Arachidonic acid in diets for early maturation stages enhances the final reproductive performances of Pacific white shrimp (Litopenaeus vannamei)

Aquaculture 479 (2017) 556–563

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Arachidonic acid in diets for early maturation stages enhances the final reproductive performances of Pacific white shrimp (Litopenaeus vannamei)

MARK

Houguo Xua,1, Yuling Zhanga,b,1, Kun Luoa, Xianhong Menga,c, Sheng Luana, Baoxiang Caoa, Baolong Chena, Mengqing Lianga,c,⁎, Jie Konga,c,⁎ a

Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, 106 Nanjing Road, Qingdao 266071, China College of marine life sciences, Ocean University of China, 5 Yushan Road, Qingdao 266003, China c Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, 1 Wenhai Road, Qingdao 266237, China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Litopenaeus vannamei Diet 20:4n-6 Reproduction

A 70-day feeding trial with Pacific white shrimp (Litopenaeus vannamei) broodstocks was conducted before eyestalk ablation to investigate the effects of arachidonic acid (ARA) in diets for early maturation stages on the final reproductive performances. Three isonitrogenous and isolipidic experimental diets were formulated to contain different ARA levels: the control diet without ARA supplementation (C, 0.87% ARA of total fatty acids (TFA)) and two diets with low (4.65% of TFA, ARA-L) or high ARA (12.39% of TFA, ARA-H) supplementation level. The diets were randomly assigned to 9 tanks of eight-month-old Pacific white shrimp broodstock (30 females and 30 males in each tank). Shrimp were reared in a flowing seawater system and fed to apparent satiation four times daily. At the end of the feeding trial, unilateral eyestalk of the females was ablated to stimulate the gonad maturation. Following the feeding trial (Period I) was a 28-day reproductive evaluation period (Period II), during which all shrimps were fed the same fresh invertebrate ingredients (a mixture of squid and polychaetes). Tissues samples of females were collected at the end of the feeding trial (Period I) and before spawning (ready to spawn) at the reproductive evaluation period (Period II) respectively to measure the gonadosomatic index (GI), hepatopancreas index (HI), estradiol level, and fatty acid composition. The spawning performance and quality of egg and larvae were assayed at Period II. The results showed that compared to the control group, ARA-L increased the GI at Period II while the ARA supplemented diets increased the estradiol synthesis at Period I. The HI was not influenced by dietary ARA level. Compared to the control group, ARA-L but not ARA-H significantly increased the spawning rate, multi-spawning rate, average spawning frequency and fecundity of female shrimp, diameter of fertilized egg, and crude metamorphosis rate of nauplii at 33 h post spawning. The hatching rate significantly ranked as follows: C < ARA-H < ARA-L. The dietary ARA supplementation significantly improved the duration of nauplius and zoea larvae in the challenge test with low salinity. The experimental diets significantly affected the fatty acid compositions in hepatopancreas and ovaries of female shrimp, as well as those in fertilized eggs. In conclusion, these results suggested that the ARA supplementation in the diets for early maturation stages enhanced the final reproductive performances of Pacific white shrimp.

1. Introduction

et al., 1997; Almansa et al., 2001; Watanabe and Vassallo-Agius, 2003; Beirão et al., 2015; Butts et al., 2015; Luo et al., 2015), the n-6 LC-PUFA arachidonic acid (ARA, C20:4n-6) has been relatively neglected. Studies with fish have demonstrated that moderate levels of dietary ARA have significant positive effects on fish reproductive performances such as spawning performance, egg quality, and offspring quality, either in marine species or in freshwater species (Bruce et al., 1999; Furuita et al., 2003; Mazorra et al., 2003; Zhou et al., 2011). However,

Lipid and fatty acid have been identified as major dietary factors that determine successful reproduction of aquatic animals (Izquierdo et al., 2001; Tocher, 2010). Among the long chain-polyunsaturated fatty acids (LC-PUFA), compared to docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the so-called n-3 LC-PUFA, of which the reproduction regulating effects have been widely studied (Abi-Ayad

⁎

1

Corresponding author at: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, 106 Nanjing Road, Qingdao 266071, China. E-mail addresses: [email protected] (M. Liang), [email protected] (J. Kong). Houguo Xu and Yuling Zhang contributed equally to this work.

http://dx.doi.org/10.1016/j.aquaculture.2017.06.037 Received 28 March 2017; Received in revised form 1 June 2017; Accepted 25 June 2017 Available online 27 June 2017 0044-8486/ © 2017 Published by Elsevier B.V.

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much less information has been available regarding the effects of ARA on reproductive performances of crustaceans although the importance of ARA to crustacean juveniles has been reported (Xu et al., 1994; González-Baró and Pollero, 1998; Izquierdo et al., 2006; Nonwachai et al., 2010; Aguilar et al., 2012). The limited studies with tiger shrimp Penaues monodon and cladoceran Daphnia magna have showed that dietary ARA improved the spawning performance and egg quality (Coman et al., 2011; Schlotz et al., 2013; Ginjupalli et al., 2015). The high ARA concentrations in maturing or mature crustacean gonads, as well as in well-performing live feeds for shrimp broodstock such as polychaetes, also indicate the importance of ARA to crustacean reproduction (Ravid et al., 1999; Wouters et al., 2001a; Leelatanawit et al., 2014; Meunpol et al., 2005; Wu et al., 2010; Taipale et al., 2011). Considering the potential of ARA in enhancing the reproductive performances of crustaceans especially shrimp, the present study is aimed at investigating the effects of dietary ARA supplementation on the reproductive performances of Pacific white shrimp Litopenaeus vannamei, which is one of the most important aquaculture shrimp species in the world. In a recent study of our lab, we have investigated the regulating effects of ARA on gonadal steroidogenesis in a marine teleost, tongue sole Cynoglossus semilaevis, finding that ARA differentially regulates the gonadal steroidogenesis depending on fish gender and maturation stage (Xu et al., 2017). Following this study, the present study is expected to extend our knowledge about the efficacy of ARA in regulating crustacean reproduction. Understanding the effects of different nutrients on reproductive performances of cultured shrimp will be helpful to improve the formulated broodstock diets and consequently to reduce the use of fresh invertebrate ingredients, which have a series of problems such as adequate nutrition, inconsistent supply, and carrying pathogens. In the present study, the whole experiment was divided into two consecutive periods, i.e., a 70-day feeding trial with the experiments diets (Period I) and a subsequent 28-day reproductive evaluation period following eyestalk ablation (Period II), during which all shrimps were fed the same fresh invertebrate ingredients. Shrimp were not fed the experimental diets during Period II for two reasons. First, because of the asynchronism of spawning activities among shrimp during the spawning season, this feeding strategy could ensure the equal feeding duration before spawning among experimental shrimp. Second, in the previous study of our lab with tongue sole C. semilaevis broodstock, it was found that the ARA efficacy varied with maturation stages, more positively regulating the gonadal steroidogenesis in early maturation stages than in mature stages (Xu et al., 2017). Thus, in the present study, we try to pay particular attention on the effects of ARA administration in early maturation stages on the final reproductive performances of Pacific white shrimp.

Table 1 Formulation and proximate composition of the experiment diets (g kg-1). Ingredients

Diet

Fish meal Casein Mussel meal Wheat gluten meal Krill meal Fish oil ARA-enriched oil Soy lecithin Cholesterol Monocalcium phosphate choline chloride L-Ascorbyl-2-polyphosphate Vitamin premixa Mineral premixb Astaxanthin Proximate composition Crude protein Crude lipid Ash

C

ARA-L

ARA-H

400 60 150 147.5 100 70 0 20 5 15 10 7.5 5 5 5

400 60 150 147.5 100 62.5 7.5 20 5 15 10 7.5 5 5 5

400 60 150 147.5 100 50 20 20 5 15 10 7.5 5 5 5

51.6 16.7 12.2

51.7 16.77 12.3

51.7 16.7 12.3

a Vitamin premix (mg or g/kg diet): thiamin 25 mg; riboflavin, 45 mg; pyridoxine HCl, 20 mg; vitamin B12, 0.1 mg; vitamin K3, 10 mg; inositol, 800 mg; pantothenic acid, 60 mg; niacin, 200 mg; folic acid, 20 mg; biotin, 1.2 mg; retinol acetate, 32 mg; cholecalciferol, 5 mg; alpha-tocopherol, 120 mg; wheat middling, 3.67 g. b Mineral premix (mg or g/kg diet): MgSO4·7H2O, 1200 mg; CuSO4·5H2O, 10 mg; ZnSO4·H2O, 50 mg; FeSO4·H2O, 80 mg; MnSO4·H2O, 45 mg; CoCl2·6H2O (1%), 50 mg; NaSeSO3·5H2O (1%), 20 mg; Ca(IO3)2·6H2O (1%), 60 mg; zoelite, 3.485 g.

Table 2 Fatty acid profiles of experimental diets (% total fatty acid)a.

2. Materials and methods 2.1. Experimental diets Three isonitrogenous (51% crude protein) and isolipidic (16.0% crude lipid) experimental diets were formulated to contain different levels of ARA (Table 1). The control diet (C) was formulated using fish meal, mussel meal, wheat gluten meal, krill meal, and casein as protein sources; and fish oil and soy lecithin as lipid sources. An ARA enriched oil (ARA concentration, 41% of total fatty acids (TFA); in the form of triglyceride; Jiangsu Tiankai Biotechnology Co., Ltd., Nanjing, China) was supplemented to the control diet, replacing fish oil, to formulate two diets with low (ARA-L, supplemented with 0.75% (dry matter) ARA enriched oil) or high (ARA-H, with 2.0% ARA enriched oil) ARA content. The diets were made, packed and stored following the common procedures in our laboratory (Xu et al., 2017). The fatty acid compositions of the experimental diets are presented in Table 2. The ARA content in C, ARA-L, and ARA-H was 0.87%, 4.65%, and 12.39% of TFA, respectively.

Fatty acid

C

ARA-L

ARA-H

C14:0 C16:0 C18:0 ∑ SFAb C16:1n-7 C18:1n-9 C18:1n-7 C20:1n-9 ∑ MUFAb C18:2n-6 C20:4n-6 ∑ n-6b C18:3n-3 C20:5n-3 C22:5n-3 C22:6n-3 ∑ n-3b ∑ PUFA ∑ n-3LC-PUFAb ∑ n-3/∑n-6 C18:1n-9/∑n-3

2.35 14.42 4.55 21.32 3.71 13.21 1.27 2.03 20.22 12.88 0.87 13.75 1.90 12.76 3.18 17.23 35.07 48.82 33.17 2.55 0.38

2.34 13.05 4.31 19.70 3.71 12.64 1.17 2.22 19.74 12.06 4.65 16.71 1.88 12.89 3.31 17.69 35.77 52.48 33.89 2.14 0.35

1.04 10.58 4.11 15.73 3.15 12.09 1.12 1.96 18.32 11.29 12.39 23.68 1.78 12.09 3.96 18.15 35.98 59.66 34.20 1.52 0.34

a Some fatty acids, of which the contents are minor, trace amount or not detected, such as C22:0, C24:0, C14:1, C20:2n-6, C20:3n-6, were not listed in the table. b SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; n-6: n-6 unsaturated fatty acid; n-3: n-3 unsaturated fatty acid; LC-PUFA: long chain-polyunsaturated fatty acid.

2.2. Experimental shrimp and feeding procedure (period I) Eight-month-old Pacific white shrimp L. vannamei broodstock, which have been reared with formulated feeds from the early juvenile stage, were used in the present study. The initial average body weight and length of female shrimp was 21.72 ± 1.20 g and 9.31 ± 0.61 cm respectively; males, 20.21 ± 1.78 g and 8.08 ± 0.52 cm respectively. The initial average hepatopancreas index of female shrimp was 3.24 ± 0.49%. The feeding trial was conducted in a flowing seawater 557

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measure the other reproductive indices, such as spawning rate, multispawning rate, average spawning frequency, fecundity, egg diameter, hatching rate, and metamorphosis rate of nauplii. Spawning rate (%) = number of spawning females in one tank/number of survived females in this tank × 100%. Multi-spawning rate (%) = number of females with more than one spawning in one tank/number of spawning females in this tank × 100%. Average spawning frequency = total number of spawning in one tank/number of spawning females in this tank. Fecundity was the average total number of eggs spawned by one female shrimp at one spawning. Fecundity was calculated by multiplying the number of eggs in 500 mL homogeneous rearing water sample (5 replicates for each determination) by the total volume of rearing water in the spawning tank. The density of nauplii and zoea in the spawning tank was determined with the same methods, to calculate the hatching rate and the crude metamorphosis rate of nauplii. Hatching rate (%) = nauplii yield/fecundity × 100%. Crude metamorphosis rates of nauplii (%) = zoea yield/nauplii yield × 100%. The artificial insemination, spawning, egg counting, nauplii counting, and zoea counting was conducted at 4: 00 PM, 12: 00 PM, 6: 00 AM the next day (6 h post spawning (HPS)), 2: 00 PM the next day (14 HPS), and 9: 00 AM the third day (33 HPS), respectively. After spawning, the rearing water was stirred every one hour until the hatching to prevent the egg sinking. The diameters of fertilized eggs were measured under a microscope with a micrometer. 100 eggs were used for each determination (each spawning).

system in Xinhai Aqua Biotechnology Co. Ltd., Huanghua, Hebei, China (N38°37′, E117°33′). Prior to the start of the feeding trial, experimental shrimp were reared in concrete tanks and fed the control diet for 7 days to acclimate to the experimental conditions. At the onset of the feeding trial, experimental shrimp were distributed into 9 concrete tanks (3 m2; water depth: 60 cm) and each diet was randomly assigned to triplicate tanks. Each tank had 60 shrimp (30 females and 30 males) and the water was changed by 100% daily. Shrimp were hand-fed to satiation four times daily (6: 00 AM, 11: 00 AM, 5: 00 PM, and 10: 30 PM). When ovaries of appr. 20–30% of females were mature, the feeding trial was terminated. The feeding trial lasted for 70 days, from 20th April to 1st July 2016. Shrimp were reared under the local natural photoperiod. During the experiment, the water temperature was control at 27–28 °C; salinity, 28–33; pH, 8.0–8.5; dissolved oxygen, > 5 mg L− 1; ammonia nitrogen < 0.5 mg L− 1; and nitrite < 0.1 mg L− 1. The residual feed and feces were cleaned daily. At the end of the feeding trial, after being fasted for 24 h, the body weight of shrimp in each tank were measured. 2.3. The reproductive evaluation period (period II) At the end of the feeding trial, unilateral eyestalk of each female shrimp was ablated to stimulate the ovary maturation. Once a female shrimp was mature (Fig. 1), the artificial insemination was operated with mature spermatophores from the same tank, and then the female shrimp was transferred to an individual spawning tank (plastic, 300 L) and allowed to spawn, after which the quality of egg and offspring were subsequently measured. Because of the asynchronism of spawning activities among shrimp, this period lasted for 28 days, from 3rd to 30th July 2016. During this period, all the shrimp were fed squid and polychaetes at the same frequency with the previous feeding trial (by appr. 20% of shrimp weight daily). The rearing conditions were also kept as the same with the previous period.

2.5. Challenge test with low salinity Nauplii and zoea from 3 random spawning activities (but from different shrimp) in each tank were selected to test their tolerance to low salinity (10). In each determination, 2000–3000 larvae were captured with a mesh (250 μm) and then transferred to a beaker (100 mL) filled with low salinity water, of which the temperature had been adjusted to be the same as the spawning tank. A simultaneous timing began with the transfer. Then 2 mL low salinity water with larvae from the beaker was took and observed under microscope. After all the larvae stopped moving, the timing was stopped and recorded. The observation was repeated 3 times for each determination. This duration of the larvae in low salinity water was used to indicate their resistance to low salinity stress.

2.4. Sampling and assay of reproductive indices Tissue samples of female shrimp were obtained at two time points, i.e., at the end of the feeding trial (Period I) and before spawning (ready to spawn) at the reproductive evaluation period (Period II), to determine the hepatopancreas index (HI), gonadosomatic index (GI), haemolymph estradiol concentration, and tissue fatty acid compositions. At each Period, 6 mature female shrimp from each tank were selected. Haemolymph was taken from cardiocoelom using sterile syringes for the determination of estradiol, and then the shrimp were dissected to collect the hepatopancreas and gonads, which were weighted subsequently to calculate the HI and GI. HI (%) = weight of hepatopancreas/shrimp body weight × 100%. GI (%) = weight of ovaries/shrimp body weight × 100%. All the tissue samples were immediately frozen with liquid nitrogen and stored at −80 °C for the further assay of fatty acid compositions. The spawning performance and quality of egg and larvae were assayed at Period II. For all the mature female shrimp at Period II except those which have been sacrificed, after artificial insemination every spawning activity and the subsequent hatching and metamorphosis was observed and recorded individually in individual spawning tanks to

2.6. Analysis of proximate composition, fatty acids, and estradiol The proximate composition analyses of experimental diets were performed in accordance with the standard methods of AOAC (AOAC, 2000). Moisture content of feed samples was determined by drying to a constant weight at 105 °C. Protein content was estimated by measuring nitrogen content via the Kjeldahl method and multiplying by a factor of 6.25. Lipid content was determined via Soxhlet extraction using petroleum ether as the solvent. Ash content was determined by combustion at 550 °C. The fatty acid compositions of diet and fish tissue lipids were analyzed via a gas chromatograph, using a flame ionization detector (FID). Fatty acids in freeze-dried samples were esterified first with KOH-methanol and then with HCL-methanol, on 72 °C water bath. Fatty acid methyl esters were extracted with hexane and then separated via gas chromatography (HP6890, Agilent Technologies Inc., Santa Clara, California, USA) with a fused silica capillary column (007-CW, Hewlett Packard, Palo Alto, CA, USA). The column temperature was programmed to rise from 150 °C up to 200 °C at a rate of 15 °C min− 1, and then from 200 °C to 250 °C at a rate of 2 °C min− 1. Both the injector and detector temperatures were 250 °C. Results are expressed as the percentage of each fatty acid with respect to total fatty acids. Estradiol levels were determined using an electrochemiluminescence method with a Roche COBAS-6000 automatic electrochemiluminescence immunoassay analyzer located in the affiliated hospital of Qingdao University (Qingdao, China).

Fig. 1. A typical muture female shrimp with a full orange gonad.

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Fig. 2. Growth of experimental shrimp at the end of the feeding trial. Values (means ± standard error, n = 3) in bars that do not have the same letter are significantly different (P < 0.05).

Fig. 4. Gonadosomatic index of female experimental shrimp. Values (means ± standard error, n = 3) in bars that do not have the same letter are significantly different (P < 0.05).

2.7. Statistical methods All data were subjected to one-way analysis of variance (ANOVA) in SPSS 16.0 for Windows. All percentage data were arcsine transformed prior to analysis. Significant differences between the means were detected by Tukey's multiple range test. The level of significance was chosen at P < 0.05 and the results are presented as means ± standard error. 3. Results 3.1. Growth, hepatopancreas index (HI) and gonadosomatic index (GI) At the end of the feeding trial, the weight gain of female shrimp was not significantly influenced by diets (P > 0.05, Fig. 2); however, the weight gain of males from group ARA-H was significantly lower compared to the other groups (P < 0.05). Males from group ARA-L showed the highest weight gain, but not significantly higher compared to group C. The HI of female shrimp at both Period I and Period II was not significantly influenced by diets (P > 0.05, Fig. 3). The GI of female shrimp at Period I was also not significantly influenced by diets (P > 0.05, Fig. 4); however, at Period II females from group ARA-L showed significantly higher GI than those from group C (P < 0.05), but no significant difference was observed either between groups C and ARA-H or between the two ARA supplemented groups (P > 0.05).

Fig. 5. Estradiol concentrations in haemolymph of female experimental shrimp. Values (means ± standard error, n = 3) in bars that do not have the same letter are significantly different (P < 0.05).

shrimp from groups ARA-L and ARA-H was significantly higher compared to the control group (P < 0.05, Fig. 5), but no significant difference was observed between the two ARA supplemented groups (P > 0.05). At Period II, the estradiol concentration in haemolymph of female shrimp was not significantly different among dietary groups (P > 0.05). 3.3. Spawning performance, fecundity, hatching rate, metamorphosis rate of nauplii, and resistance of larvae to low salinity challenge

3.2. Estradiol concentration in haemolymph

The spawning rate in group ARA-L was significantly higher than that in groups C and ARA-H (P < 0.05, Table 3), but no significant difference was observed between the latter two groups. The multispawning rate is the highest in group ARAeH, significantly higher (P < 0.05) compared to group C, while group ARA-L showed an intermediate level. Group ARA-L showed a significantly higher (P < 0.05) average spawning frequency than the other two groups. The fecundity and diameter of fertilized egg showed similar trends with average spawning frequency. The hatching rate significantly ranked as follows: C < ARA-H < ARA-L (P < 0.05). The crude metamorphosis rate of nauplii at 33 h post spawning in group ARA-L was significantly higher (P < 0.05) than that in groups C and ARA-H. The crude metamorphosis rate of nauplii was the lowest in group ARA-H, but not significantly different from group C. In the challenge test with low salinity, the duration of nauplius significantly (P < 0.05) ranked as follows: C < ARA-L < ARA-H (Fig. 6). The duration of zoea from group C was significantly lower (P < 0.05) compared to groups ARA-L and ARA-H, but no significant difference was observed between the latter two groups.

At Period I, the estradiol concentration in haemolymph of female

Fig. 3. Hepatopancreas index of female experimental shrimp. Values (means ± standard error, n = 3) in bars that do not have the same letter are significantly different (P < 0.05).

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the estradiol level in female shrimps at Period I, indicating the enhancement of estradiol production by dietary ARA. However, the estradiol level was not different among dietary groups at Period II. This discrepancy between sampling periods might be partly due to that the feeding with fresh invertebrate ingredients instead of experimental diets at Period II attenuated the ARA effects on sex steroid production. Another possibility is ARA has more significantly positive effects on gonadal steroidogenesis in early maturation stages than in mature stages, as observed in our previous study with tongue sole C. semilaevis (Xu et al., 2017). The different ARA effects among different maturation stages might be related to the feedback regulation of ARA effects by the sex steroid hormones and/or gonadotropin (Lee et al., 2001; Sohn et al., 2001; Yamaguchi et al., 2003, 2006). Mercure and Van Der Kraak (1996) reported that at low dosages of human chorionic gonadotropin, ARA potentiated the steroidogenic actions in ovarian follicles of goldfish while with maximal gonadotropin stimulation, ARA attenuated the steroid production. In contrast with estradiol level, the gonadosomatic index of female, however, was influenced by ARA only at Period II, showing significantly higher values in group ARA-L compared to the control group. This might be due to that the gonadosomatic index was still low at Period I and did not yet show the complete effects of ARA. At both Periods, the hepatopancreas index of female was not significantly different among dietary groups. This indicates that ARA may have less impact on the nutrient digestion in Pacific white shrimp. Besides the different effects among maturation stages, different effects of ARA between females and males have also been indicated in our previous study with tongue sole. However, in this study the sperm quality of male shrimp was not assayed due to the lack of labor, and thus the ARA effects between female and male shrimp cannot be compared. Nevertheless, at the end of the feeding trail, different ARA effects on growth existed between female and male shrimp. The moderate ARA level increased the weight gain of the male shrimp but not that of females. ARA might favor the final maturation of female shrimp at the expense of other physiological processes including growth (Hurtado et al., 2009; Schlotz et al., 2013). With respect to the spawning performance and egg and larvae quality, most of the indices such as spawning rate, spawning frequency, fecundity, diameter of fertilized egg, hatching rate, and metamorphosis rate of nauplii were promoted by the diet with a moderate ARA level. This demonstrated that a moderate dietary ARA level have beneficial effects on reproduction of Pacific white shrimp. The promoting effects of dietary ARA on shrimp reproduction have also been observed in tiger prawn P. monodon, which showed that 5.0 g/kg (5.8% of total fatty acids) ARA supplement in the diet increased the cumulative percentage of females spawning, number of spawnings per female, and eggs per female (Coman et al., 2011). Another study with pond-reared P. monodon reported that the ARA content of eggs was highly correlated with fecundity and egg production (Huang et al., 2008). The study with cladoceran D. magna also showed that enriching nature food sources with ARA enhanced the fecundity (Schlotz et al., 2013; Ginjupalli et al., 2015). With marine shrimps, no previous feeding trial has been conducted to evaluate the effects of dietary ARA on reproductive performances. Lipid composition analysis of Pacific white shrimp showed that ARA is one of the predominant fatty acids in lipids of ovaries (Wouters et al., 2001b), as observed in other marine shrimp P. semisulcatus (Ravid et al., 1999), L. setiferus (Middleditch et al., 1980), Marsupenaeus japonicus (Teshima et al., 1989), Melicertus kerathurus (Mourente and Rodriguez, 1991), and Pleoticus muelleri (Jeckel et al., 1989). Moreover, ARA content in Pacific white shrimp ovaries decreased during the sexual maturation. This provides new evidence for a more important role of ARA in immature ovaries than in mature ovaries. Contrarily, however, in ovaries of kuruma prawn M. japonicas the ARA content was significantly lower at stages I and II than at stage V (Tahara and Yano, 2004). The effects of ARA on shrimp reproduction might be specie specific. Moreover, results of this study showed that ARA affected most

Table 3 Spawning performance and quality of egg and larvae. Indices

C

ARA-L

ARA-H

Spawning rate (%) Multi-spawning rate (%) Average spawning frequency Fecundity (104 per shrimp per spawning) Diameter of fertilized egg (μm) Hatching rate (%) Crude metamorphosis rate of nauplii (%) at 33 HPS

67.14 ± 12.61b 57.74 ± 4.33bc

89.59 ± 4.95a 67.30 ± 4.68ab

62.99 ± 10.15bc 75.16 ± 11.55a

2.27 ± 0.15b

2.91 ± 0.16a

2.50 ± 0.24b

20.53 ± 0.28b

23.23 ± 1.02a

21.98 ± 0.89ab

259.77 ± 4.34b

271.49 ± 5.05a

263.29 ± 4.34b

38.83 ± 1.74c 78.67 ± 1.87bc

55.09 ± 2.10a 84.23 ± 0.62a

44.65 ± 2.06b 77.13 ± 2.09c

Values (means ± standard error, n = 3) in rows that do not have the same letter are significantly different (P < 0.05). HPS: hours post spawning.

Fig. 6. Duration of nauplius and zoea larvae in the challenge test with low salinity. Values (means ± standard error, n = 3) in bars that do not have the same letter are significantly different (P < 0.05).

3.4. Fatty acid composition in hepatopancreas, gonad, and egg The fatty acid compositions in hepatopancreas, gonad, and egg were presented in Tables 4, 5, and 6, respectively. The ARA contents in all shrimp tissues increased significantly with increasing dietary ARA levels irrespective of sampling periods (P < 0.05). In hepatopancreas of female shrimp, the contents of saturated fatty acid (SFA) and linolenic acid (LNA, C18:3n-3) at both Period I and Period II, as well as the monounsaturated fatty acid (MUFA) content at Period II, decreased significantly with increasing dietary ARA levels (P < 0.05, Table 4). At Period I, the EPA (C20:5n-3) content increased but the DPA (C22:5n-3) content decreased with increasing dietary ARA levels. At both Periods, the DHA (C22:6n-3) content tended to be higher in ARA supplemented groups than in group C. In ovaries, the SFA contents at both Periods significantly decreased with increasing dietary ARA levels (P < 0.05, Table 5). The MUFA content of group ARA-H at Period I was significantly higher compared to group ARA-L (P < 0.05). Compared to group C, the ARA supplemented groups tended to have higher levels of EPA and DPA at both Periods but lower levels of LNA and DHA at Period I. In fertilized eggs, besides the ARA content, only the contents of C18:1n-7 and C18:2n-6 was significantly influenced by diets, which were significantly decreased in group ARA-L compared to the other two groups (P < 0.05, Table 6).

4. Discussion In this study, dietary ARA supplementation significantly increased 560

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Table 4 Fatty acid profiles of hepatopancreas from female shrimp (% total fatty acid)a. Fatty acid

C14:0 C16:0 C18:0 ∑ SFAb C16:1n-7 C18:1n-9 C18:1n-7 C20:1n-9 ∑ MUFAb C18:2n-6 C20:4n-6 ∑ n-6b C18:3n-3 C20:5n-3 C22:5n-3 C22:6n-3 ∑ n-3b ∑ n-3/∑n-6 ∑ n-3LC-PUFAb

Period I

Period II

C

ARA-L

ARA-H

C

ARA-L

ARA-H

1.27 ± 0.03a 27.78 ± 1.87a 10.21 ± 0.30a 39.27 ± 2.13a 8.18 ± 0.27a 11.87 ± 0.04 7.64 ± 0.43 4.71 ± 0.53 32.42 ± 0.74 2.68 ± 0.21c 3.60 ± 0.24c 6.28 ± 0.45c 3.65 ± 0.00a 2.45 ± 0.19c 0.47 ± 0.01a 2.56 ± 0.45c 9.14 ± 0.63b 1.45 ± 0.00a 5.49 ± 0.63c

1.17 ± 0.06ab 25.37 ± 1.06ab 9.70 ± 0.13b 36.25 ± 1.13ab 7.40 ± 0.01b 11.92 ± 0.03 7.51 ± 0.47 4.77 ± 0.51 31.60 ± 1.01 3.17 ± 0.05b 4.64 ± 0.11b 7.81 ± 0.06b 3.38 ± 0.09ab 2.83 ± 0.06b 0.46 ± 0.01a 3.37 ± 0.18b 10.05 ± 0.33ab 1.29 ± 0.05b 6.67 ± 0.24b

1.07 ± 0.09b 22.96 ± 0.26b 9.19 ± 0.04c 33.22 ± 0.12b 6.61 ± 0.25c 11.96 ± 0.02 7.37 ± 0.52 4.83 ± 0.49 30.78 ± 1.28 3.66 ± 0.12a 5.68 ± 0.45a 9.34 ± 0.57a 3.11 ± 0.18b 3.21 ± 0.06a 0.44 ± 0.00b 4.18 ± 0.08a 10.94 ± 0.04a 1.17 ± 0.07b 7.83 ± 0.14a

tr 23.99 ± 0.61a 11.71 ± 0.13a 35.71 ± 0.74a 7.29 ± 0.31a 10.61 ± 0.20 7.38 ± 0.18 4.13 ± 0.06 29.41 ± 0.25a 1.98 ± 0.11c 5.23 ± 0.03c 7.22 ± 0.14c 2.70 ± 0.01a 5.31 ± 0.09 1.35 ± 0.01 3.51 ± 0.08b 12.88 ± 0.20 1.79 ± 0.06a 10.18 ± 0.18

tr 22.79 ± 0.20b 11.45 ± 0.05b 34.24 ± 0.25b 6.89 ± 0.17ab 10.63 ± 0.20 7.32 ± 0.21 4.16 ± 0.05 28.99 ± 0.12ab 2.23 ± 0.03b 5.76 ± 0.15b 7.98 ± 0.12b 2.56 ± 0.03b 5.51 ± 0.16 1.34 ± 0.01 3.67 ± 0.04a 13.08 ± 0.10 1.64 ± 0.01b 10.52 ± 0.13

tr 21.58 ± 0.20c 11.19 ± 0.04c 32.78 ± 0.23c 6.50 ± 0.05b 10.65 ± 0.21 7.25 ± 0.23 4.19 ± 0.04 28.59 ± 0.02b 2.47 ± 0.05a 7.11 ± 0.05a 8.75 ± 0.37a 2.43 ± 0.07c 5.70 ± 0.22 1.33 ± 0.00 3.52 ± 0.05b 12.98 ± 0.10 1.49 ± 0.05c 10.55 ± 0.17

a Some fatty acids, of which the contents are minor, trace amount or not detected, such as C22:0, C24:0, C14:1, C20:2n-6, C20:3n-6, were not listed in the table. tr: trace. Values (means ± standard error, n = 3) in rows that do not have the same letter are significantly different (P < 0.05). b SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; n-6: n-6 unsaturated fatty acid; n-3: n-3 unsaturated fatty acid; LC-PUFA: long chain-polyunsaturated fatty acid.

have also been reported to regulate the gonadal steroidogenesis of fish (Wade et al., 1994; Mercure and Van Der Kraak, 1995, 1996). Further efforts are needed to elucidate the precise mechanisms. This study also investigated the effects of dietary ARA on offspring quality of Pacific white shrimp through a challenge test with low salinity. The results indicated that dietary ARA supplementation enhanced the stress resistence of nauplius and zoea larvae. In fish, ARA has been reported to play an important role in enhancing the stress resistance against various stressors at early development stages (Koven et al., 2001, 2003; Van Anholt et al., 2004; Rezek et al., 2009). The stress resistance promoting effect of ARA seemed consistent between larval fish and shrimp. With respect to the fatty acid profiles, generally the fatty acid compositions in hepatopancreas, ovaries, and eggs closely reflect those

of reproductive indices (as well as the growth of male shrimp) in a dosedependent manner, i.e., the higher dietary ARA level had an intermediate positive effect or even negative effects compared to the lower ARA level. The dose-dependent effects of ARA have been widely observed in previous studies (Harel et al., 2001; Khozin-Goldberg et al., 2006; Hurtado et al., 2009; Xu et al., 2010). The imbalance between ARA and EPA and the consequent imbalance between ARA- and EPAderived eicosanoids were assumed to contribute to the ineffectiveness or even inhibitory effects of excess ARA (Furuita et al., 2003). The mechanisms involved in the reproduction regulating effects of ARA seem to be complicated. The ARA-derived eicosanoids prostaglandin E2 and E2a have been showed to be associated with egg-laying behavior in insects and sperm motility and ovulation in mammals respectively (Chang et al., 1997; Stanley, 2006). The prostaglandins (eicosanoids) Table 5 Fatty acid profiles of ovaries (% total fatty acid)a. Fatty acid

C14:0 C16:0 C18:0 ∑ SFAb C16:1n-7 C18:1n-9 C18:1n-7 C20:1n-9 ∑ MUFAb C18:2n-6 C20:4n-6 ∑ n-6b C18:3n-3 C20:5n-3 C22:5n-3 C22:6n-3 ∑ n-3b ∑ n-3/∑n-6 ∑ n-3LC-PUFAb

Period I

Period II

C

ARA-L

ARA-H

C

ARA-L

ARA-H

0.71 ± 0.01b 22.66 ± 0.22a 8.28 ± 0.02a 31.66 ± 0.21a 6.44 ± 0.06a 14.27 ± 0.06b 5.66 ± 0.04 1.57 ± 0.02a 27.95 ± 0.18ab 4.68 ± 0.04 3.61 ± 0.02c 8.29 ± 0.07c 0.75 ± 0.01a 9.31 ± 0.03b 2.33 ± 0.03b 10.52 ± 0.03a 22.93 ± 0.05 2.76 ± 0.02a 22.18 ± 0.02

0.77 ± 0.01a 18.99 ± 0.11b 8.38 ± 0.01a 28.14 ± 0.10b 5.50 ± 0.17b 14.80 ± 0.01a 5.58 ± 0.01 1.54 ± 0.03b 27.44 ± 0.19b 5.29 ± 0.44 6.36 ± 0.05b 11.66 ± 0.38b 0.61 ± 0.00c 9.91 ± 0.08a 2.32 ± 0.04b 9.98 ± 0.01b 22.82 ± 0.05 1.96 ± 0.06ab 22.21 ± 0.05

0.78 ± 0.01a 17.45 ± 0.80c 7.97 ± 0.05b 26.20 ± 0.83c 6.09 ± 0.21a 14.87 ± 0.06a 5.59 ± 0.04 1.53 ± 0.00b 28.08 ± 0.23a 4.86 ± 0.04 9.31 ± 0.07a 14.17 ± 0.12a 0.63 ± 0.01b 9.74 ± 0.23a 2.57 ± 0.03a 9.94 ± 0.07b 22.89 ± 0.13 1.61 ± 0.01b 22.26 ± 0.13

0.83 ± 0.01b 21.92 ± 0.86a 7.82 ± 0.17 30.57 ± 0.70a 8.40 ± 0.79 15.14 ± 0.10a 5.62 ± 0.00b 1.62 ± 0.02 30.79 ± 0.92 1.85 ± 0.29 3.68 ± 0.28c 5.54 ± 0.01c 0.65 ± 0.01 9.33 ± 0.10c 2.92 ± 0.15ab 6.64 ± 0.10 19.55 ± 0.04c 3.53 ± 0.02a 18.89 ± 0.06c

0.91 ± 0.03a 20.12 ± 0.27b 8.07 ± 0.08 29.11 ± 0.22b 8.35 ± 0.77 14.69 ± 0.05b 5.89 ± 0.09a 1.67 ± 0.04 30.62 ± 0.86 1.81 ± 0.28 6.43 ± 0.03b 8.24 ± 0.25b 0.68 ± 0.02 9.78 ± 0.05a 2.57 ± 0.04b 6.70 ± 0.08 19.74 ± 0.02b 2.39 ± 0.07b 19.06 ± 0.00b

0.83 ± 0.01b 18.71 ± 0.20b 7.69 ± 0.21 27.52 ± 0.41c 8.19 ± 0.72 15.01 ± 0.06a 5.65 ± 0.01b 1.60 ± 0.01 30.47 ± 0.81 1.99 ± 0.34 9.72 ± 0.26a 11.72 ± 0.07a 0.68 ± 0.02 9.60 ± 0.01b 3.02 ± 0.18a 6.69 ± 0.09 20.00 ± 0.11a 1.71 ± 0.00c 19.32 ± 0.08a

a Some fatty acids, of which the contents are minor, trace amount or not detected, such as C22:0, C24:0, C14:1, C20:2n-6, C20:3n-6, were not listed in the table. Values (means ± standard error, n = 3) in rows that do not have the same letter are significantly different (P < 0.05). b SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; n-6: n-6 unsaturated fatty acid; n-3: n-3 unsaturated fatty acid; LC-PUFA: long chain-polyunsaturated fatty acid.

561

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the DHA content in ovaries was lower in ARA supplemented groups compared to the control group at Period I. It is also difficult to explain these results based on the current knowledge. Further studies are needed to investigate the nutritive values of other fatty acids, especially the LC-PUFAs, in reproduction of shrimps. In conclusion, results of this study suggested that the ARA supplementation, especially at a moderate level (4.65% of total fatty acids), in diets for early maturation stages enhanced the final reproductive performances of Pacific white shrimp.

Table 6 Fatty acid profiles of fertilized eggs (% total fatty acid)a. Fatty acid

C

ARA-L

ARA-H

C14:0 C16:0 C18:0 ∑ SFAb C16:1n-7 C18:1n-9 C18:1n-7 C20:1n-9 ∑ MUFAb C18:2n-6 C20:4n-6 ∑ n-6b C18:3n-3 C20:5n-3 C22:5n-3 C22:6n-3 ∑ n-3b ∑ n-3/∑n-6 ∑ n-3LC-PUFAb

0.95 ± 0.25 22.40 ± 0.65 7.18 ± 0.11 30.54 ± 0.79 8.66 ± 0.33 13.23 ± 0.25 6.73 ± 0.08a 1.69 ± 0.29 30.32 ± 0.31b 2.98 ± 0.16a 3.22 ± 0.37c 6.20 ± 0.53c 2.78 ± 0.17 8.60 ± 0.07 3.15 ± 0.31 3.80 ± 0.36 18.33 ± 0.92 2.96 ± 0.10a 15.55 ± 0.76

0.79 ± 0.20 22.09 ± 0.55 7.25 ± 0.09 30.14 ± 0.66 9.38 ± 0.57 13.35 ± 0.22 6.18 ± 0.26b 1.90 ± 0.23 30.82 ± 0.14b 2.26 ± 0.07b 5.53 ± 0.13b 7.79 ± 0.06b 2.85 ± 0.19 8.55 ± 0.39 3.05 ± 0.28 3.60 ± 0.30 18.06 ± 1.17 2.31 ± 0.13b 15.21 ± 0.97

0.98 ± 0.26 22.13 ± 0.56 7.27 ± 0.08 30.38 ± 0.74 9.52 ± 0.62 13.43 ± 0.19 6.80 ± 0.06a 1.65 ± 0.31 31.41 ± 0.06a 2.66 ± 0.06a 6.56 ± 0.18a 9.22 ± 0.12a 2.74 ± 0.16 8.40 ± 0.01 3.16 ± 0.31 3.98 ± 0.01 18.29 ± 0.48 1.98 ± 0.07b 15.55 ± 0.32

Acknowledgments This work was supported by Central Public-interest Scientific Institution Basal Research Fund, CAFS (2016HY-ZD04), Agricultural Superior Breeding Project of Shandong Province, and Key R & D Program of Shandong Province (2016GSF115030). References Abi-Ayad, A., Mélard, C., Kestemont, P., 1997. Effects of n–3 fatty acids in Eurasian perch broodstock diet on egg fatty acid composition and larvae stress resistance. Aquac. Int. 5, 161–168. Aguilar, V., Racotta, I.S., Goytortúa, E., Wille, M., Sorgeloos, P., Civera, R., Palacios, E., 2012. The influence of dietary arachidonic acid on the immune response and performance of Pacific whiteleg shrimp, Litopenaeus vannamei, at high stocking density. Aquac. Nutr. 18 (3), 258–271. Almansa, E., Perez, M.J., Cejas, J.R., Bada, P., Villamandos, J.E., Lorenzo, A., 1999. Influence of broodstock gilthead seabream (Sparus aurata L.) dietary fatty acids on egg quality and egg fatty acid composition throughout the spawning season. Aquaculture 170, 323–336. Almansa, E., Martín, M.V., Cejas, J.R., Badía, P., Jerez, S., Lorenzo, A., 2001. Lipid and fatty acid composition of female gilthead seabream during their reproductive cycle: effects of a diet lacking n–3 HUFA. J. Fish Biol. 59, 267–286. Association of Official Analytical Chemists (AOAC), 2000. Official Methods of Analysis of AOAC International, 17th ed. AOAC, Arlington, VA, USA. Beirão, J., Soares, F., Pousão-Ferreira, P., Diogo, P., Dias, J., Dinis, M.T., Herráez, M.P., Cabrita, E., 2015. The effect of enriched diets on Solea senegalensis sperm quality. Aquaculture 435 (435), 187–194. Bruce, M.P., Oyen, F., Bell, J.G., Farndale, B.M., Asturiano, J.F., Bromage, N.R., Carrillo, M., Zanuy, S., Ramos, J., 1999. Development of broodstock diets for the European sea bass (Dicentrarchus labrax) with special emphasis on the importance of n–3 and n–6 HUFA to reproductive performance. Aquaculture 177, 85–98. Butts, I.A.E., Baeza, R., Støttrup, J.G., Krüger-Johnsen, M., Jacobsen, C., Pérez, L., Asturiano, J.F., Tomkiewicz, J., 2015. Impact of dietary fatty acids on muscle composition, liver lipids, milt composition and sperm performance in European eel. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 183, 87–96. Chang, K.J., Kim, J.W., Im, W.B., Kang, H.M., Kwon, H.B., 1997. Differential effects of gonadotropin and orthovanadate on oocyte maturation, ovulation, and prostaglandin synthesis by Rana ovarian follicles in vitro. J. Exp. Zool. 27, 155–165. Coman, G.J., Arnold, S.J., Barclay, M., Smith, D.M., 2011. Effects of arachidonic acid supplementation on reproductive performance of tank-domesticated Penaues monodon. Aquac. Nutr. 17, 141–151. Estévez, A., McEvoy, L.A., Bell, J.G., Sargent, J.R., 1999. Grovth, survival, lipid composition and pigmentation of turbot (Scophthalmus maximus) larvae fed live-prey enriched in arachidonic and eicosapentaenoic acids. Aquaculture 180, 321–343. Fernández–Palacios, H., Izquierdo, M., Robaina, L., Valencia, A., Salhi, M., Montero, D., 1997. The effect of dietary protein and lipid from squid and fish meals on egg quality of broodstock for gilthead seabream. Aquaculture 148 (2–3), 233–246. Furuita, H., Tanaka, H., Yamamoto, T., Shiraishi, M., Takeuchi, T., 2000. Effects of n–3 HUFA levels in broodstock diet on the reproductive performance and egg and larval quality of the Japanese flounder, Paralichthys olivaceus. Aquaculture 187, 387–398. Furuita, H., Yamamoto, T., Shima, T., Suzuki, N., Takeuchi, T., 2003. Effect of arachidonic acid levels in broodstock diet on larval and egg quality of Japanese flounder Paralichthys olivaceus. Aquaculture 220, 725–735. Ginjupalli, G.K., Gerard, P.D., Baldwin, W.S., 2015. Arachidonic acid enhances reproduction in Daphnia magna and mitigates changes in sex ratios induced by pyriproxyfen. Environ. Toxicol. Chem. 34 (3), 527–535. Glencross, B.D., 2009. Exploring the nutritional demand for essential fatty acids by aquaculture species. Rev. Aquacult. 1 (2), 71–124. Glencross, B.D., Smith, D.M., 2001. A study of the arachidonic acid requirements of the giant tiger prawn, Penaeus monodon. Aquacul. Nutr. 7 (1), 59–69. González-Baró, M.D.R., Pollero, R.J., 1998. Fatty acid metabolism of Macrobrachium borellii: dietary origin of arachidonic and eicosapentaenoic acids. Comp. Biochem. Physiol. 119 (3), 747–752. Harel, M., Gavasso, S., Leshin, J., Gubernatis, A., Place, A.R., 2001. The effect of tissue docosahexaenoic and arachidonic acids levels on hypersaline tolerance and leucocyte composition in striped bass (Morone saxatilis) larvae. Fish Physiol. Biochem. 24, 113–123. Huang, J.-H., Jiang, S.-G., Lin, H.-Z., Zhou, F.-L., Ye, L., 2008. Effects of dietary highly unsaturated fatty acids and astaxanthin on the fecundity and lipid content of pond-

a Some fatty acids, of which the contents are minor, trace amount or not detected, such as C22:0, C24:0, C14:1, C20:2n-6, C20:3n-6, were not listed in the table. Values (means ± standard error, n = 3) in rows that do not have the same letter are significantly different (P < 0.05). b SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; n-6: n-6 unsaturated fatty acid; n-3: n-3 unsaturated fatty acid; LC-PUFA: long chain-polyunsaturated fatty acid.

of the diets, irrespective of the experimental periods. Typically, with increasing dietary ARA levels, in shrimp tissues and eggs the contents of saturated and monounsaturated fatty acids decreased while the ARA contents increased. Nevertheless, there were exceptions. Despite the decrease of dietary oleic acid (OA, C18:1n-9) contents with increasing ARA levels, the OA contents in the fertilized eggs, as well as in hepatopancreas and ovaries, was relatively constant. OA is a major energy source during egg and larval development (Van der Meeren et al., 1991). Significant positive correlations between the OA content of fish eggs and egg viability or hatching percentages have been observed in gilthead seabream broodstock (Fernández–Palacios, H., Izquierdo, M., Robaina, L., Valencia, A., Salhi, M., Montero, D., 1997). A more balanced and stable C18:1n-9/n-3 ratio could be beneficial to the energy supply during fish reproduction (Almansa et al., 1999; Furuita et al., 2000). This may be the reason for the insensitiveness of shrimp body OA to the fluctuation in dietary OA. Besides OA, the linoleic acid (LA, C18:2n-6) contents in hepatopancreas increased with increasing ARA levels at both sampling periods despite that dietary LA contents showed a decreasing level with increasing ARA levels. Based on the present knowledge, it was difficult to explain the positive interaction between ARA and LA contents in shrimp hepatopancreas and further efforts are needed about this point. Moreover, despite no obvious increase in EPA content in ARA supplemented diets compared to the control diet, the EPA contents in both hepatopancreas and ovaries were significantly increased in ARA supplemented groups. This was on the contrary to what observed in fish studeis, which showed that EPA contents were inversely related to ARA contents in various tissues (Estévez et al., 1999; Willey et al., 2003). This, along with the high EPA contents in eggs and ovaries, to some extent confirmed the importance of EPA to this shrimp speices. The high EPA contents in Pacific white shrimp, as well as in the well-performing live feed for Pacific white shrimp, the polychaetes, have been also been reported by other workers (Wouters et al., 2001b; Leelatanawit et al., 2014). Other shrimp species such as P. monodon and Macrobrachium borelli also require EPA to support nomal growth and metabolism (Merican and Shim, 1996; González-Baró and Pollero, 1998; Glencross and Smith, 2001; Glencross, 2009). Unexpectedly, the docosapentaenoic acid (DPA, C22:5n-3) content in hepatopancreas and 562

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