American Fisheries Society Symposium 38:257–264, 2003 © 2003 by the American Fisheries Society

Essential Fatty Acid Requirement of the Chinese Mitten

XIAO-BO WEN, LI-QIAO CHEN*, CHUN-XIANG AI, ZHONG-LIANG ZHOU, HONGBO JIANG Department of Biology, East China Normal University, Shanghai 200062, PR China

Lipids are important nutrients for the growth of ese had shown that the prawn required , not only as energy sources but also as linoleic and linolenic acids as well as EPA and essential nutrients such as sterols, essential fatty DHA as EFAs, and their nutritional values increase acids (EFAs), and phospholipids (Kanazawa et al. in this same order (Guary et al. 1976; Kanazawa et 1985). Some combination of n-3 fatty acids, n-6 al. 1977, 1978). fatty acids, or both are required in all diets, The Eriocheir sinensis is including those of important cultured and considered to be a good for cul- prawn species (Castell et al. 1972; Kanazawa and ture in China. In recent years, significant advances Teshima 1977,1979; Kanazawa et al. 1978; Watan- have been made in this industry, progressing from abe 1982; Watanabe et al. 1989; Sargent et al. extensive to intensive culture. Therefore, produc- 1990; Tskeuchi et al. 1990). Results of studies on tion has increased rapidly. For intensive culture, the nutritional requirements of marine fish and the use of formulated diets is essential. In crus- have shown that fatty acids of the n-3 fam- taceans, the nutritional requirements of Japanese ily have greater EFA value for these species than prawn had been determined through the use of fatty acids of the n-6 family (Kanazawa et al. 1979; semipurified test diets (Deshimaru and Shigeno Castell 1981; Watanabe 1982; Xu et al. 1993). 1972; Deshimaru and Kuroki 1975; Deshimaru Results of studies on EFA requirements indi- and Yone 1978a, 1978b, 1978c; Kanazawa 1985). cate that the nutritional value of lipids for marine However, limited information is available about crustaceans is also a function of the type of unsatu- the nutritional requirements of Chinese mitten rated fatty acids that they contain and their degree crab. Some information has been reported for this of unsaturation. Kanazawa and Teshima (1979) species about optimal dietary protein levels and demonstrated that unlike rainbow trout sources (Chen et al. 1994) but not about optimal Oncorhynchus mykiss, several species of marine EFA levels. crustaceans exhibit only limited ability to elongate The objectives of this study were to determine and desaturate 18-carbon polyunsaturated fatty the dietary EFA requirements of Chinese mitten acids (PUFAs) of the n-3 and n-6 series to the crab and to evaluate the relative nutritional values longer-chain, more highly unsaturated forms. In of these fatty acids. studies with Japanese prawn Penaeus japonicus (Kanazawa and Teshima 1977), Indian white Methods prawn P. indicus (Read 1981), and common prawn Oils as EFA source serratus (Martin 1980), 18:3n-3 has Sesame oil, soybean oil, rapeseed oil, sunflower oil, greater EFA value than 18:2n-6. In Japanese and anchovy oil were used as EFA sources. The prawn, longer-chain n-3 highly unsaturated fatty EFA compositions of these oils are listed in Table 1. acids (HUFAs) such as eicosapentaenoic acid (20:5n-3, EPA) (Kanazawa et al. 1978) and docosa- Experimental diets hexaenoic (22:6n-3, DHA) (Kanazawa and Teshi- The ingredient composition (%) of the basal diet is ma 1979) had greater EFA value than 18:2n-6 or shown in Table 2. All of the lipid-containing ingre- 18:3n-3. In a preliminary study with fleshy dients (e.g., gelatin, dextrin, casein, and carboxy- Penaeus chinensis, the order of nutritional value of methylcellulose) were extracted with hot isopro- the purified fatty acids (when added individually at panol and ethanol to remove traces of lipid before 1% of the diet) was 22:6n-3 > 20:4n-6 > 18:3n-3 > making the basal mixture. 18:2n-6 (Xu et al. 1993, 1994). Studies with Japan- It is difficult to separate EPA and DHA con- tained in anchovy oil, and thus they were treated as

*Corresponding author. Telephone: 86 21 62233637; Fax: 86 21 one factor in the diet formula, and the ratio between 62233754; E-mail: [email protected] the two fatty acids was about 0.53:1 in every experi-

257 258 WEN ET AL.

Table 1. Compositions of oils as EFA sources (%).

Sesame oil Soybean oil Rapeseed oil Sunflower oil Anchovy oil 18:2n-6 42.45 46.38 20.38 59.53 3.36 18:3n-3 5.17 9.33 13.46 1.64 2.78 20:5n-3 2.03 1.04 9.58 22:6n-3 2.50 18.43

Table 2. Ingredient composition of the basal diet (%). CMC, carboxymethylcellulose. Ingredient Composition Ingredient Composition Casein 42 Vitamin mixturea 4 Dextrin 25 Mineral mixtureb 2 Gelatin 15 Choline chloride 0.5 CMC 9 Glycine 0.5 Binder 1 Chitin 1 a Each 1,000 g of diet contained 2,160 IU of vitamin E, 60 mg of vitamin K, 240 mg of pantothenic acid, 60 mg of pyridox- ine, 120 mg of riboflavin, 60 mg of thiamin, 60 mg of biotin, 600 mg of niacin, 60 mg of folic acid, and 600 mg of inositol. b • • Contained 18% NaH2PO4, 26.5% KH2PO4, 18.5% CaCO3, 21.5% calcium lactate, 10% MgSO4 7H2O, 1.2% AlCl3 6H2O, • • • 0.916% ZnSO4 7H2O, 0.061% FeCl3,0.143% MnSO4 4H2O, 0.058% KI, 0.176% CuCl2 6H2O, 2.8% KCl, and 0.106% • FeC6H5O7 5H2O. mental diet. Nine dietary treatments were designed The feeding trial was conducted for 30 d. Chi- 4 by means of the gradient table L9 (3 ), with three fac- nese mitten crab megalopa grew into juvenile tors and three levels. The three factors were linoleic after molting three times; molting frequency was acid, linolenic acid, and EPA+DHA. The three lev- calculated as the mean number of molts per surviv- els were 1.5, 2.1, and 2.7% for linoleic acid, 0.6, 0.9, ing animal. The experimental crabs were fed each and 1.2% for linolenic acid, and 0.4 (0.14 + 0.26), diet to slight excess twice a day, and the feeding 0.8 (0.28 + 0.52), and 1.2% (0.42 + 0.78) for rate was adjusted daily according to feed consump- EPA+DHA. Source oils were added to the basal tion. Feces and uneaten food were siphoned out, diets according to these levels (Table 3). and one-third of the water was exchanged before The dietary ingredients of the basal diet and the first feeding each day. The water temperature source oils were mixed with distilled water (60˚C) was 19–20˚C during the experiment. and cold-extruded through a 2-mm-orifice die All were counted and weighed, and (Tables 2 and 3). The extruded diets were dried in carapace width was measured to determine the sur- an oven by forced warm air (55˚C) for 3 h, crum- vival rate (SR), weight gain rate (WTGR), and bled with a blender, sifted through sieves to yield a width gain rate (WDGR). size suitable for crabs, and then stored at –20˚C in sealed plastic bags until used. Lipid analysis Experimental animals and feeding trial One sample of each diet and pooled samples of five Chinese mitten crab megalopa were obtained from crabs from each treatment group were collected at a commercial crab hatchery in Shanghai, China, the end of the study and stored in sealed plastic and desalinized for 3 days. On arrival at the Nutri- bags at –30˚C until analysis. The total lipid con- tional Laboratory of the East China Normal Uni- tent of these samples was estimated gravimetrically versity, they were acclimated for 2 d in a cement after extraction with chloroform–methanol–water tank (1 × 1 × 1 m), during which time they were (2:2:1; Bligh and Dyer 1959). Lipids were saponi- fed the basal (EFA-free) diet. At the end of the fied by 5% KOH in 95% ethanol. The non- conditioning period, megalopa were randomly dis- saponifiable material was removed by extraction tributed into 18 plastic tanks (40 × 30 × 15 cm) at with hexane, and the lipids were acidified with 1 N a density of 200 megalopa per tank. Two tanks were HCl and the free fatty acids recovered in hexane. randomly assigned to each dietary treatment. The hexane was evaporated in a stream of nitrogen ESSENTIAL FATTY ACID REQUIREMENT 259

Table 3. Source oil content added to the test diets (g oil/Kg diet)

Diet no. Sesame oil Soybean oil Rapeseed oil Sunflower oil Anchovy oil 1 23.1 19.8 43.8 2 3.8 64.5 14.6 3 82.2 9.2 17.2 29.2 4 48.5 21.2 5 64.5 13.3 43.8 6 8.9 91.5 14.6 7 50.8 30.5 14.6 8 32.2 65.7 29.2 9 12.9 98.5 43.8

and the fatty acids methylated by 7% BF3 in Influence on WTGR, WDGR, and SR of ratios methanol, and fatty acid meythl esters were ana- between 18:2n-6 and 18:3n-3 and between lyzed by using a gas chromatograph equipped with 18:2n-6 and DHA in the diets × a Carbowax capillary column (0.32 mm 25 m) The influences of the ratios between dietary 18:2n- and a flame ionization detector. Peaks were identi- 6 and 18:3n-3 and between 18:2n-6 and DHA on fied by comparing retention times with known ref- WTGR, WDGR, and SR of juvenile Chinese mit- erence standards. The EFA compositions of the test ten crab are shown in Figures 1 and 2. The SR, diets are listed in Table 4. WTGR, and WDGR reached maximum value when the ratios between 18:2n-6 and 18:3n-3 and Statistical analysis between 18:2n-6 and DHA were 2.93 and 5.26, Data were analyzed statistically by analysis of vari- respectively; maximum values were 23.75, 271.76, ance (ANOVA) and Student–Newman–Keuls and 100%, respectively. multiple-range test. Differences were considered significant at 0.05. Influence of dietary EFA on molting The Chinese mitten crab megalopa became juve- Results nile crabs at stage I after the first molting in the Requirements of EFAs 18:2n-6, 18:3n-3, study, and the juveniles grew into stage II and stage 20:5n-3, and 22:6n-3 III after the second and third molting, respectively. The SR, WTGR, and WDGR of crabs in the nine The compositions of crab populations at different dietary treatments were used as criteria to deter- stages are shown in Table 9. The influence of mine EFA requirements (Table 5). Rates of SR, dietary EFA on the first molting of megalopa was WTGR, and WDGR varied significantly with dif- not significant; however, The molting frequency in ferent dietary treatments. the dietary treatments with dietary EFA (primarily The SR, WTGR, and WDGR of juvenile Chi- EPA and DHA) increased significantly at the sec- nese mitten crab were influenced significantly by ond and third levels. The influence of 18:2n-6 and the third level of 18:2n-6, the second level of 18:3n-3 on molting frequency was not detected. 18:3n-3, and the second level of EPA+DHA (Tables 6–8). In the eighth diet, 18:2n-6 was at the Effects of dietary EFA on body lipid content and third level, 18:3n-3 at the second level, and fatty acid composition EPA+DHA at the second level. So, diet 8 was the The effects of dietary EFA on the body lipid con- optimal diet, with requirements of 18:2n-6, 18:3n- tent and fatty acid composition of juvenile Chinese 3, 20:5n-3, and 22:6n-3 as 2,79, 0.95, 0.28, and mitten crab are listed in Table 10. The fatty acid 0.53%, respectively. compositions of the juvenile crabs reflected those The mean change range of each level decreased of the diets (Table 4). The ratios of EPA to DHA in the order A < B < C. Thus, EPA and DHA was of juvenile Chinese mitten crab were similar to the the first influencing factor on SR, WTGR, and dietary ratios. The body EFA compositions varied WDGR of juvenile Chinese mitten crab, and 18:2n- markedly along with dietary EFA content. There 6 and 18:3n-3 were the second and third factors, was little indication of any significant conversion respectively (see Tables 6–8 for more comments). of 18:2n-6 to 20:4n-6 and 18:3n-3 to 20:5n-3 or 260 WEN ET AL.

Table 4. Analyzed EFA composition of the test diets (%)

Diet 18:2n-6 18:3n-3 EPA+DHA Total lipid 1 1.54 0.61 0.46+0.86 13.13 2 1.51 0.93 0.14+0.27 11.89 3 1.68 1.20 0.29+0.54 11.65 4 2.13 0.66 0.28+0.52 11.40 5 2.08 0.98 0.43+0.83 13.57 6 2.19 1.30 0.16+0.28 11.94 7 2.83 0.67 0.13+0.26 10.93 8 2.79 0.95 0.28+0.53 12.03 9 2.71 1.29 0.45+0.85 13.34

Table 5. Growth performance of juvenile Chinese mitten crab fed different experimental diets. DT, dietary treatment; A, B, and C represent 18:2n-6, 18:3n-3, and EPA+DHA, respectively; MN, megalopa number; WB, weight at beginning; WJCS, width of juvenile crabs at stage I; MTWE, mean weight at the end of the exper- iment; MDWE, mean width at the end of the experiment; SJCE, no. of surviving juvenile crabs at the end of the experiment; SR, survival rate; WTGR, weight gain rate; WDGR, width gain rate. DT A B C MN WB(mg) WJCS (cm) MTWE (mg) MDWE (cm) SJCE SR (%) WTGR (%) WDGR (%) 1 113400 6.90 0.251 20.58 0.436 52 13.00 198.26 73.71 2 121400 6.50 0.247 21.33 0.467 53 13.25 228.15 89.07 3 132400 6.63 0.250 22.10 0.454 49 12.25 233.33 81.60 4 212400 7.23 0.268 24.69 0.497 59 14.75 241.49 85.54 5 223400 6.65 0.252 20.85 0.430 51 12.75 213.53 70.63 6 231400 7.40 0.245 23.33 0.429 33 8.25 215.27 75.10 7 311400 6.53 0.248 20.75 0.465 37 9.25 217.76 87.50 8 322400 6.55 0.238 24.35 0.476 95 23.75 271.76 100.00 9 333400 6.83 0.253 21.35 0.432 62 15.50 212.59 70.75

Table 6. Effect of dietary EFAs and content levels on WTGR of juvenile Chinese mitten crab. A, B, and C represent 18:2n-6, 18:3n-3, and EPA+DHA, respectively. Level A B C 1 219.91 219.17 220.39 2 223.43 237.81 248.86 3 234.04 220.40 208.12 Change range 14.12 18.64 40.74

Optimum A3 B2 C2

Principle order C2 B2 A3

Table 7. Effect of dietary EFAs and content levels on width gain rate of juvenile Chinese mitten crab. A, B, and C represent 18:2n-6, 18:3n-3, and EPA+DHA, respectively.

Level A B C 1 81.46 82.22 83.89 2 77.06 86.57 89.02 3 86.08 75.82 71.70 Change range 9.02 10.75 17.32

Optimum A3 B2 C2

Principle order C2 B2 A3 ESSENTIAL FATTY ACID REQUIREMENT 261

Table 8. Effect of dietary EFAs and content levels on survival rate of juvenile Chinese mitten crab. A, B, and C represent 18:2n-6, 18:3n-3, and EPA+DHA, respectively.

Level A B C 1 12.83 12.33 10.58 2 11.92 16.58 16.92 3 16.17 12.00 13.42 Change range 4.25 4.58 6.34

Optimum A3 B2 C2

Principle order C2 B2 A3

Figure 1. Relationship between the ratio of linoleic acid to linolenic acid in the diet and survival rate, weight gain rate, and width gain rate of juvenile Chinese mitten crab.

Figure 2. Relationship between the ratio of linoleic acid to DHA in the diet and survival rate, weight gain rate, and width gain rate of juvenile Chinese mitten crab.

22:6n-3. The total lipid contents of the juvenile crustaceans lack the ability for de novo synthesis of crabs were not significantly affected by dietary EFA n-6 and n-3 fatty acids (Sargent et al. 1990). (Table 10). Because these fatty acids perform essential biologi- cal and physiological functions, they ought to be Discussion supplied in the diet (Castell et al. 1972; Yu and Studies of the nutritional requirements of marine Sinnhuber 1972; Fujii and Yone 1976; Kanazawa fish and crustaceans have indicated that fatty acids and Teshima 1979; Watanabe 1982). of the n-3 family have higher EFA values than Linoleic and linolenic acid as well as EPA and those of the n-6 family and that marine fish and DHA perform as EFAs in Japanese prawn, and their 262 WEN ET AL.

Table 9. Effect of dietary EFAs on the molting of juvenile Chinese mitten crab during 30-day feeding trial. M, megalopa; CI, no. of crabs at stage I; CII, no. of crabs at stage II; CIII, no. of crabs at stage III; MF, molting fre- quency. Values with different numbers of asterisks in the same column were significantly different (P < 0.01).

Diet MN After first molting After second molting After third molting MCIMFCICII MF CI CII CII MF 1 400 12 279 0.95* 27 72 1.73** 6 10 36 2.58* 2 400 10 236 0.96* 32 65 1.67*** 11 17 25 2.26** 3 400 33 272 0.93* 17 50 1.75** 6 12 31 2.51* 4 400 33 288 0.90* 23 75 1.77** 9 12 38 2.49* 5 400 18 296 0.94* 18 65 1.78** 5 13 33 2.55* 6 400 14 348 0.96* 29 53 1.64*** 5 12 16 2.33** 7 400 8 328 0.97* 30 59 1.66*** 10 8 19 2.24** 8 400 11 347 0.97* 17 107 1.86* 13 19 63 2.53* 9 400 31 274 0.90* 28 77 1.73** 9 13 40 2.50*

Table 10. Effect of dietary EFAs on total body lipid content and fatty acid compositions (%) of juvenile Chi- nese mitten crab.

Fatty acid Diet 12 34 56 7 8 9 14:0 1.1 1.2 1.6 1.9 1.4 2.0 1.0 1.4 2.2 16:0 18.7 20.1 14.6 20.3 19.5 20.6 18.6 18.9 20.8 16:1n-7 14.5 15.4 17.8 19.2 19.7 18.4 18.9 16.8 19.2 18:0 2.7 2.5 3.6 3.6 3.3 3.7 2.6 3.7 3.8 18:1n-9 32.6 32.3 37.2 31.8 31.5 29.7 34.0 33.1 32.8 18:2n-6 5.2 6.3 6.5 9.5 8.3 10.8 15.2 13.2 14.0 18:3n-3 1.8 2.8 3.4 1.5 2.4 3.6 1.4 2.3 3.3 20:1 1.0 1.5 1.2 1.8 1.5 1.2 1.8 1.9 1.5 20:4n-6 1.4 1.3 1.7 2.4 2.8 2.2 3.9 3.7 4.2 20:5n-3 2.3 0.7 1.6 1.4 2.1 0.7 0.8 1.5 2.5 22:6n-3 4.3 1.2 2.9 2.5 4.5 1.4 1.6 2.9 4.5 EPA/DHA 0.53:1 0.58:1 0.55:1 0.56:1 0.47:1 0.50:1 0.50:1 0.52:1 0.56:1 Total lipid 11.38 10.84 10.43 11.86 10.73 10.15 14.91 11.07 13.56 nutritional values increase in this order. Crus- lead to a high weight gain (Yu and Sinnhuber 1972; taceans are capable of synthesizing 18:2n-6 and Takeuchi and Watanabe 1977; Kanazawa et al. 18:3n-3 from acetate and palmitate as well as mam- 1978; Teshima 1978). The 20:5n-3 and 22:6n-3 fatty mals and fish, but compared with fish and mam- acids derived from dietary 18:3n-3 exerted a higher mals, this synthesis is not enough for crustacean efficiency as EFAs than 18:3n-3 in aquatic animals, development (Kanazawa and Teshima 1977; similar to the efficiency of arachidonic acid (20:4n- Kanazawa et al. 1980). Studies on fish and crus- 6) derived from 18:2n-6 in mammals, and the effect taceans have indicated that linolenic acid is more of dietary 18:3n-3 on weight gain probably varies effective than linoleic acid as an EFA because it is with the capacity of aquatic animals for the biocon- capable of converting exogenous 18:3n-3 to 20:5n- version of 18:3n-3 to 20:5n-3 and 22:6n-3 (Kanaza- 3 and 22:6n-3, probably through intermediates such wa et al. 1979). A dietary study on brachyuran crab as 18:4n-3, 20:3n-3, 20:4n-3, and 22:5n-3, but these Eurypanopeus depressus showed that EPA and DHA animals probably were incapable of synthesizing were more effective than 18:2n-6 and 18:3n-3 as enough 20:5n-3 and 22:6n-3 for their requirements EFAs (Levine and Sulkin 1984). from dietary 18:3n-3 (Xu et al. 1993). Fleshy shrimp fed an EFA-free diet had a low The results of dietary experiments conducted molting frequency, poor growth, and 100% mortal- with fish and crustaceans have suggested that highly ity before the end of a 60-d experimental period, unsaturated fatty acids such as 20:5n-3 and 22:6n-3 whereas a diet containing n-3 or n-6 fatty acids ESSENTIAL FATTY ACID REQUIREMENT 263 improved weight gain, molting frequency, and sur- first molting of megalopa but that EFA (primarily vival rate (Xu et al. 1994). But high dietary lipid EPA and DHA) has a significant effect on the sec- levels had been suggested to reduce the growth ond and third moltings. Considering this phenom- rates of crustaceans. D’Abramo (1990) pointed out enon and the results of reported by D’Abramo et al. that giant river prawns Macrobrachium rosenbergii (1982) and Teshima et al. (1986), increased molt- fed diets containing more than 10% lipids had ing frequency of juvenile Chinese mitten crab fed retarded growth compared with those fed optimal diets with high levels of EPA and DHA probably dietary lipid levels. Castell and Covey (1976) sug- was due to more effective cholesterol absorption, gested that 5% cod liver oil was optimal for the whereas the experimental megalopa may accumu- growth of American Homarus americanus, late enough molting hormones for the first molting and diets containing 10 or 15% lipids resulted in before the experiment and thus are not influenced no improvement. Davis and Robinson (1986) also by dietary EFA. However, further research is need- indicated that fed diets containing 9% or ed to verify this assumption. more lipids showed lower growth rates. Thus the optimal requirements of fatty acids and lipids were Acknowledgments essential for crustaceans. This study was supported by the National Nature Similar to the results obtained in studies with Science Foundation of China (39770578, fish and crustaceans, results of the present study 30271012), Trans-Century Training Program indicate that the optimal requirements of 18:2n-6, Foundation for the Talents and Foundation for 18:3n-3, 20:5n-3, and 22:6n-3 were essential for University Key Teacher from the Ministry of Edu- the growth, survival, and molting of Chinese mit- cation of China, and the Research Fund for the ten crab. Results also indicate that EPA and DHA Doctoral Program of Higher Education of China. were more effective than 18:3n-3 and that DHA The authors thank Robert W. Murphy (Depart- was more effective than 18:2n-6 according to their ment of Animal Science, University of Toronto, nutritional physiological function. The SR, Ontario, Canada) and Chen Yong (Memorial Uni- WGTR, and WTGR reached the maximum value versity, Newfoundland, Canada) for reviewing the in the eighth of nine dietary treatments. The opti- manuscript and offering helpful suggestions. mal requirements of 18:2n-6, 18:3n-3, 20:5n-3, and 22:6n-3 in the crab diet are 2,79, 0.95, 0.28, and References 0.53%, respectively. Bligh, E. G., and W. T. Dyer. 1959. A rapid method of Wang et al. (1993) and Ren et al. (1994) indi- total lipid extraction and purification. Canadian cated that the SR, WGTR, and WTGR were influ- Journal of Biochemistry and Physiology Canadian enced not only by optimal content of 18:2n-6, Journal of Biochemistry and Physiology 37:911–917. 18:3n-3, 20:5n-3, and 22:6n-3 but also by the ratios Castell, J. D. 1981. 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