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Accepted Manuscript Lipoic acid modulates energetic metabolism and antioxidant defense systems in Litopenaeus vannamei under hypoxia/ reoxygenation co...

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Accepted Manuscript Lipoic acid modulates energetic metabolism and antioxidant defense systems in Litopenaeus vannamei under hypoxia/ reoxygenation conditions

Roberta de Oliveira Lobato, Litiele Cezar Cruz, Marcelo Estrella Josende, Patricia Brinkerhoff Tavares, Wilson Wasielesky, Fábio Everton Maciel, Juliane Ventura-Lima PII: DOI: Reference:

S0044-8486(18)30345-4 doi:10.1016/j.aquaculture.2018.08.020 AQUA 633462

To appear in:

aquaculture

Received date: Revised date: Accepted date:

20 February 2018 8 August 2018 10 August 2018

Please cite this article as: Roberta de Oliveira Lobato, Litiele Cezar Cruz, Marcelo Estrella Josende, Patricia Brinkerhoff Tavares, Wilson Wasielesky, Fábio Everton Maciel, Juliane Ventura-Lima , Lipoic acid modulates energetic metabolism and antioxidant defense systems in Litopenaeus vannamei under hypoxia/reoxygenation conditions. Aqua (2018), doi:10.1016/j.aquaculture.2018.08.020

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ACCEPTED MANUSCRIPT 1

Lipoic acid modulates energetic metabolism and antioxidant defense systems in

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Litopenaeus vannamei under hypoxia/reoxygenation conditions Roberta de Oliveira Lobato

1, 2

, Litiele Cezar Cruz 2 , Marcelo Estrella Josende

Patricia Brinkerhoff Tavares 1 , Wilson Wasielesky

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Juliane Ventura-Lima

3, 4

1, 2, 4*

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Instituto de Ciências Biológicas (ICB), Universidade Federal do Rio Grande - FURG,

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Rio Grande, RS, Brazil.

Programa de Pós-Graduação em Ciências Fisiológicas, Universidade Federal do Rio

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Grande - FURG, Rio Grande, RS, Brazil.

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Instituto de Oceanografia (IO), Universidade Federal do Rio Grande - FURG,

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Programa de Pós-Graduação em Aquicultura, Universidade Federal do Rio Grande FURG, Rio Grande, RS, Brazil.

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Rio Grande, RS, Brazil.

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, Fábio Everton Maciel

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* Corresponding author: Juliane Ventura-Lima Phone/Fax: +55 5332935249 E-mail: [email protected]

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ABSTRACT

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Lipoic acid (LA) is a known potent antioxidant, which has shown positive effects on

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energy metabolism. In this study, our objective was to evaluate the protective effects of

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LA on metabolic and biochemical alterations in hypoxia/reoxygenation injury (H/R) in

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shrimp (Litopenaeus vannamei). Two experimental groups were evaluated: non-

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supplemented and supplemented with LA (1% of body weight). The groups were

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evaluated after different periods of exposure to hypoxia (6 and 24 h) at 1.5 mg O 2 L-1

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and different periods of reoxygenation (0, 1, and 3 h). When the animals were

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supplemented with LA, glucose levels were decreased in the hemolymph and increased

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in the muscle tissue under H/R conditions. Furthermore, no change in lactate

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dehydrogenase (LDH) activity was observed, and lactate levels were either decreased or

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unchanged. In biochemical analyses related to oxidative stress, a protective effect of LA

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was shown against lipid peroxidation induced by H/R after 24 h of exposure. LA

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supplementation was shown to increase antioxidant capacity more effectively during

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reoxygenation. LA modulated glutathione-S-transferase (GST) activity differently

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according to H/R exposure time and the available oxygen concentration. Our results

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show that LA positively modulated energetic aspects and oxidative stress under H/R

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conditions. In the context of disturbance of oxygen concentration, LA was effective as a

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supplement against damage induced by hypoxia. This study corroborates the unique

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properties of LA against stressful situations and may contribute positively to existing

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problems in shrimp farming.

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Keywords : lipoic acid, hypoxia, reoxygenation, metabolic aspects, antio xidant capacity, Litopenaeus vannamei.

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1. INTRODUCTION Variations in oxygen levels directly influence the quality of the environment and

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can induce harmful effects in aquatic organisms. However, to cope with this situation,

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fishes and crustaceans modulate and regulate physiological and metabolic responses

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(Soñanez-Organiz et al., 2009). In fact, in crustaceans the distribution of oxygen may be

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increased by modulations in the ventilatory capacity as well as the increase of affinity of

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respiratory pigment by O 2 allowing the tissues oxygenation (Hill et al., 1991;

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McMahon, 2001).

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During hypoxia ATP (adenosine triphosphate) stores are rapidly depleted due to

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interruption of oxidative phosphorylation in mitochondria, and the absence of energy

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leads to a series of metabolic changes (Granger and Kvietys, 2015). For example, there

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is an increase in glycogen degradation to obtain energy, which leads to increased

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production of lactate and a consequent decrease in cellular pH. These metabolic changes

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can result in changes in structure proteins and lead to cellular insults (Pérez-Jimenéz,

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2012).

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Some studies have demonstrated that aquatic organisms tend to avoid or move

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away from hypoxic conditions whenever possible (Rosas et al., 1999). However, farm

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animals cannot use this behavior, so the development of strategies to improve the

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resistance of animals to hypoxia is of great relevance. The Pacific white shrimp

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Litopenaeus vannamei (Crustacea; Decapoda) is one of the most cultivated species in

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the world. Although L. vannamei is resistant to stressful variations in its environment,

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such as temperature, salinity, and oxygen oscillations, these factors can trigger severe

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damage to the animal, mainly in a shrimp farming situation (Kumlu et al., 2010; Chong-

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Robles et al., 2014).

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ACCEPTED MANUSCRIPT During hypoxia, especially severe hypoxia (1.0–2.0 mg O 2 L-1 ) as described by

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Levin et al. (2009), electrons are accumulated along the electron transport chain, and

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during the reoxygenation phase, oxygen reacts with these electrons accumulated in

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mitochondria, leading to a large release of reactive oxygen species (ROS) (Parrilla-

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Taylor and Zenteno-Savín, 2011; Welker et al., 2013). Although ROS act in redox

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signaling and resistance against pathogens, the imbalance between these species and

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antioxidant capacity can result in an oxidative stress situation (Fridovich, 2004;

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Zenteno-Savín et al., 2006). ROS are highly reactive with biomolecules and may cause

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damage to lipids, proteins and DNA, resulting in tissue morphological and metabolic

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dysfunctions (Halliwell and Gutteridge, 2007). The antioxidant defense system plays an

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important role in attempting to reverse or eliminate ROS, using enzymatic and non-

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enzymatic defense systems (Li et al., 2016). Among the enzymes involved in this

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defense system are glutathione-S-transferase (GST), catalase (CAT), glutathione

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peroxidase (GPx), superoxide dismutase (SOD), and others. Vitamin C (ascorbic acid),

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vitamin A (retinol), vitamin E (α-tocopherol), and lipoic acid (LA), and reduced

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glutathione (GSH), among others, are part of the non-enzymatic defense system (Zhang

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et al., 2010).

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Antioxidants are substances that act in the prevention or reduction of oxidation

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induced by reactive species (Aldini et al., 2010). Studies have shown that the

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incorporation of LA into the diets of farm animals has brought numerous benefits, such

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as increased antioxidant and detoxification capacities (Lobato et al., 2013; Martins et

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al., 2014).

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LA is derived from fatty acids produced by the body and is essential for

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metabolic processes and antioxidant defense (Packer and Cadenas, 2010). LA, as an

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important non-enzymatic antioxidant, can modulate the antioxidant defense system in 4

ACCEPTED MANUSCRIPT biological models, eliminate ROS, chelate metals, and play an important role in the

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regeneration of other antioxidants, such as GSH (Packer et al., 1995). A diet

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supplemented with LA was also shown to reduce the levels of oxidized proteins in

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muscle tissue, reduce ROS levels in the brain, and increase glutamate-cysteine ligase

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(GCL) activity in the brain and liver of the fish Corydoras paleatus (Monserrat et al.,

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2008). LA also showed to improve the detoxification capacity in liver and brain of carp

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(Cyprinus carpio) exposed to microcystin (Amado et al., 2011). A similar result also

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was observed by Kutter et al. (2014) in the brain of fish (Trachinotus marginatus)

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supplemented with LA-enriched feed.

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Therefore, the main goal of this study was to evaluate whether a LA-supplemented

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diet could improve or reverse the metabolic and oxidant effects induced by hypoxia and

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reoxygenation in L. vannamei.

2. MATERIAL AND METHODS

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2.1 Shrimp maintenance

Male and female juvenile Pacific white shrimp (Litopenaeus vannamei) with an

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average weight of 9 ± 0.7 g were obtained from the Marine Aquaculture Station,

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Oceanography Institute (IO), Federal University of Rio Grande-FURG and transferred

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to the Institute of Biological Science (ICB) of the same Institution, acclimated in tanks

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under controlled parameters (salinity 25‰, pH around 8.0, photoperiod 12 h light/12 h

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dark, temperature 20 ± 0.5 °C, and feeding twice a day) at least 2 weeks prior to the

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experiments.

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2.2 Preparation of lipoic acid-supplemented diet Shrimp were distributed into two groups: the non-supplemented group received

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a commercial diet only (Purina) with 45% crude protein, and the supplemented group

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received the same diet supplemented with LA. Both groups were fed twice daily for 4

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weeks, corresponding to 1% of the average body weight. Lipoic acid (Sigma, USA) was

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incorporated into the commercial diet at a concentration of 70 mg/kg of diet according

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to Terjesen et al. (2004).

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2.3 Experimental design

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Two groups (with and without LA) were treated separately in 12 aquariums with

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6 animals each (n = 6) according to their respective experimental groups. Figure 1

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shows the experimental design and the distribution of the times of exposure to hypoxia

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and reoxygenation. Hypoxia exposure times were chosen not to cause permanent

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damage to the animals that could not be remedied or prevented.

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2.4 Exposure to hypoxia and reoxygenation

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The shrimps (n = 6) were allocated to properly capped aquariums containing 10

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L of seawater (salinity 25‰) at a temperature of 20 ± 0.5 °C. Nitrogen gas was bubbled

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until withdrawal of the oxygen gas to the desired concentration of 1.5 ± 0.2 mg O 2 L-1

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(hypoxia) occurred. Normoxia was established at 6 ± 0.5 mg of O 2 L-1 . Oxygen

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concentration was monitored at 3-hour intervals throughout the experiment using an

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oximeter (DO-5519, Lutran Eletronic Entreprise Co). Several authors consider 1.5 mg

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O 2 L-1 as severe hypoxia for L. vannamei (Van Wyk and Scarpa, 1999; Vaquer-Sunyer

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and Duarte, 2008; Trasvinã-Arena et al., 2014; Li et al., 2016).

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2.5 Analysis of metabolic properties

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Sample preparation Hemolymph was extracted with a hypodermic syringe (1 ml) fitted with a 13x

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045 mm needle, washed with anticoagulant solution [NaCl (0.45 M), glucose (0.1 M),

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sodium citrate (0.03 M), citric acid (0.026 M), EDTA (0.01 M), pH 4.6]. The needle

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was carefully inserted into the animal at the junction between the cephalothorax and

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abdomen, and each sample obtained was stored in pre-refrigerated tubes. For analysis,

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the hemolymph was processed in cold buffer (4 °C) with EDTA (6%). Then, the

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abdominal muscle was removed, weighed, and homogenized at a ratio of 1:5 w/v in

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phosphate buffer (100 mM), EDTA (1 mM), and phenylmethylsulfonyl fluoride

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(PMSF) (10 µM) at pH 7.2. All samples were centrifuged at 8000 x g and 4 °C for 20

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minutes, and the supernatant was used or stored at −80 °C for subsequent analyses.

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Total proteins in the samples were determined following the Biuret method measured

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by spectrophotometry at 550 nm utilizing a microplate reader (Biotel ELx800).

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Lactate, lactate dehydrogenase, and glucose measurements.

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hemolymph

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spectrophotometry at 340 nm using the lactate determination kit by the enzymatic

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method (1100250K code; Kovalent - Brazil). The glucose level was verified by

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spectrophotometry at 490 nm using a glucose kit, employing the method of enzymatic

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oxidation by glucose oxidase ( 1040250K code; Kovalent - Brazil). In both tissues, the

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enzymatic method was used (2100075K code; Kovalent - Brazil) to determine the

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lactate dehydrogenase (LDH) activity, measured by spectrophotometry at 340 nm,

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utilizing a microplate reader (Biotel ELx800).

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2.6 Analysis of antioxidant properties 7

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Sample preparation After dissection, the muscle and gills were homogenized (1:4 p/v) in buffer

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containing Tris-HCl (100 mM), EDTA (2 mM), MgCl2 (5 mM), PMSF (0.05 mM), with

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the pH adjusted to 7.75. The samples were centrifuged at 10,000 x g for 20 minutes at 4

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ºC and the supernatant used for biochemical measurement. Total proteins in the samples

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were measured following the Biuret method measured by spectrophotometry at 550 nm

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utilizing a microplate reader (Biotel ELx800).

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Lipid peroxides

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Lipid peroxides were measured using the thiobarbituric acid-reactive substances

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(TBARS) assay as described by Oakes and Kraak (2003). The fluorescence was

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measured at excitation/emission wavelengths of 520/580 nm (FilterMax™ F5

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microplate reader/Molecular Devices - EUA). The results were expressed as nmol of

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MDA/mg of protein, using tetramethoxypropane (TMP) (Sigma, USA) as a standard.

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Antioxidant competence against peroxyl radicals

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This analysis was done according to the method of Amado et al. (2009). The

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peroxyl radical was generated by thermal decomposition (37 ºC) of ABAP (2,2′-azobis

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(2 methylpropionamidine) dihydrochloride). The peroxyl radical reacts with H 2 DCFDA

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(2,7 dichlorodihydrofluorescein diacetate, Molecular Probes), and the esterases present

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in the sample cause deacetylation of H2 DCFDA , which is then oxidized by ROS,

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forming a fluorescent compound (DCF) detected at excitation/emission wavelengths of

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485/535 nm (FilterMax™ F5 microplate reader/Molecular Devices - EUA). The total

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antioxidant capacity against peroxyl radical was quantified by the relative area with and

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without ABAP, where a high relative fluorescence area denote a low antioxidant

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capacity, as high fluorescence levels are obtained after addition of ABAP, indicating a

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low capacity to neutralize peroxyl radicals.

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Activity of glutathione-S-transferase (EC 2.5.1.18) Glutathione-S-transferase (GST) activity was assayed using methodology

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described by Habig and Jakoby (1981). The assay is based on the formation of the

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conjugated complex of 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 1 mM reduced

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glutathione (GSH). The absorbance generated was monitored at 340 nm, utilizing a

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microplate reader (Biotel ELx800).

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2.7 Statistical analysis

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Statistical analysis was performed by two-way ANOVA being the factors were

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treatment (normoxia or hypoxia) and antioxidant (with LA or without LA) at each time

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of reoxygenation (0, 1 and 3 h) followed by Newman-Keuls post hoc test. All data were

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expressed as mean ± standard error of the mean (SEM). Differences were considered

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significant at p < 0.05. Mathematical transformations were performed when necessary

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(Zar, 1984).

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3. RESULTS All results were compared statistically between the four conditions (normoxia,

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normoxia + LA, hypoxia, and hypoxia + LA) at each reoxygenation time (0, 1, and 3 h)

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and exposure time to hypoxia or normoxia (6 and 24 h).

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3.1 Glucose levels

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Glucose levels in the hemolymph after 6 h without reoxygenation (0 h) showed a

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significant increase in the hypoxia + LA group compared with the other groups (p <

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0.05). In the 1 hour reoxygenation time after hypoxia conditions, glucose levels

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increased compared with those of the normoxia group (p < 0.05), whereas in LA

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treatment (hypoxia + LA) glucose returned to normoxia levels. In 3 h of reoxygenation,

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the hypoxia group had decreased glucose levels compared with the normoxia group (p <

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0.05), LA in this case did not reverse this effect (Figure 2a). In the hemolymph at 24 h,

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after 0 and 1 h of reoxygenation it was observed that the hypoxic condition induced an

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increase in glucose levels (p < 0.05) compared with animals exposed to normoxic

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conditions, and the inclusion of LA reversed this increase, this result was not observed

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after 3 h of reoxygenation (Figure 2b).

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In the muscle after 6 h of exposure, hypoxia increased glucose levels after 1 hour

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of reoxygenation, and in this case, LA did not reverse this increase. After 0 a nd 3 h of

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reoxygenation, co-exposure to LA and hypoxia increased glucose levels compared with

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the other groups (Figure 2c). In 24 h of exposure, only after 3 h of reoxygenation was

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an increase in glucose levels (p < 0.05) observed in the group submitted to hypoxia, and

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LA did not reverse this increase (Figure 2d).

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3.2 Lactate levels

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ACCEPTED MANUSCRIPT Lactate levels in hemolymph after 6 h of exposure followed by 0, 1, and 3 h of

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reoxygenation showed an increase in the groups submitted to hypoxia compared with

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their respective normoxia groups (p < 0.05). However, at time 0 and 1 hour of

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reoxygenation, the inclusion of LA was shown to decrease these levels, in contrast to

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the results observed at 3 h of reoxygenation (Figure 3a). At all reoxygenation times

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evaluated in the hemolymph after 24 h of hypoxia exposure, it was observed that

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hypoxia significantly increased lactate levels compared with normoxia (p < 0.05).

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However, after 0 and 3 h of reoxygenation, LA decrease lactate levels under hypoxia

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conditions (Figure 3b).

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In muscle submitted to 6 h of hypoxia followed by reoxygenation for any length

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of time, an increase in lactate levels was observed in groups exposed to hypoxia

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compared with their respective normoxia groups (p < 0.05). However, LA was shown to

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restore normal lactate levels after 3 h of reoxygenation (Figure 3c). In the muscle after

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24 h of exposure, all groups exposed to hypoxia showed an increase in lactate levels

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when compared with the respective normoxia groups (p < 0.05). Treatment with LA

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was effective at reversing this increase only in the group exposed to 1 hour of

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reoxygenation (Figure 3d).

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3.3 LDH activity

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Lactate dehydrogenase (LDH) activity in the hemolymph in the 6-hour exposure

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groups increased in the groups exposed to hypoxia at 0 and 3 h of reoxygenation

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compared with their respective normoxia groups (p < 0.05). The inclusion of LA did not

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reverse this effect at the reoxygenation times evaluated (Figure 4a). In hemolymph, in

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the 24-hour exposure groups, the enzyme activity increased significantly in response to

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hypoxia compared with their respective normoxia groups (p < 0.05) and remained so at

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all reoxygenation times evaluated. Under these conditions, LA also did not reverse the

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activity increase (Figure 4b). The activity of LDH in the muscle after 6 h of exposure followed by 0 and 3 h of

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reoxygenation increased significantly in the hypoxia group compared with the normoxia

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group (p < 0.05). Again, the inclusion of LA did not reverse this effect ( Figure 4c).

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Muscle LDH activity increased in all groups submitted to hypoxia (24 h) compared with

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their respective normoxia groups (p < 0.05). However, in this case, LA decreased the

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enzyme activity in the deoxygenated groups reoxygenated for 1 and 3 h (Figure 4d).

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3.4 TBARS levels

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After 6 h of exposure, an increase in TBARS levels was only observed in the

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group exposed to normoxia + LA in muscle (Figure 5a), while in gills, no differences in

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lipid damage was observed (Figure 5c). However, after 24 h, LA treatment showed a

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reversal in the basal levels of lipid peroxidation in gills after 3h of reoxygenation, while

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in hypoxia + LA group showed a decrease in lipid damage in muscle tissue at all times

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of reoxygenation compared to animals exposed to hypoxia without LA (Figure 5b and

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d, respectively).

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3.5 Antioxidant competence against peroxyl radicals (ACAP)

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In the muscle after 6 h of hypoxia without reoxygenation (0 hour), the total

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antioxidant capacity decreased in relation to the respective normoxia groups (p < 0.05),

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and the inclusion of LA could not reverse this decrease. However, LA increased total

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antioxidant capacity in 3 h of reoxygenation compared with the other groups ( Figure

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6a). In muscle, in the hypoxia group a decrease was observed in total antioxidant

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capacity after 24 h at all reoxygenation times compared with the respective normoxia

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ACCEPTED MANUSCRIPT 1

groups (p < 0.05). However, LA treatment increased this capacity at 0 and 3 h of

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reoxygenation (Figure 6b). In gills from animals submitted to 6 h of hypoxia, a reduction in antioxidant

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capacity after 3 h of reoxygenation was observed compared with the respective

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normoxia groups (p < 0.05). However, LA treatment reversed this decrease (Figure 6c).

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Hypoxia in gills after 24 h was shown to decrease the antioxidant capacity in the

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animals after 0 and 1 hour of reoxygenation compared with the respective normoxia

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groups (p < 0.05). Treatment with LA reversed this decrease only after 1 hour of

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reoxygenation (Figure 6d).

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3.6 GST activity

GST activity in the muscle was decreased after 6 h of hypoxia followed by 1

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hour of reoxygenation compared with the normoxia group (p < 0.05). In this group the

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inclusion of LA did not result in a reestablishment of enzyme activity ( Figure 7a). In

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muscle after 24 h of exposure to hypoxia, no changes in GST activity were observed,

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regardless of the reoxygenation time or antioxidant treatment (Figure 7b).

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In gills exposed to 6 h of hypoxia, only after 3 h of reoxygenation was a

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modulation in GST activity observed. This modulation is evidenced by the decrease in

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enzyme activity in the group submitted to hypoxic conditions compared with the

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normoxia group (p < 0.05). In this same group, LA inclusion did not result in a

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reestablishment of enzyme activity (Figure 7c). In the gills, after 24 h of exposure to

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hypoxia, the group without reoxygenation (time 0) showed decreased enzyme activity

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compared with its respective normoxia group (p < 0.05), and LA inclusion was shown

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to increase the activity of the enzyme in relation to all other groups. After 3 h of

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reoxygenation, the groups submitted to hypoxia showed a positive modulation of GST

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activity compared with their normoxia groups (Figure 7d).

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4. DISCUSSION The Pacific white shrimp Litopenaeus vannamei is a species found naturally

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from the Eastern Pacific coast to northern Peru, and it is of great commercial

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importance. In cultivation, it is noted for being tolerant to variations in temperature,

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salinity, and oxygen (Barnabé et al., 1996). However, these changes may result in

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greater susceptibility to infections, reduction in the growth rate, and mortality (Peixoto

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et al., 2003; Wei et al., 2009).

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The large fluctuations in oxygen levels in shrimp farming lead to cycles of

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hypoxia followed by reoxygenation, and prolonged periods of exposure to

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concentrations below 1.5 mg O 2 L-1 (considered severe hypoxia) can be harmful and

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even lethal to L. vannamei (Van Wyk and Scarpa, 1999). Therefore, due to the need to

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maintain adequate productivity, dietary antioxidant supplementation has been attempted

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to improve health and growth of animals in farming (Monserrat et al., 2008).

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One response of the organism to the effects of hypoxia is the activation of

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adaptation mechanisms for survival using both physiological and biochemical strategies

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in response to this condition. For example, upregulation of glycolytic pathway genes

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can occur, since glycolysis is the main pathway by which energy is obtained when low

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oxygen levels do not support oxidative phosphorylation (Cruz et al., 2016).

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The metabolic disorders that occur during hypoxia are very well established;

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however, experimental evidence demonstrates that the major events leading to cell and

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tissue dysfunction are related to subsequent reoxygenation (Biddlestone et al., 2015).

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The reoxygenation or reperfusion of hypoxic tissues may induce rapid metabolic

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remodeling, mitochondrial reprogramming followed by high production of reactive

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oxygen species (ROS), reorganization of the ionic fluxes through the plasma membrane,

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inflammation, and consequently, cell death (Biddlestone et al., 2015; Solaini et al.,

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2010). Although a variety of molecular mechanisms have been proposed to explain such

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events, ROS production receives more attention as a critical factor in the genesis of

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reoxygenation injury. Lipoic acid is an essential cofactor of the pyruvate dehydrogenase and α-

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ketoglutarate dehydrogenase complexes, two important enzymatic complexes involved

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in energy metabolism. When administered exogenously to cells or supplemented in the

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diet, LA is a potent modulator of the redox state of cells (Packer and Cadenas, 2010).

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Enamorado et al. (2015) observed a significant improvement of the the antioxidant

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capacity in different tissues of Cyprinus carpio after supplementation with LA through

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diet. In addition to the unique antioxidant properties of LA, studies also demonstrate

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that LA stimulates weight gain and growth in the crabs Eriocheir sinensis (Xu et al.,

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2018) and modulates carbohydrate, lipid and protein metabolism in Cyprinus carpio

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(Santos et al. 2016).

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In this study, we showed metabolic changes during shorter (6- h) and longer (24-

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h) exposures to hypoxia and reoxygenation (1 a nd 3 h) in Litopenaeus vannamei, in

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both hemolymph and muscle tissue (Figures 2, 3, and 4).

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In a general analysis, we showed that supplementation with LA under exposure

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to hypoxia and reoxygenation (H/R) decreased circulating glucose (except in

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hemolymph after 6 h of hypoxia) and increased glucose levels in muscle tissue.

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Interestingly, although LDH activity increased in H/R, there was no overall difference

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between the supplemented and non-supplemented groups. However, overall lactate

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production was lower or unchanged in the LA-supplemented group when exposed to

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H/R. The results suggest that supplementation with LA modulates metabolic aspects of

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shrimp under H/R stress conditions, which may be associated with an improved

2

energetic status. However, more studies are needed to verify which pathway was being

3

used to improve the energetic state during H/R when animals received the supplemented

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diet. Our results regarding shrimp metabolism with respect to H/R status are similar

6

to those already shown in a metabolic deficiency situation. Martinèz-Quintana et al.

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(2016) showed that hypoxic condition stimulates the increase in gene expression of

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glucose transporter (GLUT1) in gills of L. vannamei concomitant to increase of glucose

9

and lactate levels in this tissue. Although the effect of LA in gene expression of glucose

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transporter in crustacean is unknown, our results showed an increase in muscle glucose

11

levels and a decrease in the hemolymph associated with LA supplementation,

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suggesting the mobilization of fuel to peripheral tissues. In mammals, LA was shown to

13

decrease the glucose levels in the blood and to increase the levels of the GLUT4

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transporter in the muscle membrane (Khamaisi et al.,1997). These results suggest that a

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reduced level of plasma glucose may result from increased glucose utilization by

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peripheral tissues, such as muscle. Increased glucose in muscle tissue may reflect an

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increased availability of glucose to meet the demand for glycolysis, providing a greater

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proportion of the energetic needs of the animal under conditions of oxygen restriction.

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The antioxidant properties of LA are very interesting. It is soluble in both

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aqueous and lipid media and can permeate cell membranes, including the blood-brain

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barrier. Lipoic acid, when incorporated by cells, can be reduced in part to dihydrilipoic

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acid (DHLA), which acts as an antioxidant in biological systems (Packer et al., 1998).

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In fact, it is known that LA can minimize oxidative damage to macromolecules such as

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lipids and proteins (Arivazhagan et al., 2002; Monserrat et al., 2008).

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ACCEPTED MANUSCRIPT Some products generated during lipid peroxidation can be used as biomarkers of

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oxidative damage in macromolecules (Hermes-Lima, 2004). Surprisingly, in this study,

3

the levels of TBARS in the muscle of L. vannamei supplemented with LA under

4

conditions of normoxia were increased at 1 h of reoxygenation ( Figure 5a). A similar

5

result was also observed by Martins et al. (2014), who, under similar conditions

6

(normoxia) showed an increase in TBARS levels in the gills of L. vannamei exposed to

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140 mg LA/kg, indicating a pro-oxidant effect of LA. However, LA showed protector

8

effect against lipid peroxidation after 24 h hypoxia followed by reoxygenation (3 h in

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muscle and 1 and 3 h in gills) (Figure 5b and 5d, respectively). This result was also

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observed in gills of L. vannamei submitted to moderate hypoxia (3 mg O 2 L-1 ) (Martins

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et al., 2014).

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Although hypoxic conditions generate several metabolic and biochemical

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changes, the greatest oxidative damage occurs during reoxygenation. When oxygen

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levels are restored, LA seems to be more effective after long periods of hypoxia (>6 h),

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as already reported in other studies using aquatic animals (Zenteno-Savín et al., 2006;

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Zhang et al., 2010; Martins et al., 2014). The antioxidant capacity of LA may be related

17

to the H/R time, since no protective effect of LA was observed at 6 h in both tissues

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(Figure 5a and c). In fact, Trasviña-Arena et al. (2014) showed that the expression of

19

antioxidant enzymes in L. vannamei was only activated after 6 h of hypoxia (1.5 mg O 2

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L-1 ) and reached a peak after 24 h in this condition. This corroborates our results that

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LA protected against increased levels of TBARS after 24 h of H/R.

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In this study, the overall antioxidant competence against pe roxyl radicals

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(ACAP) in both muscles and gills was increased in animals supplemented with LA

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(Figure 6). This behavior underscores a critical period for aquatic animals facing

25

massive production of ROS after reoxygenation. Li et al. (2016) showed that L. 18

ACCEPTED MANUSCRIPT vannamei exposed to 1.5 mg O 2 L-1 , LA increased antioxidant enzyme activity only

2

after 3 h of reoxygenation when submitted to hypoxia for 12 and 24 h. In our study, in

3

the most critical phase during reoxygenation, LA supplementation was shown to be

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efficient against the effects generated by oxygen reco very. It has been reported that the

5

accumulation of electrons in the transport chain in the mitochondria are available for

6

formation of ROS, even under hypoxic conditions using available oxygen (Storey,

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1996). We show in this study a decrease in the antioxidant capacity of the animals under

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conditions of hypoxia when compared with normoxia (Figure 6a, b and d). Li et al.

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(2016) also observed this result, in similar times and tissues, showing that the

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antioxidant defense system in L. vannamei may be more effective after reoxygenation.

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In crustaceans, the GST activity is modulated in different ways during hypoxia

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and reoxygenation (Maciel et al., 2004). A study using hepatopancreas of Neohelice

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granulata exposed to anoxia did not find any significant changes in GST activity;

14

however, after reoxygenation post-anoxia, this enzyme showed a significant reduction

15

in its activity (Oliveira et al., 2005). In the present study, the reoxygenation (1 and 3 h)

16

also showed to reduce the GST activity after 6 h of hypoxia in muscle and gills,

17

respectively; and the LA did not reverse this decrease. On the other hand, the LA

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showed to increase GST activity in gills of shrimps sub mitted to hypoxia during 24 h.

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Martins et al. (2014) also showed that the LA induces the increase in GST activity in L.

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vannamei submitted to moderate hypoxia (3 mg O 2 L-1 ). Thus, our results showed that

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H/R periods affect the capability of detoxification due to decrease of GST activity and

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this decrease may lead the organisms more vulnerable to other stressful condition. In

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this case, the LA showed poor efficiency in restore this enzyme activity.

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ACCEPTED MANUSCRIPT 1 2

5. CONCLUSION Overall, shrimp supplementation with LA positively modulated metabolic

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aspects and biochemical parameters, regarding lipid peroxidation and antioxidant

5

capacity, upon exposure to hypoxia and reoxygenation. Our results are promising

6

because they emphasize the protective and modulatory potential of LA in stress

7

situations and have the potential to contribute significantly to improving shrimp

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farming.

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ACKNOWLEDGEMENTS

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The authors would like to thank IFS for financial support (Process number

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A5318/2012 granted to Juliane Ventura-Lima, PhD and Process number A/5352-1

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granted to Fábio Everton Maciel, PhD). This project also receives support from the

13

Brazilian agency CNPq (455818/2014-2). Roberta de Oliveira Lobato was a graduate

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fellow at Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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Litiele Cruz was a recipient of a fellowship from the CAPES/PNPD Program. Wilson

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Wasielesky is a research fellow at CNPq.

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Conflict of interest statement

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The authors declare that there are no conflicts of interest.

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7. Figure legends Figure 1: Schematic representation of experimental design.

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Figure 2: Glucose levels, (a) and (b): Hemolymph with 6 and 24 h of hypoxia

4

exposure, respectively; (c) and (d): Muscle with 6 and 24 h of hypoxia exposure,

5

respectively. Different letters indicates significant differences between means of

6

interactions (treatment X antioxidant) at each time of reoxygenation (0, 1 and 3 h). Data

7

are expressed as mean ± standard error (n = 6).

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Figure 3: Lactate levels, (a) and (b): Hemolymph with 6 and 24 h of hypoxia

9

exposure, respectively; (c) and (d): Muscle with 6 and 24 h of hypoxia exposure,

10

respectively. Different letters indicates significant differences between means of

11

interactions (treatment X antioxidant) at each time of reoxygenation (0, 1 and 3 h). Data

12

are expressed as mean ± standard error (n = 6).

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Figure 4: Lactate dehydrogenase (LDH) activity, (a) and (b): Hemolymph with

14

6 and 24 h of hypoxia exposure, respectively; (c) and (d): Muscle with 6 and 24 h of

15

hypoxia exposure, respectively. Different letters indicates significant differences

16

between means of interactions (treatment X antioxidant) at each time of reoxygenation

17

(0, 1 and 3 h). Data are expressed as mean ± standard error (n = 6).

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Figure 5: Thiobarbituric acid-reactive substances (TBARS) assay, (a) and (b):

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Muscle with 6 and 24 h of hypoxia exposure, respectively; (c) and (d): Gills with 6 and

20

24 h of hypoxia exposure, respectively. Different letters indicates significant differences

21

between means of interactions (treatment X antioxidant) at each time of reoxygenation

22

(0, 1 and 3 h). Data are expressed as mean ± standard error (n = 6).

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Muscle with 6 and 24 h of hypoxia exposure, respectively; (c) and (d): Gills with 6 and

3

24 h of hypoxia exposure, respectively. Different letters indicates significant differences

4

between means of interactions (treatment X antioxidant) at each time of reoxygenation

5

(0, 1 and 3 h). Data are expressed as mean ± standard error (n = 6).

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Figure 7: Glutathione-S-transferase (GST) activity, (a) and (b): Muscle with 6

7

and 24 h of hypoxia exposure, respectively; (c) and (d): Gills with 6 and 24 h of hypoxia

8

exposure, respectively. Different letters indicates significant differences between means

9

of interactions (treatment X antioxidant) at each time of reoxygenation (0, 1 and 3 h).

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Data are expressed as mean ± standard error (n = 6).

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ACCEPTED MANUSCRIPT Highlights

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1. Hypoxia and reoxygenation (H/R) can affect both energetic metabolism and antioxidant system of farmed shrimp.

2. The supplementation of lipoic acid (LA) through diet was shown to improve both energy metabolism and the antioxidant capacity.

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3. The LA to be promising as feed supplement in shrimp farming.

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