Growth, survival and fatty acid composition of juvenile Litopenaeus vannamei fed different oils in the presence and absence of phospholipids

Aquaculture 205 (2002) 325 – 343 www.elsevier.com/locate/aqua-online

Growth, survival and fatty acid composition of juvenile Litopenaeus vannamei fed different oils in the presence and absence of phospholipids Mayra L. Gonza´lez-Fe´lix a,b,*, Addison L. Lawrence a,b, Delbert M. Gatlin III b, Martin Perez-Velazquez a,b a

TAES Shrimp Mariculture Project, Texas A&M University System, 1300 Port Street, Port Aransas, TX 78373, USA b Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX 77843, USA Received 28 February 2001; received in revised form 22 May 2001; accepted 22 May 2001

Abstract A 26 factorial study was conducted to evaluate the effect of dietary phospholipids (PL) and different neutral lipids, as well as their potential interaction, on growth, survival and fatty acid composition of hepatopancreas and muscle tissue of juvenile Litopenaeus vannamei. The lipid sources were coconut, soybean, linseed, peanut, and menhaden oils. Five diets contained 5% of each test oil and 3.1% of a commercial lecithin containing 97.6% PL. Five additional diets contained 5% of each oil but no lecithin. A control diet contained 3.1% lecithin but no other oil, and an additional control diet contained no PL but 2.21% soybean oil, 0.41% monobasic sodium phosphate, and 0.48% choline chloride (estimated equivalent of soybean oil, phosphorus and choline in 3.1% lecithin). No significant differences (at P<0.05) among treatments were observed for survival and no significant interactions were observed between the effects of PL and oil type for any of the responses after 8 weeks feeding; however, shrimp fed diets containing PL obtained significantly higher final weight and instantaneous growth rate (IGR), and lower feed conversion ratios (FCR) than those fed diets containing the same oil and no PL. Shrimp fed diets containing menhaden oil, with and without PL, showed higher final weight and IGR, and lower FCR than the rest of the treatments. These diets had a larger variety of fatty acids (FA), especially long chain highly unsaturated FA (HUFA), and showed the highest percentage of arachidonic, eicosapentaenoic and

* Corresponding author. TAES Shrimp Mariculture Project, Texas A&M University System, 1300 Port Street, Port Aransas, TX 78373, USA. Tel.: +1-361-749-4625; fax: +1-361-749-5756. E-mail address: [email protected] ( M.L. Gonza´lez-Fe´lix).

0044-8486/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 1 ) 0 0 6 8 4 - 6

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docosahexaenoic acids. Menhaden oil showed a higher nutritional value for juvenile L. vannamei because it provided more essential FA, particularly n 3 HUFA. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Shrimp nutrition; Litopenaeus vannamei; Lipids; Fatty acids; Phospholipids

1. Introduction Crustaceans require dietary lipids as a source of essential fatty acids (EFA) and other lipid classes like phospholipids (PL), sterols and carotenoids. In the case of marine organisms, polyunsaturated and especially highly unsaturated fatty acids (PUFA and HUFA, respectively) are important and essential because these animals possess a limited ability to synthesize them (Kanazawa et al., 1979a,b). In early studies, Kanazawa et al. (1977a, 1978, 1979c,d) demonstrated that Marsupenaeus japonicus requires linoleic (18:2n 6, LOA), linolenic (18:3n 3, LNA), eicosapentaenoic (20:5n 3, EPA), and docosahexaenoic (22:6n 3, DHA) acids as EFA, with the n 3 HUFA being most indispensable, and suggested that a combination of EPA and DHA should be included at an optimum level of 1% of the diet for M. japonicus juveniles. They also suggested that a dietary provision of 1% n 3 HUFA could be considered as a minimal value for postlarval penaeids (Kanazawa et al., 1979a). Several authors also have reported qualitative requirements for n 3 HUFA by different shrimp species like Fenneropenaeus indicus (Read, 1981), F. chinensis (Xu et al., 1993), Penaeus monodon (Catacutan, 1991), and Litopenaeus stylirostris (Leger et al., 1985). Lim et al. (1997) evaluated the growth response and fatty acid composition of juvenile L. vannamei fed different dietary lipids. They found that menhaden oil, rich in n 3 HUFA, was the most nutritious for this species, and among plant oils, those rich in LNA had a higher nutritional value than those rich in LOA. They concluded that both n 6 and n 3 fatty acids (FA) appear to be essential in the diet, although n 3 HUFA were required for maximum growth, feed efficiency, and survival. Many studies also have demonstrated the beneficial effect of supplementing PL to the diet of shrimp, which generally has been attributable to the transport of dietary lipids like triglycerides (TG) and cholesterol in the body. Kanazawa et al. (1985) indicated that phosphatidylcholine (PC) and phosphatidylinositol (PI) containing high levels of n 6 and n 3 FA probably serve as the lipid moieties of high density lipoproteins that transport lipids through the hemolymph of M. japonicus. The total PL content recommended by Akiyama et al. (1992) is of 2% of diet. Coutteau et al. (1996) reported that the growth response of early L. vannamei postlarvae was significantly improved by addition of 1.5% soybean PC to the diet. With increasing dietary level of soybean PC, higher proportions of 20:1n 9, 20:5n 3, and total n 6 PUFA were observed in the total FA of shrimp tissue. Gong et al. (2000) observed a significant interaction between dietary PL and cholesterol on growth of juvenile L. vannamei. Shrimp growth was enhanced as the level of PL increased from 0% to 5% of diet, but as the level of dietary cholesterol increased from 0% to 0.4% of diet, the growth-promoting effect of PL diminished. Kontara et al. (1997) proposed that PL may possibly improve the utilization efficacy of EFA supplied in the diet as neutral lipid, mostly triacylglycerol,

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and thus reduce the quantitative requirements for n 3 HUFA in shrimp diets. The purpose of this study was to evaluate the effect of PL and different kinds of dietary lipid on growth, survival, and fatty acid composition of hepatopancreas and muscle tissue of juvenile L. vannamei.

2. Materials and methods 2.1. Diets Twelve semi-purified diets were tested in total (Table 1). All diets contained 35% crude protein, 4% crude fiber, and 0.97% available phosphorous. A 26 experimental design with two PL levels (0% and 3.1%) and five different lipid sources, including one control with no oil was applied. The lipid sources were coconut oil, soybean oil, linseed oil, peanut oil, and menhaden oil. Each one of them showed a characteristic fatty acid profile, for instance, coconut oil was rich in saturated FA, particularly lauric acid (12:0); peanut oil was rich in the monounsaturated oleic acid (18:1n 9); linseed oil was rich in the n 3 polyunsaturated LNA, whereas menhaden oil was rich in n 3 HUFA (e.g., 20:5n 3, 22:5n 3, 22:6n 3); soybean oil was rich in the n 6 polyunsaturated LOA, and so was soybean lecithin, the source of PL. Five diets contained 5% of each oil and 3.1% de-oiled lecithin, which contained 97.6% acetone insolubles, with 25.7% phosphatidylethanolamine (PE), 21.7% PC, and 8.8% PI (Riceland Foods, Stuttgart, AR). Five additional diets contained 5% oil but no PL; instead, they were supplemented with 0.41% monobasic sodium phosphate, 0.48% choline chloride, and 2.21% soybean oil, which were the estimated equivalents of soybean oil, phosphorus and choline provided by the 3.1% lecithin supplement. A control diet containing no supplemental oil, but an increased level of wheat starch (41.06% instead of 36.06%) and 3.1% lecithin was used as the conditioning diet. An additional control diet contained no oil or PL, but had an increased level of wheat starch and supplemental monobasic sodium phosphate, choline chloride, and soybean oil to replace PL (Table 1). Diets were prepared by cold extrusion with a Hobart A-200 extruder (Hobart, Troy, OH) and dried overnight at 45
C. Pellets were then ground to different sizes appropriate for juvenile shrimp, and stored frozen ( 20
C) until used. 2.2. Experimental system An 8-week growth trial was conducted in two indoor recirculating water systems with 8% daily water exchange. A total of 96 square plastic tanks (bottom area 0.09 m2, 32 l) in each system were used, with a water exchange rate of 0.6 l/min/tank. A randomized block design with systems as block was used, and each dietary treatment was assigned to eight tanks in each system. A 12-h light and 12-h dark photoperiod was provided by fluorescent lights controlled by timers. Temperature and salinity were controlled at 32.8F1.0
C and 24.8F1.0x, respectively. Dissolved oxygen averaged 5.28F0.22 mg/ l. Ammonia, nitrite, nitrate and pH were monitored weekly, and averaged 0.10F0.08 mg

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Table 1 Composition of experimental dietsa Ingredients Wheat starchb Soybean protein isolatedb Casein vitamin freec Mineral mixtureb Wheat glutenb Carboxymethyl celluloseb Monobasic sodium phosphated Krill meale Fish solublesf Cholesterolb Vitamin mixtureg Methionineb Lysineb Arginineb Vitamin C stableh Zinc sulfateb Cupric chlorided Soybean lecithini Soybean oilb Choline chlorided Test oilj Total

Percent (%) PL+test oil

Test oil

PL+control

Control

36.06 24.79 6.63 6.00 6.00 4.00 2.53 2.00 2.00 0.50 0.50 0.35 0.15 0.02 0.30 0.06 0.01 3.10 – – 5.00 100.00

36.06 24.79 6.63 6.00 6.00 4.00 2.94 2.00 2.00 0.50 0.50 0.35 0.15 0.02 0.30 0.06 0.01 – 2.21 0.48 5.00 100.00

41.06 24.79 6.63 6.00 6.00 4.00 2.53 2.00 2.00 0.50 0.50 0.35 0.15 0.02 0.30 0.06 0.01 3.10 – – – 100.00

41.06 24.79 6.63 6.00 6.00 4.00 2.94 2.00 2.00 0.50 0.50 0.35 0.15 0.02 0.30 0.06 0.01 – 2.21 0.48 – 100.00

a

Values represent % of dry weight. ICN Pharmaceutical, Costa Mesa, CA, USA. The mineral mixture had the following composition (g/kg): Calcium phosphate dibasic, 500.0; chromium potassium sulfate, 0.55; cupric carbonate, 0.3; ferric citrate, 6.0; manganous carbonate, 3.5; magnesium oxide, 24.0; potassium citrate monohydrate, 220.0; potassium iodate, 0.01; potassium sulfate, 52.0; sodium chloride, 74.0; sodium selenite, 0.01; sucrose, 118.0; zinc carbonate, 1.6. c United States Biochemical, Cleveland, OH, USA. d Fisher Scientific, Fair Lawn, NJ, USA. e Inual, Santiago, Chile. f Omega Protein, Reedville, VA, USA. g Dawes Laboratories, Arlington Heights, IL, USA. The vitamin mixture had the following composition (g/kg): Retinol (A), 22.2; cholecalciferol (D), 1.1; tocopherol (E), 10.3; menadione (K), 2.3; ascorbic acid (C), 300.0; thiamin (B1), 5.0; riboflavin (B2), 5.7; pyridoxine (B6), 10.1; niacin, 10.9; pantothenic acid, 10.9; biotin, 0.2; choline, 0.8; folic acid, 3.5; cyanocobalamine (B12), 0.02; dextrin, 617.0. h Roche Vitamins and Fine Chemicals, Pendergrass, GA, USA. i Riceland Foods, Suttgart, AR, USA. j Test oil: Coconut oilb, Soybean oilb, Linseed oilb, Peanut oilk, Menhaden oilk. k SIGMA Chemical Co., St. Louis, MI, USA. b

NH4 –N/l, 0.06F0.04 mg NO2 – N/l, 5.76F1.16 mg NO3 – N/l, and 8.05F0.07, respectively. 2.3. Experimental shrimp, feeding and maintenance Specific-pathogen-free L. vannamei postlarvae were obtained from Harlingen Shrimp Farm (Los Fresnos, Texas). They were fed live Artemia nauplii twice a day and a

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commercial postlarval feed (Rangen, Buhl, ID) for approximately 3 weeks while acclimating to laboratory conditions at the Nutrition Laboratory of the Texas A&M University System Shrimp Mariculture Project (Port Aransas, TX). For an additional week, they were fed the conditioning diet containing no oil but 3.1% soybean lecithin. After conditioning, shrimp were stocked into 32-l tanks at a density of four shrimp per tank. Shrimp, very similar in size, were blotted dry, weighed as a group, and stocked in the tanks; average initial group weight was 3.57 gF0.23 SD, with no significant differences among treatments. Initial and final weights were calculated by dividing the group weight by the number of shrimp weighed. The experimental diets were fed to shrimp 15 times a day using automatic feeders, and the feeding rate adjusted to provide feed slightly in excess. Uneaten feed, fecal waste and molt exuviae were daily removed before the first feeding. After termination of the feeding trial, shrimp were stored under nitrogen at 80
C, and prior to lipid analysis they were thawed and pooled into three composite samples per dietary treatment. Samples of hepatopancreas (mid-gut gland) and tail muscle were removed. Each sample consisted of tissues from three shrimp. 2.4. Lipid analyses Samples of hepatopancreas and muscle tissue were analyzed for lipid and fatty acid composition; duplicate samples of each diet were also analyzed. Lipids were extracted by the method of Folch et al. (1957) and quantified gravimetrically after drying an aliquot under nitrogen. Total lipid content was expressed as percent of wet tissue or dry diet. Lipid fractions were separated and quantified using an Iatroscan MK-5 TLC/ FID analyzer according to the method of Fraser et al. (1985) with minor modification (Gong et al., 2000). Lipid class analysis of the test diets, shrimp muscle tissue, and

Table 2 Total lipid, triglycerides (TG), phosphatidylcholine (PC), and other phospholipids (Other PL) of test diets supplemented with different lipid sources in the presence or absence of lecithina Diet

Total lipid (%)

TG (mg/g diet)

PC (mg/g diet)

Other PL (mg/g diet)

PL/Menhaden PL/Coconut PL/Soybean PL/Linseed PL/Peanut PL/Control Menhaden Coconut Soybean Linseed Peanut Control

10.38 10.54 10.87 10.38 10.69 5.88 10.53 10.62 10.38 10.74 10.35 5.97

70.56 75.22 86.01 72.77 82.99 7.49 96.87 108.83 111.81 115.80 120.70 45.15

6.96 7.20 7.69 6.87 7.39 7.28 3.92 3.86 3.86 3.66 3.80 2.53

11.32 12.37 12.23 12.08 11.41 14.91 4.89 4.09 4.40 3.18 4.59 3.67

a

Values represent averages of duplicate samples.

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Table 3 Fatty acid composition (% of total) of experimental dietsa,b Fatty acid

Experimental diets PL/ Menhaden

PL/ Coconut

PL/ Soybean

PL/ Linseed

PL/ Peanut

PL/ Control

8:0 10:0 12:0 14:0 14:1 16:0 16:1 16:2 16:3 16:4 18:0 18:1d 18:2n 6 18:3n 6 18:3n 3 18:4n 3 20:0 20:1 20:2n 6 20:3n 6 20:4n 6 20:3n 3 22:0 20:5n 3 22:1 22:2 22:3 22:4 22:5n 3 22:6n 3 Saturatese Monounsaturatesf PUFA and HUFAg Total n 3h Total n 6i

–c – 0.11 5.26 0.24 20.33 6.41 1.25 0.92 0.51 3.87 12.11 21.26 0.31 3.46 0.34 2.11 1.15 0.30 0.18 0.45 0.20 1.03 7.08 1.41 0.42 0.06 0.27 1.34 7.63 32.71 21.32 45.97 20.04 22.50

2.03 3.97 31.22 11.52 – 13.09 0.45 0.12 0.10 – 3.18 9.70 20.07 – 2.39 – 0.10 0.17 – – – – – 0.71 0.62 – – – 0.12 0.42 65.12 10.94 23.94 3.65 20.07

– – 0.00 0.50 – 13.93 0.48 0.11 0.17 – 4.48 19.69 51.33 – 6.80 – 0.15 0.30 – – – – – 0.73 0.42 0.14 – – 0.27 0.49 19.06 20.90 60.04 8.29 51.33

– – 0.05 0.45 – 11.28 0.49 0.13 0.13 – 3.72 17.78 28.64 – 34.85 – 0.11 0.31 – – – – – 0.66 0.45 0.14 – – 0.31 0.51 15.60 19.02 65.37 36.33 28.64

– – – 0.41 – 14.20 0.49 0.10 0.16 – 0.83 38.65 37.98 – 2.74 0.05 0.10 0.99 0.05 – 0.03 – – 0.74 0.56 0.14 – – 1.27 0.52 15.54 40.70 43.77 5.31 38.06

– – – 1.32 – 20.52 1.13 0.31 0.27 – 3.96 14.13 46.86 – 5.78 – 0.10 0.30 – – – – 0.33 2.30 0.92 0.12 – – 0.47 1.18 26.22 16.48 57.30 9.74 46.86

Fatty acid

Experimental diets Linseed

Peanut

Control

– – 0.05 0.47 – 9.29 0.46 0.11

– – 0.04 0.43 – 12.15 0.46 0.08

– – 0.06 0.99 – 14.27 0.84 0.25

8:0 10:0 12:0 14:0 14:1 16:0 16:1 16:2

Menhaden

Coconut

Soybean

–c – 0.11 4.50 0.20 16.33 5.60 1.10

2.01 3.72 28.96 10.88 – 11.23 0.41 0.11

– – 0.12 0.53 – 12.26 0.45 0.11

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Table 3 (continued) Fatty acid

16:3 16:4 18:0 18:1d 18:2n 6 18:3n 6 18:3n 3 18:4n 3 20:0 20:1 20:2n 6 20:3n 6 20:4n 6 20:3n 3 22:0 20:5n 3 22:1 22:2 22:3 22:4 22:5n 3 22:6n 3 Saturatese Monounsaturatesf PUFA and HUFAg Total n 3h Total n 6i

Experimental diets Menhaden

Coconut

Soybean

Linseed

Peanut

Control

0.81 0.44 3.78 16.21 20.16 0.25 3.41 0.32 1.95 1.39 0.32 0.28 0.70 0.43 1.38 7.72 1.17 0.48 0.30 0.36 1.56 8.76 28.05 24.56 47.39 22.20 21.71

0.09 – 3.45 13.53 20.86 – 2.52 – 0.04 0.21 – – 0.08 – 0.11 0.74 0.33 0.04 0.05 – 0.17 0.45 60.40 14.50 25.10 3.88 20.94

0.17 – 4.38 22.85 50.11 – 6.64 – 0.00 0.33 – – – – 0.17 0.71 0.39 – – – 0.21 0.56 17.47 24.02 58.52 8.12 50.11

0.13 – 4.04 20.73 29.17 – 32.85 – 0.13 0.32 0.07 – 0.04 0.07 0.10 0.79 0.45 0.08 – – 0.22 0.43 14.07 21.97 63.96 34.37 29.28

0.14 0.04 0.49 39.04 38.77 – 3.28 – 0.12 0.86 0.05 0.04 0.05 – 0.14 1.77 0.47 0.09 0.07 – 0.96 0.47 13.37 40.82 45.80 6.48 38.90

0.18 – 4.37 22.21 46.55 – 5.96 – 0.14 0.38 0.06 – 0.06 – 0.17 1.46 0.75 0.14 – – 0.33 0.84 20.00 24.18 55.82 8.59 46.67

a

Values represent averages of duplicate samples. Fatty acid values (% of total fatty acid methyl esters) were adjusted to express a percent of the total area identified in the chromatograms, unidentified peaks were not considered in the computations. c – = 0.0, not detected. d 18:1 is mostly oleic acid (18:1n 9), but may possibly include small quantities of 18:1n 11. e Saturates: 8:0, 10:0, 12:0, 14:0, 16:0, 18:0, 20:0, 22:0. f Monounsaturates: 14:1, 16:1, 18:1, 20:1, 22:1. g PUFA and HUFA: 16:2, 16:3, 16:4, 18:2n 6, 18:3n 6, 18:3n 3, 18:4n 3, 20:2n 6, 20:3n 6, 20:4:n 6, 20:3n 3, 20:5n 3, 22:2, 22:3, 22:4, 22:5n 3, 22:6n 3. h Total n 3: 18:3n 3, 18:4n 3, 20:3n 3, 20:5n 3, 22:5n 3, 22:6n 3. i Total n 6: 18:2n 6, 18:3n 6, 20:2n 6, 20:3n 6, 20:4n 6. b

hepatopancreas separately quantified PC and other phospholipids (other PL), TG, free FA (FFA), and cholesterol (CHOL). Because PL fractions were not well separated, except for PC, the other PL fraction included PE, PI and/or others. Concentration of lipid fractions was expressed as mg per g of wet tissue or mg per g of dry diet. FA were transesterified with boron trifluoride and fatty acid methyl esters (FAME) were analyzed with a Varian 3400 gas chromatograph equipped with an autosampler, a 30 m1.53 mm Supelcowaxk fused silica capillary column, and a flame-ionization de-

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Table 4 Initial and final weight, instantaneous growth rate (IGR), survival, and feed conversion ratio (FCR) of juvenile L. vannamei fed different oils in the presence or absence of phospholipids (PL)1 Dietary treatment PL/Menhaden PL/Coconut PL/Soybean PL/Linseed PL/Peanut PL/Control Menhaden Coconut Soybean Linseed Peanut Control ANOVA (Pr > F) System PL Oil type PLOil type

Initial weight (g)

Final weight (g) a

IGR (%/day) a

Survival (%)

FCR2

1.00F0.28 0.92F0.12 0.94F0.20 0.91F0.09 0.95F0.14 0.93F0.14 0.92F0.14 0.97F0.25 0.98F0.25 0.93F0.10 0.98F0.16 0.94F0.12

12.03F0.68 9.71F0.82cd 9.82F0.65cd 9.43F0.68de 9.49F0.72de 10.05F0.72c 10.75F0.85b 8.97F0.59ef 9.01F0.59ef 9.02F0.65ef 8.51F0.48f 8.97F0.77ef

4.56F0.38 4.29F0.19bcd 4.29F0.29bcd 4.26F0.17cde 4.19F0.21cde 4.34F0.24bc 4.47F0.21ab 4.07F0.38ef 4.06F0.28ef 4.14F0.15cdef 3.95F0.24f 4.10F0.23def

95.3F10.1 94.8F11.3 98.4F6.3 95.3F10.1 96.9F8.5 93.8F11.2 100F0.0 93.8F11.2 95.0F14.0 96.4F10.1 100F0.0 94.8F11.3

1.06 1.25 1.23 1.28 1.30 1.27 1.14 1.44 1.52 1.35 1.44 1.39

0.1308 0.6879 0.8765 0.7262

0.1011 < 0.0001 < 0.0001 0.1618

0.4895 < 0.0001 < 0.0001 0.7712

0.9514 0.5027 0.4157 0.6708

– – – –

1 Values are means of 16 replicatesFSD. Means within columns with the same letter are not significantly different (Duncan’s alpha = 0.05). 2 Values for each dietary treatment were estimated from FRC = Total dry feed fed (g)/Final biomass (g) Initial biomass (g).

tector as previously described (Lochmann and Gatlin, 1993). FA were identified by comparison of retention times to those of known standards and expressed as percent of the total FAME. 2.5. Statistical analysis Final weight, instantaneous growth rate (IGR), survival, and feed conversion ratio (FCR) were the indices used to evaluate shrimp performance. IGR was calculated from the

Table 5 Total lipid, triglycerides (TG), phosphatidylcholine (PC), and other phospholipids (other PL) of L. vannamei hepatopancreas and muscle tissue before conditioning and 1 week after feeding the conditioning dieta Tissue

Total lipid (%)

TG (mg/g wet tissue)

PC (mg/g wet tissue)

Other PL (mg/g wet tissue)

Before conditioning Hepatopancreas Muscle

2.93 1.58

4.22 0.16

6.72 7.31

4.01 3.46

1 week conditioning Hepatopancreas Muscle

2.36 1.36

1.61 0.13

7.94 6.04

4.49 3.14

a

Values represent a single pooled sample of 9 shrimp at each time point.

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Table 6 Total lipid, triglycerides (TG), phosphatidylcholine (PC), and other phospholipids (other PL) of hepatopancreas from L. vannamei fed diets supplemented with different lipid sources in the presence or absence of phospholipids (PL)1 Dietary treatment

Total lipid (%)

TG (mg/g wet tissue)

PC (mg/g wet tissue)

Other PL (mg/g wet tissue)

PL/Menhaden PL/Coconut PL/Soybean PL/Linseed PL/Peanut PL/Control Menhaden Coconut Soybean Linseed Peanut Control ANOVA Pr > F PL Oil type PLOil type

10.96F3.94 11.39F1.96 9.73F2.54 11.32F4.40 10.86F1.94 5.60F2.50 11.50F4.50 7.44F2.38 10.61F2.88 10.05F2.65 12.26F3.56 7.31F1.73

51.98F26.16bcd 54.90F6.87abcd 58.85F36.75abcd 45.50F15.94cd 67.64F14.01abcd 23.06F8.39d 101.41F46.07a 73.26F24.26abc 85.21F22.22abc 64.25F25.67abcd 98.23F25.30ab 36.99F7.96cd

4.79F0.94 5.49F0.09 4.61F0.99 5.40F0.90 5.07F0.77 4.29F0.57 5.90F0.42 4.68F0.73 5.05F0.57 5.07F0.99 5.40F1.43 4.37F0.49

8.02F2.70 8.71F0.34 7.02F2.04 8.16F2.83 6.66F2.01 6.74F0.87 8.49F0.37 6.60F1.76 6.91F1.85 7.82F2.39 7.81F6.29 6.49F0.52

0.9117 0.0848 0.6003

0.0048 0.0206 0.8424

0.6224 0.2935 0.4611

0.7995 0.8164 0.8810

1 Values represent means of three composite samples each consisting of tissue from three shrimpFSD. Means within columns with the same letter are not significantly different (Duncan’s alpha = 0.05).

Table 7 Total lipid, phosphatidylcholine (PC), and other phospholipids (other PL) of muscle tissue from L. vannamei fed diets supplemented with different lipid sources in the presence or absence of phospholipids (PL)1 Dietary treatment

Total lipid (%)

PC (mg/g wet tissue)

Other PL (mg/g wet tissue)

PL/Menhaden PL/Coconut PL/Soybean PL/Linseed PL/Peanut PL/Control Menhaden Coconut Soybean Linseed Peanut Control ANOVA Pr>F PL Oil type PLOil type

1.36F0.06ab 1.52F0.04a 1.43F0.19ab 1.25F0.06b 1.39F0.14ab 1.26F0.08b 1.51F0.15a 1.37F0.05ab 1.36F0.06ab 1.24F0.12b 1.33F0.05ab 1.26F0.06b

6.87F1.36 6.23F0.84 6.38F0.97 6.25F2.18 5.91F0.56 5.95F0.70 6.89F0.40 6.84F0.94 6.26F0.29 5.94F0.83 6.41F0.69 6.43F0.75

3.31F0.90 3.29F0.25 3.25F0.48 3.30F1.01 2.84F0.22 2.65F0.42 3.38F0.15 3.20F0.50 2.95F0.15 3.03F0.40 3.09F0.66 2.92F0.15

0.5108 0.0065 0.2474

0.5612 0.7837 0.9473

0.9518 0.4854 0.8599

1 Values represent means of three composite samples each consisting of tissue from three shrimpFSD. Means within columns with the same letter are not significantly different (Duncan’s alpha = 0.05).

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Table 8 Fatty acid composition (% of total) of L. vannamei hepatopancreas1,2 Fatty acid

Dietary treatments PL/ Menhaden**

PL/ Coconut

PL/ Soybean

PL/ Linseed**

PL/ Peanut

PL/ Control**

10:0 12:0 14:0 14:1 16:0 16:1 16:2 16:3 16:4 18:0 18:14 18:2n 6 18:3n 6 18:3n 3 18:4n 3 20:0 20:1 20:2n 6 20:3n 6 20:4n 6 20:3n 3 22:0 20:5n 3 22:1 22:2 22:3 22:4 22:5n 3 22:6n 3 Saturates5 Monounsaturates6 PUFA and HUFA7 Total n 38 Total n 69

–3 – 2.46c 0.21a 23.26bc 5.01a 0.63a 0.45a 0.10 3.52e 17.92fgh 21.74f 0.37a 2.22e 0.46a 0.66a 2.02a 2.12bc 0.37a 0.57a 0.48bcd 1.32a 5.31a 0.17 0.37a 0.18a 0.22a 1.22a 6.66a 31.22c 25.33cd 43.45c 16.34c 25.17e

0.16a 8.01a 7.36a 0.19ab 25.78a 2.99b 0.04b 0.27bc 0.16 4.63cde 19.09efg 24.44e – 2.23e 0.19d – 0.95cd 1.41d 0.11de 0.16bc 0.22ef – 0.80c 0.07 0.10bcd – – 0.07e 0.58d 45.93a 23.30de 30.77e 4.08f 26.12e

0.03c – 0.33e 0.09cd 15.41efg 0.49d 0.01b 0.15d 0.16 7.09ab 17.39fgh 47.45a – 4.57c – 0.05b 0.83d 3.55a 0.10de 0.14bc 0.64b 0.01d 0.74c 0.09 0.09bcd – – 0.08de 0.50d 22.93ef 18.89f 58.18a 6.53d 51.24a

0.02c 0.05d 0.31e 0.12abcd 14.56fg 0.96d – 0.18cd 0.10 8.88a 16.10gh 28.92d – 21.21a – – 0.87d 2.38b 0.09de 0.15bc 3.51a 0.02cd 0.74c 0.09 0.09bcd 0.03bc – 0.10de 0.51d 23.84e 18.14f 58.02a 26.07a 31.54d

0.02c – 0.32e 0.09cd 16.59ef 0.68d – 0.22bcd 0.17 – 39.35a 35.09c – 1.91e 0.13e – 1.20b 2.22bc 0.11de 0.13c 0.24ef – 0.61c 0.12 0.11bcd – – 0.25c 0.43d 16.94g 41.44a 41.62c 3.57f 37.54c

– 0.05d 0.83de 0.16abcd 24.15ab 2.49bc 0.08b 0.28b 0.17 6.47bc 15.08h 39.43b – 3.64cde 0.21c 0.09b 0.83d 2.42b 0.17c 0.23b 0.38def 0.07c 1.27b 0.14 0.15b 0.10ab 0.05b 0.18cd 0.85c 31.67c 18.71f 49.62b 6.54d 42.26b

Fatty acid

Dietary treatments Soybean**

Linseed

Peanut

Control**

Menhaden 10:0 12:0 14:0 14:1 16:0 16:1 16:2 16:3 16:4

3

– – 2.14c 0.21a 21.66cd 4.50a 0.54a 0.39a 0.21

Coconut** b

0.09 4.21b 3.95b 0.18abc 21.21cd 2.42bc – 0.18bcd 0.21

– 0.03d 0.37e 0.11bcd 16.03efg 0.65d – 0.17cd 0.09

– 0.01d 0.32e 0.08d 13.55g 0.56d 0.01b 0.15d 0.06

c

0.03 1.32c 1.27d 0.08cd 17.61e 1.00d 0.01b 0.20bcd 0.01

– 0.06d 0.70de 0.08cd 20.60d 2.17c 0.07b 0.26bc 0.15

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Table 8 (continued) Fatty acid

Dietary treatments Menhaden

18:0 18:14 18:2n 6 18:3n 6 18:3n 3 18:4n 3 20:0 20:1 20:2n 6 20:3n 6 20:4n 6 20:3n 3 22:0 20:5n 3 22:1 22:2 22:3 22:4 22:5n 3 22:6n 3 Saturates5 Monounsaturates6 PUFA and HUFA7 Total n 38 Total n 69

de

4.03 20.50ef 22.43ef 0.32b 2.12e 0.39b 0.60a 1.92a 2.48b 0.32b 0.55a 0.46bcde 1.15b 5.07a 0.20 0.31a 0.19a 0.21a 1.06b 6.03b 29.59cd 27.32c 43.08c 15.13c 26.10e

Coconut** bcde

5.43 24.56cd 29.68d – 2.66de – – 1.07bc 1.69cd 0.12de 0.17bc 0.21f – 0.90c 0.10 0.13bc 0.07bc – 0.23c 0.52d 34.89b 28.34c 36.77d 4.53ef 31.65d

Soybean** bcd

5.81 18.68efgh 46.85a – 4.28cd – – 0.96cd 3.60a 0.08ef 0.13c 0.62bc – 0.76c 0.10 0.06cd – – 0.08de 0.54d 22.24ef 20.50ef 57.26a 6.28d 50.66a

Linseed f

1.07 26.68c 29.96d – 19.04b – 0.02b 1.06bc 2.62b 0.04f 0.11c 3.30a 0.01d 0.67c 0.11 0.04d – – 0.09de 0.46d 14.98g 28.48c 56.54a 23.54b 32.74d

Peanut f

0.10 35.80b 34.69c – 2.31e – 0.01b 1.17b 2.29bc 0.08ef 0.11c 0.28def 0.01d 0.63c 0.17 0.08bcd 0.08bc 0.02bc 0.19cd 0.45d 20.36f 38.23b 41.41c 3.85f 37.17c

Control** 6.09bcd 21.70de 37.56b – 3.46cde – 0.04b 1.08bc 2.72b 0.13cd 0.18bc 0.40cdef 0.03cd 1.19b 0.17 0.12bc 0.10ab – 0.12de 0.83c 27.51d 25.21cd 47.28b 6.00de 40.59b

1 Values represent means of three composite samples or two (**) where indicated, each consisting of tissue from three shrimp. Means within rows with the same letter are not significantly different (Duncan’s alpha = 0.05). 2 Fatty acid values (% of total fatty acid methyl esters) were adjusted to express a percent of the total area identified in the chromatograms, unidentified peaks were not considered in the computations. 3 – = 0.0, not detected. Means with the same letter are not significantly different (Duncan’s alpha = 0.05). 4 18:1 is mostly oleic acid (18:1n 9), but may possibly include small quantities of 18:1n 11. 5 Saturates: 10:0, 12:0, 14:0, 16:0, 18:0, 20:0, 22:0. 6 Monounsaturates: 14:1, 16:1, 18:1, 20:1, 22:1. 7 PUFA and HUFA: 16:2, 16:3, 16:4, 18:2n 6, 18:3n 6, 18:3n 3, 18:4n 3, 20:2n 6, 20:3n 6, 20:4:n 6, 20:3n 3, 20:5n 3, 22:2, 22:3, 22:4, 22:5n 3, 22:6n 3. 8 Total n 3: 18:3n 3, 18:4n 3, 20:3n 3, 20:5n 3, 22:5n 3, 22:6n 3. 9 Total n 6: 18:2n 6, 18:3n 6, 20:2n 6, 20:3n 6, 20:4n 6.

equation: IGR = 100[ln (Final weight/Initial weight)]/Duration of feeding trial in days (Cushing, 1968). Survival was transformed by arcsine square root before statistical analysis. The FCR for each dietary treatment was estimated from FRC = Total dry feed fed/(Final biomass Initial biomass). Data were analyzed by three-way factorial ANOVA using system as a block factor to determine the effects of PL and oil type and their interaction. In the absence of interactions, one-way ANOVA of single factors was performed. Duncan’s multiple range test was used as the mean separation procedure

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Table 9 Fatty acid composition (% of total) of L. vannamei muscle tissue1,2 Fatty acid

Dietary treatments PL/ Menhaden

PL/ Coconut

PL/ Soybean**

PL/ Linseed

PL/ Peanut**

PL/ Control

12:0 14:0 14:1 16:0 16:1 16:2 16:3 16:4 18:0 18:14 18:2n 6 18:3n 6 18:3n 3 18:4n 3 20:0 20:1 20:2n 6 20:3n 6 20:4n 6 20:3n 3 22:0 20:5n 3 22:1 22:2 22:3 22:4 22:5n 3 22:6n 3 Saturates5 Monounsaturates6 PUFA and HUFA7 Total n 38 Total n 69

–3 0.88c 0.12 21.96b 2.01a 0.24a 0.31 2.61a 11.69 13.16bcd 15.45g 0.16a 1.19gh 0.20bc 0.17a 0.82a 1.59g 0.14a 1.52a 0.30gh 0.28a 14.76a 0.22c 0.18a 0.03 0.15a 0.68a 9.20a 34.99abcd 16.32cde 48.69de 26.32a 18.87g

0.49a 2.43a 0.10 21.28b 1.32c 0.02b 0.30 2.35ab 12.42 12.71bcd 28.36e – 2.44de 0.28b 0.01b 0.49cd 1.87f 0.08abc 0.88b 0.27gh 0.12b 6.81b 0.23c 0.13ab – 0.11ab 0.33cd 4.17b 36.75a 14.84cde 48.41de 14.32c 31.19e

– 0.31c 0.19 19.43d 0.26e – 0.22 2.05abc 11.50 12.81bcd 36.35a – 3.22c 0.13cd – 0.36ef 3.14ab 0.05abc 0.81b 0.51c 0.04bc 4.82c 0.53bc 0.12ab – 0.11ab 0.31cd 2.74c 31.28def 14.14de 54.58a 11.72de 40.36a

0.03c 0.34c 0.17 18.84d 0.30e 0.01b 0.22 2.36ab 13.90 11.36d 23.16f – 14.26a – 0.01b 0.47cd 2.42e 0.06abc 0.83b 2.43a 0.04bc 4.97c 0.41bc 0.12ab – 0.06abc 0.27d 2.95c 33.16abcdef 12.71e 54.13a 24.88ab 26.48f

– 0.29c 0.16 19.64d 0.34e – 0.29 1.98abc 11.80 19.50a 30.79d – 1.64fg 0.14c – 0.68b 2.35e 0.11ab 0.75b 0.22h 0.04bc 5.33c 0.31c 0.12ab – 0.10abc 0.34cd 3.08c 31.77cdef 20.98ab 47.24ef 10.76e 33.99d

– 0.37c 0.27 23.50a 1.17d – 0.33 1.18bc 10.50 11.60cd 35.58a – 3.09cd 0.21bc – 0.35f 2.52de 0.11ab 0.86b 0.38ef 0.09bc 5.51c 0.46bc 0.18a – 0.08abc 0.40c 3.07c 34.46abcde 13.85de 53.50ab 12.66d 39.07b

Fatty acid

Dietary treatments

12:0 14:0 14:1 16:0 16:1 16:2 16:3 16:4 18:0 18:14

Menhaden**

Coconut

Soybean**

Linseed

Peanut

Control

–3 0.88c 0.11 21.60b 1.85b 0.22a 0.32 2.46ab 12.84 13.74bcd

0.19b 1.50b 0.11 20.92bc 1.12d – 0.27 1.23bc 12.52 13.53bcd

– 0.45c 0.34 19.42d 0.27e – 0.26 0.96c 10.97 15.03bc

– 0.45c 0.33 18.69d 0.28e – 0.29 0.94c 10.73 15.92b

– 0.50c 0.33 19.85cd 0.34e – 0.27 0.81c 9.63 21.19a

– 0.36c 0.27 21.84b 1.10d – 0.35 1.20bc 9.90 15.17b

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Table 9 (continued) Fatty acid

Dietary treatments Menhaden**

18:2n 6 18:3n 6 18:3n 3 18:4n 3 20:0 20:1 20:2n 6 20:3n 6 20:4n 6 20:3n 3 22:0 20:5n 3 22:1 22:2 22:3 22:4 22:5n 3 22:6n 3 Saturates5 Monounsaturates6 PUFA and HUFA7 Total n 38 Total n 69

h

13.70 0.16a 0.96h 0.21bc 0.17a 0.89a 1.98f 0.14a 1.57a 0.32fg 0.24a 14.85a 0.53bc 0.17a – 0.14ab 0.56b 9.40a 35.73ab 17.12cd 47.15ef 26.29a 17.55h

Coconut c

32.64 – 2.89cd 0.43a – 0.48cd 2.08f 0.03bc 0.80b 0.32fg 0.08bc 4.74c 0.68abc 0.17a 0.01 0.09abc 0.34cd 2.83c 35.21abc 15.93cde 48.87de 11.56de 35.54c

Soybean** b

34.12 – 3.08cd – – 0.43def 3.20a – 0.85b 0.46cd – 5.23c 1.46a – – – 0.33cd 3.14c 30.85ef 17.52bcd 51.63c 12.24de 38.17b

Linseed f

23.52 – 13.16b – – 0.54c 2.68cd – 0.76b 2.23b 0.03bc 4.94c 1.11abc 0.04bc – 0.04bc 0.33cd 3.00c 29.89f 18.18bc 51.93bc 23.66b 26.96f

Peanut d

30.61 – 1.89ef 0.11cd – 0.65b 2.86c – 0.79b 0.28gh – 5.04c 1.28ab 0.04bc – 0.04bc 0.35cd 3.16c 29.98f 23.78a 46.24f 10.83e 34.26d

Control 32.01c – 2.72cd 0.12cd – 0.44de 2.92bc – 0.85b 0.43de 0.05bc 5.60c 1.05abc 0.05bc – – 0.34cd 3.25c 32.15bcdef 18.02bc 49.83d 12.46d 35.78c

1 Values represent means of three composite samples or two (**) where indicated, each consisting of tissue from three shrimp. Means within rows with the same letter are not significantly different (Duncan’s alpha = 0.05). 2 Fatty acid values (% of total fatty acid methyl esters) were adjusted to express a percent of the total area identified in the chromatograms, unidentified peaks were not considered in the computations. 3 – = 0.0, not detected. Means with the same letter are not significantly different (Duncan’s alpha = 0.05). 4 18:1 is mostly oleic acid (18:1n 9), but may possibly include small quantities of 18:1n 11. 5 Saturates: 12:0, 14:0, 16:0, 18:0, 20:0, 22:0. 6 Monounsaturates: 14:1, 16:1, 18:1, 20:1, 22:1. 7 PUFA and HUFA: 16:2, 16:3, 16:4, 18:2n 6, 18:3n 6, 18:3n 3, 18:4n 3, 20:2n 6, 20:3n 6, 20:4:n 6, 20:3n 3, 20:5n 3, 22:2, 22:3, 22:4, 22:5n 3, 22:6n 3. 8 Total n 3: 18:3n 3, 18:4n 3, 20:3n 3, 20:5n 3, 22:5n 3, 22:6n 3. 9 Total n 6: 18:2n 6, 18:3n 6, 20:2n 6, 20:3n 6, 20:4n 6.

( P < 0.05). Statistical analyses were done using the SAS software package (SAS Institute, 1999– 2000).

3. Results 3.1. Lipid class and fatty acid analyses of test diets Total lipid content of experimental diets was relatively constant, therefore, they were considered isolipidic, except for the two control diets where total lipid content was

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approximately half of the other diets, as expected from their formulation. TG were the major lipid fraction in the diets, mainly contributed by the supplemented oils. This fraction decreased when PL were added to the diets because 2.21% soybean oil was excluded at the same time. In general, TG were higher in diets containing oil but no lecithin, and PC and other PL were higher in diets containing lecithin (Table 2). The test diets reflected the fatty acid profile of the oils used in their formulation. Diets with menhaden oil had a larger variety of FA compared to other diets, especially long chained HUFA (Table 3). They also contained the highest percentage (0.45%) of arachidonic acid (20:4n 6, AA), EPA (7.08%), and DHA (7.63%). 3.2. Biological performance of shrimp No significant differences among treatments were observed for survival and no significant interactions were observed for any of the responses. However, significant differences in final weight and IGR were observed at the end of the trial. Shrimp fed diets containing PL obtained significantly higher final weight and IGR, but lower FCR than those fed diets containing the same oil type but no PL. Results also showed that shrimp fed diets containing menhaden oil had significantly higher final weight and IGR, but lower FCR than those fed the rest of the dietary treatments (Table 4). 3.3. Lipid class analysis of shrimp tissues Feeding the conditioning diet to shrimp decreased total lipid content in hepatopancreas and muscle, particularly in TG of hepatopancreas, and PC and other PL of muscle, compared to preconditioned shrimp. Total lipid in hepatopancreas of preconditioned and conditioned shrimp was approximately two times higher than that of muscle (Table 5). After the 8-week feeding trial, total lipid content of hepatopancreas was considerably higher, but in the muscle, it did not change much. TG in hepatopancreas were significantly affected by dietary PL ( P = 0.0048) and oil type ( P = 0.0206), reflecting the composition of experimental diets. TG were lower in shrimp fed supplemental PL, compared to those without supplementation, and lower in control diets, compared to the rest of the dietary treatments; however, no effect of PL supplementation was observed in the PL fractions of hepatopancreas (Table 6). Total lipid content in shrimp muscle tissue was significantly affected by dietary oil ( P = 0.0065); lower lipid content was observed in shrimp fed control diets or diets supplemented with linseed oil. No effect of PL supplementation was observed in the PL fractions of muscle (Table 7). 3.4. Fatty acid analysis of shrimp tissues The fatty acid composition of the test diets was reflected to a certain extent in the fatty acid composition of hepatopancreas and muscle tissue of shrimp (Tables 8 and 9). For instance, DHA, EPA and AA were always significantly higher in tissues of shrimp fed diets with menhaden oil. Oleic acid was significantly higher in hepatopancreas and muscle tissue of shrimp fed diets with peanut oil. LNA was significantly higher in hepatopancreas and muscle tissue of shrimp fed diets with linseed oil.

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4. Discussion Recommended lipid levels for commercial shrimp feeds range from 6% to 8%, and should not exceed 10%. According to Akiyama et al. (1992), decreased shrimp growth and increased mortalities are associated with lipid levels exceeding 10%, probably due to nutrient imbalances/deficiencies as they relate to energy and quality standards for lipid sources. In the present study, the lipid level of experimental diets averaged 10.5% and was contributed not only by test lipids but also by other basal ingredients (e.g., fish solubles, krill meal, etc.); however, negative effects on growth or survival were not evident, most likely because dietary lipid levels were not excessively high for L. vannamei. In addition, the lipid level judged to be best for a number of shrimp species may be influenced by a number of factors: the quality and quantity of dietary protein, the quality, quantity, and availability of other energy sources, and the quality of the dietary oil (D’Abramo, 1997). Results of the present study showed no significant interaction between dietary PL and the supplemental oils; however, significant effects of dietary PL and the test oils on final weight and IGR were observed. Increased growth is one of the beneficial effects of supplementing PL to the diet of shrimp, and has been well documented for many species (e.g., M. japonicus: Kanazawa et al., 1979e, 1985; Teshima et al., 1982, 1986a,b; P. monodon: Piedad-Pascual, 1986; F. chinensis: Kanazawa, 1993), including L. vannamei (Coutteau et al., 1996). The present study also demonstrated that growth of juvenile L. vannamei increased significantly when soybean lecithin was included in the diet regardless of the other lipid. Survival, however, was not significantly affected; according to Coutteau et al. (1996), significant effects of PL supplementation on survival has only been demonstrated in larval M. japonicus (Kanazawa et al., 1985; Teshima et al., 1986c), as juvenile penaeid shrimp (initial wet weight, 0.09 –1 g) appear to be less sensitive to deficiency of PL. Furthermore, all water quality parameters were very stable throughout the current trial, and within the recommended ranges for commercial shrimp production (Samocha et al., 1993), which also may have contributed to high survival in this feeding trial. Results of the present study showed that shrimp fed diets containing menhaden oil, with or without PL, had significantly higher final weight, although the growth response or the FCR values were not significantly improved by adding coconut, soybean, linseed, or peanut oil, compared to the control treatments; thus, growth was not only affected by the inclusion of soybean lecithin in the diet, but also by the addition of menhaden oil, contrary to what Piedad-Pascual (1986) observed with juvenile P. monodon. They fed increasing lecithin levels and different oils like cod liver oil, crude degummed soybean oil and purified soybean oil, but weight gain was significantly affected by the levels of lecithin rather than by oil type. Lim et al. (1997) also found that menhaden oil was better utilized than linseed, soybean, corn, safflower, and coconut oils by juvenile L. vannamei in the presence of 1% soybean lecithin. Crustaceans are able to synthesize PL such as PC from phosphorylcholine, diglycerides, and PE (Shieh, 1969; Ewing and Finamore, 1970). Teshima et al. (1986b) also suggested the conversion of dietary TG to PL classes such as PC and PE in the hepatopancreas of M. japonicus. It is also known that HUFA are preferentially incorporated and conserved in the polar lipid of crustacean tissue (Clarke, 1970; Kanazawa et al.,

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1977b; D’Abramo et al., 1980; D’Abramo and Sheen, 1993). According to this, a possible explanation for higher growth and lower feed conversion values of shrimp fed diets supplemented with fish oil is that the more unsaturated nature of menhaden oil, or the better availability of HUFA in that oil compared to the others, provided the elements for the synthesis of PL that constitute shrimp tissue. On the other hand, the fact that dietary PL further improved shrimp performance, regardless of the oil type, suggests that the rate of PL synthesis for incorporation into new tissue may not meet the metabolic demands of shrimp and/or specific PL that are used as constituents of lipoproteins for transport of cholesterol and other lipids, may not be sufficiently synthesized (Teshima, 1997). In addition, dietary PL has been shown to improve the properties of artificial diets by markedly reducing the leaching of watersoluble nutrients, in particular manganese and B vitamins (Castell et al., 1991), and also may act as emulsifiers, facilitating the digestion and absorption of FA, bile salts and other lipid-soluble substances (Coutteau et al., 1997), thus contributing to the superior performance of shrimp. However, even the inclusion of PL to diets with either coconut, soybean, peanut or linseed oils, did not improved the performance of shrimp compared to those fed the diet with menhaden oil but no PL, so probably as important as the presence of PL for achieving maximum growth and survival, is the presence of HUFA. According to Kanazawa et al. (1985), effective PL for shrimp are characterized by the possession of choline or inositol groups in addition to unsaturated fatty acid moieties. They observed that soybean PC, bonito-egg PC, and PI containing high proportions of n 6 and n 3 EFA such as LOA, LNA, or DHA were effective in improving growth and survival of M. japonicus. Teshima et al. (1986b) also suggested that the presence of PC containing EPA and DHA at the b-position in the hemolymph of M. japonicus indicated the importance of this PL class in lipid transport. Thus, shrimp fed the PL-control diet performed as well, or better than the rest of the dietary treatments, except for the menhaden oil treatments, because, in spite of no addition of oil to this diet, the formulation not only included 3.1% of PL, but also 2% of fish solubles and 2% of krill meal, all these ingredients could have provided EFA and helped improving the performance of shrimp, confirming that dietary PL may serve as a source of choline, inositol, EFA or even energy for early stages of crustaceans (Coutteau et al., 1997), and could potentially reduce the dietary requirement for additional EFA sources. The fatty acid composition of the test diets was reflected to a certain extent in the fatty acid composition of hepatopancreas and muscle tissue. These results are in agreement with other studies reporting that the fatty acid pattern of shrimp tissue reflects that of dietary lipids (P. monodon: Deering et al., 1997; Millamena, 1989; F. indicus: Colvin, 1976; M. japonicus: Guary et al., 1976; Kayama et al., 1980). The fatty acid composition of the test diets, hepatopancreas, and muscle tissue looked very much alike. However, certain FA appeared to be actively synthesized and/or retained, because they were present in small amounts in some diets and hepatopancreas, but in relatively higher amounts in muscle tissue, such as the case of AA, EPA and DHA. In contrast, lauric acid, which was abundant in coconut oil diets, was limited in muscle tissue. Sparing or retention of specific HUFA at the expense of saturated and monounsaturated FA has been demonstrated in other shrimp. In a study with F. chinensis, Xu et al. (1994) suggested that the relatively high levels of HUFA such as AA, EPA and DHA in the body lipids of shrimp fed essential fatty acid-free

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diets were probably the result of preferential utilization of short- and medium-chained FA as energy sources for metabolism rather than an increase in the absolute content of the HUFA. Deering et al. (1997) also reported that AA, EPA, and DHA occurred at slightly higher levels in the lipids of P. monodon than in the diets, particularly in polar lipids, which could be an indication of low levels of chain elongation and desaturation, but may also reflect the use of shorter-chain FA in energy metabolism and selective retention of longer-chain unsaturates. Araujo and Lawrence (1991) suggested that juvenile (4– 6 g) L. vannamei was capable of elongating LNA to synthesize EPA and DHA when fed a diet supplemented with linseed oil, but the FA profile of shrimp fed a diet supplemented with menhaden oil was the most similar to that of a wild shrimp population. In the present study, shrimp fed diets with linseed oil showed no indication of EPA or DHA synthesis from LNA. It is noteworthy, however, that the elongation product of LOA, 20:2n 6, and the elongation product of LNA, 20:3n 3, were both increased by the levels of the precursor in the diet; for instance, 20:2n 6 was significantly higher in hepatopancreas of shrimp fed diets supplemented with soybean oil, while 20:3n 3 was significantly higher in hepatopancreas of shrimp fed diets supplemented with linseed oil, indicating FA elongase activity. Under these experimental conditions, this study reconfirmed that the presence of PL in the diet of juvenile L. vannamei effectively improved growth. In addition, we observed that the fatty acid pattern of shrimp tissue reflected that of dietary lipids, and marine oils such as menhaden oil have a higher nutritional value for juvenile L. vannamei compared to the other lipid sources evaluated because they provide EFA, particularly n 3 HUFA such as EPA and DHA, which are required for maximum growth, although more studies to elucidate individual fatty acid requirements for this species need to be performed. Acknowledgements This research was funded in part by Project H-8158 of the Texas Agricultural Experiment Station, United States Department of Commerce Marine Shrimp Farming Program CSREES Grant No. 95-38808-1424, to Dr. Addison Lee Lawrence, Principal Investigator. Funding for Mrs. Gonza´lez-Fe´lix was partly provided by the Consejo Nacional de Ciencia y Tecnologia (CONACYT-Mexico). References Akiyama, D.M., Dominy, W.G., Lawrence, A.L., 1992. Penaeid shrimp nutrition. In: Fast, A.W., Lester, L.J. (Eds.), Marine Shrimp Culture: Principles and Practices. Elsevier, Amsterdam, pp. 535 – 568. Araujo, M.A., Lawrence, A.L., 1991. Fatty acids of muscle and hepatopancreas of juvenile Penaeus vannamei after non-terminal starvation and subsequent feeding with semi-purified diets: evaluation of fatty acid synthesis capabilities. J. World Aquacult. Soc. 22, 13A. Castell, J.D., Boston, L.D., Conklin, D.E., Baum, N.A., 1991. Nutritionally induced molt death syndrome in aquatic crustaceans: II. The effect of B vitamin and manganese deficiencies in lobster (Homarus americanus). Crustacean Nutr. Newsl. 7, 108 – 114. Catacutan, M.R., 1991. Growth and fatty acid composition of Penaeus monodon juveniles fed various lipids. Isr. J. Aquacult. Bamidgeh 43, 47 – 56.

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