Aquaculture 356–357 (2012) 310–316

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The effects of dietary n−3 series fatty acid on the fatty acid composition, cholesteroland fat-soluble vitamins of pleopodal eggs and stage 1 juveniles in a freshwatercrayfish, Astacus leptodactylus (Eschscholtz)

Muzaffer Mustafa Harlioğlu a,⁎, Kenan Köprücü a, Ayşe Gül Harlioğlu a, Ökkeş Yilmaz b, Sevinç Aydin b,Serpil Mişe Yonar a, Tuba Çakmak Duran a, Sinan Özcan a

a Fisheries Faculty, Fırat University, 23119, Elazığ, Turkeyb Department of Biology, Faculty of Science, Fırat University, 23169, Elazığ, Turkey

⁎ Corresponding author. Tel.: +90 424 237 0000x454E-mail addresses: mharlioglu@firat.edu.tr, mharliogl

(M.M. Harlioğlu).

0044-8486/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.aquaculture.2012.05.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 November 2011Received in revised form 1 May 2012Accepted 2 May 2012Available online 9 May 2012

Keywords:Astacus leptodactylusCholesterolPleopodal eggFat-soluble vitaminFatty acidJuvenile

To investigate the effects of dietary n−3 series fatty acid levels on the fatty acid composition, cholesterol andfat-soluble vitamins of pleopodal eggs and stage 1 juveniles in Astacus leptodactylus one control (D1) and threeexperimental diets (D2, D3 and D4) were prepared. n−3 series fatty acids were not included in the controldiet, however, D2, D3 and D4 diets were supplemented with n−3 series fatty acids (n−3 series fatty acid con-centrated from anchovy oil and contains 54.3% eicosapentaenoic acid, 37.1% docosahexaenoic acid and 8.6%other n−3 series fatty acid; docosapentaenoic, linolenic, stearidonic acid) at 1%, 2% and 3% level, respectively.Crayfish were fed these isonitrogenous (35% crude protein) and isocaloric (3600 kcal kg−1 gross energy) for-mulated diets for 251 days. The results showed that the dietary n−3 series fatty acids improved the chemicalquality of pleopodal eggs and stage 1 juveniles (i.e., fatty acid composition, cholesterol and fat-soluble vitamincontent). The results also showed that the fatty acid profiles of pleopodal eggs and juveniles were similar tothe fatty acid profiles of D1, D2, D3 and D4. In general, increasing concentrations of dietary n−3 series fattyacids lowered the deposition of linoleic acid (18:2n−6) and total n−6 PUFA (polyunsaturated fatty acids),but simultaneously improved the deposition of linolenic acid (18:3n−3), stearidonic acid (18:4n−3),eicosapentaenoic acid (20:5n−3), docosapentaenoic acid (22:5n−3), docosahexaenoic acid (22:6n−3) andtotal n−3 PUFA in the eggs and juveniles. However, the levels of cholesterol and fat-soluble vitamins inpleopodal eggs and stage 1 juveniles were unaffected by the dietary n−3 series fatty acids (except δ-tocopherol, vitamin K2 and cholesterol levels of stage 1 juveniles). The best chemical quality in eggs and juve-niles was obtained from the crayfish fed D4. In conclusion, n−3 series fatty acid concentrations in broodstockdiets significantly improved the quality of pleopodal eggs and stage 1 juveniles in A. leptodactylus.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Lipids and fatty acids play important roles on the growth and repro-ductive parameters of fish species such as gonadal development, eggquality, spawning success, hatchability and larval quality (Izquierdoet al., 2001; Ling et al., 2006; Thompson et al., 2010). For example, es-sential fatty acids, especially n−3 series fatty acids, are one of themost important nutritional factors which greatly affect egg and larvalquality in fish species (Furuita et al., 2007; Mourente and Odriozola,1990; Navas et al., 1997; Watanabe et al., 1984a,b). The effects ofeicosapentaenoic acid (EPA, 20:5n−3), docosahexaenoic acid (DHA,22:6n−3) and arachidonic acid (AA, 20:4n−6) on various aspects of re-production, such as oocyte maturation, ovulation, spawning, hatching

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success and larval quality have been determined in some studies (AnnSorbera et al., 2001; Bell and Sargent, 2003; Jaya-Ram et al., 2008;Rodriguez-Gonzalez et al., 2006a; Zakeri et al., 2011). In addition, the ef-fect of dietary highly unsaturated fatty acids (HUFAs) on the reproduc-tive performance, egg and larval quality was found in Sparus aurata byFernandez-Palacios et al. (1995), Rodriguez et al. (1998) and Almansaet al. (1999) in Plectorhynchus cinctus by Li et al. (2005), in Dicentrarchuslabrax by Bell et al. (1997) and Bruce et al. (1999), in Paralichthysolivaceus by Furuita et al. (2002, 2003), and in Japanese eel, Anguilla ja-ponica by Furuita et al. (2007).

In general, low levels of n−3HUFA in the broodstock diet reduce thequality of the eggs (Furuita et al., 2002). Negative effects have also beenobserved in egg quality as a result of excessive n−3HUFA in broodstockdiets (Fernandez-Palacios et al., 1995; Furuita et al., 2000; Lavens et al.,1999). Furuita et al. (2002), working with the Japanese flounder,established that 20–25% of the fatty acids in the broodstock diet shouldbe n−3 HUFA in order to produce high-quality eggs and larvae.

Table 1Composition of the experimental diets.(Modified from Reigh et al., 1990).

Food ingredients Experimental diets (%) (diet no.)

Control (diet D1) Diet D2 Diet D3 Diet D4

Casein (83% crude protein) 36 36 36 36Gelatin (83% crude protein) 6.2 6.2 6.2 6.2Dextrin 24.7 22.4 20.2 18Wheat starch 12 12 12 12Sunflower oil 8 8 8 8N−3 series fatty acida – 1 2 3Dicalcium phosphate 1.00 1.00 1.00 1.00Sodium phosphate 0.40 0.40 0.40 0.40Antioxidantb 0.10 0.10 0.10 0.10Vitamin mixturec 0.50 0.50 0.50 0.50Mineral mixtured 0.18 0.18 0.18 0.18Alfa-cellulose 10.92 12.22 13.42 14.62

a N−3 series fatty acid concentrated from anchovy oil and contains 54.3% eicosapentaenoicacid (EPA), 37.1% docosahexaenoic acid (DHA) and 8.6% other n−3 series fatty acid (doco-sapentaenoic, linolenic, stearidonic acid).

b Antioxidant (mg kg−1): butylated hydroxytoluene 12.5.c Vitaminmixture: (IU ormg kg−1 diet): Vitamin A 10,000 IU, vitamin D3 1000 IU, vitamin

E 100 IU, vitamin K 15mg, vitamin B1 5mg, vitamin B2 15mg, niacin 150mg, calcium d-pantothenat50mg, vitaminB610mg, vitaminB12 0.02mg, folic acid3 mg,D-biotin 1mg, cho-line chloride 500mg, vitamin C 300mg.

d Mineral mixture (mg kg−1 diet): Mn 80, Fe 35, Zn 50, Cu 5, I 2, Co 0.4, Se 0.15.

311M.M. Harlioğlu et al. / Aquaculture 356–357 (2012) 310–316

Similarly, numerous studies have suggested the importance of incorpo-rating AA (20:4n−6) into the broodstock diet in order to improve eggquality (Furuita et al., 2003; Mazorra et al., 2003; Salze et al., 2005).

The dietary regime impacts condition and reproductive perfor-mance in crustaceans (Harrison, 1997; Ribeiro et al., 2011; Wuet al., 2010). Therefore, energy and nutritional requirements mustbe adequately satisfied for the onset of gonad maturation. Accordingto Harrison (1990) nutrient reserves are mobilized and metabolizedduring maturation until oocyte enlargement and sufficient and essen-tial nutrients in the diet are critical. For example, highly unsaturatedfatty acids (HUFA) are known as essential nutrients for many crusta-ceans (Cavalli et al., 1999; Sheen and Wu, 2003; Sui et al., 2007; Wuet al., 2007a, b); thus, it was stated that the dietary supplementationof HUFA is crucial for many cultured crustaceans (Gong et al., 2000;Gonzalez-Felix et al., 2002a,b; Suprayudi et al., 2004; Wu et al., 2010).

In addition, several studies have shown that supplementation ofdietary HUFA not only enhances survival and growth in larvae andearly juveniles but also improves ovarian development and reproduc-tive performance of adult Chinese mitten crab, Eriocheir sinensis(Cheng et al., 1998a; Sui et al., 2007; Wu et al., 2007a). However, itis still unclear whether dietary HUFA supplementation may alsolead to a higher incidence of precocious puberty due to their well-known ejects of enhancing ovarian maturation in crustaceans.

Astacus leptodactylus is accepted as a cold-water living specieswith high economic value in the world (Harlıoğlu, 2004; Holdich,1993; Wickins and Lee, 2002). However, little is known on the im-provement of reproductive efficiency (i.e., egg and juvenile quality)of this species. The positive effects of dietary vitamin E on thepleopodal egg and stage 1 juvenile number of A. leptodactylus wasfound by Harlıoğlu et al. (2002), Harlıoğlu and Barım (2004). In addi-tion to these studies, the best pleopodal eggs and stage 1 juvenile num-ber of A. leptodactylus broodstock was achieved at 35% dietary crudeprotein and 3% dietary n−3 HUFAs concentrations by Harlıoğlu et al.(2012).

In addition, it is necessary to obtain n−3 HUFA concentrationssuitable for developing formulated broodstock diets. It is also neces-sary to determine the ways in which the egg and larval qualitychanges with increasing concentrations of dietary n−3 HUFAs. There-fore, the present study was carried out to investigate the effects of di-etary n−3 series fatty acids on the pleopodal eggs and stage 1juveniles quality (fatty acid composition, cholesterol and fat-solublevitamin content) of A. leptodactylus.

2. Material and methods

2.1. Crayfish and experimental design

This study was conducted between October 17, 2010 and June 24,2011 (251 days) at the crayfish reproduction unit of the Fisheries Fac-ulty of Fırat University, Elazığ, Turkey. The crayfish used in the pre-sent study were obtained from the Keban Dam Lake population of A.leptodactylus.

Four dietswere prepared to investigate the effect of different levels ofn−3 series fatty acids on the number and size of pleopodal eggs, andnumber of stage 1 juveniles in A. leptodactylus. For this aim, one control(D1) and three experimental diets (D2, D3 and D4) were prepared(Table 1). n−3 series fatty acids were not included to the controlgroup. However, D2, D3 and D4 groups were supplemented with n−3series fatty acids at 1%, 2% and 3% level, respectively. The practical controldiet used in the present study was modified from Reigh et al. (1990).

The experimental diets were formulated to contain approximately35% crude protein, on a dry-weight basis and 3600 kcal/kg gross ener-gy. The crude protein content was analyzed by Kjeldahl's method.Gross energy was calculated based on physiological fuel values of0.038 MJ/g for lipid and 0.017 MJ/g for protein and carbohydrate.Dry matter was determined after drying at 105 °C for 6 h. Ash content

was calculated after 24 h at 550 °C in the furnace, and lipid analyzedby an ether extraction method (AOAC, 1997).

Casein, gelatin, dextrin, wheat starch, α-cellulose, vitamin-mineralmixture, dicalcium phosphate, sodium phosphate, antioxidant, concen-trated anchovy n−3 series fatty acids and sunflower oil were donatedby a local food producer firm (Ürün Veren Su Ürünleri, Turizm,Medikal,Hayvancılık, Yem Sanayi Limited Cooperation, Elazığ, Turkey).

Each test ingredient was ground to a particle size of 500 μm. Dietswere mechanically mixed with distilled water (40/100 g diet, mix)and pressure-pelleted. The resultant moist pellets were then oven-dried at 60 °C for approximately 12 h and then allowed to cool to am-bient temperature in the oven. The feeds were then broken up andsieved into convenient pellet sizes (approximately 4 mm) for thecrayfish and were stored in polythene bags at 4 °C until used. Theamount of diet needed weekly was then kept at room temperature.

The experiment was carried out with 3 replicates for each dietarytreatment. Forty-eight females and 12 males were used for each rep-licate (576 females and 144 males in total). Crayfish were housed in12 concrete tanks (2×2×0.5 m) on October 17, 2010. Stocking densi-ty was 15 individuals/m2 (4 females:1 male). Sixty plastic pipes(20 cm in length and 7 cm in diameter) were provided as sheltersfor the crayfish in each tank. Supplemental water flow was 1.5 l/sper 1 m2 of tank.

Frequency and amount of feeding were adjusted according to sea-son and food consumption level. Amount of food fed was calculatedaccording to the following equation (NRC, 1987) and offered inthree separate meals:

Daily food amount to feed (g food/day)=(water temperature °C/10)×(crayfish weight/100).

During the trial, dissolved oxygen, pH and water temperaturewere measured daily. Ammonia, iron, copper, alkalinity, hardness,calcium and water flowwere measured twice a week. Mean dissolvedoxygen was 7.5±1.0 mg/l; mean ammonia, iron and copper contentwere less than 0.001 mg/l (for each parameter); mean calcium was39.2±1.6 mg/l; mean alkalinity was 217.3±2.5 mg CaCO3/l; meanhardness was 34±4 FSº; mean pH was 7.8±0.3 (APHA, 1985).Mean water temperature was 9.6±5.3 °C.

In the present study, crayfish in outdoor tanks were exposed tonatural photoperiod from October to June.

The mating and spawning of A. leptodactylus started on January 15,2011. To determine the effect of dietary n−3 series fatty acids on A.

Table 2Fatty acid composition of the experimental diets.

Fatty acids Control(diet D1)1

Diet D2 Diet D3 Diet D4

(% total fatty acids)

16:0 5.64±0.07a 5.37±0.17b 5.29±0.16b 5.43±0.11b

16:1n−7 0.76±0.01b 1.08±0.17a 1.05±0.04a 1.22±0.12a

18:0 3.33±0.04b 3.48±0.16ab 3.44±0.05ab 3.57±0.10a

18:1n−9 27.71±0.09a 24.98±0.96b 24.04±0.02b 24.73±0.91b

18:2n−6 60.37±0.37a 51.55±4.76b 48.60±0.95bc 45.39±0.51c

18:3n−6 0.19±0.02b 0.27±0.01a 0.30±0.01a 0.30±0.02a

20:0 nd5 1.30±0.55 1.10±0.45 1.60±0.2022:0 nd 0.32±0.01 0.26±0.01 0.32±0.0120:4n−3 0.54±0.02b 1.21±0.34a 1.35±0.07a 1.44±0.23a

20:4n−6 nd 0.30±0.10 0.38±0.02 0.34±0.0720:5n−3 0.85±0.12c 5.08±1.62b 7.19±0.18a 8.07±0.16a

22:5n−3 nd 1.25±0.10 1.80±0.07 2.03±0.0222:6n−3 0.58±0.08c 3.32±0.65b 4.82±0.10a 5.21±0.14a

24:0 nd 0.32±0.01 0.28±0.02 0.33±0.01∑SFA2 8.97±0.09b 10.79±1.11a 10.37±0.65ab 11.25±0.63a

∑MUFA3 28.47±0.08a 26.06±0.79b 25.09±0.05b 25.95±0.79b

∑PUFA4 62.53±0.14a 62.98±2.00a 64.44±0.59a 62.78±0.47a

∑ n−3 PUFA 1.97±0.22c 10.86±2.70b 15.16±0.27a 16.75±0.07a

∑ n−6 PUFA 60.56±0.28a 52.12±3.69b 49.28±0.86bc 46.03±0.54c

n−3/n−6 ratio 0.03±0.01c 0.20±0.07b 0.30±0.01a 0.36±0.01a

1Control diet.2Saturated fatty acids.3Monounsaturated fatty acids.4Polyunsaturated fatty acids.5Not determined.Note: within rows, values (mean±SD, N=3) followed by the different letters aresignificantly different (Pb0.05).

312 M.M. Harlioğlu et al. / Aquaculture 356–357 (2012) 310–316

leptodactylus pleopodal egg number and size, seven ovigerous femaleswere selected randomly from each replicate on January 29, 2011 andeggs stripped and number and size recorded. The rest of the femaleswere maintained for development of stage 1 juveniles. On February 15,2011, after all females laid their eggs the males were removed from thetanks. No entire egg clutches were lost from fertilization to hatching.

Stage 1 juveniles were found from June 20, 2011. To determine theeffect of dietary n−3 series fatty acids on stage 1 juvenile number inA. leptodactylus, seven females were selected randomly from eachreplicate on June 24, 2011 and juveniles were enumerated.

Pleopodal eggs and stage 1 juveniles were removed from the se-lected females by forceps and counted. Carapace length and bodyweight of the females, as well as pleopodal egg size and stage 1 juve-nile weight of each crayfish were recorded. The size of pleopodal eggs(n=10 eggs per female) was measured by use of a light microscopeand graticule.

2.2. Fatty acid, cholesterol and fat-soluble vitamin analysis

2.2.1. Extraction of lipidsThe lipids of the tissue samples were extracted with hexane–iso-

propanol (3:2 v/v) according to the method of Hara and Radin(1978). One g tissue sample was homogenized with 10 ml hexane–isopropanol mixture. The homogenate was centrifuged at 5000 rpmfor 5 min at 4 °C and parts of tissue remnants were precipitated. Thesupernatant part was used in the ADEK, cholesterol and fatty acidanalysis.

2.2.2. Preparation of fatty acid methyl estersFatty acids in the lipid extracts were converted into methyl esters

including 2% sulfuric acid (v/v) in methanol (Christie, 1992). Themix-ture was vortexed and then kept at 50 °C for 12 h. Then, after beingcooled to room temperature, 5 ml of 5% sodium chloride was addedand then it was vortexed. Fatty acid methyl esters were extractedwith 2×5 ml hexane. Fatty acid methyl esters were treated with5 ml 2% KHCO3 solution and then the hexane phase was evaporatedby the nitrogen flow and then by dissolving in 0.5 ml fresh hexane(Christie, 1992), they were taken to auto sampler vials.

2.2.3. Gas chromatographic analysis of fatty acid methyl estersMethyl esters were analyzed with the Shimadzu GC-17 Ver. 3 gas

chromatography (Kyoto, Japan). For this analysis, 25 m of long Per-mobond® FFAP–0.1 μm capillary column (Machery-Nagel, Germany)with an inner diameter of 0.25 μm and a thickness of 25 micron filmwas used. During the analysis, the colon temperature was kept at120–220 °C and the increment of temperature was 3 °C/min, injectiontemperature was kept at 240 °C and the detector temperature waskept at 280 °C. The nitrogen carrier gas flowwas 1 ml/min. The meth-yl esters of fatty acids were identified by comparison with authenticexternal standard mixtures analyzed under the same conditions.After this process, the necessary programming was made and theClass GC 10 software version 2.01 was used to process the data.

2.2.4. HPLC analysis of ADEK vitamins and cholesterolFive milliliters of supernatant was taken to 25 ml tubes with caps

and 5% KOH solution was added and immediately vortexed for 20 s.The tubes were placed in a water bath at 85 °C for 15 min. The tubeswere then taken and cooled to room temperature and 5 ml of distilledwater was added and mixed. Lipophilic molecules that did not saponifywere extractedwith 2×5 ml hexane. The hexane phasewas evaporatedwith nitrogen flow. It was dissolved in 1 ml (50+50%, v v−1) acetoni-trile/methanol mixture and then was taken to auto sampler vials andwas analyzed.

The analysis was made with the Shimadzu brand HPLC device. HPLCconditions were as follows: mobile phase 60:38:2 (v/v/v): acetonitrile/methanol/water; the mobile phase flow rate was determined to be 1 ml

A UV detector was used for the analysis and as a column the SupelcosilLC 18 (15×4.6 cm 5 μm; Sigma USA) column was used. For vitamin Eand cholesterol 202 nm, retinol, 326 nm and for vitamins D and K,265 nm was used (Katsanidis and Addis, 1999; Lopez-Cervantes et al.,2006).

2.3. Statistical analyses

The data on the fatty acid composition, cholesterol and fat-solublevitamins obtained from the pleopodal eggs and stage 1 juveniles ofA. leptodactylus were analyzed statistically using one-way analysis ofvariance (ANOVA), followed by Duncan's new multiple range test.Significant differences were based on the Pb0.05 level.

3. Results

Fatty acid composition of the experimental diets is shown inTable 2.

3.1. Pleopodal egg's fatty acid composition, fat soluble vitamins and cho-lesterol content

The fatty acid composition, fat soluble vitamins and cholesterollevels of pleopodal eggs are shown in Tables 3 and 4, respectively. Thelinolenic acid (LNA, 18:3n−3), stearidonic acid (SDA, 18:4n−3), EPA(20:5n−3), docosapentaenoic acid (DPA, 22:5n−3), DHA (22:6n−3),AA (20:4n−6) and total n−3 PUFA concentrations of A. leptodactyluseggs increased significantly with an increase in dietary n−3 seriesfatty acids (Pb0.05). In addition, n−3:n−6 ratio in pleopodal eggsalso increased with an increased concentration of dietary n−3 PUFA.

The proportion of AA (20:4n−6) in the pleopodal eggs of femalesfed on each of the diets was lower than that in the stage 1 juveniles ofA. leptodactylus (Table 5). However, cholesterol and fat-soluble vita-min levels of pleopodal eggs unaffected by the dietary n−3 seriesfatty acids (Table 4). The finest fatty acid composition, cholesteroland fat-soluble vitamin values in the pleopodal egg were obtainedfrom the crayfish fed D4.

Table 3Fatty acid composition of A. leptodactylus pleopodal eggs fed the experimental diets.

Fatty acids Control(diet D1)1

Diet D2 Diet D3 Diet D4

(% total fatty acids)

14:0 0.69±0.10a 0.61±0.16a 0.58±0.11a 0.61±0.03a

14:1 0.15±0.01b 0.10±0.01b 0.22±0.14ab 0.35±0.04a

15:0 0.66±0.08a 0.52±0.11a 0.69±0.12a 0.51±0.04b

15:1 0.98±0.14b 1.51±0.31a 1.20±0.26b 1.47±0.62a

16:0 13.84±0.67a 13.87±1.80a 13.20±0.72a 13.88±1.0a

16:1n−7 10.51±0.81b 10.67±1.26ab 12.05±1.91a 9.22±1.09b

17:0 0.55±0.19ab 0.31±0.08b 0.79±0.33a 0.59±0.06a

17:1 0.41±0.06b 1.26±0.27a 1.27±0.32a 1.22±0.53a

18:0 1.91±0.25b 1.96±0.47b 2.06±0.54b 2.89±0.58a

18:1n−9 25.9±1.32a 24.70±1.48a 24.02±0.81a 24.86±0.66a

18:2n−6 17.22±0.8a 15.74±1.85a 12.30±1.17b 11.66±1.35b

18:3n−3 0.37±0.03b 0.41±0.24b 0.46±0.21b 1.27±0.22a

18:4n−3 0.28±0.12c 0.37±0.27c 0.84±0.05b 1.35±0.25a

20:0 1.62±0.10 2.38±2.74 0.86±0.31 nd5

20:1n−9 1.04±0.11 0.86±0.23 1.21±0.61 nd20:3n−6 0.07±0.08 nd 0.15 nd20:4n−6 5.85±0.29ab 5.52±0.26b 5.89±0.59ab 6.14±0.36a

20:4n−3 0.66±0.08a 0.43±0.09b 0.66±0.09a 0.71±0.01a

20:5n−3 6.78±0.43d 7.67±0.29c 8.71±0.45b 9.6±0.51a

22:0 0.65±0.04 0.57±0.16 0.45±0.09 nd22:1 0.38±0.07a 0.21±0.02b 0.29±0.11ab 0.35±0.04a

22:5n−3 1.35±0.28c 1.59±0.39bc 1.85±0.40b 2.38±0.21a

22:6n−3 6.96±0.40b 7.13±1.99b 8.92±0.85a 9.80±0.74a

24:1n−9 1.09±0.08b 1.59±0.34a 1.26±0.25b 0.97±0.39b

∑SFA2 19.92±0.86a 20.22±3.44a 18.63±0.65a 18.48±0.75a

∑MUFA3 40.46±1.04b 40.90±3.62b 41.52±0.71a 38.44±0.51c

∑PUFA4 39.54±1.32b 38.86±1.53b 39.78±1.58b 42.99±1.95a

∑ n−3 PUFA 16.4±1.03c 17.6±2.89c 21.44±1.74b 25.11±1.37a

∑ n−6 PUFA 23.14±0.90a 21.26±3.71a 18.34±0.87b 17.8±1.48b

n−3/n−6 ratio 0.70±0.05b 0.82±0.41b 1.16±0.13a 1.41±0.14a

1Control diet.2Saturated fatty acids.3Monounsaturated fatty acids.4Polyunsaturated fatty acids.5Not determined.Note: within rows, values (mean±SD, N=8) followed by the different letters aresignificantly different (Pb0.05).

313M.M. Harlioğlu et al. / Aquaculture 356–357 (2012) 310–316

3.2. Stage I juvenile's fatty acid composition, fat soluble vitamins andcholesterol content

The fatty acid composition of stage 1 juvenile A. leptodactylus isshown in Table 5. The results showed that an increase in dietary n−3series fatty acids caused a significant increase (Pb0.05) in the LNA(18:3n−3), stearidonic acid (SDA, 18:4n−3), EPA (20:5n−3), DPA(22:5n−3), DHA (22:6n−3) and the total n−3 PUFA concentrationsof stage 1 juvenile A. leptodactylus. The n−3:n−6 ratio of stage 1 juve-niles was also increased with an increased concentration of dietaryn−3 PUFA. In contrast, the content of total n−6 PUFA in stage 1 juve-niles obtained from the crayfish fed D1, D2, D3 and D4 were muchlower than their respective dietary concentrations (Table 2).

The total PUFA content of stage 1 juveniles showed significant differ-ences (Pb0.05) among the treatments. However, the content of most

Table 4The fat soluble vitamins and cholesterol of A. leptodactylus eggs.

Vitamin and cholesterol (μg/g)* Control (diet D1) Di

Retinol 0.21±0.05a

Retinol ast. 0.40±0.11a

D2 0.96±0.45a

D3 2.08±0.43a

δ-Tocopherol 0.32±0.22a

α-Tocopherol 44.48±32.8a 4K1 0.45±0.05a

K2 21.58±2.79a 1Cholesterol 1267.7±110.38a 13

*Each value is an average of 8 samples, with its standard deviation (mean±SD). Within ro

fatty acids or fatty acid classes of the lipid was directly influenced bythe dietary n−3 series fatty acid concentrations, and displayed signifi-cant differences among the diets (Pb0.05).Moreover, the fat-soluble vi-tamin levels (except retinol ast., δ-tocopherol and vitamin K2) of stage 1juveniles unaffected from the dietary n−3 series fatty acids (Table 6),but the cholesterol level of stage 1 juveniles significantly affected fromthe dietary n−3 PUFA (Pb0.05). The best chemical quality in stage 1 ju-venile was obtained from the crayfish fed D4.

4. Discussion and conclusion

Contributions of dietary HUFA concentrations on egg quality andchemical composition in several fish species were reported in previ-ous studies (Furuita et al., 2000, 2002; Li et al., 2005; Navas et al.,1997; Pickova et al., 1997; Rodriguez et al., 1998; Watanabe et al.,1984b; Zakeri et al., 2011). It was also reported that lipids play amajor role in many reproductive processes of crustaceans. Studieshave shown that the levels and composition of dietary lipids greatlyaffect ovarian maturation and reproductive performance in crusta-ceans (Harrison, 1990). However, the dietary lipid level can be lowif it provides sufficient levels of essential fatty acids; using a 2:1 codliver oil/corn oil semi purified diet Sheen and D'Abramo (1991)found weight gain in juvenile Macrobrachium rosenbergii to behighest at 6%. Among them, the polyunsaturated fatty acids (PUFA)are particularly important. The content of PUFA in artificial and natu-ral diets impacts survival, growth, feed conversion, fecundity, egghatchability, molting and osmotic stress tolerance in crustaceans(Breet and Muller-Navarra, 1997; Cavalli et al., 1999; D'Abramo andSheen, 1993). An increase of n−3 and n−6 PUFA in broodstockdiets enhanced fecundity and egg hatchability in freshwater prawnM. rosenbergii (Cavalli et al., 1999). However, D'Abramo (1997) statedthat the ratio of n−3 and n−6 fatty acids may be more importantthan their absolute value to crustacean nutrition.

The nutritional reserves and metabolic processes that occur duringvitellogenesis are essential to understanding the reproduction of the fe-male decapods, and these topics have been examined for a number ofcrustacean species (Palacios et al., 2000; Rodriguez-Gonzalez et al.,2006b;Wen et al., 2001). The reproductive cycle is influenced bymulti-ple physiological processes as well as food sources and themobilizationof energy reserves, and understanding these factors is necessary to de-termine how these processes affect reproduction. For example, evalua-tion of amino acid and fatty acid composition during vitellogenesisprovides information about gonadal performance, nutrient flow anduse of nutritional reserves during vitellogenesis (Cavalli et al., 1999;Cheng et al., 1998b;Wen et al., 2001). On the other hand, optimum die-tary lipid levels, aswell as essential fatty acid requirement, have not beendetermined for broodstock of A. leptodactylus. The present results, there-fore, supply the first proof of dietary n−3 series fatty acids on the chem-ical quality of pleopodal eggs and stage 1 juveniles in A. leptodactylus.

The present study showed that the inclusion of dietary n−3 seriesfatty acids into the diets of A. leptodactylus improved the chemical qual-ity (fatty acid composition, cholesterol and fat-soluble vitamins) of

et D2 Diet D3 Diet D4

0.22±0.17a 0.28±0.15a 0.31±0.17a

0.31±0.09a 0.39±0.11a 0.47±0.19a

0.95±0.22a 0.97±0.07a 0.95±0.22a

2.02±0.77a 2.35±1.12a 2.27±0.59a

0.26±0.12a 0.50±0.07a 0.51±0.06a

6.67±15.79a 51.54±15.62a 62.46±33.57a

0.38±0.18a 0.35±0.19a 0.47±0.05a

9.15±4.58a 20.57±4.70a 18.71±2.55a

25.2±406.27a 1064±211.96a 1336.8±116.4a

ws, values followed by the different letters are significantly different (Pb0.05).

Table 5Fatty acid composition of stage 1 juvenile A. leptodactylus fed the experimental diets.

Fatty acids Control(diet D1)1

Diet D2 Diet D3 Diet D4

(% totalfatty acids)

14:0 0.61±0.06ab 0.66±0.03a 0.65±0.07a 0.57±0.06b

14:1 0.56±0.09a 0.46±0.02b 048±0.01b 0.49±0.04b

15:0 0.65±0.04a 0.66±0.03a 0.65±0.07a 0.68±0.08a

15:1 1.14±0.08a 0.98±0.09b 1.06±0.22ab 0.93±0.05b

16:0 15.69±0.57b 16.69±0.2a 16.54±0.6a 16.46±0.44a

16:1n−7 10.44±0.19a 10±0.43a 10.09±1.02a 9.64±1.23a

17:0 0.58±0.12a 0.42±0.05b 0.42±0.01b 0.44±0.01b

17:1 0.93±0.19a 0.67±0.19b 0.70±0.19b 0.64±0.2b

18:0 2.58±0.11a 2.45±0.32a 2.67±0.56a 2.60±0.45a

18:1n−9 24.72±0.46a 24.26±0.34a 23.78±0.36b 24.31±0.51a

18:2n−6 15.05±0.47a 14.46±1.08a 11.86±1.19b 11.57±0.5b

18:3n−3 1.42±0.32ab 1.49±0.07ab 1.53±0.18a 1.28±0.15b

20:1n−9 1.69±0.14b 1.95±0.17a 1.97±0.21a 1.86±0.19ab

20:2n−6 0.94±0.01a 0.99±0.04a 1.01±0.12a 0.99±0.11a

20:4n−6 6.08±0.32ab 5.88±0.62b 6.67±0.91a 6.47±0.52ab

20:5n−3 7.48±0.28b 7.45±0.53b 8.38±1.25ab 8.88±1.27a

22:0 0.16±0.01b 0.21±0.01ab 0.24±0.02a 0.24±0.05a

22:1 0.36±0.02b 0.33±0.02b 0.36±0.02b 0.43±0.08a

22:5n−3 0.44±0.02b 0.46±0.03b 0.54±0.04b 1.03±0.47a

22:6n−3 7.54±0.37c 8.60±0.24b 9.34±0.53a 9.58±0.55a

24:0 0.94±0.05ab 0.90±0.04ab 1.02±0.06a 0.89±0.12b

∑SFA2 21.21±0.41b 21.99±0.17a 22.19±0.47a 21.88±0.33a

∑MUFA3 39.84±0.35a 38.65±0.78ab 38.44±1.18b 38.30±1.80b

∑PUFA4 38.95±0.61a 39.33±0.66a 39.33±1.53a 39.80±1.90a

∑ n−3 PUFA 16.88±0.59b 18±0.64b 19.79±1.48a 20.77±1.38a

∑ n−6 PUFA 22.07±0.67a 21.33±0.70a 19.54±0.79b 19.03±0.72b

n−3/n−6 ratio 0.76±0.04d 0.84±0.05c 1.01±0.09b 1.09±0.05a

1Control diet.2Saturated fatty acids.3Monounsaturated fatty acids.4Polyunsaturated fatty acids.Note: within rows, values (mean±SD, N=8) followed by the different letters aresignificantly different (Pb0.05).

314 M.M. Harlioğlu et al. / Aquaculture 356–357 (2012) 310–316

pleopodal eggs and stage 1 juveniles. In addition, it was found that thefatty acid profiles of pleopodal eggs and stage 1 juveniles reflected theprofiles of experimental diets (D1, D2, D3 and D4). In general, theincreasing concentrations of dietary n−3 series fatty acids in dietslowered the deposition of linoleic acid (LA, 18:2n−6) and total n−6PUFA, and simultaneously improved the deposition of LNA (18:3n−3),SDA (18:4n−3), EPA (20:5n−3), DPA (22:5n−3), DHA (22:6n−3)and total n−3 PUFA in eggs and juveniles obtained from the crayfishfed experimental diets (D1, D2, D3 and D4), which probably affectedthe spawning performance of this species. However, cholesterol andfat-soluble vitamin levels of pleopodal eggs and stage 1 juveniles unaf-fected from dietary n−3 series fatty acids (except δ-tocopherol, vitaminK2 and cholesterol levels of stage 1 juvenile). The best chemical qualityin egg and stage 1 juveniles was obtained from the crayfish fed D4.These results imply that the high concentrations of n−3 series fattyacids in the broodstock diets had positive effects on the pleopodal eggsand stage 1 juveniles quality in A. leptodactylus. Similarly, positive effects

Table 6The fat soluble vitamins and cholesterol of stage 1 juveniles of A. leptodactylus.

Vitamin and cholesterol (μg/g)* Control (diet D1) Di

Retinol 0.2±0.14a

Retinol Ast. 0.18±0.06a

D2 0.23±0.15a

D3 1.6±0.32a

δ-Tocopherol 0.7±0.34ab

α-Tocopherol 79.91±28.81a 7K1 0.5±0.3a

K2 21.23±3.27a 1Cholesterol 918.25±196.5a 72

*Each value is an average of 8 samples, with its standard deviation (mean±SD). Within ro

of dietary n−3 series fatty acids on the number of pleopodal eggsand stage 1 juveniles in A. leptodactylus were found by Harlıoğlu et al.(2012). They also obtained the best result from the crayfish fed D4.

However, it was reported that inadequate essential fatty acids inbroodstock diets reduce egg and larval quality in fish species (Furuitaet al., 2000). In the present study, the quality of stage 1 juveniles was sig-nificantly higher in the crayfish fed D4 than the crayfish fed other diets(D1, D2 and D3). This finding is in agreement with the results fromsome fish species, in which deficiency of n−3 HUFAs in broodstockdiet reduced egg and larval quality in Pagrus major (Watanabe et al.,1984a), S. aurata (Zohar et al., 1995), P. olivaceus (Furuita et al., 2000)and P. cinctus (Li et al., 2005). Despite these studies, Furuita et al.(2002) revealed that a high level of dietary n−3 HUFA may also have anegative effect on the egg production, egg quality and larval survival inJapanese flounder. Similar result has also been reported for gilthead seabream by Fernandez-Palacios et al. (1995) for turbot by Lavens et al.(1999) and for P. cinctus by Li et al. (2005).

Chemical composition of fish eggs is one of themost important factorsin evaluating egg quality. So, eggs must satisfy nutritional needs for em-bryonic and larval development (Bell et al., 1997; Furuita et al., 2002; Liet al., 2005). The results of present study showed that the concentrationsof n−3 series fatty acids in eggs were related to the dietary treatment.Thiswas also noticed for red sea bream (Watanabe et al., 1984b), Europe-an sea bass (Navas et al., 1997), and P. cinctus (Li et al., 2005). In addition,profiles and classes of fatty acids including n−3 HUFAs in eggs reflect therespective dietary fatty acid compositions. The findings of present studyrevealed that an increasing trend with increase of dietary n−3 HUFAconcentration. occurs in the LNA (18:3n−3), SDA (18:4n−3), EPA(20:5n−3), DPA (22:5n−3), DHA (22:6n−3), AA (20:4n−6) and totaln−3 PUFA values of eggs.

According to Furuita et al. (2007) AA is themajor precursor of eicos-anoids in fish cells and eicosanoids are important in the control ofovulation. They also stathed that AA are probably involved in embryo-genesis, development of the immune system, hatching and early larvaldevelopment. Therefore, EPA ratio, probably due to the competition ofEPA with enzyme systems producing eicosanoids from AA, and thusexerting a modulating influence over the quantity and efficacy of AA-derived eicosanoids. Hence, the ratio of AA to EPA is a vital nutritionalfactor for embryos and larvae (Furuita et al., 2007). In the presentstudy, it was found that the DHA concentration in the pleopodal eggsof crayfish fed D4 was higher than the crayfish fed control (D1), D2and D3. These may be one of the main reasons for the high egg qualityproduced by broodstock fed D4 because of the fact that these fattyacids are the main component of phospholipids, function as criticalstructural and physiological components of cell membranes. Moreover,DHA, in particular, has a high biological value during larval develop-ment (Furuita et al., 2007; Takeuchi, 1997).

HUFAs are essential nutrients for many crustaceans (Gong et al.,2000; Gonzalez-Felix et al., 2002a,b; Holme et al., 2007; Wu et al.,2010). Previous research carried out on some juvenile freshwatercrustaceans, such as the giant freshwater prawn M. rosenbergii andred claw crayfish Cherax quadricarinatus, generally reported a lower

et D2 Diet D3 Diet D4

0.2±0.14a 0.2±0.26a 0.2±0.14a

0.13±0.05ab 0.18±0.03a 0.11±0.03b

0.18±0.09a 0.24±0.9a 0.22±0.08a

1.61±0.34a 1.55±0.9a 1.58±0.2a

0.62±0.35b 0.9±0.2ab 1.11±0.23a

6.01±23.17a 71.15±21.65a 70.16±20.74a

0.48±0.39a 0.46±0.43a 0.48±0.25a

5.77±2.14c 18.21±2.04b 15.61±1.51c

4.75±119.48b 877.5±144.05ab 738±115.98b

ws, values followed by the different letters are significantly different (Pb0.05).

315M.M. Harlioğlu et al. / Aquaculture 356–357 (2012) 310–316

HUFA requirement than that of marine species (D'Abramo and Sheen,1993; Reigh and Stickney, 1989; Thompson et al., 2003). In addition,Wu et al. (2010) reported that the precocious E. sinensis femaleshad lower ARA, EPA and HUFA contents in hepatopancreas than thenormal juvenile females regardless of being fed diet. This result mayreflect selective absorption and preferential accumulation of HUFAduring ovarian development of precocious E. sinensis, leading to ahigh proportion of hepatic HUFA being transferred to the ovaries(Chen et al., 2003).

However, Ribeiro et al. (2011) reported that the dietary propor-tion of PUFA affected Macrobrachium amazonicum fecundity. A ratioof 1:2 n−3:n−6 fatty acids induced the highest GSI of mature femalesand number of eggs spawned. Raising the level of both n−3 and n−6fatty acids from ~0.6% to ~1.2% of diet did not produce any effect onGSI and fecundity, suggesting that the ratio is more important thanthe absolute value of these two families of fatty acids. It is wellknown that desaturase enzymes have higher affinity for long-chainPUFA n−3 fatty acids than for equivalent chain length n−6 PUFA se-ries (Harrison, 1990). According to Ribeiro et al. (2011), the addition-al amount of long-chain n−3 PUFA present in the 1.2 n−3/1.2 n−6diet competed with n−6 PUFA for available desaturase enzymesand impaired the conversion of 18:2 (linoleic acid) to other PUFAfamily metabolites. Hence, although a dietary level of 1.2% n−6PUFA appears to increase ovary development and fecundity inM. amazonicum, the excess of n−3 long chain PUFA may break thiseffect. Conversely, Cavalli et al. (1999) observed that an increase ofboth n−3 and n−6 PUFA groups from ~0.5 to ~1.4% of diet increasedGSI and fecundity in M. rosenbergii. According to Ribeiro et al. (2011),this suggests that the metabolism of PUFA is species-specific forfreshwater prawns. It may be an evolutionary response to differentfood availability in the environment where each species evolved.

Few studies have been carried out to research effects of dietaryfatty acid content on hatchling success and larval lipid compositionin fish species. In the present study, the beneficial effects of dietaryn−3 HUFA concentrations on the fatty acid profiles in both pleopodaleggs and stage 1 juveniles was found. The results also indicated thatthe content of most fatty acids and fatty acid classes display correla-tion between pleopodal eggs and stage 1 juveniles quality and the re-spective dietary fatty acid compositions. This suggests that the dietaryfatty acid composition affected egg fatty acid content. Similar resultsfor fish larvae were also found by Rennie et al. (2005) and Linget al. (2006). Ling et al. (2006) stated that although maternal transfercould be the major pathway for AA deposition in larvae, the possibil-ity of HUFA biosynthesis in larva cannot be discarded. Furthermore,higher AA concentrations in larva as compared to eggs were alsoreported in several marine species (Li et al., 2005; Ling et al., 2006).

However, in the present study, it was observed that the DHA con-centration of pleopodal eggs was similar to that of stage 1 juvenile foreach treatment. DHA was the main n−3 HUFA in both pleopodal eggsand stage 1 juveniles of A. leptodactylus, and there was a significant dif-ference in the favour of D4 between the broodstocks fed experimentaldiets (D1, D2, D3 and D4). This may be the principal reason of higherpleopodal eggs and stage 1 juvenile quality of the broodstocks fed D4.

In conclusion, the present study demonstrated that the n−3 HUFAconcentrations in broodstock diet improved the quality and fatty acidscomposition of pleopodal eggs and stage 1 juveniles in A. leptodactylus.The best chemical quality in pleopodal eggs and stage 1 juveniles wasobtained from the broodstock fed D4. However, the cholesterol andfat-soluble vitamin levels of pleopodal eggs and stage 1 juveniles (ex-cept δ-tocopherol, vitamin K2 and cholesterol levels) unaffected fromdietary n−3 HUFA.

Acknowledgment

This studywas carried out as a part of a research project, “Increasing repro-ductive efficiency of freshwater crayfish (Astacus leptodactylus Eschscholtz)”

supported by the Scientific and Technological Research Council of Turkey(Project No: TÜBİTAK-TOVAG 110O418).

References

Almansa, E., Jose Perez, M., Cejas, J.R., Badıa, P., Villamandos, J.E., Lorenzo, A., 1999. In-fluence of broodstock gilthead sea bream (Sparus aurata L.) dietary fatty acids onegg quality and egg fatty acid composition throughout the spawning season. Aqua-culture 170, 323–336.

American Public Health Association (APHA), 1985. Standard Methods for the Examina-tion of Water and Wastewater, 16th. Edition. Washington, DC.

Ann Sorbera, L., Francisco Asturiano, J., Carrillo, M., Zanuy, S., 2001. Effects of poly un-saturated fatty acids and prostaglandins on oocyte maturation in a marine teleost,the European sea bass (Dicentrarchus labrax). Biology of Reproduction 64,382–389.

AOAC, 1997. Official Methods of Analysis of Association of Official Analytical Chemists,16th ed. AOAC, Arlington, VA, p. 1298.

Bell, J.G., Sargent, J.R., 2003. Arachidonic acid in aquaculture feeds: current status andfuture opportunities. Aquaculture 218, 515–528.

Bell, J.G., Farndale, B.M., Bruce, M.P., Navas, J.M., Carillo, M., 1997. Effects of broodstockdietary lipid on fatty acid compositions of eggs from sea bass (Dicentrarchuslabrax). Aquaculture 149, 107–119.

Breet, M.T., Muller-Navarra, D.C., 1997. The role of highly unsaturated fatty acids inaquatic foodweb processes. Freshwater Biology 38, 483–499.

Bruce, M., Oyen, F., Bell, G., Asturiano, J.F., Farndale, B., Carrillo, M., Zanuy, S., Ramos, J.,Bromage, N., 1999. Development of broodstock diets for the European sea bass(Dicentrarchus labrax) with special emphasis on the importance of n−3 and n−6highly unsaturated fatty acid to reproductive performance. Aquaculture 177,85–97.

Cavalli, R.O., Lavens, P., Sorgeloos, P., 1999. Performance of Macrobrachium rosenbergiibroodstock fed diets with different fatty acid composition. Aquaculture 179,387–402.

Chen, Z.Z., Cheng, Y.X., Wang, W., 2003. Changes of hepatopancreas index, lipid contentand fatty acid composition in Eriocheir sinensis during precocity. Journal of Fisheryof China 27, 52–61.

Cheng, Y.X., Yan, S.L., Wang, W., Shi, Z.F., Tan, Y.J., 1998a. Effect of dietary polyunsatu-rated fatty acids and phospholipids on survival and growth of Eriocheir sinensisfrom megalopa to juvenile. Journal of Fishery of China 22, 9–15.

Cheng, Y., Du, N., Lai, W., 1998b. Lipid composition in hepatopancreas of Chinese mit-ten crab Eriocheir sinensis at different stages. Acta Zoologica Sinica 44, 420–429.

Christie, W.W., 1992. Gas Chromatography and Lipids. The Oil Pres, Glaskow, p. 302.D'Abramo, L.R., 1997. Triacylglycerols and fatty acids. In: D'Abramo, L.R., Conklin, D.E.,

Akiyama, D.M. (Eds.), Crustacean Nutrition, Advances in World Aquaculture, Vol. 6.World Aquaculture Society, Baton Rouge, Louisiana, USA, pp. 71–84.

D'Abramo, L.R., Sheen, S.S., 1993. Polyunsaturated fatty acid nutrition in juvenile fresh-water prawn Macrobrachium rosenbergii. Aquaculture 115, 63–86.

Fernandez-Palacios, H., Izquierdo, M.S., Robaina, L., Valencia, A., Salhi, M., Vergara, J.,1995. Effects of n−3 HUFA level in broodstock diets on egg quality of giltheadsea bream (Sparus aurata L.). Aquaculture 132, 325–337.

Furuita, H., Tanaka, H., Yamamoto, T., Shiraishi, M., Takeuchi, T., 2000. Effects of n−3HUFA levels in broodstock diet on the reproductive performance and egg and lar-val quality of the Japanese flounder, Paralichthys olivaceus. Aquaculture 187,387–398.

Furuita, H., Tanaka, H., Yamamoto, T., Suzuki, N., Takeuchi, T., 2002. Effects of highlevels of n−3 HUFA in broodstock diet on egg quality and egg fatty acid composi-tion of Japanese flounder, Paralichthys olivaceus. Aquaculture 210, 323–333.

Furuita, H., Yamamoto, T., Shima, T., Susuki, N., Takeuchi, T., 2003. Effect of arachidonicacid levels in broodstock diet on larval and egg quality of Japanese flounder Para-lichthys olivaceus. Aquaculture 220, 725–735.

Furuita, H., Hori, K., Suzuki, N., Sugita, T., Yamamoto, T., 2007. Effects of n−3 and n−6fatty acids in broodstock diet on reproduction and fatty acid composition ofbroodstock and eggs in the Japanese eel Anguilla japonica. Aquaculture 267, 55–61.

Gong, H., Lawrence, A.L., Jiang, D.H., Castille, F., Gatlin, D.M.I.I.I., 2000. Lipid nutrition ofjuvenile Litopenaeus vannamei I. Dietary cholesterol and de-oiled soy lecithin re-quirement and their interaction. Aquaculture 190, 305–324.

Gonzalez-Felix, M.L., Gatlin III, D.M., Lawrence, A.L., Perez-Velazquez, M., 2002a. Effectof dietary phospholipids on essential fatty acid requirements and tissue lipid com-position of Litopenaeus vannamei juveniles. Aquaculture 202, 151–167.

Gonzalez-Felix, M.L., Lawrence, A.L., Gatlin III, D.M., Perez-Velazquez, M., 2002b.Growth, survival and fatty acid composition of juvenile Litopenaeus vannamei feddifferent oils in the presence and absence of phospholipids. Aquaculture 205,325–343.

Hara, A., Radin, N.S., 1978. Lipid extraction of tissues with a low-toxicity. AnalyticalBiochemistry 90 (1), 420–426.

Harlıoğlu, M.M., 2004. The present situation of freshwater crayfish, Astacus leptodactylus(Eschscholtz, 1823) in Turkey. Aquaculture 230, 181–187.

Harlıoğlu, M.M., Barım, Ö., 2004. The effect of dietary vitamin E on the pleopodal eggand stage-1 juvenile numbers of freshwater crayfish Astacus leptodactylus(Eschscholtz, 1823). Aquaculture 236, 267–276.

Harlıoğlu, M.M., Köprücü, K., Özdemir, Y., 2002. The effect of vitamin E on thepleopodal egg number of Astacus leptodactylus (Eschscholtz, 1823). AquacultureInternational 10, 391–397.

Harlıoğlu, M.M., Çakmak, M.N., Köprücü, K., Aksu, Ö., Harlıoğlu, A.G., Mişe Yonar, S.,Çakmak Duran, T., Özcan, S., Gündoğdu, H., 2012. The effect of dietary n−3 seriesfatty acids on the number of pleopodal egg and stage 1 juvenile in freshwater crayfish,

316 M.M. Harlioğlu et al. / Aquaculture 356–357 (2012) 310–316

Astacus leptodactylus Eschscholtz. Aquaculture Research, http://dx.doi.org/10.1111/j.1365-2109.2012.03090.x.

Harrison, K.E., 1990. The role of nutrition in maturation, reproduction and embryonicdevelopment of decapod crustaceans: a review. Journal of Shellfish Research 9,1–28.

Harrison, K.E., 1997. Broodstock nutrition and maturation diets. In: D'Abramo, L.R.,Conklin, D.E., Akiyama, D.M. (Eds.), Crustacean Nutrition, Advances inWorld Aqua-culture, Vol. 6. World Aquaculture Society, Baton Rouge, Louisiana, USA, pp.390–408.

Holdich, D.M., 1993. A review of astaciculture freshwater crayfish farming. Aquatic Liv-ing Resources 6 (3), 307–317.

Holme, M.H., Southgate, P.C., Zeng, C.S., 2007. Assessment of dietary lecithin and cho-lesterol requirements of mud crab, Scylla serrata, megalopa using semi-purifiedmicrobound diets. Aquaculture Nutrition 13, 413–423.

Izquierdo, M.S., Fernandez-Palacios, H., Tacon, A.G.J., 2001. Effect of broodstock nutri-tion on reproductive performance of fish. Aquaculture 197, 25–42.

Jaya-Ram, A., Kuah, M.K., Lim, P.S., Kolkovski, S., Shu-Chien, A.C., 2008. Influence of di-etary HUFA levels on reproductive performance, tissue fatty acid profile and desa-turase and elongase mRNA expression in female zebrafish Danio rerio. Aquaculture277, 275–281.

Katsanidis, E., Addis, P.B., 1999. Novel HPLC analysis of tocopherols and cholesterol intissue. Free Radical Biology & Medicine 27, 1137–1140.

Lavens, P., Leregue, E., Jaunet, H., Brunel, A., Dhert, Ph., Sorgeloos, P., 1999. Effect of di-etary essential fatty acids and vitamins on egg quality in turbot broodstock. Aqua-culture International 7, 225–240.

Li, Y.Y., Chen, W.Z., Sun, Z.W., Chen, J.H., Wu, K.G., 2005. Effects of n−3 HUFA content inbroodstock diet on spawning performance and fatty acid composition of eggs andlarvae in Plectorhynchus cinctus. Aquaculture 245, 263–272.

Ling, S., Kuah, M.K., Muhammad, T.S.T., Kolkovski, S., Shu-Chien, A.C., 2006. Effect of di-etary HUFA on reproductive performance, tissue fatty acid profile and desaturaseand elongase mRNA in female swordtail Xiphpphorus helleri. Aquaculture 261,204–214.

Lopez-Cervantes, G., Sanchez-Machado, D.I., Rios-Vazquez, N.J., 2006. High perfor-mance liquid chromatography method for the simultaneous quantification of reti-nol, tocopherol and cholesterol in shrimp waste hydrolysate. Journal ofChromatography. A 1105, 135–139.

Mazorra, C., Bruce, M., Bell, J.G., Davie, A., Alorend, E., Jordan, N., Rees, J., Papanikos, N.,Porte, M., Bromage, N., 2003. Dietary lipid enhancement of broodstock reproduc-tive performance and egg and larval quality in Atlantic halibut (Hippoglossushippoglossus). Aquaculture 227, 21–33.

Mourente, G., Odriozola, J.M., 1990. Effect of broodstock diets on lipid classes and theirfatty acid composition in eggs of gilthead sea bream Sparus aurata L. Fish Physiol-ogy and Biochemistry 8 (2), 93–101.

National Research Council (NRC), 1987. Predicting feed intake of food-producing ani-mals. Subcommittee on Feed Intake Committee on Animal Nutrition Board on Ag-riculture. National Academy Press, Washington, D.C.

Navas, J.M., Bruce, M., Thrush, M., Farndale, B.M., Bromage, N., Zanuy, S., Carrillo, M.,Bell, J.G., Ramos, J., 1997. The impact of seasonal alternation in the lipid composi-tion of broodstock diets on egg quality in the European sea bass. Journal of Fish Bi-ology 51, 760–773.

Palacios, E., Ibarra, A.M., Racotta, I.S., 2000. Tissue biochemical composition in relationto multiple spawning in wild and pond-reared Penaeus vannamei broodstock.Aquaculture 185, 353–371.

Pickova, J., Dutta, P.C., Larsson, P.O., Kiessling, A., 1997. Early embryonic cleavage pat-tern, hatching success, and egg-lipid fatty acid composition: comparison betweentwo cod (Gadus morhua) stocks. Canadian Journal of Fisheries and Aquatic Sciences54 (10), 2410–2416.

Reigh, R.C., Stickney, R.R., 1989. Effects of purified dietary fatty acids on the fatty acidcomposition of freshwater shrimp, Macrobrachium rosenbergii. Aquaculture 77,157–174.

Reigh, R.C., Braden, S.L., Craig, R.J., 1990. Apparent digestibility coefficients for commonfeedstuffs in formulated diets for red swamp crayfish, Procambarus clarkii. Aqua-culture Society 84, 321–334.

Rennie, S., Huntingford, F.A., Loeland, A.L., Rimbach, M., 2005. Long term partial re-placement of dietary fish oil with rapeseed oil; effects on egg quality of Atlanticsalmon Salmo salar. Aquaculture 248, 135–146.

Ribeiro, K., Franceschini-Vicentini, I.B., Papa, L.P., New, M.B., Valenti, W.C., 2011. Effectof polyunsaturated fatty acids on the fecundity of the Amazon river prawn Macro-brachium amazonicum (Heller, 1862). Aquaculture Research 1–8, http://dx.doi.org/10.1111/j.1365-2109.2011.02980.x.

Rodriguez, C., Cejas, J.R., Martín, M.V., Badia, P., Samper, M., Lorenzo, A., 1998. Influenceof n−3 highly unsaturated fatty acid deficiency on the lipid composition ofbroodstock gilthead sea bream (Sparus aurata L.) and on egg quality. Fish Physiol-ogy and Biochemistry 18, 177–187.

Rodriguez-Gonzalez, H., Hernandez-Llamas, A., Villarreal, H., Saucedo, P.E., Garcia-Ulloa, M., Rodriguez-Jaramillo, C., 2006a. Gonadal development and biochemicalcomposition of female crayfish Cherax quadricarinatus (Decapoda: Parastacidae)in relation to the gonadosomatic index at first maturation. Aquaculture 254,637–645.

Rodriguez-Gonzalez, H., Garcia-Ulloa, M., Hernandez-Llamas, A., Villarreal, H., 2006b.Effect of dietary protein level on spawning and egg quality of redclaw crayfishCherax quadricarinatus. Aquaculture 257, 412–419.

Salze, G., Tocher, D.G., Roy, W.J., Robertson, D.A., 2005. Egg quality determinants in cod(Gadus morhua L.): egg performance and lipids in eggs from farmed and wildbroodstock. Aquaculture Research 36, 1488–1499.

Sheen, S.S., D'Abramo, L.R., 1991. Response of juvenile freshwater prawn, Macro-brachium rosenbergii, to different levels of a cod liver oil/corn oil mixture in a semi-purified diet. Aquaculture 93, 121–134.

Sheen, S.S., Wu, S.W., 2003. Essential fatty acid requirements of juvenile mud crab,Scylla serrata (Forskal, 1775). Crustaceana 75, 1387–1401.

Sui, L.X., Mattieu, W., Cheng, Y.X., Sorgeloos, P., 2007. The effect of dietary n−3 HUFAlevels and DHA/EPA ratios on growth, survival and osmotic stress tolerance of Chi-nese mitten crab Eriocheir sinensis larvae. Aquaculture 273, 139–150.

Suprayudi, M.A., Takeuchi, T., Hamasaki, K., 2004. Essential fatty acids for larval mudcrab Scylla serrata: implication of lack of the ability to bioconvert C18 unsaturatedfatty acids to highly unsaturated fatty acids. Aquaculture 231, 403–416.

Takeuchi, T., 1997. Essential fatty acid requirements of aquatic animal with emphasison fish larvae and fingerlings. Reviews in Fisheries Science 5, 1–25.

Thompson, K., Muzinic, L., Christian, T., Webster, C., Manomatis, L., Rouse, D., 2003. Lec-ithin requirements of juvenile American red claw crayfish Cherax quadricarinatus.Aquaculture Nutrition 9, 223–230.

Thompson, K.R., Bailey, T.J., Metts, L.S., Brady, Y.J., Webster, C.D., 2010. Growth re-sponse and fatty acid composition of juvenile red claw crayfish (Cheraxquadricarinatus) fed different sources of dietary lipid. Aquaculture Nutrition 16,604–615.

Watanabe, T., Arakawa, T., Kitajima, C., Fujita, S., 1984a. Effects of nutritional quality ofbroodstock diets on reproduction of red sea bream. Bulletin of the Japanese Societyof Scientific Fisheries 50, 495–501.

Watanabe, T., Ohashi, S., Itoh, A., Kitajima, C., Fujita, S., 1984b. Effect of nutritional ofdiets on chemical components of red sea bream broodstock and eggs produced.Nippon Suisan Gakk 50 (3), 503–515.

Wen, X., Chen, L., Ai, C., Zhou, Z., Jiang, H., 2001. Variation in lipid composition of Chi-nese mitten-handed crab, Eriocheir sinensis during ovarian maturation. Compara-tive Biochemistry and Physiology Part B, Biochemistry & Molecular Biology 130,95–104.

Wickins, J.F., Lee, D.O'C., 2002. Crustacean Farming Ranching and Culture. BlackwellScience, Oxford. 446 pp.

Wu, X.G., Cheng, Y.X., Sui, L.Y., Zeng, C.S., Southgate, P.C., Yang, X.Z., 2007a. Effect of di-etary supplementation of phospholipid and highly unsaturated fatty acids onreproductive performance and offspring quality of the Chinese mitten crab,Eriocheir sinensis (H. Milne Edwards), female broodstock. Aquaculture 273,602–613.

Wu, X.G., Cheng, Y.X., Tang, B.P., Zhou, B., Hu, B., Yu, Z.Y., Yang, X.Z., 2007b. Changes inlipid and fatty acid composition of pre- and post spawning Onchidium struma. ActaZoologica Sinica 53, 1089–1100.

Wu, X., Wang, Z., Cheng, Y.X., Zeng, C., Yang, X., Lu, J., 2010. Effects of dietary phospho-lipids and highly unsaturated fatty acids on the precocity, survival, growth and he-patic lipid composition of juvenile Chinese mitten crab, Eriocheir sinensis (H.Milne-Edwards). Aquaculture Research 1–12, http://dx.doi.org/10.1111/j.1365-2109.2010.02643.x.

Zakeri, M., Kochanian, P., Marammazi, J.G., Yavari, V., Savari, A., Haghi, M., 2011. Effectsof dietary n−3 HUFA concentrations on spawning performance and fatty acidscomposition of broodstock, eggs and larvae in yellowfin sea bream, Acanthopagruslatus. Aquaculture 310, 388–394.

Zohar, Y., Harel, M., Hassin, S., Tandler, A., 1995. GiltHead seabream (Sparus aurata). In:Bromage, N.R., Roberts, R.J. (Eds.), Broodstock Management and Egg and LarvalQuality. Blackwell Science Ltd., Oxford, London, UK, pp. 94–118.