. Studies on a purified diet for prawn-II, optimum contents of cholesterol and glycosamine in the diet. Bull. Jpn. Soc. Sci. Fish. 40, 421–424. Imai, Y., Sakagami, T., 1966. Metabolism of compound lipids. In: Biochemistry of lipids, Tokyo: Asakurashoten, Cited by Kanazawa, A., Teshima, S., Sakamoto, M., 1985. Effects of dietary lipids, fatty acids, and phospholipids on growth and survival of prawn (Penaeus japonicus) larvae. Aquaculture Vol. 50, pp. 39–49. Kanazawa, A., 1983. Effects of dietary phospholipids on growth of the larval red sea bream and knife jaw. Mem. Fac. Fish. Kagoshima Univ. 32, 109–114. Kanazawa, A., Tanaka, N., Teshima, S., Kashiwada, K., 1971. Nutritional requirement of prawn-II, requirement of sterols. Bull. Jpn. Soc. Sci. Fish. 37, 211–215.
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Kanazawa, A., Teshima, S., Sakamoto, M., 1985. Effects of dietary lipids, fatty acids, and phospholipids on growth and survival of prawn (Penaeus japonicus) larvae. Aquaculture 50, 39–49. Kean, J.C., Castell, J.D., Boghen, A.G., D’Abramo, L.R., Conklin, D.E., 1985. A re-evaluation of the lecithin and cholesterol requirements of juvenile lobster (Homarus americanus) using crab protein-based diets. Aquaculture 47, 1431–1449. Sheen, S.S., Liu, P.C., Chen, S.N., Chen, J.C., 1994. Cholesterol requirement of juvenile tiger shrimp Ž Penaeus monodon.. Aquaculture 125, 131–137. Steffens, W., 1989. Principles of Fish Nutrition. Ellis Horwood, West Sussex. Tacon, A.G.J., 1990. Standard Methods for the Nutrition and Feeding of Farmed Fish and Shrimp. Vol. 1: The Essential Nutrients. Argent Laboratories Press, Washington. Teshima, S., Kanazawa, A., 1980a. Lipid constituents of serum lipoproteins in the prawn. Bull. Jpn. Soc. Sci. Fish. 46, 57–62. Teshima, S., Kanazawa, A., 1980b. Transport of dietary lipids and role of serum lipoproteins in the prawn. Bull. Jpn. Soc. Sci. Fish. 46, 51–55. Teshima et al., 1983. Wilkenfeld, J.W., Lawrence, A.L., Kuban, F.D., 1983. Rearing penaeid shrimp in a small scale system for experimental purposes. In: Rogers, G.L., Day, R., Lim, A. ŽEds.., Proceedings of the First International Conference on Warm Water Aquaculture Crustacea. Brigham Young University Hawaii Campus, Office of Continuing Education, Laie, HI, USA, pp. 72–81.
Optimal dietary levels of lecithin and cholesterol for black tiger prawn Penaeus monodon larvae and postlarvae Chalee Paibulkichakul a , Somkiat Piyatiratitivorakul a,b,) , Prasat Kittakoop c , Voranop Viyakarn a , Arlo W. Fast d , Piamsak Menasveta a,b,c a
Department of Marine Science, Faculty of Science, Chulalongkorn UniÕersity, Bangkok 10330, Thailand b Aquatic Resources Research Institute, Chulalongkorn UniÕersity, Bangkok 10330, Thailand c Marine Biotechnology Research Unit, National Genetic Engineering and Biotechnology Center, Chulalongkorn UniÕersity, Bangkok 10330, Thailand d Hawaii Institute of Marine Biology, UniÕersity of Hawaii, Kaneohe, HI 96744, USA Accepted 13 July 1998
Abstract The effect of lecithin and cholesterol on growth and survival of larval and postlarval Penaeus monodon was evaluated using semi-purified diets containing four levels of lecithin Ž0.0, 0.5, 1.0 and 1.5%. and three levels of cholesterol Ž0.0, 0.5 and 1.0%.. Three early stages Žzoeal, mysid and postlarval. of P. monodon were fed the experimental diets. Growth and survival of shrimp fed diets containing 1.0 and 1.5% lecithin were not significantly different Ž P ) 0.05. but these groups had significantly greater growth and survival than those fed 0.0 and 0.5% lecithin diets. Shrimp fed diets containing 1.0% cholesterol had significantly greater Ž P - 0.05. growth and survival than that of shrimp fed diets containing 0.0 and 0.5% cholesterol. There was no interaction between lecithin and cholesterol on growth and survival of P. monodon. During a low salinity stress test, PL-15 shrimp fed diets containing 1.0% cholesterol had significantly greater Ž P - 0.05. tolerance to low salinity exposure. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Lecithin; Cholesterol; Larval feed; Salinity stress test; Penaeus monodon; Black tiger prawn; Marine shrimp
)
Corresponding author. Tel.: q66 2 2188161 fax: q66 2 2544259; e-mail: [email protected]
0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 3 2 7 - 5
274
C. Paibulkichakul et al.r Aquaculture 167 (1998) 273–281
1. Introduction Currently, shrimp larviculture primarily depends on live food organisms such as micro-algae, rotifers, copepods and Artemia sp., all of which are often seasonally available. Furthermore, the quality of live feed is not consistent and declines after harvest and storage. A high quality artificial feed that can serve as a replacement for live feeds is needed. An essential step in the development of artificial feed for larval shrimp is defining nutrient requirements. Lecithin and cholesterol are two such nutrients. Phospholipids can be synthesized by shrimp, but the rate of synthesis is too slow to satisfy metabolic requirements of young crustaceans ŽD’Abramo et al., 1981; Kanazawa, 1983; Kanazawa et al., 1985.. All crustaceans, including marine shrimp, are incapable of synthesizing steroid ring compounds ŽAkiyama et al., 1992.. Lecithin plays important roles in lipid and carbohydrate metabolism in the liver of fish, and as an emulsifying agent in biological systems associated with transportation and absorption of fatty acids within the body. Lecithin is an essential component of biomembrane systems in all eukaryotic cells. Cholesterol is an essential precursor of bile acids, steroid hormones, molting hormones, vitamin D 3 and prostaglandins ŽSteffens, 1989; Tacon, 1990; Akiyama et al., 1992.. Appropriate phospholipid and cholesterol supplementation is known to promote growth and survival of many marine and freshwater crustaceans ŽCastell et al., 1975; D’Abramo et al., 1981, 1984, 1985; Kanazawa et al., 1985; Briggs et al., 1988; Chen and Jenn, 1991.. Most studies of dietary lecithin and cholesterol requirements have focused on juvenile and larger shrimp rather than early life stages of marine shrimp. Therefore, our present study was designed to determine optimal dietary levels of lecithin and cholesterol for growth, survival and low salinity tolerance of postlarval Penaeus monodon.
2. Materials and methods P. monodon eggs were obtained from the Andaman Sea broodstock and were hatched at the Aquaculture Laboratory, Department of Marine Science, Chulalongkorn University in Bangkok, Thailand. After hatching, shrimp larvae were cultured in a static water hatchery system as described by Wilkenfeld et al. Ž1983.. This system had a 50% water exchange every two days. Salinity was maintained at 30‰, temperature at 28 " 18C, pH at 7.75 " 0.25, dissolved oxygen at 6.5–7.7 mgrl, and ammonia concentration at - 0.5 mgrl. Twelve hours of dim light, and 12 h of darkness were provided. Two larval stages Žzoeal and mysid. and postlarvae were used to evaluate the effects of dietary lecithin and cholesterol on growth and survival, while 15 day old postlarvae ŽPL-15. were used in the low salinity stress tests. The experiment was 4 = 3 factorials in a completely randomized design with three replicates per dietary treatment combination. Diets included combinations of four levels of lecithin Ž0.0, 0.5, 1.0 and 1.5%. and three levels of cholesterol Ž0.0, 0.5 and 1.0%.. Diets were semi-purified with mean protein and lipid levels of 50 and 8%, respectively. These diets were produced using methods modified from Kanazawa et al. Ž1985. and
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Table 1 Composition of the test diets before addition of lecithin or cholesterol Ingredients
Dry matter Ž%.
Casein Dextrin Semi-refined fish oil Mineral mixturea Vitamin mixtureb Lecithinc Cholesterold Carrageenan a-cellulose
55.0 15.5 8.0 8.0 4.0 0–1.5 0–1.0 5.0 2.0–4.5
a
Mineral mixture 100 g contains: K 2 HPO4 2.0 g, Ca 3 ŽPO4 . 2 2.720 g, MgSO4 .7H 2 O 3.041g, NaH 2 PO4 .2H 2 O 0.790 g. b Vitamin mixture 100 g contains: r-aminobenzoic acid, 10.0 mg; biotin, 0.40 mg; inositol, 400.0 mg; nicotinic acid, 40.0 mg; Ca-pantothenate, 60.0 mg; pyridoxine-HCl, 12.0 mg; riboflavin, 8.0 mg; thiamin-HCl, 4.0 mg; menadione, 4.0 mg; a-tocopherol, 20.0 mg; cyanocobalamine, 0.08 mg; calciferol, 1.20 mg; folic acid, 0.80 mg; choline chloride, 120.0 mg; ascorbic acid, 20 mg; astaxanthin, 10 mg. c Soy lecithin, feed grade. d Ninety five percent cholesterol, laboratory grade, Sigma.
ingredient composition is presented in Table 1. All diets were ground and sieve sorted into three sizes; - 53 m for the zoeal diet, 53–125 m for the mysid diet, and ) 125–500 m for the postlarval diet. All diets were prepared at a temperature - 408C and stored in dark containers at y208C until used. In preparation for the zoeal trials, stage VI nauplii were separated into 4-l, cylindrical rearing units at a density of 100 naupliirl. The trial was terminated after larvae reached mysis stage I. For the mysid and postlarval trials, mysis stage I and PL-1 were prepared the same as in the zoeal trial, but densities were 30 and 10 individualsrl, respectively. During each trial, shrimp were fed four times daily Ž0800, 1200, 1600 and 2000 h.. Each morning Ž0900–1000 h. 10 larvae from each replicate of the zoeal and mysid trials were randomly sampled for growth determination and then returned. Growth index was calculated using the method of Kanazawa et al. Ž1985.. The total length of postlarvae was determined 15 days after metamorphosis Žat PL-15.. During the low salinity stress tests, ten PL-15 from each dietary feed group were placed in 500 ml of dilute seawater Ž2‰.. Shrimp mortalities were observed every 5 min for 2 h. Data were analyzed using a probit analysis to determine median tolerance time for each treatment group. 3. Results There was no significant Ž P ) 0.05. interaction between dietary lecithin and cholesterol on growth and survival of larval and postlarval P. monodon. Therefore, the effects of lecithin and cholesterol on growth and survival are discussed separately. Mean zoeal growth rate was not significantly different Ž P ) 0.05. for the four levels of dietary lecithin ŽFig. 1.. For the mysid stage, the growth rate of shrimp fed the diet
276
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Fig. 1. Effects of lecithin and cholesterol on growth of P. monodon zoeal, mysid and postlarval stages. Mean values are shown at the top of each bar. Means with the same superscript are not significantly different.
containing 1.5% lecithin was significantly greater Ž P - 0.05. than that of shrimp fed diets containing 0.0 and 0.5% lecithin. Postlarval shrimp fed diets containing 0.5, 1.0 and 1.5% lecithin had a growth rate that was significantly greater Ž P - 0.05. than that of postlarvae fed a diet containing no lecithin.
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The growth rates of larval and postlarval P. monodon fed diets containing each level of cholesterol were significantly different Ž P - 0.05. ŽFig. 1.. In all cases, the highest dietary cholesterol content Ž1.0%. always resulted in the greatest growth rate.
Fig. 2. Effects of lecithin and cholesterol on survival of P. monodon zoeal, mysid and postlarval stages. Mean values are shown at the top of each bar. Means with the same superscript are not significantly different.
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Fig. 3. Effects of lecithin and cholesterol on survival of P. monodon PL-15 exposed to low salinity Ž2‰. stress. Median tolerance times ŽTL 50 . in minutes are shown at the top of each bar. Values with the same superscript are not significantly different.
The effects of lecithin and cholesterol on survival of larval and postlarval P. monodon followed essentially the same patterns as those for growth rate ŽFig. 2.. Each larval stage fed diets containing 1.0 and 1.5% lecithin had significantly greater Ž P - 0.05. survival than that for 0.0 and 0.5% lecithin containing diets. Shrimp fed diets containing 1.0% cholesterol had the greatest survival, significantly greater than that of shrimp fed a diet containing no cholesterol. As found in survival and growth responses, there was no significant Ž P ) 0.05. interaction between lecithin and cholesterol content of diets on PL-15 tolerance of P. monodon to low salinity stress tests ŽFig. 3.. Survival of P. monodon fed diets containing different levels of lecithin was not significantly different Ž P ) 0.05.. However, regression analysis indicates that shrimp fed diets containing higher levels of lecithin survived significantly Ž r s 0.96; P - 0.05. longer than shrimp fed low concentrations of lecithin when exposed to low salinity. Survival of PL-15 shrimp fed diets containing 1.0% of cholesterol was significantly greater Ž P - 0.05. than that PL-15 shrimp fed other diets ŽFig. 3.. Regression analysis revealed a significant correlation Ž r s 0.99; P - 0.05. between dietary levels of cholesterol and median tolerance time of shrimp exposed to low salinity. 4. Discussion Our findings of no interactions between dietary lecithin and cholesterol relative to survival, growth and salinity tolerance of larval and postlarval P. monodon agree with published results for other crustaceans, including; Homarus americanus ŽKean et al., 1985., Macrobrachium rosenbergii ŽBriggs et al., 1988., Penaeus penicillatus ŽChen and Jenn, 1991. and juvenile P. monodon ŽChen, 1993.. Although lecithin and cholesterol are essential nutrients, the nutritional functions of these compounds are independent. The dietary phospholipid requirement for P. penicillatus was reported as 1.25% of the total diet ŽChen and Jenn, 1991., and 0.4–2.0% of the total diet for other marine
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shrimp ŽAkiyama et al., 1992.. Reported dietary cholesterol requirements for Penaeus japonicus vary from 0.5% ŽKanazawa et al., 1971. to 2.0% ŽDeshimaru and Kuroki, 1974., and 0.5% or higher for P. penicillatus ŽChen and Jenn, 1991.. In our present study, P. monodon larvae and postlarvae fed a diet containing either 1.0 or 1.5% of lecithin had significantly better growth and survival, whereas 1.0% cholesterol added to the diet significantly increased growth and survival of P. monodon. Greater tolerance to low salinity stress was also associated with higher survival and increased growth of shrimp. Mammals synthesize phospholipids from 1,2-diglycerides with the aid of CDP-choline ŽImai and Sakagami, 1966, cited by Kanazawa et al., 1985.. Larvae of P. japonicus, however, are believed to have a limited ability to biosynthesize phospholipid because of the inefficacy of CDP-choline ŽKanazawa et al., 1985.. Our study indicates that P. monodon larvae are similar to P. japonicus larvae in their ability to biosynthesize phospholipids, based on our observed growth and survival of P. monodon. This indicates that dietary phospholipids such as phosphatidylcholine and phosphatidylinositol improved growth and survival of P. monodon larvae and PL by effects other than enhancement of dietary lipid emulsification in the digestive tract. Previous work has shown that the hemolymph of juveniles of P. japonicus contains high-density lipoproteins rich in phospholipids as the major lipoproteins ŽTeshima and Kanazawa, 1980a., and also that the high-density lipoproteins were involved in the transport of lipids through the hemolymph ŽTeshima and Kanazawa, 1980b.. We, therefore, suspect that phosphatidylcholine and phosphatidylinositol containing high levels of n-3 and n-6 fatty acids serve as the lipid moieties of high-density lipoproteins in P. japonicus larvae. Conklin et al. Ž1980. and D’Abramo et al. Ž1981. demonstrated a positive effect of phospholipid on survival of juvenile Homarus americanus. Kanazawa et al. Ž1985. demonstrated the relative efficacy of different sources of phospholipids in improving growth and survival of P. japonicus. The relative nutritional value of phospholipids on growth and survival of crustaceans seems to vary with the quality of esterified fatty acids, in addition to the kinds of compounds esterified with phosphoric acid at the C-3 position. Studies of the effects of lecithin and cholesterol on juvenile, Macrobrachium rosenbergii ŽBriggs et al., 1988. and lobster H. americanus ŽKean et al., 1985. have found that there is no interaction between lecithin and cholesterol in influencing growth or survival. They used semi-purified diets such as crab protein rather than purified diet such as casein as the primary protein source. D’Abramo et al. Ž1982. indicated that the absence of soy lecithin in purified diets fed to juvenile lobsters caused a significant decrease in the concentration of total cholesterol and phospholipid in the serum, possibly owing to insufficient levels of certain dietary amino acids when casein was the main source of protein. Kanazawa et al. Ž1971. demonstrated that diets containing 0.5% cholesterol significantly improved the growth and survival of P. japonicus. Teshima et al. Ž1983. reported that diets containing 1.0% cholesterol improved growth and survival of P. japonicus larvae. At these cholesterol levels, larval shrimp grew as well as those fed natural diets. Sheen et al. Ž1994. reported that diets containing 0.2–0.8% cholesterol improved growth and survival of shrimp.
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During our trials, we observed greater mortality of zoea during the first three days of this stage, compared with that for the last three days. This higher mortality was associated with incomplete ecdysis and feed residue in the digestive tract. This mortality syndrome has been previously observed and may be related to problems with sterol metabolism ŽConklin et al., 1980; Bowser and Rosemark, 1981.. Additional amounts of dietary cholesterol may overcome this problem. In our study survival, growth and salinity tolerance continued to increase even at the highest cholesterol levels used Ž1.0%. ŽFigs. 1–3.; therefore optimal dietary cholesterol levels may well exceed 1.0%.
Acknowledgements Financial support for this research was provided by the Thailand Research Fund ŽTRF..
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