Arachidonic acid effects on the overall performance, fatty acid profile, hepatopancreas morphology and lipid-relevant genes in Litopenaeus vannamei juveniles

Journal Pre-proof Arachidonic acid effects on the overall performance, fatty acid profile, hepatopancreas morphology and lipid-relevant genes in Litopenaeus vannamei juveniles

Bruno Cavalheiro Araújo, Krishna Flores-Galvez, Renato Massaaki Honji, Vitalina Magalhães Barbosa, María Teresa Viana, Aurora Tinajero, José Antonio Mata-Sotres PII:

S0044-8486(20)30083-1

DOI:

https://doi.org/10.1016/j.aquaculture.2020.735207

Reference:

AQUA 735207

To appear in:

aquaculture

Received date:

10 January 2020

Revised date:

6 March 2020

Accepted date:

7 March 2020

Please cite this article as: B.C. Araújo, K. Flores-Galvez, R.M. Honji, et al., Arachidonic acid effects on the overall performance, fatty acid profile, hepatopancreas morphology and lipid-relevant genes in Litopenaeus vannamei juveniles, aquaculture (2019), https://doi.org/10.1016/j.aquaculture.2020.735207

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© 2019 Published by Elsevier.

Journal Pre-proof Arachidonic acid effects on the overall performance, fatty acid profile, hepatopancreas morphology and lipid-relevant genes in Litopenaeus vannamei juveniles.

Bruno Cavalheiro Araújoa,b, Krishna Flores-Galvezc, Renato Massaaki Honjib, Vitalina

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Magalhães Barbosac, María Teresa Vianac, Aurora Tinajeroc, José Antonio Mata-Sotresd*

Núcleo Integrado de Biotecnologia, Universidade de Mogi das Cruzes. Av. Dr. Cândido

Centro de Biologia Marinha da Universidade de São Paulo (CEBIMar/USP) Rodovia

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Xavier de Almeida Souza, 200, 08780-911, Mogi das Cruzes, SP, Brasil

Instituto de Investigaciones Oceanológicas, Universidad Autónoma de Baja California

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Manoel Hipólito do Rego, km 131,5, São Sebastião, SP, 11612-109, Brasil;

CONACYT, Instituto de Investigaciones Oceanológicas, UABC, Ensenada, BC, México

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d

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(UABC), km 107 carretera Tij/Eda, 22860 Ensenada, Baja California, México

__________ *Corresponding author: [email protected] (J.A. Mata-Sotres)

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Abstract The effect of arachidonic acid (ARA) was evaluated on Litopenaeus vannamei juveniles. Growth performance; hepatopancreas and muscle fatty acid compositions; hepatopancreatic cells morphology; and expression of lipid-relevant genes were estimated after six-week experiment. A basal fat-reduced diet was manufactured and subsequently coated with different fatty acids sources, distinct levels of ARA (0, 0.3, and 0.6%), fish and soybean oil (50:50 Control diet). L. vannamei juveniles (1.10 ± 0.22 g) were randomly divided into twelve

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tanks with thirty animals each. The animals were fed three times per day, with 8% of the total biomass. The results showed that animals fed with diet containing 0.3% of ARA presented a

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lower final weight compared to the Control. Different levels of ARA inclusion in the

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experimental diets directly influenced in the tissues fatty acid profile with lower ARA levels

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in Control and 0% followed by 0.3% and 0.6%. In muscle and hepatopancreas tissues, a higher accumulation of docosahexaenoic acid (DHA) was revealed in the Control compared

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to the rest of the experimental treatments. The expression pattern of lipoxygenase (alox5),

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prostaglandin E synthase (pges2), sterol regulatory element-binding protein-1 (srebp1) and

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cycloxygenase (cox2) were significantly affected by the ARA inclusion, showing the highest expression in the Control treatment with a decrease according to the increasing levels of dietary ARA. It is concluded that ARA inclusion in shrimp diets result in a negative impact on the overall performance, probably modulated by a differential gene expression related to the eicosanoids synthesis, lipolytic and lipogenic pathways. Besides, the lipid deposition on the hepatopancreatic cells was reduced.

Keywords: Lipid nutrition; 20:4n-6; eicosanoids; prostaglandin;

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1. Introduction Marine fish and shrimp are highly fish oil dependent, mainly due to the nutritional requirement by long-chain polyunsaturated fatty acids (LC-PUFA), especially from n-3 series. Docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3) are the most important LC-PUFAs for marine species. Both play essential physiological functions, and are target of several nutritional studies that aim the fish oil replacement by lower costs and more sustainable ingredients, such as terrestrial fats and vegetable oils (Tocher, 2003;

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Glencross, 2009; Turchini et al., 2009). Previous studies showed that entirely fish oil replacement by alternative sources, negatively impacted the performance and muscle fatty

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acid composition in marine fish and shrimp (Chen et al., 2015; Xu et al., 2016a; Jin et al.,

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2017; Soller et al., 2018). However, an adequate LC-PUFA supplementation usually provides

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by the marine algae extract inclusion can reduce these impacts (Salini et al., 2016; Jin et al., 2017; Mata-Sotres et al., 2018; Araújo et al., 2019a,b).

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Although considered promising ingredients for the marine aquaculture diets, due to the

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high DHA and EPA levels, marine algae extracts are deficient in arachidonic acid (ARA,

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20:4n-6), other physiological important LC-PUFA. ARA nutrition studies in marine organisms are scarce compared to the n-3 LC-PUFA, probably due to its low requirement compared to the other LC-PUFAs. However, recent studies carried out with fish and shrimp species presented promisors results in the growth performance (Nayak et al., 2017; Ding et al., 2018; Torrecillas et al., 2018; Araújo et al., 2019a), survival and stress resistance (Boglino et al., 2014; Yuan et al., 2015; Torrecillas et al., 2017), disease resistance (Koven et al., 2001; Xu et al., 2010) and reproduction (Furuita et al., 2003; Xu et al., 2017). Among several functions, ARA is essential for the immunological system. ARA synthesize the eicosanoids, being physiologically active for most aquatic organisms (Bell and Sargent, 2003; Tocher, 2003; Arts and Kohler, 2009). Eicosanoids, such as prostaglandins,

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leukotrienes, and thromboxanes, act as local hormones or signaling molecules modulating the immune and inflammatory processes (Bell et al., 1995; Bell and Sargent, 2003; Arts and Kohler, 2009). Eicosapentaenoic acid and ARA have similar biochemical structure competing for binding to the enzymes in the eicosanoids synthesis, such as cyclooxygenase (COX) and lipoxygenase (ALOX). Therefore, an adequate EPA/ARA ratio in aquaculture diets need to be always considered especially to the marine organisms to cope with a proper metabolism and growth performance (Bell and Sargent, 2003; Turchini et al., 2009; Arts and Kohler, 2009;

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Salini et al., 2016).

Pacific white shrimp (Litopenaeus vannamei) is an economically important worldwide

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species, being the main crustacean species cultured, producing nearly four million metric tons

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per year (FAO, 2018). Despite the importance of L. vannamei to the aquaculture sector, few

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studies have been performed aiming to evaluate the influence of different LC-PUFA levels in fish oil-free diets on the overall performance and lipid metabolism (González-Félix et al.,

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2009; Kumar et al., 2018; Araújo et al., 2019b). Due to the relevance of ARA in the marine

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organism nutrition, the present study aimed to investigate the effects of different ARA

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inclusion levels in fish oil-free diets on the growth performance, hepatopancreas and muscle fatty acid profile, hepatopancreatic cells morphology and expression of genes related to the fatty acids and eicosanoids metabolism in L. vannamei juveniles. Information that will be crucial for fish oil-free diets manufacturing for the Pacific white shrimp.

2. Material and methods 2.1. Experimental design Three thousand juveniles of L. vannamei (0.80 ± 0.10), obtained from a commercial hatchery (Maricultura del Pacífico, Sinaloa, México), were transported to the facilities of the Instituto de Investigaciones Oceanológicas at the Universidad Autónoma de Baja California

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(IIO - UABC, Mexico). The organisms were stocked in a recirculating system (RAS) composed of two 1,000-L circular tanks, pump, biofilter and protein skimmer. Shrimp were fed twice daily (10% of the total biomass) with diets designed to contain approximately 41, 9% of crude protein, and crude lipid, respectively. When shrimp reached a size of 1.10 ± 0.22 g, mean ± SE, were randomly distributed in twelve 300-L circular tanks (RAS) equipped with biofilter (PolyGeyser®; PneumaticDrop Bead Filter model PG7 International Filter Solutions, TX, USA), UV, heater and home-made protein skimmer, with a final density of 30 individuals

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per tank. Shrimps were fed with the respective experimental diets (triplicate treatments) three times a day (08:00, 12:00 and 17:00 h) for six weeks (at 8% from total biomass per day).

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Water temperature (27.9 ± 0.94ºC), salinity (35.8 ± 0.85 ppt) and dissolved oxygen (5.60 ±

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0.65 mg L-1 ) were monitored daily (YSI-55, YSI Inc., Yellow Springs, OH, USA) and total

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ammonia (0.06 ± 0.01 mg L-1 ), nitrite (0.20 ± 0.01 mg L-1 ) and alkalinity (104.7 ± 4.22 mg L−1 ) were measured every three days (API test kits, Mars Fishcare Inc., Chalfont, PA, USA)

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2.2. Experimental Diets

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(mean ± SE).

The ingredients, proximal composition and fatty acid profile from the experimental diets are presented in Tables 1 and 2. The fishmeal and poultry by-product meals were previously defatted (fish and poultry meal defatted by 3:1 hexane:meal) to properly formulate the experimental diets to the lipids required. Four isoproteic and isolipidic diets were formulated to contain 43% of crude protein, and 8.5% of crude lipid, as shown in Table 1. The lipid content in the Control diet was composed of a blend of fish oil and soybean oil (1:1). An oil mix that is usually present in the commercial diet for this species. The rest of the experimental diets contained a source of DHA and EPA concentrated oils (in the same proportion), saturated (SFA) and monounsaturated (MUFA) fatty acid-rich oils (coconut and

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peanut oils, respectively) and different ARA levels (0, 0.3, and 0.6%). The diets were formulated and balanced to contain all fatty acid classes (SFA, MUFA, and PUFA), trying to reduce the undesirable linoleic acid (18:2n-6), which is higher (approximately 55%) in the soybean oil. The experimental diets were produced at the facilities of the IIO - UABC, following the internal protocols. Firstly, the macronutrients were pulverized to 0.5 mm (Inmimex M300, Mexico), and sifted (Kemutek-Gardner K300, USA). Thereafter were mixed using a

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vertical cutter-mixer (Robot Coupe R-60, USA) until a homogeneous mass was obtained. Simultaneously, the micronutrients were incorporated into the bulk meal. The oil blend of

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each respective experimental diet was added and throughout mixed, and finally, the water was

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added until the desired texture was achieved. Diets were mixed (Robot-Coupe, model R10,

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USA), pelleted at 5 mm in a meat grinder (Tor-Rey, Model M32-5, Mexico) and dried at 60 °C in a forced air oven for 24 h. Once dried, diets were kept cooled (4°C) throughout the

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feeding trial.

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2.3. Growth performance and samples collection At the end of the six-week feeding trial, shrimps were counted, and weight. The individual weight was calculated by dividing the whole biomass per experimental unit dividing the bulk weight by the number of individuals, performance response indices and somatic indices were calculated as follows: Specific growth rate (SGR, %) = 100 x [(ln Wt - ln Wo)/days of feeding]; Survival rate (SR, %) = Nt × 100/No; Where Wt and Wo were final and initial shrimp weight, respectively; Nt and No, corresponds to final and initial number of shrimps, respectively.

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The organisms were handled to minimize stress, and three animals per tank were euthanized by hypothermia and posteriorly stored at -80ºC prior analysis according to the ethics committee protocols at UABC.

2.4. Proximate composition and fatty acid analysis All experimental diets and whole shrimp were analyzed in triplicate according to AOAC (2015). Dry weight and the ash content were determined by drying ground samples at

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60ºC for 24 h followed by carbonization in a muffle furnace at 550ºC for 6 h. Crude protein was analyzed by the micro-Kjeldahl method (UDK 129, Velp, Italy), and the content was

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calculated by nitrogen conversion (%N x 6.25). Fat content was analyzed by the Soxhlet

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method according to the AOAC using petroleum ether as a carrier. While those lipids used to

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analyze the fatty acid profile from the diets; hepatopancreas and muscle tissue were directly methylated by the transmethylation methodology described by Parrish et al. (2014), using

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dichloromethane and methanol as a base solvent. Fatty acids methyl esters (FAMEs) were

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analyzed using gas chromatography equipped with flame ionization detector (Agilent GC

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6880, Agilent Technologies, Santa Clara, CA, USA) using hydrogen as the carrier gas. The GC column (60 m x 0.25 mm with 0.25 µm film thickness; Agilent 122-2362 dB-23) conditions were: oven temperature initial of 50ºC for 1 min, 50 to 140°C at 30ºC min-1 , held at 140ºC for 5 min, from 140 to 240ºC at 4ºC min-1 , and finally 240ºC for 20 min. The injector and detector temperature were kept at 230 and 260ºC, respectively. FAMEs were identified and quantified comparing retention times between a standard mix (37 Component FAME mix, PUFA 1 and PUFA 3, Supelco/Sigma-Aldrich, St. Louis, MO, USA).

2.5. Hepatopancreatic cell morphology

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The hepatopancreas samples used to perform the histological analyzes were initially fixed in formalin solution during 24 hours (n=3 per tank, 9 per treatment). Thereafter, samples were dehydrated through a series of increasing ethanol concentrations, cleared in a dimethyl benzene solution and embedded in Paraplast® according to routine histological procedures. Hepatopancreas semi-serial sections were sliced at 5-µm thick using a microtome (Leica HistoCore AutoCut), and mounted on a Poly-L-Lysine solution-coated slides, stained with hematoxylin-eosin, examined and documented using a computerized image analyzer (Leica

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DM1000 LED light microscope, Leica MC170HD camera, and computer image capture, Leica LAS Interactive Measurements) to evaluate the hepatopancreatic cell profiles and

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average area.

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The methodology used for the images was following the methods published for fish

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(Honji et al., 2015, 2019; Birba et al., 2015) and shrimp (Han et al., 2018a,b) species. The images were captured from samples using the LAS system (1260 pixels by 960 pixels), and

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for the morphological hepatopancreas tissues analysis the average area of blasenzellen and

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restzellen cells were used. Therefore, semi-serial sections of the hepatopancreas tissue were

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performed every 100m of the distance between sections. Five cells per section and three sections per sample (with 15 cells per animal) were analyzed, totalizing 500m distance from the first section to the last part. A software Leica LAS Interactive Measurements was used to obtain the measurements.

2.6. RNA extraction and quantitative real-time PCR Six hepatopancreas samples per treatment were preserved in RNAlater (Ambion) and individually processed for total RNA extraction using the NucleoSpin® RNA kit (MachereyNagel). Genomic DNA (gDNA) was removed via on-column DNase digestion at 37°C for 30 min using DNase (RNase-free) included in the kit. A micropistill was used to homogenize the

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tissue before the extraction. The quantity and quality of RNA were measured using gel electrophoresis and spectrophotometer (Nanodrop® LITE, Thermo Fisher Scientific INC., Wilmington, USA). Only RNA samples with OD260nm-OD280nm ratios between 1.90 and 2.10 were used for expression quantification. Total RNA (500 ng) was reverse-transcribed in a 20 μL reaction using the HighCapacity cDNA Reverse Transcription kit (Applied Biosystems; Carlsbad CA, USA) in a Verity 96 well thermal cycler (Applied Biosystems). The reverse transcription program

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consisted of 10 min at 25°C, 120 min at 37°C, 5 min at 85°C and finally kept at 4°C. qRTPCR reactions were performed with one ng of cDNA, sense and antisense primers (200 nM

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each, indicated in Table 3) and SYBR® Select Master Mix (Applied Biosystems). Reactions

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were conducted in 10 μL, in MicroAmp® Fast Optical 96-well reaction plates (Applied

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Biosystems) covered with MicroAmp® Optical Adhesive Film (Applied Biosystems). Relative gene quantification was calculated by the

ΔΔ

CT method (Livak and

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Schmittgen, 2001) using automated threshold and walking baseline for determining the CT

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values. PCR conditions were: an initial denaturation and polymerase activation step during 10

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min at 95°C; 40 cycles of denaturing for 15 s at 95°C, annealing and extension for 45 s at 60°C; and a final melting curve from 60°C to 95°C for 20 min to check for primer-dimer artifacts. Optimization of qRT-PCR conditions was made on primer annealing temperature (60°C), primer concentration (200 nM) and template concentration (five 1:10 dilution series from 10 ng to 100 fg of input RNA). The cDNA from three individuals from the Control treatment was used as calibrator for all experimental conditions and genes studied, including the reference gene, and being repeated for all analyzed plates. β-actin was used as the internal reference gene (GenBank acc. no AF300705). GenBank accession numbers for the studied genes are: XM_027368952 for prostaglandin E synthase 2 (pges2), DQ836128 for lipoxygenases-5 (alox5), MG770374.1 for sterol regulatory element-binding protein-1

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(srebp1), XM_027371896.1 for Cyclooxygenase-2 (cox2), and XM_027373671 for carnitine palmitoyltransferase 1a (cpt1a).

2.7. Statistical analysis Data were presented as mean ± SE (standard error of the mean). Results of growth performance, muscle and hepatopancreas fatty acid profile, hepatopancreas gene expression and hepatopancreatic average area among the experimental groups were performed by one-

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way analysis of variance (ANOVA), followed by the Tukey's HSD test using the software SIGMASTAT for Windows version 3.5 (SigmaStat Software, CA, USA). A significance level

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of P < 0.05 was used for all statistical tests.

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3. Results

3.1. Biological index, performance and proximal composition

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The overall performance is presented in Table 4. The animals fed with a diet

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containing 0.3% of ARA presented a lower final weight compared to the Control group.

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However, no significant differences in SGR and survival were observed among the experimental treatments. The animals fed with 0.6% diet had significantly lower protein deposition in the whole body compared to those fed with rest of the treatments. Further, no significant differences were observed in moisture, lipid and ash content among the experimental groups (Table 4).

3.2. Hepatopancreatic cell morphology The hepatopancreas sections and the hepatopancreatic cells average size of L. vannamei fed with the experimental diets for six weeks are given in the Figure 1 and Table 4, respectively. The average area of B (blasenzellen) cells was higher in the Control group than

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that obtained in the other experimental treatments. In the same way the average area of R (restzellen) cells was higher in the Control group compared to the other groups. However, the Control diet without ARA resulted in higher average area of R cells than 0.6% and no differences were observed between 0.3% compared to the 0 and 0.6% groups.

3.3. Fatty acid profile Fatty acids hepatopancreas and muscle profile are presented in Tables 5 and 6

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respectively. In general, different levels of ARA inclusion in the experimental diets directly influenced in the tissues fatty acid profile. Animals fed with 0.6% diet presented higher ARA

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deposition in hepatopancreas compared to the others experimental groups, while in the muscle

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was observed a gradual ARA increase with lower levels in 0% followed by Control, 0.3% and

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0.6% respectively. Significant differences were also observed in the other LC-PUFAs (DHA and EPA) in hepatopancreas. Eicosapentaenoic acid percentage was higher in Control than

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0.3% and 0.6%, while DHA percentage in 0.3% was lower compared to Control and 0%, and

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no differences were observed between 0.6% and the other experimental groups.

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Docosahexaenoic acid levels in muscle were higher in Control compared to 0.6%, and, no differences were observed in the EPA percentage between the experimental groups. Differences observed in 18:2n6, ARA, EPA and DHA levels in hepatopancreas influenced in the n-6 and n-3 total PUFA, with higher n-6 percentage in Control compared to the others groups (total PUFA followed exactly the same profile) and higher n-3 percentage in Control compared to 0.3% and 0.6% groups. However, no differences were observed in n-3 and n-6 PUFA in the muscle. The differences in the ARA, EPA and DHA percentages also influenced in significant changes in DHA/ARA and EPA/ARA in both tissues (hepatopancreas and muscle), with lower ratio in Control and 0%, followed by 0.3% and 0.6% groups.

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Few punctual differences were observed in the others fatty acid levels, such as a higher 18:1n-9 deposition in Control compared to the others groups in both tissues, and this variation influenced in a higher total MUFA in the same group compared to the others treatments. In the same way, differences were observed in 18:2n-6 that was more representative in Control, while in the muscle this fatty acid was less representative in 0.3% and 0.6% compared to Control and 0% treatments.

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3.4. Gene expression

At the end of the experiment, the expression of cox2 and srebp1 resulted in the same

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profile, being up-regulated in the Control compared to the experimental groups (P˂0.001).

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The pges2 expression decreased gradually, with higher expression in the Control group

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followed by 0% and 0.6%, while the 0.3% treatment was lower compared only to the Control (P˂0.001). The expression of cpt1a was higher in 0% treatment than the rest of the

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experimental groups, being lower in the Control compared to 0.6% (P˂0.001). In contrast, no

4. Discussion

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differences in alox5 expression were observed among the experimental groups (Figure 2).

This study demonstrated the suitability of formulating with alternative lipids sources such as coconut and peanut oil, supplemented with LC-PUFA (i.e., DHA and EPA) levels in the diets for L. vannamei juveniles. In this way, the final fatty acid profile from shrimp results in a lower amount of linoleic acid, considered as detrimental for human health (Levitan et al., 2012; Patterson et al., 2012). Previous studies performed primarily with marine fish species have also shown promising results in growth performance in animals fed with fish oil-free diets containing high SFA and MUFA levels and supplemented with LC-PUFA (Xu et al., 2016a; Mata-Sotres et al., 2018; Araújo et al., 2019a). In an earlier study performed with

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L.vannamei (Araujo et al., 2019b) it was demonstrated that using beef tallow (rich in SFA and MUFA) as lipid base, but supplemented with DHA and low content of EPA it was possible to remove the fish oil without a detrimental effect on the overall performance. However, since those sources often contain a negligible amount of ARA in their composition and due to their physiological importance, the inclusion of this fatty acid in diets for marine organisms should be always evaluated (Bell and Sargent, 2003). In several previous studies performed with fish juveniles and even shrimp species such

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as, Psetta máxima (Castell et al., 1994), Paralichthys olivaceus (Estévez et al., 1997), Dicentrarchus labrax (Atalah et al., 2011); Siganus rivulatus (Nayak et al., 2017),

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Rachycentron canadum (Trushenski et al., 2012; Araújo et al., 2019a), Penaeus chinensis (Xu

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et al., 1994) and Macrobrachiu nipponense (Ding et al., 2018), have presented better growth

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performance when ARA is present in the diets. However, the results of this study showed that the growth performance of L. vannamei juvenile (using this ARA inclusion levels, weight

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range and conditions), was negatively affected by the ARA inclusion since the animals from

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0.3% group (1.82% of the total fatty acids) had a lower weight gain than animals from the

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Control (0.75% of the total FA). Likewise, no significant changes were observed between animals fed with 0.6% diet (3.14% of the total fatty acids) compared to the rest of the dietary treatments. Similar results were observed in studies performed with Penaeus monodon juveniles (Glencross and Smith, 2001), with lower growth in animals fed with ARA richdiets, and in L. vannamei juveniles that was not observed differences in the growth performance between animals fed with lower (0.8% of the total fatty acids) and higher (3.2% of the total fatty acids) ARA levels (Aguilar et al., 2012). These results suggest that others LC-PUFA such as EPA and DHA can influence more effectively in the marine shrimp growth compared to ARA. For example, according to Kanazawa et al. (1979), the nutritional fatty acid ranking for Penaeus japonicus is: EPA > DHA > 18:3n-3 > 18:2n-6 > 18:1n-9, and ARA

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was not considered essential. Even though some studies point out to a low ARA requirement in marine shrimp, González-Félix et al. (2003) demonstrated that L. vannamei juveniles fed with 0.5% of ARA had a better growth performance than animals fed with diets containing the same inclusion level of 18:3n-3 and 18:2n-6. In a similar way, Xu et al. (1993) observed in P. chinensis juveniles a higher weight gain of 120% (after 8 weeks) in animals fed with diets containing 0.5% of ARA, than those fed with 0.5% of 18:2n-6 (weight gain of 81%) and 18:3n-3 (weight gain of 95%). However in the same work it was observed that animals fed

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with a diet containing 0.5% of DHA had weight gain of 260%. Those previous studies demonstrated that ARA is more effective for the marine shrimp growth than others PUFA,

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being less essential than DHA and EPA. Certainly, others factors need be always considered

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in the growth performance evaluation facing to the ARA levels inclusion for aquatic

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organisms such as, the species specificity, life phase, culture system and conditions, genetic linage, and mainly the diet composition, since is know that LC-PUFA ratios (mainly between

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DHA, EPA and ARA) and different fatty acid series (mainly n-3/n-6) can directly influence in

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metabolic processes and consequently in the growth performance (Bell and Sargent, 2003;

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González-Félix et al., 2003; Glencross, 2009). In general, LC-PUFAs are usually preserved more than metabolized being this profile observed in marine crustaceans (González-Félix et al., 2003, 2010; Aguilar et al., 2012; Ding et al., 2018; Kumar et al., 2018; Araújo et al., 2019b) and fish species (Trushenski et al., 2012; Araújo et al., 2016, 2019a; Mata-Sotres et al., 2018), while other fatty acid incorporated in high proportion in the diet, such as 12:0, 16:0 are probably metabolized to produce energy by β-oxidation in a sparing effect (Turchini et al., 2011). However, in most works reported, the tissues composition in general (hepatopancreas and muscle) reflect the experimental diets FA profile, being common for shrimp and fish species (Turchini et al., 2011; Rombenso et al., 2016; Salini et al., 2016; Araújo et al., 2016, 2019 a,b; Mata-Sotres et al., 2018). Here this

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was also evident considering the ARA muscle concentration, observing a gradual increase along the experimental groups. Surprisingly, a lower DHA deposition was observed accordingly to the ARA enrichment in the experimental diets in hepatopancreas and in less extends in the muscle tissue. This effect is difficult to explain, however, it could be due to a DHA and ARA competition in the phospholipids esterification process, resulting in an inverse proportion of these fatty acids in the tissues (Tocher and Sargent, 1986; Bell et al., 1995; Bell and Sargent, 2003). Similar results were observed in a study performed with L. vannamei

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juveniles fed with diets containing different EPA/ARA ratio levels (Aguilar et al., 2012). The EPA/ARA ratio need always be considered in the marine fish and shrimp

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nutrition. These fatty acids are highly biologically relevant for most organism, due to an

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eicosanoids synthesis competition pathway, mainly by the prostaglandins (PGs) synthesis

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(Bell and Sargent, 2003; Tocher, 2003). The mechanisms that modulate the growth in response to the ARA/EPA levels in the diets have not been described as far as our knowledge.

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However, previous studies point out a direct relationship between PGs synthesized from EPA

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(3-series PGs) and ARA (2-series PGs) (Bell and Sargent, 2003; Arts and Kohler, 2009).

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According to Palmer (1990), PGF2α synthesized from ARA, stimulates the muscle fiber formation and inhibit protein degradation, promoting growth. Theoretically, the higher ARA is included in the diet an up-regulation in the expression of genes related to the eicosanoids synthesis should result. Interestingly the cox2 and pges2 expression (and higher trendy in alox5 expression) were higher in the Control group. COX2 synthesizes the prostanoids PGH2 and PGH3 from ARA and EPA, respectively without specificity between them. However, PGES2 acts specifically in the ARA eicosanoids pathway synthesizing PGH2 into PGE2 which is posteriorly converted into PGF 2α (Calder, 2007). It has been reported that the higher amount of n-3 LC-PUFA deposition (mainly EPA) in the cell membranes results in a decrease of ARA eicosanoids synthesis (PGE2 and PGF2α) (Calder, 2007; Arts and Kohler, 2009). The

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increase in cox2, and mainly the pges2 expression in the Control group probably is related to the higher EPA deposition in the cell membranes, influenced by the higher EPA percentage in the Control diet. According to Arts and Kohler (2009), n-3 rich diets can directly neutralize the eicosanoid synthesized from ARA by three factors: displacement (changes in the n-3/n-6 ratio in the cell membranes); competition (EPA compete for COX and ALOX action); and opposition (eicosanoids from EPA prevents the action of ARA-derived eicosanoids). In an in vitro experiment carried out in the kidney cells of Salmo salar, Araujo et al., (2014) observed

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that ARA presence in the culture media inhibited the PGE2 synthesis. The latter suggesting that ARA incorporation in the cell membranes resulted in higher n-3 LC-PUFA release,

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mainly EPA and DHA that may have been converted by COX2 in 3-series PGs (PGE3 and

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PGF3α).

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As observed for the cox2 and pges2, the fatty acid composition of diets had a direct influence in cpt1a and srebp1 expression. CPT1a can be directly related to the fatty acid

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catabolism transferring the long-chain FA into mitochondria to produce energy through the β-

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oxidation processes (Jump, 2002; Torstensen et al., 2009). Apparently, the cpt1a expression

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observed in L. vannamei hepatopancreas does not show a direct relationship with the ARA levels inclusion. Few studies have evaluated the specific effects of LC-PUFA in the lipolytic processes in crustacean. However, Shu-Chien et al. (2017) in a study carried out in the Sagmariasus verreauxi observed an up-regulation on the expression of genes related to the FA catabolism such as cpt1a, acyl-CoA oxidase (aox) and acid co-A ligase3 (co-A ligase3) influenced by MUFA-rich diets (composed by vegetable oils). According to Tocher (2003) the preferential fatty acid oxidation order for the most organisms, is SFA > MUFA > PUFA. However, the inclusion levels of each fatty acid in the diet will influence directly on the oxidative profile of different fatty acid classes. Apparently, the cpt1a expression results here observed in L. vannamei hepatopancreas is related to the SFA and MUFA levels in the diets,

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since 0% diet presented higher expression, whereas in the Control diet a lower percentage of these fatty acid classes were observed, correspondingly to the same profile observed in the expression of this gene. SREBP1 is a membrane-bound transcriptional factor involved in the regulation of genes related to the lipid (phospholipids and triglyceride) synthesis (Jump, 2002). As noted in the cox2, srebp1 expression, a noticeable increase was observed in Control compared to the other experimental groups. According to Jump (2002), the fatty acid hierarchy that regulates srebp1 expression is EPA = ARA > 18:2n-6 > 18:1n-9, and the higher

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expression observed in Control can be related to the higher EPA and 18:2n-6 percentage in this diet compared to the rest of the treatments. Similarly, in an experiment carried out with

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Portunus trituberculatus, Yuan et al. (2019) observed a higher expression of srebp1 and other

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genes related to the lipogenesis, when fed diets containing krill and soybean oil, being rich in

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EPA and 18:2n-6, respectively. However, the relationship between different fatty acid ratios in the transcription of genes for lipid synthesis is not clear and it could be influenced by other

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2008).

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variables such as the triglycerides/phospholipids ratio in the diet (Jump, 2002; Leaver et al.,

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Among several functions, the hepatopancreas is responsible for absorbing and storing metabolic substrates and synthesizing and secreting digestive enzymes (Johnston et al., 1998). Basically, the hepatopancreas is formed by four different cells, being the most abundant the R cells, where the lipid and glycogen are stored; and the B cells, responsible for intracellular digestion with a concomitant secretory function (Loizzi, 1971; Caceci et al., 1988). In general, the hepatopancreatic cells morphology, such as hepatic cells in fish, commonly reflect the nutritional status. In an experiment carried out with L. vannamei juveniles fed with different lipids level, Xu et al., (2016b) observed a higher lipid deposition in R cells in animals fed diets containing 120 g kg-1 of lipids compared to animals fed diets containing 60 e 90 g kg-1 . The latter work is an evidence of the influence of the diet composition on the hepatopancreas

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morphological in this species. Clearly, R and B cells were more abundant with a larger area in the shrimp fed the Control group. Studies related to the hepatopancreatic cell morphology influenced by the fatty acid composition in shrimp nutritional studies are scarce. However, for most organisms, EPA-rich diets showed a lipogenic inhibition process where the β-oxidation process is stimulated, with a lower hepatocytes vacuolization (Frøyland et al., 1996; Clarke, 2001; Caballero et al., 2004). In an experiment performed with R. canadum juveniles, Araújo et al. (2019a) observed that animals fed with ARA-rich diets, resulted in higher lipid

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deposition on the hepatocytes than those animals fed with fish oil, which is the opposite than that here observed. Based on the srebp1 expression, it is suggested that the higher EPA

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concentration influenced in a higher lipid synthesis, and consequently higher lipid deposition

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on the hepatopancreatic cells in the Control group. Although in fish the EPA/ARA ratio has

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been directly related to the lipid deposition in the liver, other fatty acid may have directly influence on the increase of hepatocyte vacuolization. Studies carried out with S. aurata

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juveniles showed that when high 18:2n-6 levels in the diets, influenced in a higher hepatic

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steatosis. A fact that in the present work could be related to the higher lipid deposition in the

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shrimp from de Control group that received higher 18:2n-6 than those from the rest of the experimental treatments.

In conclusion, ARA inclusion in fish oil-free diets to L. vannamei juvenile result in a negative impact on the growth performance and influenced in a differential transcription of lipid-relevant genes, mainly those related to the eicosanoids synthesis, additionally a lower hepatopancreas lipid deposition on the ARA fed animals was verified. Clearly, the results of this study, such as few previous studies, point out to the higher requirement for other LCPUFA, especially EPA and DHA, since these fatty acid have a greater influence on the growth performance in the marine shrimp species compared to ARA. However, due to the ARA physiological importance for the most organisms, further research is recommended

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mainly related to the ARA effect in L. vannamei keep in conditions that extend out the optimal physiological ranges of salinity, water temperature, oxygen, and density. The results presented in this study can directly contribute to the manufacturing of low cost and more sustainable specific diets to L. vannamei juveniles.

Acknowledgments We would like to thanks the Fundação de Amparo a Pesquisa do Estado de São Paulo

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(FAPESP) for the postdoctoral fellowship of Bruno Araújo (project number: 2018/13000-2), and technical assistance (project number: 2017/06765-0) mainly to morphological analysis.

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This project was financed by CONACYT (Project PN-2016- 2293) and UABC.

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5. References

Aguilar, V., Racotta, I.S., Goytortúa, M., Wille, M., Sorgeloos, P., Civera, R., Palacios, E.,

al

2012. The influence of dietary arachidonic acid on the immune response and performance

rn

of Pacific whiteleg shrimp, Litopenaeus vannamei, at high stocking density. Aquac. Nutr.

Jo u

18, 258–271.

AOAC, 2015. Official Methods of Analysis. Association of Analytical Chemists, Arlington, VA, USA.

Araujo, P., Lucena, E., Yang, Y., Ceemala, B., Mengesha, Z., Holen, E., 2014. The impact of exogenous n-6 and n-3 polyunsaturated fatty acids on the induced production of pro- and anti-inflammatory prostaglandins and leukotrienes in Atlantic salmon head kidney cells using full factorial design and LC–MS/MS. J. Chromatogr. B. 964, 164–171. Araújo, B., Salini, M., Glencross, B., Wade, N., 2016. The influence of dietary fatty acid and fasting on the hepatic lipid metabolism of barramundi (Lates calcarifer). Aquac. Res. 48 (7), 3879–3893.

Journal Pre-proof

20

Araújo, B., Honji, R.M., Rombenso, A.N., Souza, G.B., Mello, P.H., Hilsdorf, A.W.S., Moreira, R.G., 2019a. Influence of different arachidonic acid levels and temperature on the growth performance, fatty acid profile, liver morphology and expression of lipid genes in cobia (Rachycentron canadum) juveniles. Aquaculture 511, doi.org/10.1016/j.aquaculture.2019.734245. Araújo, B., Mata-Sotres, J.A., Viana, M.T., Tinajero, A., Braga, A., 2019b. Fish oil-free diets for Pacific white shrimp Litopenaeus vannamei: The effects of DHA-EPA

oo

f

supplementation on juvenile growth performance and muscle fatty acid profile. Aquaculture 511, doi.org/10.1016/j.aquaculture.2019.734276.

pr

Arts, M.T., Kohler, C.C., 2009. Health and condition in fish: the influence of lipids on

e-

membrane competency and immune response. In Lipids in Aquatic Ecosystems. Edited

Pr

by M.T. Arts, M.T. Brett, and M.J. Kainz. Springer, New York. pp. 237–256. Atalah, E., Hernández-Cruz, C.M., Ganuza, E., Benítez-Santana, T., Ganga, R., Roo, J.,

al

Montero, D., Izquierdo, M., 2011. Importance of dietary arachidonic acid for the growth, survival and stress resistance of larvae European sea bass (Dicentrarchus labrax) fed

rn

high dietary docosahexaenoic and eicosapentaenoic acids. Aquac. Res. 42, 1261–1268.

Jo u

Bell, J.G., Sargent, J.R., 2003. Arachidonic acid in aquaculture feeds: current status and future opportunities. Aquaculture 218, 491–499. Bell, J.G., Castell, J.D., Tocher, D.R., MacDonald, F.M., Sargent, J.R., 1995. Effects of different dietary arachidonic acid: docosahexaenoic acid ratios on phospholipid fatty acid compositions and prostaglandin production in juvenile turbot (Scophthalmus maximus). Fish Physiol. Biochem. 14, 139–151. Birba, A., Ramallo, M.R., Lo Nostro, F., Moreira, R.G., Pandolfi, M. 2015. Reproductive and parental care physiology of Cichlasoma dimerus males. Gen. Comp. Endocrinol., 221: 193-200.

Journal Pre-proof

21

Boglino, A., Wishkerman, A., Darias, M.J., de la Iglesia, P., Andree, K.B., Gisbert, E., Estévez, A., 2014. Senegalese sole (Solea senegalensis) metamorphic larvae are more sensitive to pseudo-albinism induced by high dietary arachidonic acid levels than postmetamorphic larvae. Aquaculture 433, 276–287. Caballero, M.J., Izquierdo, M.S., Kjørsvik, E., Fernández, A.J., Rosenlung. G., 2004. Histological alterations in the liver of sea bream, Sparus aurata L., caused by short-or-

oo

oil as the sole lipid source. J. Fish Dis. 27, 531-541.

f

long-term feeding with vegetable oils. Recovery of normal morphology after feeding fish

pr

Caceci, T., Neck, K.F., Lewis, D.D.H., Sis, R.F., 1988. Ultrastruture of the hepatopancreas of the Pacific white shrimp, Penaeus vannamei (Crustacea: Decapoda). J. Mar. Biol. Assoc.

e-

UK 68 (2), 323-337.

Pr

Calder, C.C., 2007. Immunomodulation by omega-3 fatty acids. Prostag. Leukotr. Ess. 77, 327–335.

al

Castell, J.D., Sinnhuber, R.O., Wales, J.H., Lee, J.D., 1994. Effects of purified diets

rn

containing different combinations of arachidonic and docosahexaenoic acid on survival,

Jo u

growth and fatty acid composition of juvenile turbot (Scophthalmus maximus). Aquaculture 128, 315-333.

Chen, K., Li, E., Xu, C., Wang, X., Lin, H., Qin, J.G., Chen, L., 2015. Evaluation of different lipid sources in the diet of pacific white shrimp Litopenaeus vannamei at low salinity. Aquacult. Rep. 2, 163 – 168. Clarke S.D., 2001. Nonalcoholic steatosis and steatohepatitis. Molecular mechanism for polyunsaturated fatty acid regulation of gene transcription. Am. J. Phys. 281, 865-869. Ding, Z, Zhou, J., Kong, Y., Zhang, I., Cao, F., Luo, N., Ye, J., 2018. Dietary arachidonic acid promotes growth, improves immunity, and regulates the expression of immune-

Journal Pre-proof

22

related signaling molecules in Macrobrachium nipponense (De Haan). Aquaculture 484, 112–119. Estévez, A., Ishikawa, M., Kanazawa, A., 1997. Effects of arachidonic acid on pigmentation and fatty acid composition of Japanese flounder, Paralichthys olivaceus (Temminck and Schlegel). Aquac. Res. 28, 279–289. FAO 2018. The State of World Fisheries and Aquaculture 2018 - Meeting the sustainable development goals. Food and Agriculture Organization of the United Nations (FAO),

oo

f

Rome. Italy. 210pp.

Frøyland, L., Helland, K., Totland, G.K., Kryvi, H., Berge R.K., 1996. A hypolipidemic

pr

peroxisome proliferating fatty acid induces polydispersity of rat liver mitochondria. Biol.

e-

Cell 87, 105–112.

Pr

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

al

olivaceus. Aquaculture 220, 725–735.

rn

Glencross, B.D., Smith, D.M., 2001. A study of the arachidonic acid requirements of the giant

Jo u

tiger prawn, Penaeus monodon. Aquac. Nutr. 7, 59–69. Glencross, B.D., 2009. Exploring the nutritional demand for essential fatty acids by aquaculture species. Rev. Aquac. 1, 71–124. González-Félix, M., Gatlin, D.M.I.I.I., Lawrence, A.L., Perez-Velazquez, M., 2003. Nutritional evaluation of fatty acids for the open thelycum shrimp, Litopenaeus vannamei: II. Effect of dietary n-3 and n-6 polyunsaturated and highly unsaturated fatty acids on juvenile shrimp growth, survival, and fatty acid composition. Aquac. Nutr. 9, 115–122. González-Félix, M.L., Perez-Velazquez, M., Quintero-Alvarez, J.M., 2009. Effect of various dietary levels of docosahexaenoic and arachidonic acids and different n-3/n-6 ratios on

Journal Pre-proof

23

biological performance of Pacific white shrimp, Litopenaeus vannamei raised in low temperature. Aquaculture 309,152 – 158. J. World Aquacult. Soc. 40, 194 – 206. González-Félix, M.L., da Silva, F.S.D., Davis, D.A., Samocha, T.M., Morris, T.C., Wilkenfeld, J.S., Perez-Velazquez, M., 2010. Replacement of fish oil in plant based diets for Pacific white shrimp (Litopenaeus vannamei). Aquaculture 309,152 – 158. Han, S., Wang, B., Liu, M., Wang, M., Jiang, K., Liu, X., Wang, L., 2018b. Adaptation of the white shrimp Litopenaeus vannamei to gradual changes to a low-pH environment.

oo

f

Ecotoxicol. Environ. Saf., 149, 203-210.

Han, S., Wang, M., Liu, M., Wang, B., Jiang, K., Wang, L., 2018a. Comparative sensitivity of

pr

the hepatopancreas and midgut in the white shrimp Litopenaeus vannamei to oxidative

e-

stress under cyclic serious/medium hypoxia. Aquaculture. 490, 44-52.

Pr

Honji, R.M., Caneppele, D., Pandolfi, M., Lo Nostro, F.L., Moreira, R.G., 2015. Gonadotropins and growth hormone Family characterization in na endangered Siluriform

al

species, Steindachneridion parahybae (Pimelodidae): rrelationship with annual

rn

reproductive cycle and induced spawning in captivity. Anat. Rec., 298: 1644-1658.

Jo u

Honji, R.M., Caneppele, D., Pandolfi, M., Lo Nostro, F.L., Moreira, R.G., 2019. Characterization of the gonadotropin-releasing hormone system in the Neotropical teleost, Steindacheneridion parahybae during the annual reproductive cycle in captivity. Gen. Comp. Endocrinol., 273: 73-85. Jin, M., Lu, Y., Yuan, Y., Li, Y., Qiu, H., Sun, P., Ma, H.N., Ding, L.Y., Zhou, Q.C., 2017. Regulation of growth, antioxidant capacity, fatty acid profiles, hematological characteristics and expression of lipid related genes by different dietary n−3 highly unsaturated fatty acids in juvenile black seabream (Acanthopagrus schlegelii). Aquaculture 471, 55–65.

Journal Pre-proof

24

Johnston, D.J., Alexander, C.G., Yellowhees, D. (1998). Epithelial cytology and function in the digestive gland of Thenus orientalis (Decapoda, Scyllaridae). J. Crust. Biol. 18(12), 271-278. Jump, B.D., 2002. Dietary polyunsaturated fatty acids and regulation of gene transcription. Genet. Mol. Biol. 13, 155–164. Levitan, E.B., Wolk, A., Hakansson, N., MItleman, M.A., 2012. Α-Linolenic acid, linoleic acid and heart failure in women. Br. J. Nutr. 108, 1300-1306.

oo

f

Kanazawa, A., Teshima, S.-i, Endo, M. 1979. Requirements of prawn, Penaeus Japonicus for essential fatty acids. Memoirs of the Faculty of Fisheries, 28. Kagoshima University, pp.

pr

27–33.

e-

Koven, W., Barr, Y., Lutzky, S., Ben-Atia, I., Weiss, R., Harel, M., Behrens, P., Tandler, A.,

Pr

2001. The effect of dietary arachidonic acid (20:4n−6) on growth, survival and resistance to handling stress in gilthead seabream (Sparus aurata) larvae. Aquaculture 193, 107–

al

122.

rn

Kumar, V., Habte-Tsion, H., Allen, K.M., Bowman, B.A., Thompson, K.R., El-Haraon, E.,

Jo u

Filer, K., Tidwell, J.H., 2018. Replacement of fish oil with Schizochytrium meal and its impact on the growth and lipid metabolism of Pacific white shrimp (Litopenaeus vannamei). Aquac. Nutr. 24, 1769 – 1781. Leaver, J. M., Villeneuve, L.AN., Obach, A., Jensen, L., Bron, J.E., Tocher, D.R., Taggart, J.B., 2008. Functional genomics reveals increases in cholesterol biosynthetic genes and highly unsaturated fatty acid biosynthesis after dietary substitution of fish oil with vegetable oils in Atlantic salmon (Salmo salar). BMC Genomics 9, 299. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2- ΔΔCT method. Methods 25, 402–408.

Journal Pre-proof

25

Loizzi, R.F., 1971. Interpretation of crayfish hepatopancreatic function based on fine structural analysis of epithelial cell lines and muscle net-work. Cell Tissue Res, 113, 420440. Mata-Sotres, J.A., Tinajero-Chavez, A., Barreto-Curiel, F., Pares-Serra, G., Rio-Zaragoza, O.B.D., Viana, M.T., Rombenso, A.N., 2018. DHA (22:6n3) supplementation is valuable in Totoaba mcdonald fish oil-free diets containing poultry by-product meal and beef tallow. Aquaculture 497, 440–451.

oo

f

Nayak, S., Koven, W., Meiri, I., Khozin-Goldberg, I., Isakov, N., Zibbeh, Mohammad, Z., Zilberg, D., 2017. Dietary arachidonic acid affects immune function and fatty acid

pr

composition in culture rabbitfish Siganus rivulatus. Fish Shellfish Immun. 68, 46–53.

e-

Palmer, R.M., 1990. Prostaglandins and the Control of muscle protein synthesis and

Pr

degradation. Prostag. Leukotr. Ess. 39, 95–104.

Parrish, C.C., Nichols, P.D., Pethybridge, H., Young, J.W., 2014. Direct determination of

rn

95.

al

fatty acids in fish tissues: quantifying top predator trophic connections. Methods 177, 85-

Jo u

Patterson, E., Wall, R., Fitzgerald, G.F., Ross, R.P., Stanton, C., 2012. Health implications of high dietary omega-6 polyunsaturated fatty acids. J. Nutr. Metab. 2012, 539426. Rombenso, A.N., Trushenski, J.T., Jirsa, D., Drawbridge, M., 2016. Docosahexaenoic acid (DHA) and arachidonic acid (ARA) are essential to meet LC-PUFA requirements of juvenile California yellowtail (Seriola dorsalis). Aquaculture 463, 123–134. Salini, M.J., Wade, N.M., Araújo, B.C., Turchini, G.M., Glencross, B.D., 2016. Eicosapentaenoic acid, arachidonic acid and eicosanoid metabolism in juvenile barramundi Lates calcarifer. Lipids 51, 973–988.

Journal Pre-proof

26

Soller, F., Roy, L.A., Davis, D.A., 2018. Replacement of fish oil in plant-based diet for Pacific white shrimp, Litopenaeus vannamei, by stearine fish oil and palm oil. J. World Aquacult. Soc. 50, 186 – 203. Shu-Chien, A.C., Han, W., Carter, C.G., Fritzgibbon, Q.P., Simon, C.J., Kuah, M., Battaglene, S.C., Codabaccus, B.M., Ventura, T., 2017. Effect of dietary lipid source on expression of lipid metabolism genes and tissue lipid profile in juvenile spiny lobster Sagmariasus verreauxi. Aquaculture 479, 342-351.

oo

f

Tocher, D.R., 2003. Metabolism and functions of lipids and fatty acids in teleost fish. Rev. Fish. Sci. 11 (2), 107–184.

pr

Tocher, D.R., Sargent, J.R., 1986. Incorporation of [1- 14 C] arachidonic and [1- 14 C]

e-

eicosapentaenoic acids into the phospholipids of peripheral blood neutrophils from the

Pr

plaice, Pleuronectes platessa L. Bba-Lipid Lipid Met. 876 (3), 592-600. Torrecillas, S., Román, L., Rivero-Ramírez, F., Caballero, M.J., Pascual, C., Robaina, L.,

al

Izquierdo, M.S., Acosta, F., Montero, D., 2017. Supplementation of arachidonic acid rich

rn

oil in European sea bass juveniles (Dicentrarchus labrax) diets: effects on leucocytes and

Jo u

plasma fatty acid profiles, selected immune parameters and circulating prostaglandins levels. Fish Shellfish Immunol. 64, 437–445. Torrecillas, S., Betancor, M.B., Caballero, M.J., Rivero, F., Robaina, L., Izquierdo, M., Dontero, D., 2018. Supplementation of arachidonic acid rich oil European sea bass juvenile (Dicentrarchus labrax) diets: effects on growth performance, tissue fatty acid profile and lipid metabolism. Fish Physiol. Biochem. 44, 283–300. Trushenski, J., Schwarz, M., Bergman, A., Rombenso, A., Delbos, B., 2012. DHA is essential, EPA appears largely expendable, in meeting the n-3 long-chain polyunsaturated fatty acid requirements of juvenile cobia Rachycentron canadum. Aquaculture 326-329, 81–89.

Journal Pre-proof

27

Turchini, G.M., Torstensen, B.E., Ng, W.K., 2009. Fish oil replacement in finfish nutrition. Rev. Aquac. 1, 10–57. Turchini, G.M., Ng, W.K., Tocher, D.R., 2011. Fish Oil Replacement and Alternative Lipid Sources in Aquaculture Feeds. Taylor and Francis, CRC Press, Boca Raton, FL USA. Xu, X., Ji, W., Castell, J.D., O’Dor, R., 1993. The nutritional value of dietary n-3 and n-6 fatty acids for the Chinese prawn (Penaeus chinensis). Aquaculture 118, 277–285. Xu, X.L., Ji, W.J., Castell, J.D., O'Dor, R.K., 1994. Essential fatty acid requirements of the

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f

Chinese prawn, Penaeus chinensis. Aquaculture 127, 29–40.

Xu, H., Ai, Q., Mai, K., Xu, W., Wang, J., Ma, H., Zhang, W., Wang, X., Liu, Z., 2010.

pr

Effects of dietary arachidonic acid on growth performance, survival, immune response

e-

and tissue fatty acid composition of juvenile Japanese seabass, Lateolabrax japonicus.

Pr

Aquaculture 307, 75–82.

Xu, H., Dong, X., Zuo, R., Mai, K., Ai, Q., 2016a. Response of juvenile Japanese seabass

al

(Lateolabrax japonicus) to different dietary fatty acid profiles: Growth performance,

rn

tissue lipid accumulation, liver histology and flesh texture. Aquaculture 461, 40–47.

Jo u

Xu, C., Li, E., Liu, Y., Wang, S., Wang, X., Chen, K., Qin, J.G., Chen, L., 2016b. Effect of dietary lipid level on growth, lipid metabolism and health status of the Pacific white shrimp Litopenaeus vannamei. Aquacult. Nutr .24, 204 – 214. Xu, H., Cao, L., Zhang, Y., Johnson, R.B., Wei, Y., Zheng, K., Liang, M., 2017. Dietary arachidonic acid differentially regulates the gonadal steroidogenesis in the marine teleost, tongue sole (Cynoglossus semilaevis), depending on fish gender and maturation stage. Aquaculture 468, 378–385. Yuan, Y.H., Li, S.L., Mai, K.S., Xu, W., Zhang, Y.J., Ai, Q.H., 2015. The effect of dietary arachidonic acid (ARA) on growth performance, fatty acid composition and expression of

Journal Pre-proof

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ARA metabolism-related genes in larval half-smooth tongue sole (Cynoglossus semilaevis). Br. J. Nutr. 113, 1518–1530. Yuan, Y., Sun, P., Jin, M., Wang, X., Zhou, Q., 2019. Regulation of dietary lipid sources on tissue lipid classes and mitochondrial energy metabolism of juvenile swimming crab,

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Portunus trituberculatus. Front. Physiol. 10, 454.

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Table legends Table 1 Formulation and proximate composition (g kg-1 on a dry matter basis, DM) of experimental diets formulated to contain different ARA (arachidonic acid, 20:4n-6) levels. Table 2 Fatty acid composition of the experimental diets and oils (% total fatty acids). Table 3 Primers pairs used for q-PCR. Primer sequences, amplicon sizes in base pairs (bp), reaction efficiencies (E) and Pearson's coefficients of determination (R2) are indicated. Table 4 Effect of ARA on growth performance, survival, whole body proximal composition

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(mg g1 ) and hepatopancreatic cells average area (µm2 ) of L. vannamei fed with different experimental diets.

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different experimental diets (% total fatty acids).

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Table 5 Hepatopancreas fatty acid composition of L. vannamei juvenile fed with

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Table 6 Muscle fatty acid composition of L. vannamei juvenile fed with different

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experimental diets (% total fatty acids).

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Figure legends Figure 1. Transverse section through the hepatopancreas of L. vannamei juvenile fed with the experimental diets. Hepatopancreatic cells profile observed in animals from (a) Control group, (b) 0%, (c) 0.3% and (d) 0.6%. Arrow indicates the B (blasenzellen) cells and arrowhead indicates R (restzellen) cells. Scale bar 25 µm.

Figure 2. Relative expression pattern of alox5, cox2, pges2, cpt1a and srebp1 in L.

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vannamei hepatopancreas at six weeks fed diets containing different ARA levels (Control, 0%, 10%, 15%, 20%). Different letters represent significantly different values (P < 0.05, n =

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6 by treatment).

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Author Contribution

Bruno Cavalheiro Araújo: Conceptualization, trial conduction, analysis, project administration, writhing (original draft).

Krishna Flores-Galvez: Conceptualization, trial conduction, analysis, writing (review).

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Renato Massaaki Honji: Conceptualization, trial conduction, analysis, writing (review).

Vitalina Magalhães Barbosa: Trial conduction, analysis, writing (review).

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Aurora Tinajero: Conceptualization, trial conduction, analysis, writing (review).

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María Teresa Viana: Conceptualization, project administration, writing (review).

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José Antonio Mata-Sotres: Conceptualization, trial conduction, analysis, project administration,

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writhing (original draft).

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Table 1 Formulation and proximate composition (g kg -1 on a dry matter basis, DM) of experimental diets formulated to contain different A RA (arachidonic acid, 20:4n-6) levels. Dietary ARA levels (g kg 1 ) Ingredient Control 0% 0.3% 0.6% a 290.4 290.4 290.4 290.4 Poultry Meal b 145.4 145.4 145.4 145.4 Fish Meal c 150.0 150.0 150.0 150.0 Soy Protein (42%) d 80.0 80.0 80.0 80.0 Gelatin 225.8 225.8 225.8 225.8 Maizena (corn starch) e 20.0 20.0 20.0 20.0 Rovimix f 10.0 10.0 10.0 10.0 Soy Lecithin g 5.0 5.0 5.0 5.0 Cholesterol Taurine 5.0 5.0 5.0 5.0 Sodium Benzoate 2.3 2.3 2.3 2.3 BHT 1.0 1.0 1.0 1.0 Stay C 0.3 0.3 0.3 0.3 h 32.5 Fish oil Soybean oil 32.5 Coconut oil 29.0 27.0 26.0 Peanut oil 26.0 25.0 23.0 DHA oilo 7.0 7.0 7.0 EPA oilp 3.0 3.0 3.0 ARA oilq 0.0 3.0 6.0 Proximate composition (g kg -1 )

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Crude Lipid 84.72 85.31 82.16 83.61 Crude Protein 442.02 447.80 456.90 448.60 Moisture 10.82 12.61 12.19 11.17 Ash 69.11 68.35 68.90 68.29 NFE 394.33 385.93 379.85 388.33 NFE (g kg -1 ) = 100% – (crude protein + crude lipid + moisture + ash). a Pet food grade from Proteínas Marinas y Agropecuarias SA de CV, Guadalajara, Jalisco, Mexico. b Sardine fishmeal from Proteínas Marinas y Agropecuarias SA de CV, Guadalajara, Jalisco, Mexico. c Alimentos COLPAC, Hermosillo, Sonora, México. d Progel Mexicana SA de CV, Léon, Guanajuato, México. e DSM Nutritional Products México SA de CV, Guadalajara, Jalisco, Mexico, contains in g kg ρ-aminobenzoic acid 1.45; biotin 0.02; myo-inositol 14.5; nicotinic acid 2.9; Capantothenate 1.0; pyridoxine-HCl 0.17; riboflavin 0.73; thiamine-HCl 0.22; menadione 0.17; α-tocopherol 1.45; cyanocobalamine 0.0003; calciferol 0.03; L-ascorbyl-2phosphate-Mg 0.25; folic acid 0.05; choline chloride 29.65; retinol 0.015; NaCl 1.838; MgSO4·7H2O 6.85; NaH2PO4·2H2O 4.36; KH2PO4 11.99; Ca(H2PO4)2·2H2O 6.79; Fe-citrate 1.48; Ca-lactate 16.35; AlCl3·6H2O 0.009; ZnSO4·7H2O 0.17; CuCl2 0.0005; MnSO4·4H2O 0.04; KI 0.008; CoCl2 0.05 and Stay–C (Vitamin C). f Interquimica SA de CV, Atizapán de Zaragoza, Mexico, Mexico. g Mitsui & Co, LTD. h Scoular de México, S. de R.L. de C.V. o EPA oil (>EPA 45%). Phosphotech Laboratories. ZAC de la Lorie. France; p DHA oil (>50% DHA). Jangsu Tiankai Biotechnology Co.. Ltd. Nanjing. China; q ARA oil (>40% ARA). Jangsu Tiankai Biotechnology Co.. Ltd. Nanjing. China;

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Table 2 Fatty acid composition of the experimental diets and oils (% total fatty acids). Diets Oils Fatty acids Control 0% 0.3% 0.6% ARA EPA 12:0 n.d. 15.27 14.83 14.49 n.d. n.d. 14:0 3.48 7.09 6.75 6.59 2.29 0.49 15:0 0.36 0.11 0.11 0.11 n.d. 0.16 16:0 19.28 15.15 14.93 14.61 8.96 20.44 18:0 5.30 4.14 4.24 4.37 9.70 0.38 20:0 0.79 0.54 0.53 0.54 0.87 n.d. 24:0 0.87 0.53 0.52 0.55 n.d. n.d. ∑SFA 30.47 42.83 41.91 41.24 21.82 21.47 16:1 4.31 1.76 1.69 1.64 0.11 0.36 18:1n-9 19.75 26.78 26.23 25.49 28.02 1.35 18:1n-7 1.67 0.77 0.72 0.75 1.46 0.79 20:1n-9 0.93 1.00 0.98 0.95 0.18 n.d. ∑MUFA 26.66 30.31 29.61 28.83 29.77 2.50 18:2n-6 26.67 15.73 16.08 16.09 n.d. n.d. 20:4n-6 0.75 0.40 1.82 3.14 44.95 n.d. ∑PUFA n-6 27.43 16.12 17.91 19.23 44.95 n.d. 18:3n-3 3.91 0.91 0.95 0.92 n.d. n.d. 20:5n-3 5.05 2.58 2.58 2.67 n.d. 53.15 22:6n-3 4.86 4.47 4.34 4.26 n.d. 12.59 ∑PUFA n-3 13.82 7.96 7.87 7.85 n.d. 65.74 ∑PUFA 41.25 24.08 25.78 27.08 44.95 65.74 n-3/n-6 0.50 0.49 0.44 0.41 n.d. n.d. DHA/ARA 6.48 11.17 2.38 1.36 n.d. n.d. EPA/ARA 6.73 6.45 1.42 0.85 n.d. n.d. Others 1.62 2.78 2.69 2.85 3.46 10.29 ΣSFA, ΣMUFA, ΣPUFA, Σn-3 PUFA, Σn-6 PUFA are the sum of saturated, monounsaturated, polyunsaturated, polyunsaturated n3 and polyunsaturated n6, respectively.

DHA n.d. 0.78 0.23 18.96 0.75 0.21 n.d. 20.93 0.28 0.34 n.d. 0.19 0.81 n.d. n.d. n.d. 0.26 11.53 60.27 72.06 72.06 n.d. n.d. n.d. 6.10

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Table 3 Primers pairs used for q-PCR. Primer sequences, amplicon sizes in base pairs (bp), reaction efficiencies (E) and Pearson's coefficients of determination (R2 ) are indicated. Fwd sequence (5´ - 3´)

Rv sequence (5´ - 3´)

Size (bp)

E

R2

alox5

CGT TCA ACC CAA TGA CAC AG

TAT CGC TGG GCA GAA AGA CT

161

1.02

0.92

cox2

GGG AGG CCT ACT CCA ATC TC

AGA ACT GGT GGG TGA AGT GC

210

1.01

0.95

pges2

GGC AAG AAC CTG AAG AGA CG

TCG ACA GCA GTC AGA ACA CC

170

0.97

0.99

srebp1

ACT GTA TCC CAC CGT TGA GC

CCT AGG TTG AGG CTG TCT CG

183

0.98

0.98

cpt1a

TGC AAG ATC AGT CCA GAT GC

CGT CTC ATT GTT GGT CAT GG

189

0.99

0.93

β-actin

GCT AAC CGC GAG AAG ATG AC

CAG GGC ATA TCC CTC GTA GA

174

1.00

0.99

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Table 4 Effect of ARA on growth performance, survival, whole body proximal composition (mg g 1 ) and hepatopancreatic cells average area (µm2 ) of L. vannamei fed with different experimental diets. 0.3% 1.10±0.03 2.53±0.31b 3.42±0.76 96.67±5.77

0.6% 1.10±0.03 2.77±0.35ab 3.93±0.79 88.89±5.09

P-value 1.00 0.047 0.414 0.330

Proximal composition Moisture Protein Lipid Ash

662.81±11.87 73.57±0.52a 10.70±4.58 57.21±9.64

650.11±14.55 72.67±0.27a 11.08±5.36 66.65±20.97

656.65±9.98 71.20±1.00b 14.77±5.88 81.01±8.94

0.463 0.001 0.682 0.363

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649.06±8.30 73.86±0.44a 10.37±355 61.48±21.01

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Index Initial Weight (g) Final Weight (g) SGR1 Survival (%)2

Dietary DHA level Control 0% 1.10±0.01 1.10±0.01 3.03±0.12a 2.93±0.57ab 4.64±0.51 4.31±1.27 91.11±3.85 90.00±5.77

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Hepatopancreatic cells area Blasenzellen cells (B) 82.26±8.19a 55.58±9.04b 54.61±11.82b 46.81±7.13b ˂0.001 a bc b Restzellen cells (R) 30.69±2.69 19.37±3.12 21.90±2.74 15.42±2.89c ˂0.001 Values represent means ± standard error. a,b Different letters indicate statistical differences between experimental diets, by Tukey's test (P>0.05). 1 Specific growth rate (SGR, %d) = (LnWt-LnWo) × 100/t 2 Survival rate (SR, %) = Nt × 100/No Where Wt and Wo were final and initial fish weight, respectively; Nt and No were final and initial number of fish, respectively; t was the duration of experimental in days

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Table 5 Hepatopancreas fatty acid composition of L. vannamei juvenile fed with different experimental diets (% total fatty acids).

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Fatty Acids Control 0% 0.3% 0.6% P-value 12:0 n.d. 3.40±1.00 4.90±0.87 3.93±0.59 0.164 14:0 1.38±0.13a 2.55±0.81ab 2.93±0.62b 2.36±0.41ab 0.017 16:0 19.27±1.11 17.72±0.53 18.20±0.59 17.15±2.32 0.327 18:0 7.21±0.22a 5.64±0.57b 5.51±0.26b 5.81±0.39b 0.002 ∑SFA 27.85±0.98 29.31±1.81 31.54±1.03 29.34±1.67 0.073 16:1 1.98±0.24 1.70±0.26 1.75±0.15 1.56±0.17 0.180 18:1n-9 20.07±1.56a 27.23±0.55b 28.92±0.59b 28.95±3.38b 0.001 18:1n-7 3.63±0.10a 2.45±0.13b 2.10±0.26b 2.37±0.25b ˂0.001 20:1n-9 n.d. 0.62±0.04 0.61±0.06 0.61±0.17 0.993 ∑MUFA 25.68±1.67a 32.00±0.57b 33.38±0.74b 33.49±3.14b 0.002 18:2n-6 17.46±0.85a 14.17±0.75b 14.84±1.09b 13.88±1.23b 0.004 18:3n-6 1.81±0.08 1.80±0.07 1.75±0.22 1.82±0.25 0.648 20:2n-6 2.49±0.06a 1.49±0.34b 1.41±0.38b 1.51±0.35b 0.008 20:4n-6 2.67±0.36a 2.09±0.48a 2.83±0.38a 4.87±1.18b 0.004 ∑PUFA n-6 24.43±0.70a 19.55±0.64b 20.83±0.49b 22.08±0.61b 0.002 18:3n-3 1.51±0.17a 0.70±0.07b 0.70±0.03b 0.56±0.04b ˂0.001 20:5n-3 7.01±0.81a 5.44±0.33ab 3.64±0.45b 4.37±1.34b 0.006 22:5n-3 n.d. 0.91±0.08 0.99±0.16 0.85±0.20 0.648 22:6n-3 8.41±1.21a 8.11±1.02a 6.04±0.41b 6.35±1.19ab 0.045 ∑PUFA n-3 16.94±2.07a 15.16±1.02ab 11.37±0.62b 12.13±2.75b 0.018 ∑PUFA 41.37±1.99a 34.71±1.37b 32.20±0.58b 34.21±3.34b 0.030 n-3/n-6 0.69±0.09ab 0.78±0.05a 0.55±0.04b 0.55±0.11b 0.016 DHA/ARA 3.15±0.21a 3.88±0.23a 2.13±0.18b 1.30±0.08c ˂0.001 EPA/ARA 2.62±0.01a 2.60±0.09a 1.29±0.01b 0.90±0.07c ˂0.001 Others 5.10±0.93ª 3.98±0.79a 2.88±0.49b 2.96±1.72c 0.113 Values represent means ± standard error (n=3 by treatment). ΣSFA, ΣMUFA, ΣPUFA, Σn-3 PUFA, Σn-6 PUFA are the sum of saturated, monounsaturated, polyunsaturated, polyunsaturated n3 and polyunsaturated n6 respectively. a,b Different letters indicate statistical differences between experimental diets, by Tukey's test (P<0.05).

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Table 6 Muscle fatty acid composition of L. vannamei juvenile fed with different experimental diets (% total fatty acids). Fatty Acids Control 0% 0.3% 0.6% P-value 12:0 n.d. 0.36±0.15ª 0.72±0.37a 1.60±0.09b 0.002 14:0 0.41±0.08 0.56±0.19 0.64±0.20 0.71±0.01 0.148 16:0 18.00±0.10 17.85±0.61 17.52±0.28 18.16±0.63 0.417 18:0 10.74±0.50 10.09±0.47 10.18±0.43 9.55±1.62 0.492 ∑SFA 29.15±0.48 28.85±0.09 29.05±0.85 30.01±1.05 0.280 16:1 0.97±0.05 0.94±0.07 0.79±0.10 0.68±0.24 0.088 18:1n-9 12.89±0.05a 17.41±0.81b 17.15±1.24b 16.53±0.69b ˂0.001 18:1n-7 3.19±0.38ª 2.50±0.11ab 2.20±0.06b 2.83±0.40ab 0.013 20:1n-9 1.44±0.26 1.70±0.18 1.09±0.16 1.46±0.31 0.073 ∑MUFA 18.49±0.65a 22.55±1.13b 21.23±1.50b 21.50±0.652b 0.008 18:2n-6 12.62±0.53a 12.05±0.36a 10.95±0.73b 9.53±0.66b ˂0.001 18:3n-6 1.55±0.35 1.93±0.31 1.54±0.21 2.64±0.89 0.097 20:2n-6 2.57±0.02a 2.21±0.21b 1.91±0.35b 2.41±0.25ab 0.049 20:4n-6 4.56±0.07a 4.17±0.18b 6.32±0.06c 8.28±1.43d ˂0.001 ∑PUFA n-6 21.30±0.65 20.36±0.45 20.72±0.25 22.85±1.89 0.073 18:3n-3 1.58±0.11ª 0.98±0.10ab 1.36±0.90ab 0.74±0.20b 0.047 20:5n-3 12.38±0.51 11.47±0.65 11.54±1.61 10.41±0.21 0.148 22:6n-3 14.35±0.84ª 14.05±0.86ª b 13.78±0.86ª b 12.50±0.67b 0.042 ∑PUFA n-3 28.31±1.50 26.56±1.05 26.68±3.32 23.65±1.53 0.117 ∑PUFA 49.61±0.86 46.85±0.80 47.40±3.13 46.51±0.36 0.183 n-3/n-6 1.33±0.11 1.30±0.08 1.29±0.17 1.04±0.15 0.091 DHA/ARA 3.15±0.12a 3.37±0.09a 2.18±0.03b 1.51±0.28c 0.005 EPA/ARA 2.71±0.11ª 2.75±0.21a 1.82±0.07b 1.26±0.17c ˂0.001 Others 2.75±0.52 1.75±0.42 2.32±0.78 1.98±0.30 0.626 Values represent means ± standard error (n=3 tanks by treatment). ΣSFA, ΣMUFA, ΣPUFA, Σn-3 PUFA, Σn-6 PUFA are the sum of saturated, monouns aturated, polyunsaturated, polyunsaturated n3 and polyunsaturated n6 respectively. a,b Different letters indicate statistical differences between experimental diets, by Tukey's test (P<0.05).

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Highlights •

First study that investigated the arachidonic acid influence in fish oil-free diets on the performance and

lipid metabolism of L. vannamei;

Arachidonic acid inclusion resulted in a negative impact on growth performance;

•

Arachidonic acid reduced significantly the DHA deposition in the muscle;

•

Supplementation of arachidonic acid in fish oil-free diets to Pacific white shrimp is dispensable.

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•

Figure 1

Figure 2A

Figure 2B