Variation in lipid composition of Chinese mitten-handed crab, Eriocheir sinensis during ovarian maturation

Variation in lipid composition of Chinese mitten-handed crab, Eriocheir sinensis during ovarian maturation

Comparative Biochemistry and Physiology Part B 130 Ž2001. 95᎐104 Variation in lipid composition of Chinese mitten-handed crab, Eriocheir sinensis dur...

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Comparative Biochemistry and Physiology Part B 130 Ž2001. 95᎐104

Variation in lipid composition of Chinese mitten-handed crab, Eriocheir sinensis during ovarian maturation XiaoBo Wen1, LiQiao ChenU , ChunXiang Ai, Zhongliang Zhou, Hongbo Jiang Department of Biology, East China Normal Uni¨ ersity, Shanghai 200062, PR China Received 20 January 2001; received in revised form 23 April 2001; accepted 2 May 2001

Abstract This experiment was conducted to investigate the variation in lipid composition during the ovarian maturation of the crab Eriocheir sinensis. The Chinese mitten-handed crab broodstock was divided into six different maturation periods according to the size and color of ovary. Ovary, hepatopancreas, muscle, and hemolymph of broodstock in different maturation periods were analyzed for total lipid and fatty acids using gas chromatography, and lipid classes by thin-layer chromatography. The ovarian lipid concentration Žexpressed as percent wet ovarian weight. increased steadily from stage II Ž5.4%. to stage IV Ž19.1%., and decreased to the lowest levels after spawning Žstage V, 6.6%.. The hepatopancreatic lipid concentration Žexpressed as percent wet hepatopancreatic weight. increased with maturity of the ovaries, reached a maximum at stage III 2 Ž29.9%., and decreased during the subsequent period to spawning Ž16.7%.. The muscular and hemolymph lipid concentration did not change markedly during the ovarian development. These results suggest the possible movement of hepatopancreatic lipids to the ovaries during the ovarian maturation. Both triacylglycerol and phosphatidylcholine were responsible for the increase in ovarian lipid concentration during sexual maturation. The fatty acids of total lipid, triacylglycerol, and phosphatidylcholine of the ovaries did not vary systematically during the ovarian maturation, but the ratio between n-3PUFA Žpolyunsaturated fatty acid. and n-6PUFA did change regularly with the ovarian lipid. These suggest that enough PUFA, especially n-3PUFA, should be supplied to the crab during ovarian maturation. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Eriocheir sinensis; Fatty acid composition; Lipid class; Lipid concentration; Maturation; Phosphatidylcholine; Polyunsaturated fatty acid; Triacylglycerol; Variation

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Corresponding author. Tel.: q86-21-62233637; fax: q86-21-62233754. E-mail addresses: [email protected] ŽX. Wen., [email protected] ŽL. Chen.. Present address: Department of Animal Science, Hubei Agriculture College, Jingzhou 434103, Hubei Province, PR China.

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1096-4959r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 1 . 0 0 4 1 1 - 0

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1. Introduction The Chinese mitten-handed crab Eriocheir sinensis, is considered to be a good crustacean species for culture in China. During the last years, significant advances have been made in this industry, progressing from extensive to intensive culture. Therefore, the production of this species increased rapidly. There are, however, still many constraints to further development of crab culture in China, particularly shortage of larvae and postlarvae supplies. Because the wild larvae and postlarvae are almost extinct, the current larvae and postlarvae supplies are largely gained from artificial hatcheries. The usual practice in the crab hatcheries involves rearing larvae and postlarvae from wild broodstock or female crabs matured in captivity. However, the broodstock and egg quality are poor, which result in very low fecundity, egg hatchability and larvae survival rate in the artificial hatcheries of the crab. So the production of larvae and postlarvae is low and inconsistent with the request for further development of culture. In an attempt to improve broodstock and egg quality, recent studies on maturation of shrimp have focused on the nutritional requirements of the broodstock during ovarian development. Researchers have suggested that proper ovarian development and normal maturation of broodstock are related to nutritional status ŽBrown et al., 1980; Middleditch et al., 1980; Xu et al., 1994a.. However, less is known of the relation between nutrition and ovarian maturation of crab in comparison with those of shrimp. Lipids are important nutrients for growth of crustaceans not only as energy sources but also as essential nutrients such as sterols, fatty acids, and phospholipids ŽKanazawa et al., 1985., and are believed to be one of the key nutritional factors influencing egg hatching rates and larval survival ŽXu et al., 1994b.. In crustaceans, the hepatopancreas is generally regarded as a major lipid storage organ analogous to the fat body in insects and the adipose tissue and liver in vertebrates. The stored lipid is transported to some organs and tissues during a certain period such as premolt stage. In the case of female crustaceans, ovaries also contain higher levels of lipid than other organs ŽAndo et al., 1977., and this suggests that lipid is important for maturation of crustacean ovaries. Research on

broodstock of Penaeus monodon has shown that ovarian lipid levels increased from 5.8% in immature prawn to 17% in fully mature wild Žunablated. females and from 7.5% to 21% in wild ablated females ŽMilliamena and Quinito, 1985., and the content of highly unsaturated fatty acids ŽHUFAs, 20- and 22-carbon fatty acids. ranged from 12 to 25% of the total fatty acids of both unablated and ablated wild prawn. The high content of HUFAs in the broodstock prawn and eggs indicate their importance in the prawn reproductive process. Teshima and Kanazawa Ž1983. found the ovarian lipid concentrations of Penaeus japonicus increased during the slight mature and yellow ovarian periods, remained at roughly constant levels during the subsequent ovarian period to spawning, and then decreased to low levels at the spent ovarian period. This implies that large quantities of lipid are necessary for development of ovaries. However, little information is available for lipid metabolism during the ovarian maturation of crab. The current study was designed to clarify the variation in lipid and fatty acid composition of the ovary, hepatopancreas, muscle, and hemolymph during the development of ovaries, evaluate the relation between lipid metabolism and the ovarian maturation of Eriocheir sinensis, and furnish suggestions for crab broodstock culture.

2. Materials and methods 2.1. Experimental broodstock crab The specimens of female crab were caught by fishermen in Yangchen lake near Shanghai, PR China, on 8 September and 10 November, and transported to the laboratory. Some living crabs were pooled for lipid analysis, and some crabs were held in a 1 = 1 = 1-m cement tank in a laboratory of East China Normal University until they spawned in order to choose the individuals with the spent ovaries. All the crabs used for this experiment were weighed and dissected, and after ovary, hepatopancreas, muscle, and hemolymph were obtained and weighed, these tissues were stored at y70⬚C until extracted. Ovarian development was divided into the following six stages according to the size and color of the ovary ŽXue et al., 1987.: stage I Ž25᎐31 mm, transparent milky white.; stage II Ž34 mm, milky white.; stage

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III 1 Ž40᎐50 mm, milky white.; stage III 2 Ž60᎐90 mm, lightly brown.; stage IV Ž92᎐100 mm, purplish brown.; stage V Žspent ovaries..

The amount of each lipid class was measured by the acidic dichromate oxidation method of Amenta Ž1964..

2.2. Lipid analysis

2.2.3. Fatty acid analysis After TG and PC had been prepared, they were saponified in a solution of 5% KOH dissolved in 95% ethanol. After removal of the nonsaponifiable material by extraction with hexane and acidification with 1 N HCl, the saponifiable materials were recovered in hexane. The hexane was evaporated in a stream of nitrogen and the fatty acids were methylated in 7% BF3 in methanol ŽMorrison and Smith, 1964.. Fatty acid methyl esters were analyzed using a gas chromatograph ŽHewlett-Packerd model HP 5890., equipped with a 0.32-mm= 25-m Carbowax capillary column and flame ionization detector. Peaks were identified by comparing retention times with known reference standards ŽAckman and Burger, 1965.. Identity of fatty acids was not confirmed by mass spectrometry.

2.2.1. Lipid preparation Total lipid was extracted with chloroform᎐ methanol᎐water Ž2:2:1. ŽBligh and Dyer, 1959. from the ovary, hepatopancreas, muscle, and hemolymph of Eriocheir sinensis. The purified lipid extract was freed of solvent in a rotating vacuum evaporator. Total lipid Ž% wet wt.. was determined gravimetrically for the ovary, hepatopancreas, and muscle, and photometrically for the hemolymph. Some total lipid was used for fatty acid analysis immediately Žmethod is the same as in Section 2.2.3., and some total lipid of ovary and hepatopancreas was separated into neutral lipid and polar lipid by the solvent method with petroleum ether as basal phase and 95% methanol as recovery phase ŽSkipski and Barclay, 1969.. 2.2.2. Thin-layer chromatography of neutral lipids and phospholipids Neutral lipids were separated into lipid classes such as steryl esters ŽSE., triacylglycerol ŽTG., cholesterol ŽCHL., diacyglycerol ŽDG., free fatty acids ŽFFA., and monoacyglycerol ŽMG. using the two-step development system of Skipski et al. Ž1965.: Ž1. isopropyl ether᎐acetic acid, 96:4 Žvrv.; and Ž2. petroleum ether᎐diethyl ether᎐acetic acid, 90:10:1 Žby vol... Polar lipids were separated into phosphatidylcholine ŽPC., phosphatidylethanolamine ŽPE., phosphatidylinositol ŽPI., and phosphatidylserine ŽPS. by the method of Skipski et al. Ž1963. using a solvent system of chloroform᎐methanol᎐acetic acid᎐water, 25:15:4:2 Žby vol...

2.3. Statistical analysis Statistical analysis of data on lipid concentration was conducted by an analysis of variance at the 1% or 5% level.

3. Results 3.1. Variation in lipid concentration of crab during o¨ arian maturation Fig. 1 and Table 1 show the variation in total lipid concentration of the ovary, hepatopancreas, muscle, and hemolymph of crab in II᎐V ovarian development stages. The lipid concentration of

Table 1 Variation in lipid concentration of ovary, hepatopancreas, muscle, and hemolymph during ovarian maturation of Eriocheir sinensis Ovary stage II III1 III2 IV V a b

GIS Ž%.a

Body wt. Žg.

b

51.9" 5.7 Ž4. 72.9" 4.7 Ž6. 89.0" 7.8 Ž4. 99.8" 6.5 Ž6. 86.5" 3.7 Ž5.

0.3" 0.1 1.8" 0.6 5.3" 1.2 12.4" 1.1 1.4" 0.2

Gonadosomatic index s Žovary wt.=100.rbody wt. S.D. and the number of crabs analyzed.

Lipid concentration Ž% wet wt.. Ovary

Hepatopancreas

Muscle

5.4" 0.9 8.3" 1.2 17.2" 1.4 19.1" 1.8 6.6" 2.7

6.5" 2.5 19.7" 1.9 29.9" 1.8 21.0" 1.5 16.7" 2.1

2.5" 0.3 2.4" 0.4 3.7" 0.1 2.6" 0.2 3.9" 0.3

Hemolymph 1.7" 0. 2.1" 0.3 2.1" 0.2 2.7" 0.1 1.8" 0.4

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muscle and hemolymph was lower than that of ovary and hepatopancreas, and it did not change markedly during the ovarian development. On the other hand, the lipid concentration of ovary and hepatopancreas varied markedly with the degree of ovarian maturation. The ovarian lipid concentration rose steadily from stage II to stage IV, and decreased to their lowest levels after spawning Žstage V.. Statistical analysis showed that the average concentration of ovarian lipid was significantly different between the following ovarian stages: stage II᎐stage III 1 Ž P- 0.01., stage III 1 ᎐stage III 2 Ž P- 0.01., stage III 2 ᎐stage IV Ž P- 0.05., and stage IV᎐stage V Ž P- 0.01.. There was no major difference in the lipid concentration between stage II and stage V. The hepatopancreatic lipid concentration also increased with increasing GSI values, reached the highest level at stage III 2 , and decreased until spawning. The average concentration of hepatopancreatic lipid was significantly Ž P- 0.01. different between the following stages: stage II᎐stage III 1 , stage III 1 ᎐stage III 2 , stage III 2 ᎐stage IV and stage IV᎐stage V. There was also a major difference in the lipid concentration between stage II and stage V. This trend was inconsistent with that of the ovarian lipid.

3.2. Variation in fatty acid composition of crab during o¨ arian maturation

Tables 2 and 3 show the variation in fatty acid composition of lipid extracted from the ovary, hepatopancreas, muscle, and hemolymph during ovarian maturation. Palmitic acid Ž16:0., palmitoleic acid Ž16:1n-7., and oleic acid Ž18:1n-9. predominated in the lipid of ovary, hepatopancreas, muscle, and hemolymph. The ovarian lipid contained higher proportions of polyunsaturated fatty acids ŽPUFA. than that of hepatopancreas at every stage of ovarian maturation. However, the hepatopancreatic lipid contained higher 16:0, 18:1n-9, and saturated fatty acid ŽSAF. than that of ovary at every stage. The muscular lipid contained higher 16:0, 18:1n-9, and 18:2n-6 than that of hemolymph at every stage, and the hemolymph lipid contained higher 14:0, 16:1n-7, 18:0, and 20:1n-9 than that of muscle. In the case of one tissue: ovary, hepatopancreas, muscle or hemolymph, there was no regular variation of fatty acid composition between different maturation periods except the ratio of n-3PUFA to n-6PUFA. The ratios in the ovarian and muscular lipid increased steadily from stage II to stage IV, and decreased to the lowest level after spawning Žstage

Fig. 1. Variation in lipid concentration Ž% wet wt.. of ovary, hepatopancreas, muscle, and hemolymph during ovarian maturation of E. sinensis.

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Table 2 Variation in fatty acid composition Ž% of total fatty acids. of total lipid of ovary and hepatopancreas during ovarian maturation of E. sinensis Main fatty acida

14:0 16:0 16:1n-7 18:0 18:1n-9 18:2n-6 18:3n-3 20:1n-9 20:4n-6 20:5n-3 22:6n-3 ⌺SFA ⌺MUFA ⌺PUFA ⌺n-3PUFA ⌺n-6PUFA n-3rn-6ratio

Ovary

Hepatopancreas

II

III1

III2

IV

V

II

III1

III2

IV

V

2.2 15.7 15.2 5.6 31.8 7.3 3.8 1.8 7.8 5.8 2.6 3.5 48.8 27.3 12.2 15.1 0.81

3.1 15.6 20.7 4.6 30.4 9.0 4.8 1.7 1.7 5.6 2.3 23.4 52.9 23.5 12.7 10.8 1.18

1.9 16.0 17.7 5.6 27.6 10.1 5.2 3.5 1.8 9.0 1.2 23.5 48.8 27.3 15.4 11.9 1.29

4.8 16.0 17.3 9.8 24.5 6.3 2.5 3.8 2.6 7.6 4.3 30.7 45.7 23.5 14.5 9.0 1.61

2.4 15.4 13.6 8.3 28.0 8.6 5.7 2.5 5.7 6.1 3.2 26.2 44.2 29.5 15.1 14.4 1.04

3.6 23.4 11.7 4.0 37.5 7.6 4.1 0.8 0.9 3.2 2.9 31.0 50.1 16.3 7.8 8.5 0.91

1.5 24.5 11.2 4.4 35.4 7.5 2.8 1.4 2.6 4.3 4.0 30.5 48.1 21.3 11.2 10.1 1.11

2.3 21.8 11.7 5.2 40.6 6.4 3.6 1.6 1.0 4.3 1.1 29.5 53.9 16.6 19.1 7.5 1.21

1.8 25.3 14.2 4.9 30.9 6.9 5.7 2.6 2.5 3.3 1.3 32.0 47.8 29.9 10.4 9.5 1.09

2.3 22.2 16.2 2.0 38.7 12.2 2.1 1.2 0.4 0.6 1.9 26.5 57.1 17.3 4.6 12.7 0.36

a Identity of fatty acids was not confirmed by mass spectrometry; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; n-3rn-6 ratio, ratio between ⌺n-3PUFA and ⌺n-6PUFA. Means of all abbreviations in the latter tables are the same as those in this one.

3.3. Variation in lipid classes composition during o¨ arian maturation

V., and the ratios in the hepatopancreatic and hemolymph lipid also increased continuously with the ovarian maturation, reached the highest level at stage III 2 , and decreased until spawning.

Table 4 and Fig. 2 showed variation in lipid

Table 3 Variation in fatty acid composition Ž% of total fatty acids. of total lipid of muscle and hemolymph during ovarian maturation of E. sinensis Main fatty acid

14:0 16:0 16:1n-7 18:0 18:1n-9 18:2n-6 18:3n-3 20:1n-9 20:4n-6 20:5n-3 22:6n-3 ⌺SAF ⌺MUFA ⌺PUFA ⌺n-3PUFA ⌺n-6PUFA n-3rn-6ratio

Muscle

Hemolymph

II

III1

III2

IV

V

II

III1

III2

IV

V

3.1 16.9 5.7 10.1 32.0 12.8 2.5 3.5 3.6 7.1 2.2 30.3 41.2 28.4 11.9 16.5 0.72

8.9 18.2 7.0 9.4 31.8 5.6 2.4 3.2 2.5 5.6 4.6 36.7 42.2 20.9 12.7 8.2 1.54

7.0 20.2 5.9 11.0 28.2 8.1 7.7 0.7 1.4 5.6 3.7 38.3 34.9 26.6 17.1 9.5 1.79

6.4 21.8 8.0 6.5 24.9 6.1 4.9 4.2 3.0 6.7 6.9 34.8 37.2 27.8 15.6 12.2 1.88

6.0 21.5 11.4 3.4 27.7 7.2 3.6 2.8 6.0 4.3 5.5 31.0 42.0 26.7 13.5 12.0 1.12

10.7 11.4 7.5 12.6 14.0 5.4 4.0 15.7 4.5 4.5 9.2 34.7 37.3 27.8 17.8 10.0 1.78

11.9 4.2 9.2 10.5 16.8 4.2 5.3 17.3 5.5 8.4 5.5 26.6 43.3 28.9 19.2 9.7 1.98

8.2 4.6 11.3 11.1 24.4 6.8 7.4 3.1 2.9 7.4 7.2 23.9 38.8 31.7 22.0 9.7 2.26

8.4 12.4 13.1 9.8 22.3 5.8 8.0 7.5 3.3 3.5 5.4 30.6 42.9 26.0 16.9 9.1 1.85

11.2 15.8 11.8 13.9 18.7 6.9 3.6 5.0 3.6 5.5 3.6 40.9 35.5 23.2 12.7 10.5 1.20

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Table 4 Variation in lipid class composition of ovary and hepatopancreas during ovarian maturation of E. Sinensis Lipid class a

Composition Ž% of total lipid. Ovary

NL

DG TG CHL FFA SE MG PS PI PC PE

PL

Hepatopancreas

II

III1

III2

IV

V

II

III1

III2

IV

V

3.0 44.4 17.7 10.4 21.2 2.9 tb t 67.4 24.3

4.2 46.8 11.3 10.4 20.4 6.7 1.3 2.2 70.5 20.9

2.0 54.1 14.6 7.8 18.0 3.4 2.1 1.5 73.1 23.3

1.3 69.4 10.7 4.6 10.0 3.8 0.8 0.2 88.5 10.3

3.0 28.2 18.7 17.4 28.6 3.9 1.1 2.5 61.2 25.1

6.7 42.7 ᎐ 24.4 12.4 6.7 3.8 11.4 72.3 10.4

7.5 49.6 ᎐ 26.5 14.2 8.9 5.5 13.5 73.2 5.6

5.9 61.5 ᎐ 13.5 11.1 7.6 6.0 6.9 79.9 5.9

8.4 46.3 ᎐ 21.4 11.1 12.7 7.4 11.3 73.9 5.3

5.2 38.2 ᎐ 29.7 11.2 15.6 14.3 8.3 65.6 6.6

a

NL, neutral lipid; PL, polar lipid; DG, diacylglycerol; TG, triacylglycerol; CHL, cholesterol; FFA, free fatty acid; SE, steryl esters; MG, monoacyglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine; PE, phosphatidylethanolamine. b Trace.

classes of ovary and hepatopancreas during ovarian maturation. Neutral and polar lipid of the ovary and hepatopancreas were separated into different lipid classes. The ovaries contained TG Ž28.2᎐69.4%, as % total neutral lipid., CHL Ž10.7᎐18.7%., and SE Ž10.0᎐28.6%. as the major neutral lipid classes and PC Ž61.2᎐79.5%, as % total polar lipid. and PE Ž21.3᎐28.7%. as the major phospholipid classes at every stage of ovarian maturation. As shown in Table 4 and Fig. 2, the concentration of PC and TG in the ovaries

increased with the progress of ovarian maturation. Therefore, the increase in the concentration of ovarian lipid during ovarian development ŽTable 1 and Fig. 1. are due mainly to both TG and PC. However, the fatty acid composition of ovarian PC and TG remained roughly constant throughout the developmental stages of the ovaries ŽTable 5.. The hepatopancreas contained TG Ž38.2᎐ 61.5%, as % total neutral lipid. and FFA Ž13.5᎐29.7%. as the major neutral lipid classes

Table 5 Variation in fatty acid composition Ž% of total fatty acids. of ovarian TG and PC during ovarian maturation of E. sinensis Main fatty acid

14:0 16:0 16:1n-7 18:0 18:1n-9 18:2n-6 18:3n-3 20:1n-9 20:4n-6 20:5n-3 22:6n-3 ⌺SAF ⌺MUFA ⌺PUFA ⌺n-3PUFA ⌺n-6PUFA n-3rn-6ratio

TG

PC

II

III1

III2

IV

V

II

III1

III2

IV

V

0.8 18.1 13.2 3.2 39.2 9.6 2.9 0.5 3.4 4.6 3.9 22.2 53.1 24.6 11.6 13.0 0.89

1.0 19.2 12.9 3.0 38.0 9.4 2.9 1.0 3.3 4.4 4.3 23.3 52.0 24.5 11.8 12.7 0.92

2.1 17.1 10.4 4.2 35.4 8.2 6.9 3.6 3.2 5.5 2.9 23.5 49.5 26.8 15.3 11.5 1.33

1.2 17.4 19.1 3.2 29.9 10.7 6.8 0.7 1.8 5.3 3.2 21.9 49.9 28.0 15.4 12.6 1.22

1.8 19.8 16.5 3.1 33.8 11.7 3.8 1.5 3.3 2.7 1.6 24.8 51.8 23.2 8.2 15.0 0.54

1.2 15.4 10.9 10.1 34.3 7.7 3.9 2.5 3.6 6.6 3.1 26.8 47.8 25.2 13.8 11.4 1.21

1.4 13.1 19.6 7.3 28.4 10.3 7.1 2.0 2.2 5.2 3.0 21.8 50.1 27.8 15.3 12.5 1.22

1.2 10.8 15.7 7.3 3.2 9.5 7.3 2.7 2.3 5.7 3.8 19.4 43.3 28.7 16.9 11.8 1.43

1.2 10.4 12.7 8.8 36.8 7.2 3.5 1.9 3.1 6.0 7.9 20.5 51.5 27.8 17.4 10.8 1.67

1.3 13.5 9.2 9.9 34.2 9.8 4.6 3.9 3.1 5.5 4.5 24.8 47.3 27.7 14.7 13.0 1.13

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Fig. 2. Variation in lipid class composition of ovary and hepatopancreas during ovarian maturation of E. sinensis.

with PC Ž70.3᎐79.9%, as % total polar lipid. as the major polar lipid during all the maturation periods. TG and PC were mainly responsible for the increase in hepatopancreatic lipid concentration during stages III 1 ᎐III 2 ŽFig. 2.. However, the decrease in the lipid concentration of hepatopancreas between stages III 2 ᎐IV was mostly attributable to that in the hepatopancreatic TG.

4. Discussion There are two types of variation of ovarian lipid during ovarian maturation in crustaceans. This study suggests that the total ovarian lipids increase steadily from stage II to stage IV and reach a maximum in stage IV Ž full ripe period.. The same results were reported for Penaeus japonicus and Parathelphusa hydrodromus ŽTeshima and Kanazawa, 1983., and Macrochium borelli ŽGonazalez-Baro and Pollero, 1988.. Chung et al. Ž1975. indicated much lipid and protein were required for biosynthesis of lipovitellin by the crustacean during ovarian maturation, so ovarian lipids increased at the same time. Vitellogenesis, the process of yolk deposition, is a crucial event in the female gametogenesis of crustaceans. Lipovitellin ŽLv. is the major density

lipoprotein that accumulates within the ovary during this process ŽAdiyodi and Subramoniam, 1983.. The Lv has a molecular weight of 500 000 and a protein to lipid ratio of approximately 2:1. The lipid portion of the Lv includes phosphatidylcholine, phosphatidylethanolamine, and some unidentified carotenoids ŽLui et al., 1975.. In crustaceans, discordant views are expressed on the synthetic site of the Lv. Convincing evidence is available for the synthesis of Lv by the hepatopancreas and its transport to the ovary of several decapod crustaceans ŽPaulus and Laufer, 1987; Quackenbush, 1989; Fainzilber et al., 1992.. De novo synthesis of Lv in the ovary has also been shown for several other decapods ŽEastman-Recks and Fingerman, 1985; Browdy et al., 1990.. Du et al. Ž1999. indicated that the origin of the Lv in Eriocheir sinensis was dual. The oocytes produced endogenous Lv by autosynthesis, and obtained exogenous Lv of heterosynthesis directly or indirectly. This exogenous Lv was possibly synthesized by the hepatopancreas and sequestered into oocytes through the hemolymph. The lipoidal portion of the endogenous Lv may also come from hepatopancreas and be transported to and absorbed into the crab ovary. In crustaceans, vitellogenesis occurs in two stages: Ž1. a primary vitellogenesis which extends for several months

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and results in slow increase in the size of the oocyte; and Ž2. a secondary vitellogenesis which results in rapid increase in oocyte size leading to oviposition ŽAiken and Waddy, 1980.. With a corresponding increase in the size of the oocytes, lipid as a component of Lv is transported to the ovary from the hepatopancreas following the transport of Lv, and some lipid is transported to the ovary for the synthesis of the endogenous Lv. So, the total ovarian lipids increase during the ovarian maturation. In crustaceans, another type of variation of ovarian lipid was reported in studies of Penaeus durorarum ŽGehring, 1974.. The ovarian lipid of P. durorarum decreased from 7.7% in stage 3 Žnearly ripe. to 5.7% in stage 4 Žfully ripe.. Gehring Ž1974. indicated there were several possible explanations for the decrease of lipids between stage 3 and 4 in this species. Lipid might be converted into other compounds associated with the rod-like bodies that first appear in stage 4, it might provide the energy source for this dramatic morphological change, or stage 4 might be an early resorptive stage. As shown in Table 4 and Fig. 2, the concentrations of PC and TG in the ovaries increased during the ovarian maturation and reached a maximum in stage IV. Meanwhile, the concentration of TG in the hepatopancreas decreased from 61.5% in stage III 2 to 46.3% in stage IV, and PC decreased from 79.9% to 73.9%. This assumes that the hepatopancreatic TG and PC are transported to the developing ovaries during the period of stage III 2 ᎐IV. In many crustaceans, such as Cancer magister ŽAllen, 1972., Penaeus setiferus ŽCastille and Lawrence, 1989., Macrobrachium borelli ŽGonazalez-Baro and Pollero, 1988., Penaeus japonicus ŽTeshima and Kanazawa, 1983., Pleoticus muelleri ŽJeckel et al., 1989., and Penaeus aztecus ŽCastille and Lawrence, 1989., the lipid concentration in the hepatopancreas decreased markedly with ovarian lipid reaching a maximum during ovarian maturation, with the hepatopancreatic lipid moved to the ovaries as a form of lipoproteins through hemolymph. Thus, it can be postulated that in Eriocheir sinensis, the hepatopancreatic TG and PC may move to the ovaries as a form of lipoprotein. The hemolymph TG and PC concentration must be studied during stage III 2 to stage IV, and tracer experiments must be employed for further clarification. As shown in Table 4, no cholesterol can be

found in the lipid class composition of the hepatopancreas during every ovarian development. However, cholesterol is a major lipid class in ovaries. Similar results were reported in studies of Carcinus maenas ŽChapelle, 1977. and Pachygrapsus marmoratus ŽLautier and Lagarrigue, 1988.. Young et al. Ž1992. indicated that sterol ester was a storage form of cholesterol and fatty acids. Polyenoic fatty acids from the diet, along with dietary free cholesterol, were combined to form steryl ester in the hepatopancreas and then transported in the hemolymph to the other tissues and it could be speculated that there is a role of lipoproteins Žas mammals. in this process. Table 4 indicates steryl esters can be seen in both the ovary and hepatopancreas, and SE concentration in ovarian lipid classes composition was largely higher than that in hepatopancreas. Considering this fact and Young’s opinion, it can be postulated that cholesterol is rapidly esterified in the hepatopancreas, and then transported into the ovaries in a form of SE, and SE in the ovary are hydrolyzed during vitellogenesis to release free cholesterol and fatty acids which may be necessary for aspects of ovarian development. Table 4 indicated there were high FFA in ovary Ž4.6᎐17.4%, as % total neutral lipid. and hepatopancreas Ž13.5᎐29.7%.. For the samples were properly treated in this experiment, high FFA in ovary and hepatopancreas is possibly an identity of the crab, and the similar results were reported in studies of Penaeus japonicus ŽTeshima and Kanazawa, 1983.. Tables 2 and 3 showed that no regular variation in the fatty acid composition was seen in the lipid of ovary, hepatopancreas, muscle, and hemolymph, respectively, but the ratio of n-3PUFA to n-6PUFA varied regularly as the total lipid Žfatty acids identifies were not confirmed by mass spectrometry.. PUFA, especially n-3 PUFA, are essential fatty acids of shrimp and crab. They cannot be biosynthesized in vivo, and must be supplied from exogenous diets ŽKanazawa et al., 1977, 1985; Xu et al., 1994a,b.. So in the crab broodstock culture, enough PUFA, especially n-3 PUFA, must be supplied for high quality broodstock and eggs. In the future, we will conduct further studies on physiological and reproductive influences of different essential ingredients added in the compound feed on the crab according to these results.

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Acknowledgements This study was supported by grant no. 39770578 from the National Nature Science Foundation of China, Trans-Century Training Program Foundation for the Talents and Foundation for University Key Teacher from the ministry of Education of China. The authors thank Prof. Robert W. Murphy, of the Department of Animal Science, University of Toronto, Canada, and Prof. Chen Yong, of Memorial University of Newfoundland, Canada, for reviewing the manuscript and their helpful suggestions. References Ackman, R.G, Burger, R.D., 1965. Cod liver oil fatty acids as secondary reference standards in the GLC of polyunsaturated fatty acids of animal origin: Analysis of a dermal oil of the Atlantic leather back turtle. J. Am. Oil. Chem. Soc. 42, 38᎐42. Adiyodi, R.G, Subramoniam, T., 1983. Arthropoda ᎏ Crustacea. In: Asiyodi, K.G., Adiyodi, R.G. ŽEds.., Reproductive Biology of Invertebrates. Oogenesis, Oviposition and Oosorption, 1. John Wiley & Sons, New York, NY, pp. 443᎐495. Aiken, D.E., Waddy, S.L., 1980. Reproductive biology. In: Cobb, J.S., Phillips, B.E. ŽEds.., The Biology and Management of Lobsters, 1. Academic Press, New York, NY, pp. 215᎐276. Allen, W.B., 1972. Lipid transport in Dungeness crab, Cancer magister Dana. Comp. Biochem. Physiol. 43B, 193᎐207. Amenta, J.S., 1964. A rapid chemical method for quantification of lipids separated by thin-layer chromatography. J. Lipid Res. 5, 270᎐272. Ando, T., Kanazawa, A., Teshima, S., 1977. Variation in the lipids of tissues during the molting cycle of prawn. Bull. Jpn. Soc. Sci. Fish 43 Ž12., 1445᎐1449. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911᎐917. Browdy, C.L., Fainzilber, M., Tom, M., Loya, Y., Lubzens, E., 1990. Vitellin synthesis in relation to oogenesis in vitro incubated ovaries of Penaeus semisulcatus ŽCrustacea, Decapoda, Penaeidae.. J. Exp. Zool. 255, 205᎐215. Brown, A., McVey, J., Middleditch, B.S., 1980. Preliminary results on the maturation and spawning of Penaeus stylirostris under controlled laboratory conditions. Proc. World Maricult. Soc. 11, 463᎐470. Castille, F.L., Lawrence, A.L., 1989. Relationship between maturation and biochemical composition of the gonads and digestive glands of the shrimps Pe-

103

naeus aztecus and Penaeus setiferus. J. Crusta. Biol. 9, 202᎐211. Chapelle, S., 1977. Lipid composition of tissues of marine crustaceas. Biochem. Syst. Ecol. 5, 241᎐248. Chung, W.L., Becky, A., J.D., O’Connor, 1975. Biosythesis of lipovitellin by the crustacean ovary. J. Exp. Zool. 188, 289᎐296. Du, N.S., Lai, W., Chen, Y.X., Nan, C.R., 1999. Studies on vitellogenesis of Eriocheir sinensis ŽCrustacea, Decapoda.. Acta Zool. Sin. 45 Ž1., 88᎐92. Eastman-Recks, S.B, Fingerman, M., 1985. In vitro synthesis of vitellin by the ovary of the fiddler crab, Uca pugilator. J. Exp. Zool. 233, 111᎐116. Fainzilber, M., Tom, M., Shafir, S., Applebaum, S.W., Lubzens, E., 1992. Is there extraovarian synthesis of Vg in penaeid shrimp? Biol. Bull. 183, 233᎐241. Gehring, W.R., 1974. Maturational changes in the ovarian lipid spectrum of the pink shrimp, Penaeus duorarum Burkenroad. Comp. Biochem. Physiol. 49A, 511᎐524. Gonazalez-Baro, M., Pollero, R.G., 1988. Lipid characterization and distribution among tissues of the freshwater crustacean Macrochium borelli during an annual cycle. Comp. Biochem. Physiol. 91B, 271᎐276. Jeckel, W.H., Moreno, J.E.A., Moreno, V.J., 1989. Biochemical composition, lipid classes and fatty acids in the ovary of the shrimp Pleoticus muelleri Bate. Comp. Biochem. Physiol. 92B, 271᎐276. Kanazawa, A., Teshima, S., Sakamoto, M., 1985. Effects of dietary lipids, fatty acids, and phospholipids on growth and survival of prawn Ž Penaeus japonicus. larvae. Aquaculture 50, 39᎐49. Kanazawa, A., Tokiw, S., Sakamoto, M., 1977. Essential fatty acids in the diet of prawn ᎏ I Effects of linoleic and linolenic acids on growth. Bull. Jpn. Sco. Sci. Fish. 43, 1111᎐1114. Lautier, J., Lagarrigue, J.G., 1988. Lipid metabolism of the crab Pachygrapsus marmoratus during vitellogenesis. Biochem. Syst. Ecol. 16, 203᎐212. Lui, C.W, Sage, B.A., O’Connor, J.D., 1975. Biosynthesis of lipovitellin by the crustacean ovary. J. Exp. Zool. 188, 289᎐296. Middleditch, B.S., Missller, S.R., Ward, D.G., 1980. Metabolic profiles of penaeid shrimp: Dietary lipids and ovarian maturation. J. Chromatogr. 195, 359᎐368. Milliamena, O.M., Quinito, E.T., 1985. Lipids and essential fatty acids in the nutrition of Penaeus monodon larvae. Proceeding 1st Conference on Culture of Pinaeid PrawnrShrimp, Iloilo City, The Philippines, p. 181. Morrison, W.R, Smith, L.M., 1964. Preparation of fatty acid methyl esters and dimethyl acetals from lipids with boron trifluoride᎐methanol. J. Lipid Res. 5, 600᎐608.

104

X. Wen et al. r Comparati¨ e Biochemistry and Physiology Part B 130 (2001) 95᎐104

Paulus, J.E., Laufer, H., 1987. Vitellogenocytes in the hepatopancreas of Carcinus maenas and Libinia emarginata ŽDecapoda, Brachyura.. Int. J. Invertebrate Reprod. Dev. 11, 29᎐44. Quackenbush, L.S., 1989. Yolk protein production in the marine shrimp Penaeus ¨ annmei. J. Crustacean Biol. 9, 509᎐516. Skipski, V.P., Barclay, M., 1969. Thin-layer chromatography of lipids. In: Lowenstein, J.M. ŽEd.., Methods in Enzymology, 14. Academic Press, New York, pp. 530᎐598. Skipski, V.P., Peterson, R.F., Sander, J., 1963. Thinlayer chromatography of phospholipids using Silica gel without calcium sulfate binder. J. Lipid Res. 4, 227᎐230. Skipski, V.P., Smolowe, A.F., Sullivan, R.C., 1965. Separation of lipid classes by thin-layer chromatography. Biochim. Biophys. Acta 106, 386᎐402. Teshima, S., Kanazawa, A., 1983. Variation in lipid

composition during the ovarian maturation of the prawn. Bull. Jpn. Soc. Sci. Fish. 49, 957᎐962. Xu, X.L., Ji, W.J., Castell, J.D., O’Dor, R.K., 1994aa. Influence of dietary lipid sources on fecundity, egg hatchability and fatty acid composition of Chinese prawn Ž Penaeus chinensis. broodstock. Aquaculture 119, 359᎐370. Xu, X.L., Ji, W.J., Castell, J.D., 1994bb. Essential fatty acid requirement of the Chinese prawn, Penaeus chinensis. Aquaculture 127, 29᎐40. Xue, J.Z, Du, N.S., Lai, W., 1987. Histology of female reproductive system in Chinese mitten-handed crab, Eirocheir sinensis ŽCrustacean, Dacapoda.. J. East China Normal Univ. ŽNatural Science Edition. 3, 88᎐97. Young, N.J., Quinlan, P.T., Goad, L.J., 1992. Cholesteryl esters in the decapod crustacean, Penaeus monodon. Comp. Biochem. Physiol. 102B Ž4., 761᎐768.