i

UNIVERSIDADE FEDERAL DO RIO GRANDE – FURG

PROGRAMA DE PÓS- GRADUAÇÃO EM AQUICULTURA – PPGAq

INSTITUTO DE OCEANOGRAFIA – IO

RESPOSTAS ANTIOXIDANTES E DANO OXIDATIVO NO CAMARÃO

Litopenaeus vannamei: EFEITOS DA SUPLEMENTAÇÃO COM

ANTIOXIDANTE E USO DE TECNOLOGIA DE BIOFLOCOS

ÁTILA CLIVEA DA SILVA MARTINS

RIO GRANDE, RS

AGOSTO, 2015

1

i

UNIVERSIDADE FEDERAL DO RIO GRANDE – FURG

PROGRAMA DE PÓS- GRADUAÇÃO EM AQUICULTURA – PPGAq

INSTITUTO DE OCEANOGRAFIA – IO

TESE DE DOUTORADO

RESPOSTAS ANTIOXIDANTES E DANO OXIDATIVO NO CAMARÃO

Litopenaeus vannamei: EFEITOS DA SUPLEMENTAÇÃO COM ANTIOXIDANTE E

USO DE TECNOLOGIA DE BIOFLOCOS

ÁTILA CLIVEA DA SILVA MARTINS

Orientador: Dr. José María Monserrat (FURG)

Coorientador: Dr. Wilson Wasielesky Jr. (FURG)

Rio Grande – RS – Brasil

Agosto, 2015

Tese apresentada ao Programa de Pós-

graduação em Aquicultura da

Universidade Federal do Rio Grande,

como requisito parcial à obtenção do título

de DOUTOR.

ii

1

ii

SUMÁRIO

DEDICATÓRIA................................................................................... v

AGRADECIMENTOS......................................................................... vii

RESUMO.............................................................................................. viii

ABSTRACT.......................................................................................... x

1 INTRODUÇÃO GERAL..................................................................... 1

2 HIPÓTESES......................................................................................... 10

3 OBJETIVOS......................................................................................... 10

3.1 OBJETIVO GERAL.............................................................................. 10

3.2 OBJETIVOS ESPECÍFICOS................................................................. 10

4 MATERIAL E MÉTODOS GERAL.................................................. 11

4.1 DESENHO EXPERIMENTAL.............................................................. 11

4.1.1 Preparação das nanocapsulas e cápsulas vazias................................ 14

4.1.2 Diluição do ácido lipóico em hidróxido de sódio............................... 14

4.2 ANÁLISE DA ÁGUA........................................................................... 15

4.3 COLETA E HOMOGENEIZAÇÃO DAS AMOSTRAS...................... 16

4.4 DETERMINAÇÃO DA ATIVIDADE DA ENZIMA GLUTATIONA

S-TRANSFERASE (GST) ....................................................................

16

4.5 DETERMINAÇÃO DA ATIVIDADE DA GLUTATIONA

REDUZIDA (GSH) ...............................................................................

17

4.6 DETERMINAÇÃO DOS NÍVEIS DE PEROXIDAÇÃO LIPÍDICA.. 17

4.7 DETERMINAÇÃO DA CAPACIDADE ANTIOXIDANTE TOTAL

CONTRA RADICAIS PEROXIL (ACAP) ..........................................

18

4.8 CONTAGEM DIFERENCIAL DE HEMÓCITOS................................ 19

4.9 ANÁLISE ESTATÍSTICA DOS RESULTADOS.................................. 19

REFERÊNCIAS................................................................................... 21

CAPÍTULO I: Antioxidant and oxidative damage responses in

different organs of Pacific white shrimp Litopenaeus vannamei

(Boone, 1931) reared in a biofloc technology system........................

28

ABSTRACT.......................................................................................... 29

1 INTRODUCTION................................................................................ 30

2 MATERIALS AND METHODS......................................................... 31

iii

2.1 DETERMINATION OF GST ACTIVITY............................................. 32

2.2 DETERMINATION OF TOTAL ANTIOXIDANT CAPACITY........... 32

2.3 DETERMINATION OF CONCENTRATION OF REDUCED

GLUTATHIONE (GSH)........................................................................

33

2.4 DETERMINATION OF LIPID PEROXIDATION............................... 33

2.5 DETERMINATION OF TOTAL AMMONIA....................................... 34

2.6 STATISTICAL ANALYSIS................................................................... 34

3 RESULTS.............................................................................................. 34

4 DISCUSSION....................................................................................... 38

REFERENCES..................................................................................... 41

CAPÍTULO II: Antioxidant effects of nanoencapsulated lipoic

acid in tissues and immune condition in hemolymph of shrimp

Pacific Litopenaeus vannamei (Boone, 1931).....................................

45

ABSTRACT.......................................................................................... 46

1 INTRODUCTION................................................................................ 47

2 MATERIALS AND METHODS......................................................... 50

2.1 DETERMINATION OF GLUTATHIONE S-TRANSFERASE

ACTIVITY (GST)..................................................................................

53

2.2 DETERMINATION OF CONCENTRATION OF REDUCED

GLUTATHIONE (GSH)........................................................................

53

2.3 DETERMINATION OF LIPID PEROXIDATION............................... 53

2.4 DETERMINATION OF TOTAL AMMONIA....................................... 54

2.5 DIFFERENTIAL HEMOCYTE COUNT (DHC) ................................. 54

2.6 STATISTICAL ANALYSIS................................................................... 55

3 RESULTS.............................................................................................. 55

4 DISCUSSION....................................................................................... 56

5 CONCLUSÃO...................................................................................... 61

REFERENCES..................................................................................... 62

CAPÍTULO III: Effects of lipoic acid in the total antioxidant

capacity in biofloc.................................................................................

71

ABSTRACT.......................................................................................... 72

1 INTRODUCTION................................................................................ 72

iv

2 MATERIALS AND METHODS......................................................... 74

2.1 EXPERIMENTAL DESIGN.................................................................. 74

2.2 DILUTION LIPOIC ACID IN SODIUM HYDROXIDE..................... 74

2.3 WATER QUALITY ANALYSIS............................................................ 75

2.4 COLLECTION AND HOMOGENIZATION OF SAMPLES............... 75

2.5 DETERMINATION OF TOTAL ANTIOXIDANT CAPACITY

AGAINST PEROXYL RADICAL (ACAP) .........................................

76

2.6 STATISTICAL ANALYSIS................................................................... 76

3 RESULTS.............................................................................................. 77

4 DISCUSSION....................................................................................... 80

5 CONCLUSION..................................................................................... 81

6 FUTURE PERSPECTIVE.................................................................. 81

REFERENCES..................................................................................... 82

CONCLUSÃO GERAL....................................................................... 85

v

DEDICATÓRIA

À minha mãe (Célia Santana) e a meu pai (Antônio Carlos Martins), entre erros e

acertos não permitiram que o amor incondicional provocasse vícios e dependências, me

deram autonomia, confiança e independência para traçar rumos, fazer escolhas, superar

frustações e cometer meus próprios erros e acertos. Em cada nova fase de minha vida

tivemos perdas e ganhos dos dois lados, porque o amor de pai e mãe é processo de

libertação permanente e esse vínculo não para de se transformar ao longo da vida.

Dando-me dando a certeza a cada dia de que eles, até quando puderem, estarão lá firmes

na concordância ou na divergência, no sucesso ou no fracasso, com o peito aberto para

um aconchego, abraço apertado e conforto em todas as horas. A natureza nos ajuda a

enxergar e compreender a importância das raízes. Quanto mais vigorosas são, maior e

mais forte é a árvore. Para voar alto e livre é preciso ter um terreno firme de onde

decolar. É tudo isso que meus pais representam para mim.

À minha vó (Maria Madalena Santana – em memória), que estava sempre preocupada

com as horas que estávamos sem comer, se andávamos descalços. A mulher que a seu

modo através de olhares e sorrisos conseguia dizer o quanto nos amava. Ainda tenho

vivo na memória nossos últimos momentos juntas. Presenciei alguns sorrisos, alguns

olhares atentos, e percebi, ela ainda estava ali, estava vendo e observando todos nós, por

trás daquela mulher fraca estava ela: A minha vó, a mesma de sempre!

À minha irmã (Carla Martins), que me ajudou a enfrentar o mundo com inteligência,

coragem e sabedoria. Inteligência para encontrar no mundo a oportunidade de mudança

e aprendizado. Coragem para aceitar mudanças. Sabedoria para sorrir, chorar, sem

perder a linha, sem perder o passo. Ensinou-me que algumas situações na vida servem

como cinzel que esculpe, que talha, que faz o bloco amorfo de mármore se transformar

em estátua, em obra de arte.

A Carlos Eduardo Winievisck, quem tem sorriso mais contagiante que já conheci.

Carrega consigo a alegria de viver, que ainda conserva um sotaque irresistivelmente

encantador. E quando estamos juntos a paz chega bem pertinho de mim, os problemas

que embalam os dias e que roubam as energias misteriosamente se escondem como se

vi

por aqueles instantes não mais existissem. Somos apenas nós diante de um cenário

escolhido, jogando palavras, formando conversas e confissões. Quem me trouxe os mais

lindos instantes e me completou com a magnitude de cada olhar. "Como tudo o que não

pode ser tocado com a mão e nem visto como os olhos, e ainda se torna mais forte. As

únicas coisas que importam são as feitas de verdade e alegria, não as de lata e vidro..."

(Fernão Capelo Gaivota).

A Ivis Winievisck, meu amigo foram tantas as vezes que você apareceu no momento

certo, e que mesmo sem querer dizia exatamente o que eu precisava ouvir, como se

pudesse me ler. Nos conhecemos sem que sejam necessárias as palavras. É tudo tão

grande que as diferenças se tornaram pequenos detalhes. Homem de frases lindas que

tanto respondi com brincadeiras tão sinceras, mas que hora ou outra me faz dizer

olhando nos olhos frases tão difíceis de serem ditas pessoalmente por mim. Que me

ensinou a arte de ver a mim mesmo com minhas forças e fraquezas, mas sem máscaras,

sem ilusões. A arte de perceber que as feridas cicatrizam sempre, e que ali a pele se

torna mais resistente.

Ao Prof. Dr. José María Monserrat, que foi meu orientador acadêmico e na vida pessoal

que me ensinou a ver que o que me acontece no presente não vai definir meu futuro nem

quem sou e sim a maneira como eu reajo a tudo que me acontece é o que vai definir

quem eu serei e quem eu quero ser.

vii

AGRADECIMENTOS

Ao meu co-orientador Prof. Dr. Wilson Wasielesky.

A CAPES pelo suporte financeiro através da concessão de bolsa de estudos.

A todos do grupo do Projeto Camarão.

A todos do grupo de Piscicultura Estuarina e Marinha.

Ao grupo EAOx (Espécies ativas de oxigênio), em especial a Josencler, Camilla Porto,

Juliana Artigas.

Agradeço a Jani (Ivanildo), pela ajuda no processo de qualificação do doutorado, pela

companhia na preparação e análise do terceiro trabalho e aos passeios de bicicleta.

Aos amigos que aqui fiz: Paula Maicá, Mércia, Vita, André Braga, Yorleys (Miss

Colômbia), Adriana (Shakira), Viviana Lisboa, Paola, Alain (Aladin), Cecilia (Ceci),

agradeço por todas as vezes que me fizeram fortes, que me fizeram sorrir que me

abraçaram no momento exato, que me ajudaram academicamente e assim renovaram

minhas energias para seguir sempre em frente.

Aos meus amigos de sempre: Thays, Gisa, Aline, Guto, Bernardo, Mila, cada um em seu

momento soberam me dar incentivo a continuar, lutar e superar a distância.

viii

RESUMO 1

Em aquicultura o aumento da intensificação está diretamente ligado a aplicação de 2

novas tecnologias que aumente o volume de produção ao mesmo tempo que causa o 3

mínimo de impacto ao meio ambiente que circunda a produção. Deste modo a 4

tecnologia de biofloco desponta como método no qual o tratamento de qualidade de 5

água é efetuado dentro do tanque de criação de modo que organismos fotoautotrofico 6

(microalgas), autotróficos (bactéria nitrificante) e heterotróficos (bactéria heterotrófica) 7

reciclam compostos nitrogenados que podem vir a ser tóxicos para o camarão 8

Litopenaeus vannamei, além de serem capazes de transformar amônia em biomassa 9

bacteriana que servirá como fonte proteica e lipídica para o camarão, podendo reduzir 10

custo com ração. Com aumento da densidade no sistema de criação faz-se necessário 11

aumentar a resistência bioquímica do animal criado, para tanto este trabalho apresenta 12

em três capítulos meios de suplementação com antioxidante ácido lipóico (AL) que 13

auxilia na resposta bioquímica antioxidante como mecanismo de melhorar o bem estar 14

do camarão Litopenaeus vannamei. Primeiramente, através de análises bioquímicas 15

como atividade da glutationa S-transferase (GST), concentração de glutationa reduzida 16

(GSH), capacidade antioxidante total contra radicais peroxil (ACAP) e níveis de 17

peroxidação lipídica (TBARS) em brânquias, hepatopâncreas e músculo de camarão, 18

observando-se que o biofloco induz aumento da atividade da GST em brânquias, 19

aumento da concentração de GSH em músculo, aumenta a capacidade antioxidante total 20

em músculo e reduz níveis de peroxidação lipídica em hepatopâncreas. O segundo 21

trabalho, aplicou-se ácido lipóico nanoencapsulado (NCLA) e cápsula vazia (NC) na 22

ração, em água clara e em água com biofloco no qual foi observado que NCLA induzida 23

aumento da atividade de GST nos hepatopâncreas. A concentração de GSH foi maior no 24

músculo do que em brânquias e hepatopâncreas. A capacidade antioxidante também 25

mostrou um padrão tecido-específico, tendo hepatopâncreas com maior capacidade 26

antioxidante nenhuma ação evidente do desempenho do NCLA contra os radicais 27

peroxil. Níveis de peroxidação lipídica foram menores no músculo, com acentuado 28

efeito do NCLA. Nos grupos com NCLA houve um aumento na porcentagem de 29

hemócitos granulares, células com maiores quantidades de componentes 30

imunocompetentes. No trabalho 3, foi observado que o AL é capaz de aumentar a 31

capacidade antioxidante no biofloco, analisado através da determinação da capacidade 32

ix

antioxidante total contra radicais peroxil (ACAP), principalmente para concentração de 33

10 µM (2.06 mg de AL in 1 L de água destilada). 34

35

Palavras chave: Biofloco, Litopenaus vannamei, antioxidante, ácido lipoíco, análises 36

bioquímicas, nanotecnologia, contagem diferencial de hemócitos. 37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

x

ABSTRACT 57

In aquaculture, the increased intensification is directly linked to application of new 58

technologies to increase the volume of production while causing minimal impact to the 59

environment surrounding the production. Thus, the biofloc blunts as a new technology 60

method in which the treatment water quality is made inside the tank, so that creation of 61

photoautotrophic organisms (microalgae), autotrophs (nitrifying bacteria) and 62

heterotrophic (heterotrophic bacteria) recycle nitrogen compounds that may to be toxic 63

to shrimp Litopenaeus vannamei. These microorganisms are able to turn ammonia into 64

bacterial biomass that will serve as protein and lipid source for shrimp and may reduce 65

feed cost. With increased density in the build system it is necessary to increase the 66

resistance biochemistry of the animal created, therefore this work presents in four 67

chapters how of supplementation with the antioxidant lipoic acid (LA) can be assist in 68

antioxidant biochemical response as a mechanism to improve the wellness of shrimp 69

Litopenaeus vannamei. Firstly, by biochemical analyzes as gluthatione S-transferase 70

(GST) activity, reduced gluthatione (GSH) concentration, total antioxidant capacity 71

against peroxyl radicals (ACAP) and levels of lipid peroxidation (TBARS) in gills, 72

hepatopancreas and shrimp muscle, observing that the biofloc induces increased in GST 73

activity in gills, increased GSH concentration in muscle, increases the total antioxidant 74

capacity in muscle and reduces lipid peroxidation levels in hepatopancreas. The second 75

work was applied nanoencapsulated lipoic acid (NCLA) and empty nanocapsules (CN) 76

in feed in clean water (SW) and biofloc (BFT) in which it was observed that NCLA 77

induced increased GST activity in hepatopancreas. The GSH concentration was higher 78

in muscle than in gills and hepatopancreas. The antioxidant activity also showed a 79

specific pattern of tissue having higher antioxidant capacity in hepatopancreas, without 80

no obvious action of NCLA in performance against peroxyl radicals. Lipid peroxidation 81

levels were lower in the muscle, with marked effect of NCLA. In groups with NCLA 82

there was an increase in the percentage of granular hemocytes, cells with higher 83

amounts of immunocompetents components. In the work 3, it was observed that LA can 84

increase the antioxidant capacity on biofloc, analyzed by determining the total 85

antioxidant capacity against peroxyl radicals (ACAP), especially for concentration 10 86

µM (2.06 mg AL in 1 L of water distilled). 87

xi

Keywords: Biofloc, Litopenaus vannamei, antioxidant, lipoic acid, biochemistry, 88

nanotechnology, differential count of hemocytes. 89

90

1

1 INTRODUÇÃO GERAL 91

Várias pesquisas no setor de aquicultura são destinadas ao aumento da 92

intensificação da produção. Estes esforços englobam maximizar as atividades na relação 93

aquicultura e ambiente externo, como também o aumentar a sustentabilidade 94

(econômica, social e ambiental). Ainda deve ser considerada a relação entre aquicultura 95

e ambiente interno, que corresponde a potencializar o crescimento e sobrevivência do 96

camarão, em menores ciclos de produção e com rendimento e qualidade do produto 97

final. A carcinicultura se tornou uma grande atividade aquícola e sua expansão em todo 98

mundo tem aumentado a preocupação ambiental em torno desta atividade (Xu et al. 99

2013). Tendo o camarão branco do Pacífico, Litopenaeus vannamei (Boone 1931), a 100

espécie com maior volume de produção (Kim et al. 2014) por apresentar rápido 101

crescimento, maior índice de sobrevivência e ser tolerante a alta densidade e estocagem 102

(Xu et al. 2012). 103

A produção mundial cresceu a uma taxa de 15,1% entre 2000 e 2008, que se 104

deve a intensificação da produção de Litopenaeus vannamei na China, Tailândia e 105

Indonésia (FAO 2012). Em 2014, a produção mundial de camarão em cativeiro 106

aumentou para 3.680.404 toneladas, um aumento de 7% em relação aos 3.436.918 de 107

toneladas produzidas em 2013, com base em estimativas obtidas de fontes oficiais e na 108

sua ausência de fontes oficiais, as estimativas fornecidas por fontes da indústria, a 109

produção brasileira se estabilizou entre os anos de 2013 e 2014 em 90 mil toneladas 110

(Shrimp News International 2015). 111

O camarão branco Litopenaeus vannamei é a espécie de camarão mais 112

comercializada em muitas partes do mundo e sua produção em sistemas intensivos de 113

biofloco com pouca ou nenhuma troca de água tem demostrado ser uma prática 114

sustentável (Avnimelech 2012; Xu e Pan 2014), especialmente em aquicultura intensiva 115

que está ligada com a poluição da água por um excesso de materiais orgânicos e 116

nutrientes que são susceptíveis de causar efeitos tóxicos agudos e riscos ambientais de 117

longo prazo (Piedrahita 2003). Em um sistema aquícola convencional o método mais 118

comum para lidar com este tipo de poluição tem sido a substituição contínua da água do 119

tanque com água fresca externo (Gutierrez-Wing e Malone, 2006). No entanto, o 120

2

volume de água necessário para pequeno a médio tanque aquícola pode chegar a várias 121

centenas de metros cúbicos por dia. 122

Uma nova abordagem é uso da tecnologia de biofloco (BFT), que é a formação e 123

estimulação de um microecossistema que incluem microalga, bactérias autotróficas, 124

bactérias heterotróficas, detritos orgânicos e inorgânicos. Desta forma, processos de 125

renovação de água neste sistema é mínima ou zero, havendo, portanto, reutilização da 126

água e alguns riscos, como a introdução de agentes patogénicos, escapamento de 127

espécies exóticas e descarga de águas residuais (poluição) são reduzidos ou mesmo 128

eliminados (Ray 2012). 129

Estes microrganismos (biofloco) tem como funções principais: (i) manutenção 130

da qualidade da água, pela absorção de compostos nitrogenados e sua transformação em 131

proteína microbiana e (ii) nutrição que aumenta viabilidade econômica da produção, 132

reduzindo a conversão alimentar e uma diminuição dos custos de alimentação 133

(Emerenciano 2013). Os macroagregados (biofloco) é fonte natural rica em lipídeos e 134

proteínas, disponível in situ 24 horas por dia (Avnimelech 2007). Na coluna de água 135

ocorre uma complexa interação entre matéria orgânica, substrato físico e grande 136

variedade de microrganismos, como fitoplâncton, bactérias livres e aderidas, agregados 137

de partículas de matéria orgânica e herbívoros, como os rotíferos, ciliados e flagelados 138

protozoários e copépodes (Ray 2010). 139

Esta produtividade natural tem um papel importante na reciclagem de nutrientes 140

e na manutenção da qualidade de água. Bactérias autotróficas fazem a conversam de 141

amônia a nitrito e depois convertem nitrito a nitrato e, as bactérias heterotróficas 142

conseguem compostos nitrogenados e transformam em proteína microbiana. As 143

bactérias autotróficas são mais eficientes nesta conversão, porém o processo é feito de 144

forma lenta, e as bactérias heterotróficas tem crescimento mais rápido e, portanto, 145

retiram de forma mais rápida os nitrogenados e transformam em proteína microbiana, 146

por isso há a manipulação da taxa de carbono e nitrogênio na proporção 20:1 para 147

favorecer crescimento e domínio de bactérias heterotróficas (Avnimelech 1999). 148

O consumo de biofloco por camarão ou peixe tem demonstrado inúmeros 149

benefícios tais como a melhoria da taxa de crescimento, diminuição da taxa de 150

conversão alimentar e os custos associados em alimentos para animais (Buford et al. 151

3

2004; Wasielesky et al. 2006). A melhoria do crescimento tem sido atribuída a bactérias 152

e algas como componentes nutricionais, pelo qual até 30% da ração comercial pode ser 153

reduzido devido ao consumo de biofloco pelo camarão (Buford et al. 2004). E o uso de 154

biofloco pode ser uma alternativa para substituir o uso de proteínas alternativas como a 155

farinha de peixe (Azim 2008). 156

Estudo tem demostrado que além do uso manutenção da qualidade de água e 157

como fonte de proteína e lipídio, o biofloco também tem o efeito de manter o equilíbrio 158

das funções fisiológicas como sistema antioxidante, que é essencial para a manutenção 159

do bem estar do camarão e assim garantir crescimento e sobrevivência satisfatórios 160

(Castex et al. 2010; Xu e Pan 2014; Martins et al. 2014, 2015) em um ambiente de 161

criação. Estudos efetuados com Litopenaues vannamei indicaram que o biofloco pode 162

aumentar o estado antioxidante do camarão, com causa provável do biofloco ser rico em 163

microrganismos naturais e compostos bioativos de natureza antioxidante (Ju et al. 2008; 164

Xu e Pan 2013; Martins et al. 2015). 165

A espécie Litopenaeus vannamei vem largamente sendo afetada por doenças 166

(Kim et al. 2014). Muitas doenças são agravadas pela alteração do equilíbrio 167

bioquímico celular e pouco se conhece sobre os benéficos bioquímico/fisiológicos do 168

biofloco e seus efeitos no camarão. O desequilíbrio bioquímico, em espécies aeróbicas, 169

pode ocorrer quando há maior produção de espécies reativas de oxigênio (ERO), que 170

são produtos intermediários da redução parcial dos quatro elétrons do oxigênio 171

resultando em água (H2O), ânion superóxido (O2•-), radical hidroxila (OH•) e espécies 172

não radicalar como o peróxido de hidrogênio (H2O2) (Abele e Pintarulo 2004). Por 173

definição redução é a perda de oxigênio ou ganho de elétrons, desta forma o O2 sofre 174

redução tetravalente com ganho de quatro elétrons e formando H2O (Gutteridge e 175

Halliwell 2010) e neste processo os produtos intermediários reativos (O2•-, H2O2, OH•) 176

podem ser nocivos quando o sistema antioxidante não é capaz de controlá-los, situação 177

que pode derivar em estresse oxidativo (Sies 1985; Abele e Pintarulo 2004). 178

O estresse oxidativo é um estado de desbalanço entre a produção intra e 179

extracelular de ERO e o sistema antioxidante, resultando em dano oxidativo de muitos 180

tipos de moléculas como lipídios, proteínas e DNA. Desta forma, a necessidade de 181

prevenir, interceptar ou retardar as ações das ERO, no decorrer da evolução, acarretou 182

4

no desenvolvimento de defesas antioxidantes, comumente divididas em enzimáticas e 183

não-enzimáticas (Anderson 1998; Dickinson e Forman 2002). O sistema antioxidante 184

enzimático é o primeiro mecanismo de defesa celular e é composto por superóxido 185

dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx) e glutationa-S-186

transferase (GST) que são moléculas de maior peso molecular, somado a estes há a ação 187

de outros compostos antioxiantes como vitaminas A, C e E que têm menor peso 188

molecular (Hellou et al. 2012). Dentre as defesas antioxidantes não-enzimáticas o 189

tripeptídeo glutationa (γ-L-glutamil–L-cisteinil–glicina) é considerado a primeira ação 190

de defesa contra ERO (Anderson 1998; Dickinson e Forman 2002) e está presente nos 191

organismos nas formas reduzidas (GSH) e oxidada (GSSG) (Hellou et al. 2012). Além 192

deste, inclui-se também α-tocoferol, carotenóides e flavonoides (Barreiros et al. 2006) e 193

o ácido lipóico (AL) que é um dos focos desta Tese. 194

Acredita-se que as mitocôndrias consumam 90% do oxigênio celular em células 195

átonas (jovens) e são os principais locais de produção de ERO em células aeróbicas 196

(Lenaz 1998; Abele e Pintarulo 2004). Na redução univalente, o O2 (oxigênio) é 197

convertido a O2•- (radical superóxido), que por ação da enzima superóxido dismutase 198

(SOD) é convertido em H2O2 (peróxido de hidrogênio), que é um ERO mas não é um 199

radical livre, este composto é difundo livremente através da mitocôndria de forma 200

espontânea (Abele e Pintarulo 2004). Caso o H2O2 não seja decomposto 201

enzimaticamente, este pode ser convertido em OH• (radical hidroxila), que tem curta 202

vida por ser altamente reativo (Halliwell e Gutteridge 1985; Abele e Pintarulo 2004). 203

Alguns componentes (poluentes) podem ser difíceis de oxidar e, portanto, é necessário 204

que o H2O2 seja ativado por catalizadores (ferro, cobre, manganês), sendo mais comum 205

utilizar o ferro como catalizador que quando reage com H2O2 caracteriza a reação de 206

Fenton que requer pH ácido e produz radicais hidroxila (OH•) que são altamente 207

reativos que degradam poluentes orgânicos (Wang et al. 2012). O H2O2 é convertido em 208

água e oxigênio, cuja reação é catalisada pela enzima catalase (CAT) ou é utilizado para 209

oxidar substratos, como por exemplo peroxidases como glutationa peroxidase (GPx) 210

(Hellou et al. 2012). 211

Um estado redox pró-oxidante é caracterizado com uma queda relativa na 212

proporção de glutationa reduzida (GSH/GSSG) e da relação NADH/NAD (Abele e 213

5

Pintarulo 2004). A glutationa é uma molécula produzido naturalmente pelo fígado, 214

também é encontrada em frutas, verduras e carnes, sendo uma combinação de três 215

blocos de proteína ou aminoácidos (tripéptido – cisteína, glicina e glutamina) que 216

contém um grupo químico de enxofre (SH), que atua como atrativo para moléculas que 217

podem causar dano ao organismo como espécies reativas de oxigênio e xenofibiótico 218

(Nuttall et al. 1998, Huber et al. 2008). Normalmente a glutationa é reciclada no corpo, 219

exceto quando há sobrecarga com muito estresse oxidativo ou muitas toxinas, a 220

glutationa se esgota e diminui a proteção contra os radicais livres ou toxinas (Nuttall et 221

al. 1998, Huber et al. 2008). A rede antioxidante é composta por vários componentes 222

que incluem vitaminas, minerais e produtos químicos especiais chamados tióis 223

(glutationa e o ácido alfa-lipóico) (Huber et al. 2008). 224

A glutationa é um antioxidante intracelular que tem a capacidade de maximizar a 225

atividade de todos os outros antioxidantes, incluindo vitaminas C, vitamina E e ácido 226

lipóico, removendo toxinas das células e protegendo contra os efeitos nocivos da 227

radiação, produtos químicos e poluentes ambientais (Schafer e Buettner 2001; 228

Dickinson e Forman 2002; Huber et al. 2008). O AL é uma molécula com características 229

hidro e lipossolúvel, com múltiplos efeitos benéficos em doenças como diabetes, 230

Alzheimer e hipertensão, o que mostra seu grande potencial biomédico (Packer et al. 231

1995). O AL e sua forma reduzida, o ácido dihidrolipóico (DHLA), preenchem todos os 232

critérios avaliados na análise do potencial antioxidante de um composto: quelam metais, 233

são varredores de ERO, participam da reciclagem de outras moléculas antioxidantes e 234

do reparo de moléculas danificadas pelo estresse oxidativo (Packer et al. 1995). O ácido 235

lipóico a princípio foi classificado como vitamina, contudo posteriormente foi 236

constatado que este composto é sintetizado em células animais (Carreau 1979), atuando 237

como cofator em complexos multi-enzimáticos que catalisam reações de 238

descarboxilação oxidativa no ciclo de Krebs (Packer et al. 1995). 239

Um antioxidante pode ser definido como qualquer substância que quando 240

presente em baixas concentrações em relação ao substrato oxidável (que causa dano), 241

atrasa significativamente ou impede a ação danosa do referido substrato (Halliwell e 242

Getturidge 1995). Nas últimas décadas o ácido lipóico tem recebido atenção devido a 243

sua função antioxidante em organismos aquáticos (Monserrat et al. 2008). O papel do 244

6

ácido lipóico no reestabelecimento dos níveis de ácido ascórbico em pacu (Piaractus 245

mesopotamicus) foi importante do ponto de vista bioquímico e fisiológico, o pacu, 246

assim como outros teleósteos, não sintetiza o ácido ascórbico, sendo este, portanto, uma 247

vitamina. A deficiência dela pode induzir uma redução de crescimento e maior 248

suscetibilidade a vários tipos de doenças (Terjesen et al. 2004; Trattner et al. 2007). Em 249

truta arco-íris (Oncorhynchus mykiss) e carpas (Cyprinus carpio) a deficiência de 250

vitamina C provoca anorexia, lordose, escoliose, hemorragias, deformações em 251

brânquias, exoftalmia (Trattner et al. 2007). 252

A vitamina C quela o ferro e o reduz a Fe2+, subsequentemente, o Fe2+ pode 253

transferir um elétron ao oxigênio ou para outro ERO e induzir o estresse oxidativo, 254

porém em quantidades equimolares de ferro e vitamina C, ácido lipóico é capaz de 255

competir com a vitamina C para a quelação e, consequentemente, haver proteção contra 256

a peroxidação de lípidos (Biewenga et al. 1997). O ácido lipóico tem sido referido como 257

um antioxidante universal por atuar tanto na membrana quanto na fase aquosa das 258

células, proporcionando proteção a membrana, devido a sua interação com os 259

antioxidantes vitamina C e glutationa, as quais por sua vez podem reciclar a vitamina E 260

(Flora 2009). As propriedades do ácido lipóico incluem também a capacidade de varrer 261

ERO, além de regenerar antioxidantes endógenos (Packer et al. 1995; Flora 2009; 262

Külkamp-Guerreiro et al. 2009). 263

O trabalho de Amado et al. (2011) avaliou o efeito quimioprotetor do ácido 264

lipóico contra a toxidade de microcistina em carpa Cyprinus carpo. Estes autores 265

observaram o tempo necessário para indução na expressão de genes que codificam três 266

classes da glutationa-S-transferase (alfa, mu e pi). Os resultados constataram que o AL 267

foi eficaz em promover aumento na transcrição de genes da GST no fígado após duas 268

injeções de AL dadas com intervalo de 24 h. Os dados sugerem que AL pode ser útil 269

como agente quimioprotetor contra indução toxica da microcistina, estimulando a 270

desintoxicação através do incremento da atividade da GST (cérebro) ou por meio da 271

reversão da inibição da GST (fígado). 272

O trabalho de Monserrat et al. (2008), considerou os efeitos do AL em diferentes 273

órgãos (brânquias, cérebro, músculo e fígado) no peixe Corudoras paleatus 274

(Callychthyidae). O AL, na dose de 70 mg/kg de massa corporal foi adicionada na 275

7

ração, alimentados diariamente (1% do peso). Os resultados mostraram redução da 276

concentração de espécies reativas de oxigênio no cérebro e aumento da atividade do 277

glutamato-cisteína ligase (GCL) no cérebro e no fígado do mesmo grupo experimental. 278

A GCL é uma enzima que controla a velocidade da síntese de GSH. Organismos 279

suplementados com AL apresentaram maior atividade da glutationa-S-transferase no 280

cérebro, indicando que o AL melhora a capacidade de desintoxicação nas reações de 281

fase II. Foi observado também notável redução da oxidação de proteínas no músculo e 282

no fígado dos peixes suplementados com AL, indicando que o tratamento foi eficaz na 283

redução de parâmetros de estresse oxidativo. 284

O organismo do camarão, como qualquer organismo aeróbico, é susceptível a 285

estresse oxidativo, resultado da ação de ERO que podem ser provenientes do meio 286

ambiente (exógenas) ou gerada no próprio organismo (endógenas). No camarão 287

Litopenaeus vannanmei, foi verificado o efeito de 3 doses de AL (35, 70 e 140 mg de 288

AL por 1 kg de ração), que foi suplementada na ração durante 45 dias e análises de 289

espécies reativas de oxigênio foi efetuada em brânquias e hepatopâncreas. Constatou-se 290

que das 3 doses aplicadas a dose de 70 mg/kg foi a que melhor resultou no aumento da 291

atividade antioxidante, principalmente em brânquias (Martins et al. 2014). 292

A utilização do ácido lipóico é promissora, contudo esta substância é lábil e sem 293

estabilidade química e, portanto, sujeito a degradação por ação térmica, fotoquímica, 294

meio ácido e oxidação. Sendo assim, existem pesquisas desenvolvendo um complexo de 295

ácido lipóico e ciclodextrina, que apresentam maior estabilidade à temperatura e a luz, o 296

que reduz a dispersão do AL em água, aumenta a biodisponibilidade e reduz seu odor, 297

no entanto, estes complexos apresentaram ampla distribuição de tamanho e aumento de 298

tamanho das partículas após duas semanas de armazenamento à temperatura ambiente 299

(Külkamp-Guerreiro et al. 2009). A alternativa ainda pouco explorada para a estabili-300

zação do ácido lipoico é o emprego de nanocápsulas poliméricas, as quais consistem em 301

sistemas vesiculares nanoestruturados carreadores de substâncias que apresentam 302

diâmetros entre 200 e 300 nm e baixa polidispersão, e que se destacam devido às suas 303

potencialidades no controle da liberação de substâncias e à capacidade de aumentar a 304

estabilidade do composto, tanto no armazenamento quanto nos fluídos biológicos 305

(Külkamp-Guerreiro et al. 2009). O termo nanotecnologia foi inserido em 1974, para 306

8

descrever a manipulação de partículas de menos de um micrômetro. Em particular, o 307

processo de nanoencapsulado é eficiente no desenvolvimento de produtos funcionais e 308

pode auxiliar a combater a perda de funcionalidade dos antioxidantes ou produtos 309

bioativos durante o processamento ou armazenamento, geração de maus odores e 310

sabores, entre outros problemas (Quintanilla-Carvajal et al. 2010). 311

A utilização de nanocápsula é descrito para proteção de diferentes sistemas 312

aplicados em fármacos e cosméticos, especialmente em substâncias que degradam em 313

temperaturas acima de 40 ºC ou são sensíveis à oxidação em presença de água, por 314

variação de pH ou por efeito de luz ultravioleta (Müller et al. 2004, Külkamp-Guerreiro 315

et al. 2009). A membrana polimérica da nanocápsula possui efeito protetor de 316

substâncias contra danos causados por agentes externos, prevenindo a degradação. 317

(Bauchemal et al. 2006, Weiss-Angeli et al. 2008, Külkamp-Guerreiro et al. 2009). Para 318

este estudo, a análise das condições do sistema antioxidante do biofloco e do camarão 319

Litopenaues vannamei envolveram as análises da atividade da glutationa S-transferase 320

(GST), concentração da glutationa reduzida (GSH), níveis de peroxidação lipídica 321

(TBARS), análise da capacidade antioxidante contra radicais peroxil (ACAP) e 322

contagem diferencial de hemócitos para análise do estado imune. 323

A glutationa-S-transferase é uma enzima multifuncional que está envolvida na 324

desintoxicação de xenobióticos, oferecendo proteção contra danos oxidativos e efetúa 325

também transporte intracelular de hormônios, metabólitos endógenos e exógenos de 326

produtos químicos em diversos organismos. Assim a GST é componente importante de 327

várias vias de desintoxicação e tolerância ao estresse, uma vez que protege contra lesões 328

induzidas por substâncias químicas ambientais (Zhou et al. 2009). 329

A glutationa (GSH) é um antioxidante muito importante na preservação do 330

estado redox celular, na defesa contra ERO e detoxificação de xenobióticos. Está 331

molécula é um tripeptídeo composto por ácido glutâmico, cisteína e glicina, que sob 332

condições normais e niveis de cisteína adequados a taxa limitante para sua sintese é 333

determinada pela atividade da enzima glutamato cisteína ligase (GCL) (White et al. 334

2003). 335

Quando em condições normais ou anormais a produção de ERO ultrapassa a 336

proteção endógena de enzimas específicas e vitaminas antioxidantes ocorre dano 337

9

celular, fenômeno este chamado de estresse oxidativo (Oakes e Van Der Kraak 2003). 338

As EROs atacam radicais livres como ácidos graxos poliinsaturados (PUFAs) que são 339

um substrato rico em elétrons (Esterbauer 1996; Oakes e Van Der Kraak 2003). Este 340

procedimento então, é realizado pela quantificação de compostos como o 341

malondialdeído (MDA), que é um subproduto da peroxidação lipídica (Janero 1990; 342

Oakes e Van Der Kraak 2003). Portanto, a reação do MDA com o ácido 2-tiobarbitúrico 343

(TBA), ensaio este conhecido como TBARS, é um dos mais amplamente utilizados 344

como estimadores de estresse oxidativo que analise os níveis de peroxidação lipídica 345

dos tecidos (Liu et al. 1997; Oakes e Van Der Kraak 2003). 346

Embora seja importante medir a eficência de antioxidante individuais para 347

combater a produção de oxiradicais, faz-se necessário compreender a resistência dos 348

tecidos a toxidade causada por ERO e não somente medir um numero limitado de 349

antioxidantes (Amado et al. 2009). Por isto usa-se a análise da capacidade antioxidante 350

contra radicais peroxil (ACAP), que é um método simples, rápido e confiável na 351

detecção de ERO por fluorometria, se valendo do 2,7´ diclorofluresceína diacetato 352

(H2DCF-DA) como substrato o qual, após sua deacetilação, que irá a interagir com 353

radicas peroxil que são gerados pela decomposição térmica a 37°C do 2,2- azobis 354

(2metilpropianoamidina) dihidrocloreto (ABAP). Uma queda na fluorescência nestas 355

condições é interpretada como um efeito antioxidante da amostra, através da 356

interceptação ou redução dos peroxi radicais, gerando um menor sinal de fluorescência 357

emitida pela reação entre EROs e H2DCF (Amado et al. 2009). 358

359

360

361

362

363

364

365

366

367

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2 HIPÓTESES 368

- Camarões criados em sistema de biofloco terão sua maior capacidade antioxidante, em 369

função dos antioxidantes presentes no biofloco. 370

- O ácido lipóico aumentará a capacidade antioxidante e diminuir o dano oxidativo no 371

camarão branco do pacífico Litopenaus vannemei, visto os efeitos já descritos deste 372

antioxidante em espécies aquáticas. 373

- O ácido lipóico aumentará a competência antioxidante do biofloco, vista suas 374

caraterísticas descritas e definidas como de “antioxidante ideal”. 375

376

3 OBJETIVOS 377

3.1 OBJETIVO GERAL 378

Avaliar se a suplementação com ácido lipoíco na ração e no biofloco altera do 379

estado antioxidante do camarão L. vannemei e do biofloco através de análise 380

bioquímicas que contemplem respostas antioxidantes, de detoxificação e de dano 381

oxidativo. 382

383

3.2 OBJETIVOS ESPECÍFICOS 384

- Avaliar os efeitos antioxidantes do biofloco em brânquia, hepatopâncreas, músculo do 385

camarão L. vannemei por meio da determinação a atividade da glutationa S-transferase 386

(GST) e glutationa reduzida (GSH), bem como avaliar o dano oxidativo dos tecidos pela 387

análise dos níveis de peroxidação lipídica (TBARS) e capacidade antioxidante total 388

contra radicais peroxil (ACAP). 389

390

- Avaliar o estado redox dos tecidos (brânquia, hepatopâncreas, músculo) após 391

suplementação do ácido lipóico (AL) na ração do camarão L. vannemei por meio da 392

determinação a atividade da glutationa S-transferase (GST) e glutationa reduzida 393

(GSH), bem como avaliar o dano oxidativo dos tecidos pela análise dos níveis de 394

peroxidação lipídica (TBARS) e contagem diferencial de hemócitos (CDH). 395

11

- Avaliar os efeitos do ácido lipóico no biofloco através da análise da capacidade 396

antioxidante total contra radicais peroxil, capacidade dos compostos em atuar como 397

varredores de espécies reativas de oxigênio (ERO). 398

399

4 METODOLOGIA GERAL 400

4.1 DESENHO EXPERIMENTAL 401

Os camarões utilizados foram juvenis da espécie Litopenaeus vannamei, 402

provenientes dos viveiros da Estação Marinha de Aquacultura (EMA), Universidade 403

Federal do Rio Grande – FURG. Os animais foram aclimatados em 2 tanques de fibra de 404

vidro (1000 L) com volume útil de 800 L, 150 animais em cada tanque foram estocados 405

a aclimatados durante 25 dias (6 a 30 de maio). 406

Após estes dias, foram submetidos a biometria e transferidos para tanques de 407

plástico (70 L) com volume útil de 50 L. Foram separados para primeira publicação em 408

tratamentos: (i) água clara e (ii) biofloco (Figura 1). Para o segundo manuscrito os 409

tratamentos foram: (i) água clara/nanocapsula vazia, (ii) água clara/AL 410

nanoencapsulado, (iii) biofloco/nanocapsula vazia e (iv) biofloco/AL nanoencapsulado 411

(Figura 2). Os camarões foram estocados a uma densidade de 15 camarões por tanque 412

(cerca de 300 camarões por m3) e passaram 7 dias aclimatados nessa condição (31 de 413

maio a 6 de junho), a partir de então foram ofertadas as respectivas rações. Nos tanques 414

com água clara os animais apresentaram peso médio inicial de 5,91 ± 0,07 g, nos 415

tanques com biofloco o peso médio inicial foi de 5,01 ± 0,05 g e quando houve aumento 416

de amônia acima de 1 mg/L foi adicionado melaço. 417

418

Figura 1 – Desenho experimental trabalho 1. 419

12

420

Figura 2 – Desenho experimental trabalho 2. 421

422

A ração comercial SUPRA® (35% de proteína bruta) foi pesada, em seguida 423

trituradas, misturadas a respectivas soluções (Tabela 1) para obtenção de uma massa 424

homogenia, passada em seringa de 5 mL e produto foi colocado em estufa a 50°C e 425

depois peleitizada. A dieta foi administrada três vezes ao dia (8:00, 15:00 e 22:00 h) a 426

uma taxa alimentar de 3% da biomassa do camarão. O experimento teve duração de 30 427

dias (7 de junho a 6 de julho), nos quais nos tanques com água clara houve renovação 428

de 80-90 % de água a intervalo de 1 dia e nos tanques com biofloco não houve 429

renovação de água. 430

431

Tabela 1 – Quantidades de ração e solução em cada tratamento. 432

Tratamento Ração (g) Solução (mL)

Água clara 500 200 (água destilada)

Biofloco 500 200 (água destilada)

Água clara/cápsula vazia 500 200 (cápsula vazia)

Água clara/AL nanoencapsulado 500 200 (AL nanoencapsulado)

Biofloco/cápsula vazia 500 200 (cápsula vazia)

Biofloco//AL nanoencapsulado 500 200 (AL nanoencapsulado)

433

13

Para o terceiro trabalho, foi realizado a suplementação do ácido lípoico na água 434

com biofloco. Neste caso foram utilizados 15 tanques de plásticos (70 L), com volume 435

útil de 50 L cada. Os tratamentos foram fixados em: Controle (0 mg de AL/L de 436

biofloco), Concentração de 1,25 µM (0,26 mg/L), Concentração de 2,5 µM (0,51 mg/L), 437

Concentração de 5 µM (1,03 mg/L) e Concentração de 10 µM (2,06 mg/L); todos em 438

triplicada (Figura 2). 439

Figura 2 – Desenho experimental do trabalho 3. 440

441

O primeiro trabalho, comparando água clara e biofloco foi publicado na revista 442

Marine and Freshwater Behaviour and Physiology, com título “Antioxidant and 443

oxidative damage responses in different organs of Pacific white shrimp Litopenaeus 444

vannamei (Boone 1931) reared in a biofloc technology system”. O segundo trabalho 445

comparando tratamentos com nanocapsula vazia e ácido lipóico nanoencapsulado foi 446

submetido a revista Comparative Biochemistry and Physiology e é intitulado: 447

“Antioxidant effects of nanoencapsulated lipoic acid in tissues and immune condition in 448

hemolymph of shrimp Pacific Litopenaeus vannamei (Boone, 1931)”. O terceiro 449

trabalho, com título de “Effects of lipoic acid in the total antioxidant capacity in 450

biofloc”, será submetido a revista Aquaculture. 451

452

453

14

4.1.1 Preparação das Nanocapsulas e Nanocapsulas vazias 454

Foi realizado segundo Longaray-Garcia et al. (2013), no qual a suspensão de 455

nanocápsulas com ácido lipóico foram preparados pelo método de precipitação do 456

polímero pré-formado. O ácido lipóico foi pesado (70 mg) e dissolvido na fase orgânica 457

composta por triglicéridos caprílico (0,33 mL), monoestearato de sorbitano (76,6 mg), 458

poli (ξ-caprolactona) (100 mg), acetona (26,7 mL) e butil-hidroxi-tolueno (BHT) (0,01 459

g). A fase orgânica foi injetada em fase aquosa contendo polissorbato 80 (76,6 mg), 460

diazolidinil ureia (0,01 g) e água Milli-Q (53,3 mL), através de um funil e mantida sob 461

agitação magnética moderada durante 10 min. A suspensão foi preparada protegida da 462

luz, e os solventes foram evaporados em evaporador rotativo (Bu chi R-114) a uma 463

temperatura de aproximadamente 30 °C até um volume final de 10 mL, para dar uma 464

concentração final de 70 mg mL-1. As suspensões foram nanocápsulas por espessão 465

com o emulsionante de silicone DC RM2051® (4 g) e Unistab S69® (0,5g). Somente 466

suspensão foi utilizada para o tratamento com nanocapsula vazia. 467

468

4.1.2 Diluição do Ácido Lipóico em Hidróxido de Sódio 469

As concentrações fixadas de α-ácido lipóico sintético (> 99% pureza, Sigma-470

Aldrich), foram dissolvidas individualmente para cada tanque, em solução de hidróxido 471

de sódio e água destilada para garantir a diluição total do ácido lipóico seguindo a 472

metodologia de Amado et al. (2011). Após a diluição o pH foi ajustado para 7,90. As 473

concentrações utilizadas foram adicionadas a cada 24 horas (Tabela 2) 474

475

476

477

478

479

15

Tabela 2: Preparação de solução de ácido lipóico, NaOH (hidróxido de sódio; 2 mM), 480

ddH2O (água destilada), AL (ácido lipóico). A partir do cálculo geral de 300 mL de 481

NaOH para 1g de AL e 1000 mL de H2Odd para 80 g de NaOH. 482

Tratamento NaOH (g) ddH2O (mL) AL (g)

Controle

1,25 µM

2,5 µM

5 µM

10 µM

0

0,62

1,22

2,47

4,94

0

7,8

15,3

30,9

61,8

0

0,26

0,51

1,03

2,06

483

4.2 ANÁLISE DA ÁGUA 484

Diariamente os parâmetros físicos e químicos da água foram monitorados, 485

incluindo oxigênio dissolvido (mg/L) e temperatura da água (°C) com multiparâmetro 486

YSI, salinidade com refratômetro ótico (Atago 103, ±1 ppt), pH com eléctrodo Mettler 487

Toledo FEP20 – FiveEasy Plus™. As determinações de alcalinidade (mg de CaCO3/L) e 488

nitrato (mg/L) foram feitas pelo método APHA (1985) e as de amônia total (NH3 + 489

NH4+ mg/L; UNESCO 1983) e nitrito (mg/L) de acordo com Benderschneider e 490

Robinson (1952) (Trabalho 1, 2 e 3). Quando a amônia foi superior a 1 mg/L foi 491

adicionado melaço de cana de açúcar como fonte de carbono para ajustar a relação C/N 492

de 20:1, segundo métodos de Ebeling et al. (2006) e Avnimelech (1999), nos quais 493

determinaram que 6 g de carbono é necessário para converter 1 g de nitrogênio 494

amoniacal total em biomassa bacteriana. 495

Os sólidos sedimentáveis (material particulado orgânico e inorgânico) foram 496

determinados pela sedimentação dos sólidos em cones Imhoff, onde coloca-se 1 L de 497

água e deixa-se repousar por 1 h, momento o qual são lidos os sólidos suspensos em 498

mL/L, caracterizado neste estudo como biomassa do biofloco (Tovar e Erazo 2009) 499

(Trabalho 3). 500

501

502

16

4.3 COLETA E HOMOGENEIZAÇÃO DAS AMOSTRAS 503

Para o trabalho 1 e 2, após 30 dias, foi efetuada a biometria final e os camarões 504

foram colocados em água com gelo para serem eutanaziado. Depois foram armazenados 505

em ultrafreezer (-80 oC) para no dia seguinte ser retirado brânquias, hepatopâncreas e 506

músculo. As amostras foram homogeneizadas (1:5, peso/volume) em solução tampão 507

de crustáceos (pH 7,2), contendo Tris-base (20 mM), EDTA (1 mM), MgCl2 (0,05 508

mM); DTT (Ditiotreitol – 1 mM); Sacarose (5 mM), KCl (Cloreto de potássio – 1 mM), 509

dissolvidos em água Milli Q. Posteriormente os extratos foram centrifugados a 9000 x 510

g, durante 30 minutos, em temperatura de 4 ºC e retirado o sobrenadante que foi 511

congelado a -80 ºC (Trabalho 1 e 2). 512

Para o trabalho 3, as amostras foram coletadas após leitura do cone Imhoff, no 513

qual a água foi retirada por sifonamento, e o floco colocados em falcon de 50 mL e 514

armazenado em gelo, para depois serem transferidos para eppendorf de 2 mL e serem 515

centrifugadas a 800 x g, a 4 °C, por 10 minutos e armazenado a -80°C. Para 516

homogeneização, as amostras de biofloco foram pesadas em eppendorff, adicionados 517

metanol (100%) sobre a relação 1000mg/1000µL, homogeneizadas e agitadas durante 3 518

horas, em seguida centrifugadas por 10 minutos a 10000 rpm e 4°C, então retirado o 519

sobrenadante que foram utilizados para determinar a capacidade antioxidante contra 520

radicais peroxil. 521

522

4.4 DETERMINAÇÃO DA ATIVIDADE DA ENZIMA GLUTATIONA S-523

TRANSFERASE (GST) 524

O ensaio da atividade da GST seguiu o método de Habig e Jakobi (1981). Neste 525

processo a absorbância é gerada pela conjugação de 1 mM de GSH (glutationa reduzida, 526

Sigma-Aldrich) com 1 mM de CDNB (1-chloro-2,4-dinitrobenzene, Sigma-Aldrich), 527

em absorbância de 340 nm, a 25 °C. Para leitura foi adicionado em microplaca 528

transparente de fundo chato, 15 μL do sobrenadante em 235 μL de meio de reação 529

(tampão fosfato 0,1 M e CNDB 50 mM, pH 7,0), mais 10 μL de GSH 25 mM. Nas 530

amostras de branco foi utilizado 15 μL tampão de homogeneização de crustáceo. As 531

leituras foram em espectrofluorímetro com leitora de placas (Víctor 2, Perkin Elmer). A 532

17

atividade específica da GST foi expressa em nanomoles de produto CDNB-GSH por 533

minuto por mg de proteína. 534

535

4.5 DETERMINAÇÃO DA ATIVIDADE DA GLUTATIONA REDUZIDA (GSH) 536

Foi efetuado pelo método de White et al. (2003) que mensura a concentração de 537

GSH pela sua reação com NDA (2,3 naftalenedicarboxialdeido), gerando um complexo 538

fluorescente (GSH-NDA) que detectado a 485 nm de excitação e 530 nm de emissão. 539

Para o ensaio primeiro foi feita uma curva padrão em diferentes diluições de GSH (40, 540

20, 10, 5 e 2,5 µM). Depois foram adicionados a microplaca transparente de fundo 541

cônico 25 μL de sobrenadante e 25 μL de tampão de homogeneização para o branco, 25 542

μL de ácido sulfosalicílico (200 mM) e incubado por 20 minutos. Em seguida a placa 543

foi centrifugada a 2500 rpm por 5 minutos. Foram então transferidos para microplaca 544

branca 20 μL de sobrenadante e 180 μL de solução de reação (Tris-base 50 mM, NaOH 545

500 mM e NDA 10 mM). As leituras foram feitas em espectrofluorímetro com leitora de 546

placas (Víctor 2, Perkin Elmer), em temperatura ambiente. A concentração foi expressa 547

em µomoles de GSH por mg de proteína. 548

549

4.6 DETERMINAÇÃO DOS NÍVEIS DE PEROXIDAÇÃO LIPÍDICA 550

A determinação de dano oxidativo segui o protocolo descrito por Oakes & Van 551

der Kraak (2003), este método envolve a reação do malondialdeído (MDA), um 552

subproduto da peroxidação lipídica (Hermes-Lima, 2004), com o ácido tiobarbitúrico 553

(TBA) sob condições de alta temperatura e acidez, gerando um cromógeno que é 554

quantificado por fluorometria. Para o ensaio foi feito a curva padrão com 6,25; 3,125; 555

1,5625; 0,78; 0,39; 0,195; 0,0975; 0,04887; 0,0243 e 0,0121 nmol de TMP (1,1,3,3-556

tetramethoxypropano). Em tubos de vidro (em duplicata) foi colocado 10 µL de amostra 557

e 41,2 µL de tampão de homogeneização de crustáceo nos brancos. Depois 20 µL de 558

solução estoque de BHT (hidroxitoluenobutilado, 1,407 mM), apenas nos tudos das 559

amostras. Em seguida 150 µL de solução de ácido acético 20%, 150 µL da solução de 560

TBA 0,8%; 50 µL de água MilliQ, 20 µL de SDS 8,1%. A mistura foi vortexada e 561

colocada em banho-maria a 95 ºC por 30 minutos. Logo após os tubos foram esfriados 562

18

por 10 minutos a temperatura ambiente, para então adicionar 100 µL de água MilliQ. O 563

contudo dos tubos foi transferido para eppendors de 1,5 mL e adicionou-se 500 µL de n-564

butanol, sendo logo vortexado e centrifugado a 3.000 x g por 10 minutos a 15 ºC. 565

Finalmente foi removida 150 µL da fase orgânica (sobrenadante) e transferida a 566

microplacas brancas. As leituras são feitas em fluorímetro, com comprimento de 567

excitação de 520 nm e emissão de 580 nm (Víctor 2, Perkin Elmer). Os resultados 568

foram expressos em nmol de TMP (Acros Organics) por mg de tecido fresco. 569

570

4.7 DETERMINAÇÃO DA CAPACIDADE ANTIOXIDANTE TOTAL CONTRA 571

RADICAIS PEROXIL (ACAP) 572

Foi efetuada de acordo com o protocolo de Amado et al. (2009), a dosagem tem 573

início com fixação a concentração de proteína em 2 mg/ml das amostras. Para o trabalho 574

3, não houve a fixação da concentração de proteína. Depois é adicionado a microplaca 575

branca 127,5 µL de tampão de reação, composto por 0,3575 g de ácido etanosulfônico 576

4.2-hidroxietil piperazina-1 (HEPES), 0,7455 g de cloreto de potássio (KCl), 0,0102 g 577

de cloreto de magnésio (MgCl2) dissolvidos em 50 ml de água Milli Q com pH ajustado 578

em 7,2; 10 µL de extrato de tecido; 7,5 µL de água MilliQ para as amostras sem ABAP 579

(2,2- azobis 2metilpropianoamidina dihidrocloreto) ou 7,5 µL de solução de ABAP para 580

amostras com ABAP (gerador de radicais peroxil) e 10 µL de solução de H2DCF-DA 581

(diacetato de ´2,7 diclorofluresceína). A leitura é realizada no tempo zero e depois a 582

cada 5 minutos até completar 30 minutos em fluorímetro de placas (Víctor 2, Perkin 583

Elmer) utilizando comprimento de onda de 530 nm de emissão e 485 nm de excitação, a 584

37 °C, temperatura que favorece a termólise do ABAP. Este método quantifica a 585

capacidade que o tecido possui em neutralizar as ERO geradas pela decomposição do 586

ABAP, incluindo as defesas antioxidantes enzimáticas e/ou não enzimáticas, através do 587

cálculo da área relativa. A área relativa apresenta uma relação inversa com a capacidade 588

antioxidante, onde menores valores da área relativa indicam uma maior capacidade 589

antioxidante e vice-versa. Para o trabalho 3, as leituras foram feitas em fluorímetro de 590

placas (FILTERMAX F5, Multi-mode microplate reader) utilizando comprimento de 591

onda de 530 nm de emissão e 485 nm de excitação, a 37 °C e o cálculo da área relativa 592

foi realizado segundo Monserrat et al. (2014). 593

19

4.8 CONTAGEM DIFERENCIAL DE HEMÓCITOS 594

A hemolinfa foi recolhido por punção cardíaca utilizando uma seringa de 3 mL 595

contendo solução anticoagulante de crustáceo, contendo NaCl (450 mM), glucose (100 596

mM), citrato de sódio (30 mM), ácido cítrico (23 mM), EDTA (20 mM) diluídos em 597

água MilliQ, com pH fixado em 7,4. Depois, um esfregaço de hemolinfa foi feita em 598

lâminas de vidro que foram imersas em metanol durante 5 minutos e coradas com May-599

Grunwald-Giemsa. Em seguida, hemócitos foram quantificados por microscópio de 600

lente ocular Integrando Disc 1, 25 pontos-G49 (Carl Zeiss), seguindo a metodologia de 601

Weibel (1980). 602

603

4.9 ANÁLISE ESTATÍSTICA DOS RESULTADOS 604

No primeiro trabalho os dados foram expressos em média ± desvio padrão. Cada 605

variável (atividade GST, capacidade antioxidante total, concentração de GSH, 606

peroxidação lipídica) foi analisada através de bi-fatorial ANOVA, sendo os fatores o 607

tratamento (água clara e biofloco) e órgãos (brânquias, hepatopâncreas e músculos). 608

Anteriormente, os pressupostos de normalidade e homogeneidade de variância foram 609

analisados e transformações matemáticas aplicada se a menos uma suposição foi 610

violado. Comparações de médias foram feitas usando a teste Newman-Keuls ou 611

contrastes ortogonais. Foram analisados os parâmetros físico e químicos da água por 612

meio do teste t de Student para variâncias desiguais (Zar, 1984). Em todos os casos foi 613

utilizado um nível de significância de 5%. 614

Para o segundo trabalho, os dados foram expressos em média ± desvio padrão. 615

Cada variável de cada órgão, incluindo a hemolinfa (atividade GST, peroxidação 616

lipídica, capacidade antioxidante total e contagem total de hemócitos) foi analisada 617

através de bi-fatorial ANOVA, sendo os fatores os tratamentos (NC ou NCLA) e 618

condição de criação (SW ou BFT). Anteriormente foram analisados os pressupostos de 619

normalidade e homogeneidade de variância. Comparações de médias foram feitas 620

usando o teste de Newman Keuls. Parâmetros físico e químicos da água foram 621

analisados pelo teste t de Student para variâncias desiguais (Zar 1984). Em todos os 622

casos foi utilizado um nível de significância de 5%. 623

20

Para o trabalho 3, os dados foram expressos em média ± desvio padrão. Com 624

análise feita por meio de bi-fatorial ANOVA (diferentes concentrações de ácido lipóico 625

e tempo). Anteriormente, os pressupostos de normalidade e homogeneidade de variância 626

foram analisados. Comparações de médias foram feitas usando a teste Newman-Keuls. 627

Em todos os casos foi utilizado um nível de significância de 5%. 628

629

630

631

632

633

634

635

636

637

638

639

640

641

642

643

644

645

646

647

648

21

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230. 835

836

837

838

839

840

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CAPÍTULO I 841

842

843

Antioxidant and oxidative damage responses in different 844

organs of Pacific white shrimp Litopenaeus vannamei (Boone, 845

1931) reared in a biofloc technology system 846

847

848

849

850

Átila Clivea da Silva Martins, Juliana Artigas Flores, Camilla 851

Porto, Wilson Wasielesky Junior & José Maria Monserrat 852

853

854

855

856

857

858

859

860

Marine and Freshwater Behaviour and Physiology, 2015. Vol. 48, 861

No. 4, 279–288. 862

863

864

865

866

867

868

29

ABSTRACT 869

Shrimp (Litopenaeus vannamei) reared in a conventional seawater (SW) aquarium 870

system SW were compared with those raised in a biofloc technology (BFT) system. 871

After 30 days, the L. vannamei shrimp were euthanized and samples of gills, 872

hepatopancreas and muscle were dissected. Statistical analysis was performed using bi-873

factorial ANOVA, with the factors of the treatment (shrimp reared in SW or in a BFT 874

system) and organs (gills, hepatopancreas and muscles). No differences (p>0.05) in 875

glutathione-S-transferase activity were observed between shrimp reared in SW and 876

shrimp reared in BFT (p< 0.05). Glutathione levels were lower (p<0.05) in the gills and 877

hepatopancreas of shrimp reared in the BFT system, suggesting changes in the 878

antioxidant composition of these organs. Lipid peroxidation levels were higher in the 879

hepatopancreas than in muscle in shrimp reared in SW (p<0.05) and this difference was 880

not observed in organisms reared in the BFT system (p>0.05). Overall, the results 881

showed that rearing shrimp in the BFT system altered both their antioxidant and 882

oxidative damage responses. This indicates that some microbial communities in BFT 883

systems can influence the redox state of L. vannamei. 884

885

Keywords: Litopenaeus vannamei; BFT; bioflocs; antioxidant responses; lipid 886

peroxidation; total antioxidant capacity. 887

888

889

890

891

892

893

894

895

896

30

1 INTRODUCTION 897

Among many aquatic species that are cultured in the world, the white shrimp 898

Litopenaeus vannamei has a number of advantages. For example, the species has a high 899

tolerance to variations in temperature, salinity, pH, and dissolved oxygen, possesses in 900

efficient feed conversion ratio, is resistant to several types of disease and is tolerant to 901

crowding (ABCC 2010). 902

Aquacultural activity can have some negative environmental outcomes such as 903

the release of toxic nitrogen compounds like ammonia and nitrite (Xu et al. 2013). The 904

use of bioflocs technology (BFT) systems has provided a useful alternative in which the 905

presence of aerobic and heterotrophic organisms together with vigorous aeration allows 906

for the rearing of aquatic organisms at a high density without the need for constant 907

water exchange (Furtado et al. 2011; Kim et al. 2014). Recent studies have shown that 908

the benefits of BFT systems are not restricted to the reduction of the need for water 909

exchanges. Additionally, the microorganism communities are beneficial for the 910

organisms being cultured. Xu et al. (2013) showed that BFT improved activity in 911

digestive enzymes such as proteases, amylase and lipase in L. vannamei. These authors 912

proposed that the high digestive enzyme activity could be related to exogenous enzymes 913

released from microorganisms lysed in the bioflocs. Improvement in weight gain, 914

specific growth rate and the feed conversion rate of L. vannamei reared in a BFT system 915

were reported by Xu and Pan (2012). These results led the authors to postulate that 916

bioflocs are a food supplement that improves digestion and growth. Kim et al. (2014) 917

observed augmented gene expression in the prophenol oxidase system in white shrimp 918

reared in BFT, which reinforces the idea that several biological benefits are linked by 919

this rearing strategy. 920

When considering bioflocs as an additional food source (Xu et al. 2013), it is 921

important to note that they contain several biomolecules including carotenoids, amino 922

sugars and phytosterols, among others (Ju et al. 2008). Molecules such as carotenoids 923

are known to possess antioxidant properties (Hermes-Lima 2004; Halliwell & 924

Gutteridge 2007). It is thus possible that BFT may improve not only digestive, imune 925

and growth parameters, but also antioxidant responses. Several conditions can generate 926

oxidative stress in aquatic organisms, and some of these conditions are important for 927

31

aquaculture practices. Crustaceans exhibit daily variations in their enzymatic 928

antioxidant system (Fanjul-Moles et al. 2003; Maciel et al. 2004). This means that they 929

may be more susceptible to oxidative stress during certain periods. Infection by the 930

white spot virus was also shown to reduce the antioxidant competence of 931

Fenneropenaeus indicus (Mohankumar & Ramasamy 2006) and the crustacean immune 932

response itself promotes a pro-oxidant condition (Cornet et al. 2007). 933

Taking into account the properties of BFT systems that led Xu et al. (2003) to 934

consider providing a natural food supplement to organisms such as L. vannamei, this 935

study aimed to evaluate the effect of a BFT system on the antioxidant and oxidative 936

damage responses of this species and to record other beneficial changes elicited by the 937

use of this technology. To address this aim we compared the antioxidant response of the 938

shrimp L. vannamei reared in a SW system (without biofloc) and in a BFT system. 939

940

2 MATERIALS AND METHODS 941

This study employed juvenile shrimp (L. vannamei) from the nursery sector of 942

the Aquaculture Marine Station at the Federal University of Rio Grande – FURG 943

(Brazil). During acclimation period (seven days) shrimp were dispersed in six plastic 944

tanks with 50 L of sea water (SW) each. Measured physico–chemical parameters of SW 945

were: 7.79 ± 0.15 (pH), 6.5 ± 0.12 mg/L (dissolved oxygen), 26.26 ± 0.38 °C 946

(temperature), 31.00 ± 0.01 (salinity), 0.78 ± 0.47 mg/L (ammonia), 0.07 ± 0.04 mg/L 947

(nitrite) and 0.39 ± 0.53 mg/L (nitrite). Organisms were stocked at a density of 15 948

shrimp per tank (approximately 210 shrimp per m3). The diet consisted of commercial 949

food with 35% crude protein supplied three times daily (08:00, 15:00 and 22:00 h) at a 950

feeding rate of 3% of shrimp biomass. When the concentration of total ammonia 951

exceeded 1 mg/L, molasses was added to the bioflocs (Avnimelech 1999). The 952

experiment lasted for 30 days and no water exchanges were performed in the BFT 953

system treatment. In SW, 80–90% of the water was replaced every 48 h. 954

During the experiment, the physical and chemical parameters of water, including 955

dissolved oxygen (mg/L), water temperature (°C), salinity and pH, were measured daily 956

using an YSI multiparameter apparatus (Yellow Springs Instruments, model 55). Shrimp 957

32

from both treatments were weighed at the end of the experiment, frozen, and the gills, 958

hepatopancreas and muscle from the second abdominal somite were dissected. 959

Organ and tissue samples were homogenized (1:5, w/v) in a buffer, previously 960

used for crustacean species (Pinho et al. 2005; de Souza et al. 2014). The composition 961

was Tris-Base (20 mM), EDTA (1 mM), dithiothreitol (1 mM; Sigma), sucrose (500 962

mM) and KCl (150 mM) dissolved in Milli Q water with the pH adjusted to 7.2. The 963

homogenates were centrifuged (9000 × g, 30 min at 4 °C), and the supernatants were 964

kept and divided into four aliquots for subsequent analysis of glutathione-S-transferase 965

(GST) activity, levels of lipid peroxidation (TBARS assay), levels of reduced 966

glutathione (GSH) and total antioxidant capacity against peroxyl radicals. 967

968

2.1 DETERMINATION OF GST ACTIVITY 969

GST was measured according to the protocol described by Habig et al. (1974). 970

Briefly, in the assay the conjugation of 1 mM 1-chloro-2,4-dinitrobenzene (CDNB, 971

Sigma) with 1 mM reduced glutathione (GSH, Sigma) in the presence of 10 μl of 972

sample homogenates (source of GST enzyme) generated a CDNB-GSH complex. The 973

absorbance of this complex was measured at 340 nm using a spectrofluorometer with a 974

microplate reader (Victor 2, Perkin Elmer). 975

976

2.2 DETERMINATION OF TOTAL ANTIOXIDANT CAPACITY 977

Total antioxidant capacity against peroxyl radicals was measured by determining 978

the reactive oxygen species in aliquot homogenates according to the protocol of Amado 979

et al. (2009). Peroxyl radicals were generated through thermal decomposition, at 37 °C 980

of 2,2′-azobis 2 methylpropionamidine dihydrochloride (ABAP; Aldrich). The probe 981

dichlorofluorescein diacetate (H2DCF-DA, Invitrogen; 40 μM) was added to a white 982

ELISA 96-well microplate, which was immediately placed in a fluorometer with a 983

microplate reader (Victor 2, Perkin Elmer) heated at 37 °C. The fluorescence was read 984

(excitation: 485 nm; emission: 520 nm) every 5 min over a 30-min period, and the 985

relative area was calculated according to Amado et al. (2009). According to these 986

authors, a high relative area means a low antioxidant capacity indicating a low capacity 987

33

of antioxidants present in the biological sample to degrade or intercept the peroxyl 988

radicals. 989

990

2.3 DETERMINATION OF CONCENTRATION OF REDUCED GLUTATHIONE 991

(GSH) 992

The procedure was based on that described by White et al. (2003) with 993

modifications proposed by da Rocha et al. (2009). The substrate 2,3 naphthalene 994

dicarboxialdehyde (NDA, Invitrogen) reacts with GSH to form a fluorogenic 995

compound. Aliquots of organ homogenates were transferred to white ELISA 96-well 996

microplate. The NDA solution was subsequently added to all wells and, after 30 min of 997

incubation, the fluorescence intensity of the NDA–GSH complex was read at excitation 998

and emission wavelengths of 485 and 530 nm, respectively, using a plate reader 999

fluorometer (Victor 2, Perkin Elmer). A standard curve of GSH was prepared to express 1000

the data in terms of μM equivalents of GSH per mg of total proteins. 1001

1002

2.4 DETERMINATION OF LIPID PEROXIDATION 1003

Lipid peroxidation was determined by a TBARS (thiobarbituric acid reactive 1004

substances) assay following Oakes and Van der Kraak (2003), adapted to microplate by 1005

Da Rocha et al. (2009). Aliquots (10 μl) of sample homogenates were added to glass 1006

tubes, together with thiobarbituric acid (0.8%, Sigma), acetic acid (20%), Milli Q water 1007

and sodium dodecyl sulfate (8.1%). The mixture was incubated at 95 °C over 30 min 1008

and, after cooling, n-butanol was added following a centrifugation step (3000 × g for 10 1009

min at 15 °C). The n-butanol phase was pipetted to a white ELISA 96-well microplate 1010

and the fluorescence read (excitation: 520 nm; emission: 580 nm) in a fluorometer with 1011

a microplate reader (Victor 2, Perkin Elmer). The standard employed was 1,1,3,3-1012

tetramethoxypropane (TMP, Across Organics). The results were expressed as nmol 1013

equivalents of TMP per mg of fresh tissue. 1014

1015

1016

34

2.5 DETERMINATION OF TOTAL AMMONIA 1017

Total ammonia determination (NH3+ NH4+) follows the UNESCO (1983) 1018

method in which ammonia in basic pH (between 10.8 and 11.5) reacts with bleaching 1019

solution, forming monochloramine. In the presence of phenol and an excess of 1020

hypochlorite and the addition of sodium nitroprusside as a catalyst, indophenol blue is 1021

formed, a molecule that absorbs at 630 nm. 1022

1023

2.6 STATISTICAL ANALYSIS 1024

Data were expressed as the mean ± standard deviation. Each variable (GST 1025

activity, lipid peroxidation, GSH levels and total antioxidant capacity) was analyzed 1026

through bifactorial ANOVA, with factors composed of the treatment (shrimp reared in 1027

SW or BFT and organs (gills, hepatopancreas and muscles). Prior to application of 1028

ANOVA the assumptions of normality and homogeneity of variance were tested and 1029

mathematical transformations were applied if at least one assumption was violated. 1030

Means comparisons were done using the Newman–Keuls test or orthogonal contrasts. 1031

Water physico–chemical parameters were analyzed using Student’s t test for unequal 1032

variances (Zar 1984). In all cases, a significance level of 5% was used. 1033

1034

3 RESULTS 1035

The average initial weight, final weight and average weight gain were not 1036

significantly different (p>0.05) for SW and BFT reared shrimp. In both cases, survival 1037

was 100% (Table 1). Several differences in physico–chemical parameters between SW 1038

and water from BFT system were observed (Table 2). Water from the BFT system 1039

showed significantly higher values for pH, salinity and nitrate (p<0.05) compared with 1040

SW whereas oxygen concentration and nitrite levels were lower (p<0.05). 1041

1042

1043

1044

35

Table 1. Mean concentration (±1 standard error) of morphological and physiological 1045

parameters measured in shrimp L. vannamei maintained in a SW or in a BFT system. 1046

1047

Note: No significant differences were detected between treatments (p>0.05). Number of 1048

samples analyzed in each treatment: 45. 1049

1050

No significant differences were observed in GST activity between treatments 1051

(p>0.05 and Table 3) although GST activity in the gills showed an upward trend in BFT 1052

shrimp. When comparing the different organs, the gills showed the highest GST activity, 1053

followed by the muscle and the hepatopancreas (p<0.05). 1054

The muscles of BFT shrimp showed significantly higher total antioxidant 1055

capacity (low relative area) when compared with SW shrimp (high relative area) 1056

(p<0.05 and Table 4). In SW shrimp the hepatopancreas had the highest antioxidant 1057

capacity (p<0.05) when compared with the muscle and gills, which presented similar 1058

values (p>0.05). BFT shrimp showed differences in every organ: highest antioxidant 1059

capacity in the hepatopancreas, followed by the muscle with the lowest mean value in 1060

the gills (p<0.05 and Table 4). 1061

A significant GSH reduction was observed in the gills and hepatopancreas of 1062

BFT shrimp (p<0.05) indicating that glutathione levels were affected by the rearing 1063

environment. In each system, the differences in GSH content among the different organs 1064

were the same: muscle showed the highest level, followed by the hepatopancreas and 1065

then the gills (p<0.05 and Table 5A). 1066

1067

1068

1069

36

Table 2. Mean concentration (±1 standard error) of the physico-chemical parameters 1070

measured in SW or in a BFT system. 1071

1072

Note: Asterisks (*) indicate significant differences (p<0.05) between the experimental 1073

groups after performing Student’s t-test assuming unequal variances. Number of 1074

samples analyzed in each treatment: 27–186. 1075

1076

Table 3. Mean activity (±1 standard error) of GST (nmol CDNB-GSH 1077

conjugate/min/mg of proteins) in the gills, hepatopancreas and muscle of shrimp L. 1078

vannamei reared in SW or in a BFT system. 1079

1080

Note: Different letters indicate significant differences (p<0.05) between experimental 1081

groups after performing a Student–Newman–Keuls post hoc test. Number of samples 1082

analyzed in each treatment: 15. GSH: reduced glutathione. CDNB: 1-chloro-2,4-1083

dinitrobenzene. 1084

1085

Table 4. Mean values (±1 standard error) of the total antioxidant capacity against 1086

peroxyl radicals (expressed in relative area) in the gills, hepatopancreas and muscle of 1087

L. vannamei reared in SW or in a BFT system. 1088

37

1089

Note: Different letters indicate significant differences (p<0.05) between experimental 1090

groups after performing a Student–Newman–Keuls post hoc test. Number of samples 1091

analyzed in each treatment: 10–15. 1092

1093

Table 5. Mean concentration (±1 standard error) of: (A) reduced glutathione (GSH) (μM 1094

of GSH/mg of total proteins) and (B) thiobarbituric reactive substances (nmol of 1095

TMP/mg of tissue) in the gills, hepatopancreas and muscle of L. vannamei reared in SW 1096

or in a BFT system. 1097

Note: Different letters indicate significant differences (p<0.05) between experimental 1098

groups after performing a Student–Newman–Keuls post hoc test. Number of samples 1099

analyzed in each treatment: 10–15. TMP: 1,1,3,3-tetramethoxypropane, the standard 1100

employed in TBARS assay. 1101

Oxidative damage measured in terms of TBARS was similar between the organs 1102

of BFT shrimp and SW shrimp (p>0.05 and Table 5B). The TBARS concentration in 1103

each organ was, however, different depending on the rearing environment. In SW 1104

shrimp, the highest TBARS concentration was observed in the hepatopancreas, followed 1105

by the gills, with the lowest TBARS concentration observed in the muscle (p<0.05 and 1106

Table 5B). In BFT shrimp no differences in TBARS levels were detected between 1107

organs (p>0.05 and Table 5B). 1108

38

4 DISCUSSION 1109

Nitrate levels can vary from 4.52 to 26 to mg/L during the rearing of commercial 1110

shrimp (Muir et al. 1991) and can reach 500 mg/L in recirculation systems (Pierce et al. 1111

1993). In studies with juvenile Penaeus monodon, however, the highest safe nitrate level 1112

at a salinity of 35 was reported to be 158 mg/L (Tsai & Chen 2002). Lower nitrate levels 1113

were observed during the present study in both SW, where 80–90% of water was 1114

renewed every 48 h and in BFT, where no water renewal was performed. 1115

Safe nitrite levels were estimated for L. vannamei to be 15.2 mg/L (Lin & Chen 1116

2003), a value much higher than those registered in both our treatments. The study of 1117

Zhang et al. (2006) into oxygen tolerance determined that in L. vannamei, the lethal 1118

levels were below 0.5 mg/L of dissolved oxygen. This shows again that the measured 1119

O2 levels in both our treatments were far below a level that could be considered 1120

stressful. The lower O2 concentration observed here with BFT was also reported in a 1121

previous study from our group (Emerenciano et al. 2012) and is consistent with the high 1122

density of aerobic organisms. 1123

The measured parameters of water quality (temperature, salinity, pH, dissolved 1124

oxygen, ammonia, nitrite and nitrates) in a BFT system and in SW in the study of 1125

Wasielesky et al. (2006) were close to those registered in the present study. In summary, 1126

according to values cited in the literature the water quality parameters in our study were 1127

within acceptable limits for the survival and growth of L. vannamei. 1128

Different organs of aquatic species differ in antioxidant competence and 1129

oxidative damage, as found by Monserrat et al. (2008) in the fish Corydoras paleatus, 1130

where, for example, GSH levels were higher in the muscle compared with the brain or 1131

gills. In L. vannamei shrimp, Lobato et al. (2013) found higher TBARS levels in the 1132

hepatopancreas compared to the muscle. In the present study, some differences were 1133

observed between the gills, hepatopancreas and muscle of L. vannamei. For example, 1134

higher GST activity was observed in the gills of shrimp reared in both systems. Some 1135

GSTs possess peroxidase activity, catalyzing the degradation of organic hydroperoxides 1136

(Halliweel & Gutteridge 2007), and this most likely explains why the gills have 1137

moderate TBARS levels even when the total antioxidant capacity is lower than that in 1138

the other organs. The differences among the organs in terms of lipid peroxidation for 1139

39

SW reared shrimp were not observed in BFT reared shrimp which suggests that the 1140

microbial community of this system could be influencing the redox state of L. vannamei 1141

resulting in the organs presenting similar levels of oxidative damage expressed in terms 1142

of TBARS levels. 1143

Other evidence of the positive influence of BFT on the antioxidant system of L. 1144

vannamei included the higher total antioxidant competence against peroxyl radicals 1145

(lower relative area) in the muscle of shrimp reared in the BFT system compared with 1146

SW reared shrimp. The observed reduction in GSH levels in the gills and 1147

hepatopancreas of shrimp reared in BFT deserves careful consideration. These results a 1148

priori can be interpreted as a pro-oxidant action induced by BFT, lowering the 1149

concentration of the antioxidant. However, the total antioxidant capacity in these two 1150

organs was similar in shrimp reared in both systems, and the TBARS levels in the gills 1151

and hepatopancreas were also similar between shrimp reared in the two systems. It is 1152

possible that rearing the shrimp in the BFT system altered their antioxidant 1153

composition. According to Regoli and Winston (1999) and Regoli (2000), antioxidants 1154

such as GSH, ascorbic acid, uric acid and α-tocopherol accounts for almost 70% of 1155

antioxidant capacity against peroxyl radicals. In this sense, a reduction in GSH content 1156

can be compensated by other antioxidants, leaving the total antioxidant capacity 1157

unchanged. 1158

Several examples exist in nature that show how biological interactions modify 1159

the antioxidant defense system. Moraes et al. (2006) showed that the mucus secretion of 1160

the polychaete Laeonereis acuta is colonized by bacteria communities that confer 1161

antioxidant properties. In the present study, the results indicated altered antioxidant 1162

responses and ameliorated lipid peroxidation in shrimps maintained in the BFT system. 1163

Future studies will need to consider the influence of microbial communities with 1164

different species compositions on the antioxidant competence of cultured organisms. It 1165

will also be important to learn how a microbial community of a BFT system can vary its 1166

antioxidant properties such as by adding exogenous antioxidants to the system. 1167

1168

1169

40

Acknowledgements 1170

J.M. Monserrat and Wilson Wasielesky Junior receive productivity research fellowships 1171

from the Brazilian agency CNPq (process numbers PQ 307880/2013-3 and PQ 1172

310993/2013-0, respectively). J.A. Artigas and C. Porto received undergraduate 1173

fellowship from FAPERGS. The authors appreciate the commentaries and suggestions 1174

of the three anonymous reviewers that helped to improve the focus and clarity of the 1175

manuscript. The financial support of PROPESP from FURG for English revision is also 1176

acknowledged. 1177

1178

Disclosure statement 1179

No potential conflict of interest was reported by the authors. 1180

1181

ORCID: José Maria Monserrat http://orcid.org/0000-0002-9463-3049 1182

1183

1184

1185

1186

1187

1188

1189

1190

1191

1192

1193

1194

41

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Freshw Behav Physiol. 47:1–10. 1276

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diferente salinity levels. Aquaculture. 213:163–170. 1278

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Paris, Intergovernmental Oceanographic Commission; p. 53. 1280

44

Wang C, Liu H., Sun Z. 2012. Review Article: Heterogeneous Photo-Fenton Reaction 1281

Catalyzed by Nanosized Iron Oxides for Water Treatment. International Journal 1282

of Photoenergy: 1 – 10. 1283

Wasielesky Jr W, Atwood H, Stokes A, Browdy CL. 2006. Effect of natural production 1284

in a zero exchange suspended microbial floc based super-intensive culture 1285

system for white shrimp Litopenaeus vannamei. Aquaculture. 258:396–403. 1286

White CC, Viernes H, Krejsa CM, Botta D, Kavanagh TJ. 2003. Fluorescence-based 1287

microtiter plate assay for glutamate-cysteine ligase activity. Anal Biochem. 1288

318:175–180. 1289

Xu W-J, Pan L-Q. 2012. Effects of bioflocs on growth performance, digestive enzyme 1290

activity and body composition of juvenile Litopenaeus vannamei in zero-water 1291

exchange tanks manipulating C/N ratio in feed. Aquaculture. 356–357:147–152. 1292

Xu W-J, Pan LQ, Sun XH, Huang J. 2013. Effects of bioflocs on water quality, and 1293

survival, growth and digestive enzyme activities of Litopenaeus vannamei 1294

(Boone) in zero-water exchange culture tanks. Aquaculture Res. 44:1093–1102. 1295

Zar JH. 1984. Biostatistical analysis. New Jersey (NJ): Prentice Hall. 1296

Zhang P, Zhang X, Li J, Huang G. 2006. The effect of body weight, temperature, 1297

salinity, pH, light intensity and feeding condition on lethal DO levels of whiteleg 1298

shrimp Litopenaeus vannamei. Aquaculture. 256:579–587. 1299

1300

1301

1302

1303

1304

1305

1306

1307

45

CAPÍTULO II 1308

1309

1310

Antioxidant effects of nanoencapsulated lipoic acid in tissues 1311

and differential hemocyte count in hemolymph of shrimp 1312

Pacific Litopenaeus vannamei (Boone, 1931) created in biofloc 1313

system 1314

1315

1316

Átila Clivea da Silva Martins, Juliana Artigas Flores, Camilla 1317

Porto, Luis Alberto Romano, Wilson Wasielesky Junior, Sergiane 1318

S. Caldas, Ednei G. Primel, Irene Clemes Külkamp-Guerreiro, 1319

José Maria Monserrat 1320

1321

1322

Submetido a: 1323

Comparative Biochemistry and Physiology - Part A: Molecular & 1324

Integrative Physiology 1325

1326

1327

1328

46

ABSTRACT 1329

One of the limiting factors for the intensification of production in aquaculture is the 1330

welfare of reared organisms. In this study, we evaluated antioxidant and oxidative 1331

damage responses and immune parameters in shrimp Litopenaeus vannamei reared in 1332

saline water (SW) and in biofloc system (BFT). Animals were fed with ration 1333

supplemented with nanocapsules containing the antioxidant lipoic acid (NCLA) or with 1334

empty nanocapsules (NC). Oxidative stress analysis was evaluated through 1335

measurement of glutathione S-transferase activity (GST), reduced glutathione (GSH) 1336

concentration and lipid peroxidation (TBARS) in gills, hepatopancreas and muscle. 1337

Immune parameters were determined through differential hemocyte count in the 1338

hemolymph. There was an increase in the average final weight of shrimps treated with 1339

NCLA (p< 0.05). In gills, increased GST activity (p< 0.05) was verified in shrimps 1340

reared in SW in both treatments (WNC and NCLA). In the same organ, higher GSH 1341

levels were observed in shrimps maintained in BFT (p< 0.05), and decreased levels of 1342

lipid peroxidation was observed in shrimps supplemented with NCLA (p< 0.05). In 1343

hepatopancreas, both in SW and BFT, NCLA induced an increase of GST activity and 1344

GSH concentration, the lipid peroxidation was increased in BFT and NCLA treatment. 1345

The muscle was low levels of GST activity, there was no significant difference between 1346

treatments in GSH concentration, and there was an increased lipid peroxidation. There 1347

was a decrease in the percentage of hyaline hemocytes and increased granular 1348

hemocytes in all treatments. The nanoencapsulated lipoic acid had tissue-specific action, 1349

with increased antioxidant activity in gills and hepatopancreas. 1350

1351

47

Keywords: Biofloc System, Nanotechnology, Shrimp, Antioxidants supplements, 1352

Antioxidant defenses, Oxidative damage. 1353

1354

1. INTRODUCTION 1355

Intensification of production in carciniculture demand priority attention in welfare of 1356

reared organisms, as well as an integrated water management to avoid or minimize 1357

pollution of water bodies (i.e.: release of nitrogenous compounds) and a greater control 1358

over infection of pathogens (Xie and Yu, 2007; Moss et al., 2012; Xu and Pan, 2013) 1359

These actions are particularly important for the rearing of the white shrimp Litopenaeus 1360

vannamei, one of the most marketed aquatic organisms worldwide (Xu and Pan, 2013). 1361

Biofloc technology systems (BFT) appears as a sustainable solution that enables the 1362

improvement of water quality with the removal of toxic nitrogen (ammonia, nitrite), 1363

highest growth performance through food supplement and digestive stimulant by 1364

biofloc organisms (Xu and Pan, 2013). BFT are rich in natural microorganisms and 1365

bioactive compounds, forming a microbial culture through heterotrophic aggregates of 1366

organic particulate in suspended matter (Ray et al., 2010; Xu and Pan, 2013). 1367

Animal welfare is compromised by several factors, including reactive oxygen species 1368

(ROS) that are generated as oxygen metabolism products in aerobic organisms (Vergely 1369

et al., 2003; Rochette et al., 2013). However, conditions of oxidative stress are 1370

generated when exist a redox imbalance between pro-oxidants and antioxidants causing 1371

damage to macromolecules. Several conditions can lead to a scenario of oxidative 1372

stress, including factors that simultaneously increase ROS generation and reduce 1373

antioxidant activity (Jones, 2006; Oudot et al., 2006; Rochette et al., 2013). To prevent 1374

48

or ameliorate this imbalance, organisms have developed defense mechanisms like 1375

antioxidants. 1376

An antioxidant (enzymatic and non-enzymatic) is any substance that, when present in 1377

low concentrations compared to an oxidable substrate, significantly delays or inhibits 1378

oxidation of this substrate (Halliwell and Gutteridge, 1995; Rochette et al., 2013). 1379

Lipoic acid (LA), also known as thioctic acid, is a naturally compound synthesized in 1380

small amounts by plants and animals. LA makes covalent bonds with specific proteins, 1381

acting as co-factor for mitochondrial enzyme complexes (Carreau, 1979; Reed, 2001). 1382

In addition, LA neutralize ROS, chelates metals and regenerates antioxidants (Rochette 1383

et al., 2013; Monserrat et al., 2014). 1384

LA is a hydro and liposoluble antioxidant molecule, extensively distributed in cell 1385

membranes, cytosol, and extracellular spaces. When supplemented in the diet may 1386

accumulate in several tissues and a fraction is reduced to DHLA (dihydrolipoic acid), 1387

where the amount of this reduction is tissue-specific (Rochette et al., 2013; Monserrat et 1388

al., 2014). LA administered in the diet can reduce oxidative stress and restore levels of 1389

other antioxidants that may be at low levels (Monserrat et al., 2008; Martins et al., 1390

2014). 1391

However LA is an unstable compound, and being subject to degradation by thermal and 1392

photochemical degradation, acidic oxidation and thus the use of polymeric nanocapsules 1393

(lipid) for the protection and slow release of the antioxidant has been considered and 1394

applied in aquatic organisms as carp Cyprinus carpio (Longaray-Garcia et al., 2013). 1395

Nanocapsules are nanostructured vesicular systems carriers of with diameters between 1396

200 and 300 nm and low polydispersity, which stand out due to their capabilities in 1397

49

controlling the release of substances and the ability to increase the stability of the 1398

compound, both in storage and in the biological fluid (Külkamp-Guerreiro et al., 2009). 1399

The cellular stress leads to the activation of defense mechanisms resulting in increase in 1400

the detoxification capacity (Kültz, 2005; Roncalli et al., 2015). One of the detoxification 1401

enzymes belongs to the glutathione S-transferase (GST) family, proteins of 200-250 1402

amino acids that are activated in response to oxidative damage and/or exposure to a 1403

variety of xenobiotics (Frova, 2006; Sheehan et al., 2001; Roncalli et al., 2015). GST 1404

enzymes catalyze the conjugation of reduced glutathione (GSH) with hydrophobic 1405

molecules, enhancing their solubility and further excretion (Ranson and Hemingway, 1406

2005; Roncalli et al., 2015). 1407

Reduced glutathione (GSH) is an intracellular antioxidant, which assists in 1408

detoxification and elimination of ROS and maintains the cellular redox equilibrium 1409

(Rochette et al., 2013). Studies in rats suggest that LA can increase GSH synthesis in 1410

aged rats via increased expression of γ- glutamate cysteine ligase (GCL), the limiting 1411

enzyme for GSH generation (Suh et al., 2004a). 1412

According to Amado et al. (2009), it is also necessary to evaluate oxidative damage and 1413

the ability of each tissue in neutralizing reactive oxygen species. Therefore, non-specific 1414

responses as thiobarbituric acid reactive substances (TBARS) have been measured in 1415

this work. Measurement of lipid peroxidation through TBARS assay offers information 1416

about phospholipid peroxidation of cell membranes (Gorbi et al., 2008; Diaz-Jaramillo 1417

et al., 2013). 1418

The shrimp immune defense is performed by hemocytes present in the hemolymph and 1419

the measurement of number and kind of hemocytes may indicate the shrimp welfare 1420

50

(Perazzolo et al., 2002). Hyaline cells have the immune function perform phagocytosis, 1421

the semigranular cells perform encapsulation, phagocytosis (limited), storage and 1422

release of prophenoloxidase system and cytotoxic activity, and granule cells has the 1423

function of storing and releasing the prophenoloxidase system and cytotoxic activity 1424

(Johansson et al., 2000). In this study was the analysis of the immune response of the 1425

shrimp L. vannmaei by differential hemocyte count in the hemolymph, by hyaline and 1426

granular hemocyte percentage and contribute to the wellness paranorama in front of the 1427

use of nanoencapsulated lipoic acid (NCLA) or nanocapsule without lipoic acid (empty 1428

nanocapsule – NC) in shrimp. 1429

The objective of the study was therefore to determine the animal welfare conditions 1430

using NCLA in clean water and in a biofloc system, through biochemical analysis of 1431

antioxidant and oxidative damage parameters in gills, hepatopancreas and muscle, plus 1432

immune responses determined by differential hemocyte count in the hemolymph of 1433

Pacific white shrimp L. vannmaei. 1434

1435

2. MATERIAL AND METHODS 1436

It was used juvenile shrimp (L. vannamei) reared at the Marine Aquaculture Station 1437

(EMA) of the Federal University of Rio Grande – FURG (Brazil). The shrimps were 1438

stored at a density of 15 shrimp per tank (about 210 shrimp per m3), arranged in 12 1439

plastic tanks with 50 L (useful volume) each. The shrimps were submitted to four 1440

different treatments: (i) shrimps maintained in saline water and fed with ration 1441

supplemented with nanocapsules without lipoic acid (SW-NC), (ii) shrimps mantained 1442

in saline water and fed with ration supplemented with nanoencapsulated lipoic acid 1443

51

(SW-NCLA), (iii) shrimps maintained in water with bioflocs and fed with ration 1444

supplemented with nanocapsules without lipoic acid (BFT-NC, (iv) shrimps reared in 1445

water with bioflocs and fed with ration supplemented with nanoencapsulated lipoic acid 1446

(BFT-NCLA). In the two paragraphs below are described the methodologies used to 1447

prepare the nanocapsules suspensions and how they were included in the ration. 1448

Nanocapsules containing lipoic acid were prepared according to Longaray-Garcia et al. 1449

(2013). Briefly, lipoic acid (70 mg/ml) was dissolved in the organic phase composed of 1450

triglycerides of caprylic and capric acid, sorbitan monostearate, poly (-caprolactone), 1451

acetone and butylated hydroxytoluene. The organic phase was disposed over an aqueous 1452

phase in the dark that contained polysorbate 80, diazolidinilureia and Milli-Q water and 1453

stirred for 10 min. The formulation was evaporated at 35 °C until a final volume of 10 1454

ml, obtaining nanocapsules, which included lipoic acid (NCLA). Nanocapsules 1455

containing all the components cited above except the inclusion of lipoic acid were also 1456

prepared and represented the nanocapsules control (NC) (Külkamp-Guerreiro et al., 1457

2009). The mean diameter and polydispersion of suspended nanocapsules were 1458

measured through dynamic light scattering employing the equipment Zetasizer Nano 1459

series Nano-Zs (Malvern Instruments). Samples were diluted (1:500 v/v) in Milli-Q 1460

water, and the size distribution by intensity was analyzed in triplicate. Determination of 1461

lipoic acid in diet was performed through liquid chromatography with mass 1462

spectrometric detection, following the procedures described in Martins et al. (2014). 1463

Commercial diet SUPRA® with 35% crude protein was grinded and mixed with 1464

dispersions of NC or NCLA. The homogeneous mass was passed through a 5 mL 1465

syringe, and the obtained mass filaments were placed in oven at 50 °C, for 24 h before 1466

obtaining the pellets. The diet was provided three times a day (8:00, 15:00 and 22:00 h) 1467

52

at a feeding rate of 3% of shrimp biomass. The concentration of LA present in the ration 1468

was estimated in 68 ± 8.6 mg LA/Kg. 1469

Physical and chemical parameters of water were measured daily, including dissolved 1470

oxygen (mg/L), water temperature (°C), salinity and pH. Ammonia and nitrite was 1471

measured at intervals of one day and nitrate once a week. When the total ammonia 1472

concentration exceeded 1 mg/L, molasses were added to BFT water (Avnimelech, 1473

1999). The experiment lasted 30 days, where there was no water changes in BFT 1474

treatment. In the treatment of saline water, 80-90% of water was renewed every 48 h. 1475

Parameters of water quality presented significant differences (p< 0.05) for the 1476

concentration of dissolved oxygen, being lower at BFT. Nitrite concentration also was 1477

lower at BFT, although nitrate levels were augmented in BFT (Table 1). 1478

At the beginning and the end of the experiment, shrimps from the four treatment were 1479

weighed. At the end of the experiment, the hemolymph was collected from shrimp heart 1480

using a 3 mL syringe (see details in section 2.6). After, shrimps were frozen and in the 1481

next day the tissues gills, hepatopancreas and muscle from the second abdominal somite 1482

were dissected and stored in ultrafreezer at -80 oC. 1483

Organ samples were homogenized (1:5, w/v) in a buffer previously employed for 1484

crustacean species (Pinho et al., 2005; Sousa et al., 2014) composed of Tris-Base (20 1485

mM), EDTA (1 mM), dithiothreitol (1 mM; Sigma), sucrose (500 mM), KCl (150 mM) 1486

and dissolved in Milli-Q water, with pH adjusted to 7.2. Homogenates were centrifuged 1487

(9,000 x g, 30 min at 4°C) and the supernatants were kept and divided into three 1488

aliquots for later analysis of glutathione-S-transferase (GST) activity, levels of lipid 1489

peroxidation (TBARS assay) and levels of reduced glutathione (GSH). 1490

53

2.1. DETERMINATION OF GLUTATHIONE S-TRANSFERASE ACTIVITY (GST) 1491

Glutathione-S-transferases was measured according to the protocol of Habig et al. 1492

(1974). The conjugation of 1 mM of 1-chloro-2,4-dinitrobenzene (CDNB, Sigma) with 1493

1 mM of reduced glutathione (GSH, Sigma) in presence of 20 µL of sample 1494

homogenates (source of GST enzyme) generated a CDNB-GSH complex. The 1495

absorbance of this complex was measured at 340 nm in a spectrofluorometer with a 1496

microplate reader (Victor 2, Perkin Elmer). 1497

1498

2.2. DETERMINATION OF CONCENTRATION OF REDUCED GLUTATHIONE 1499

(GSH) 1500

The procedure was based on White et al. (2003). The substrate 2,3 naphthalene 1501

dicarboxialdehyde (NDA, Invitrogen) reacts with GSH to form a fluorogenic 1502

compound. Aliquots from each homogenates were transferred to white ELISA 96-well 1503

microplates. After, it was added to all wells the NDA solution, and after 30 min of 1504

incubation, the fluorescence intensity of the NDA-GSH complex was read at excitation 1505

and emission wavelength of 485 and 530 nm, respectively, using a plate reader 1506

fluorometer (Victor 2, Perkin Elmer). A standard curve of GSH was prepared to express 1507

the data in terms of µM of GSH/mg of total proteins. 1508

1509

2.4. DETERMINATION OF LIPID PEROXIDATION 1510

Lipid peroxidation was determined by TBARS (thiobarbituric acid reactive substances) 1511

assay following Oakes and Van der Kraak (2003). Aliquots (20 µl) of sample 1512

homogenates were added to glass tubes, allowing the reaction with thiobarbituric acid 1513

54

(0.8%, Sigma), 20% acetic acid, Milli Q water and 8.1 % of sodium dodecyl sulfate. 1514

The mixture was incubated at 95 oC during 30 min and after cooling it was added n-1515

buthanol. Following a centrifugation step (3,000 x g during 10 min at 15 oC), the n-1516

buthanol phase was pippeted to a white ELISA 96-well microplate and the fluorescence 1517

read (excitation: 520 nm; emission: 580 nm) in a fluorometer with a microplate reader 1518

(Victor 2, Perkin Elmer). As standard it was employed 1,1,3,3-tetramethoxypropane 1519

(TMP, Across Organics). The results were expressed as nmol equivalents of TMP per 1520

mg of fresh tissue. 1521

1522

2.5. DETERMINATION OF TOTAL AMMONIA 1523

Total ammonia determination (NH3 + NH4+) follows the UNESCO (1983) method in 1524

which ammonia in basic pH (between 10.8 and 11.5) reacts with a bleaching solution, 1525

forming monochloramine. In the presence of phenol and an excess of hypochlorite, 1526

addition of sodium nitroprusside as a catalyst generates indophenol blue, a molecule 1527

that absorbs at 630 nm. 1528

1529

2.6. DIFFERENTIAL HEMOCYTE COUNT (DHC) 1530

The hemolymph was collected by heart puncture using 3 mL syringe containing 1531

anticoagulant crustacean solution, containing NaCl (450 mM), glucose (100 mM), 1532

sodium citrate (30 mM), citric acid (23 mM), EDTA (20 mM) diluted in MilliQ water, 1533

with pH fixed at 7.4. After, a smear of hemolymph was made on glass slides that were 1534

fixed in metanol for 5 minutes a stained with May-Grunwald-Giemsa. Then, hemocytes 1535

were quantified by microscope eyepiece lens Integrating Disc 1, 25 points-G49 (Carl 1536

Zeiss), following methodology of Weibel (1980). 1537

55

2.7. STATISTICAL ANALYSIS 1538

Data were expressed as mean ± standard deviation. Each variable from each organ, 1539

including hemolymph (GST activity, lipid peroxidation, total antioxidant capacity and 1540

total hemocyte count) was analyzed through bi-factorial ANOVA, being the factors the 1541

treatments (NC or NCLA) and rearing condition (SW or BFT). Previously the 1542

assumptions of normality and homogeneity of variance were analyzed. Means 1543

comparisons were done using the Newman-Keuls test. Water physic-chemical 1544

parameters were analyzed using Student t test for unequal variances (Zar 1984). In all 1545

cases it was used a significance level of 5%. 1546

1547

3. RESULTS 1548

The initial weight was not significantly different between treatments (p> 0.05), but after 1549

30 days of experiment, shrimps that were supplemented with NCLA showed higher 1550

weight, both in SW and BFT, although the maximum increase was observed in BFT-1551

NCLA treatment. In every case survival was of 100% (Table 2). 1552

The GST activity was higher in gills of shrimp of SW-NC in SW and lower in BFT-NC 1553

when compared with the other two treatments (p< 0.05) (Figure 1a). In hepatopancreas 1554

the highest GST activity was observed in BFT-NCLA and the minimum in the treatment 1555

SW-NC (p< 0.05). In muscle, there was no significant difference in GST activity 1556

between treatments (p> 0.05) (Figure 1a). 1557

In gills, the peak of GSH concentration was observed in treatment BFT-NCLA, being 1558

different from BFT-NC treatment (p <0.05), whereas no differences were observed in 1559

SW (Figure 1b). In hepatopancreas, treatment with NCLA induced higher GSH levels 1560

56

(p< 0.05) both in SW and in BFT. However, the BFT-NCLA showed the highest GSH 1561

level (Figure 1b). Muscle was the organ that presented highest GSH levels, although no 1562

influence of treatments was verified (p> 0.05) (Figure 1b). 1563

In gills, shrimps from SW-NC presented the highest TBARS levels, an effect that was 1564

lowered in SW-NCLA treatment (p< 0.05). Shrimps maintained in BFT presented the 1565

lowest TBARS levels, both for NC and NCLA (p< 0.05; Figure. 1c). In hepatopancreas 1566

a significant (p< 0.05) peak of TBARS was registered in BFT-NCLA, being different 1567

from the rest of the treatments (Figure 1c). In muscle, the treatment SW-NC showed the 1568

highest TBARS levels, being different from the other treatments (p< 0.05) (Figure 1c). 1569

Hemocytes counts were different in the four treatments. The percentage of hyaline 1570

hemocytes was highest in SW-NC treatment, and lowest in BFT-NCLA. Values for SW-1571

NCLA and BFT-NC remained intermediate between those registered in SW-NC and 1572

BFT-NCLA (p< 0.05; Figure 2). An opposite response was observed for percentage of 1573

granular hemocytes, being highest for BFT-NCLA and lowest for SW-NC treatment, 1574

where SW-NCLA and BFT-NC responses remained intermediate (p< 0.05; Figure 2). 1575

1576

4. DISCUSSION 1577

Water quality parameters were in accordance with proposed parameters for the 1578

Litopenaeus vannamei species proposed by Samocha et al. (2010) and Ray et al. (2014). 1579

These parameters were also in according with previous experiment Martins et al. (2015) 1580

with saline water and biofloc, except that salinity in such biofloc experiment was 33. 1581

Ammonia and nitrite concentrations remained below of the levels considered toxic for 1582

this species (Lin and Chen, 2001; Lin and Chen, 2003). The low concentration of nitrite 1583

57

and high concentration of nitrate in BFT indicate heterotrophic bacteria activity in the 1584

conversion of nitrogen products (Ebeling et al., 2006). There was an increase in the 1585

average final weight of the treatments with LA, indicating that this zootechnical 1586

parameter is improved with supplementation of LA for Litopenaueus vannamei shrimp 1587

in clean water and biofloc, as previously reported by Martins et al. (2014) for the same 1588

species reared in saline water when supplemented with lipoic acid at doses higher than 1589

190 mg LA/kg of ration. 1590

Shrimps as any other aerobic organism, possess an integrated and coordinated 1591

enzymatic and non-enzymatic antioxidant systems that maintain the redox equilibrium 1592

of the organism (Ren et al., 2014). Under this context, it is expected that lipoic acid 1593

supplementation should aid in the establishment of the redox system and thus for the 1594

organism welfare. 1595

Previous studies have showed several beneficial effects of lipoic acid in aquatic 1596

organisms (for a review: Kütter et al., 2014), although the responses are strongly organ-1597

dependent (Monserrat et al., 2008; Kütter et al., 2013). Some of the positive effects 1598

include: high GST activity, low concentration in thiobarbituric reactive substances in 1599

different organs, higher levels of GSH and of the limiting enzyme in their synthesis 1600

(glutamate cysteine ligase) (Monserrat et al., 2008; Martins et al., 2014; Monserrat et 1601

al., 2014; Enamorado et al., 2015). The lower GST activity observed in SW-NCLA 1602

when compared with SW-NC fits to the previous observation of Monserrat et al. (2014), 1603

where LA also reduced GST activity in gills of Jenynsia multidentata. These authors 1604

postulated that high levels of the antioxidant in these organs should favor a reductive 1605

redox state that should inhibit the up-regulation of genes related to antioxidant and 1606

detoxification processes. This idea is also supported by the results of Kütter et al. 1607

58

(2013), where in liver of pompano fish Trachinotus marginatus, high LA doses (40 and 1608

60 mg LA/kg of body weight) lowered GST activity and at a lower one (20 mg LA/kg 1609

of body weight) it was registered a significant increase of enzyme activity. 1610

By the other side, the hepatopancreas presented high GST activity in shrimps reared in 1611

SW and BF and submitted to NCLA, a result similar to that found by Kütter et al. 1612

(2013) with low doses of free LA. A previous study of Lobato et al. (2013) also showed 1613

a similar result in the same species, L. vannamei, even when the free LA dose employed 1614

by those authors was higher than in present study. The comparison of GST results in 1615

gills and hepatopancreas suggest that the biodistribution and entry of LA in the different 1616

organs should determine strong or moderate reductive redox state and this should 1617

influence GST activity. The tripeptide-reduced glutathione (GSH) is used in conjugation 1618

reactions catalyzed by GST and also acts as a first barrier against reactive oxygen 1619

species (ROS). In this way, increased levels of this antioxidant in hepatopancreas of 1620

shrimps from experimental groups SW-NCLA and BFT-NCLA indicate the antioxidant 1621

responses triggered by LA. Up-regulation of genes that code for both modulatory and 1622

catalytic sub-units of glutamate cysteine ligase (the limiting enzyme for GSH synthesis) 1623

by LA has been described in rats (Suh et al., 2004). In gills, the same effect was 1624

observed only in shrimps reared in BFT indicating that in this case some components, 1625

including antioxidants present in bioflocs as reported by Xu et al. (2013), should aid to 1626

obtain a better antioxidant response that in saline water. Also Martins et al. (2015) 1627

reported that shrimps reared in BFT presented different antioxidant responses than 1628

shrimps maintained in saline water. In the case of muscle, the so much higher GSH 1629

levels indicate that the antioxidant system is expressed near its maximum and few 1630

59

regulations can occur even for the antioxidants present in the bioflocs or by the 1631

exogenous LA offered through ration. 1632

In terms of oxidative damage, the antioxidant treatment with LA was effective only in 1633

SW, where a reduction of TBARS levels were observed in gills and muscle, as 1634

previously reported by Lobato et al. (2013) in muscle of the same species L. vannamei. 1635

Coincident with this previous study no effect was observed in hepatopancreas. The 1636

results in BFT were quite different, since a pro-oxidant condition was verified in 1637

hepatopancreas when exposed to NCLA. The antioxidant/pro-oxidant duality of LA has 1638

been recognized by several authors like Çakatay et al. (2005), Atukeren et al. (2010), 1639

and Valdecantos et al. (2010). The pro-oxidant condition of LA is related to its ability to 1640

reduce Fe3+ to Fe2+, favoring the generation of hydroxyl radical and thus the triggering 1641

of lipid peroxidation (Goralska et al., 2003). The reduced form of lipoic acid, 1642

dihydrolipoic acid (DHLA) possess the ability to remove iron from proteins like ferritin, 1643

again favoring the Fenton reaction and hydroxyl radical generation (Çakatay et al., 1644

2005). Several enzymes, including glutathione reductase that employs GSH as reductive 1645

power, favor the reduction of LA to DHLA (for a review: Kütter et al., 2014). Note that 1646

hepatopancreas GSH levels are higher in shrimps from BFT-NCLA (824.75±34.64 µM 1647

of GSH mg-1 of total proteins) than the values presented by shrimps from SW-NCLA 1648

group (526.36 µM of GSH mg-1 of total proteins). Thus, a greater reductive power in 1649

shrimps from BFT-NCLA group should favor the generation of DHLA and thus 1650

promoting iron release from proteins. 1651

On granular hemocytes (GH) are found degradative and coagulation enzymes, 1652

antimicrobial peptides, apopitotics factors and prophenoloxidase system, these have 1653

higher amount immunoefetoras and immunoregulatory molecules are considered more 1654

60

immunocompetent (Barracco et al., 2008). Some immune responses are associated with 1655

the production of reactive oxygen species (ROS) and are highly microbicides (Bachère 1656

et al., 1995; Muñoz et al., 2000; Campa-Cordova et al., 2002). The process begins when 1657

stimulation leads to an increase in oxygen consumption, whose reduction, catalyzed by 1658

NADPH-oxidase bound to membrane yields superoxide anion (Campa-Cordova et al., 1659

2002). The antioxidant superoxide dismutase (SOD) converts this metabolite 1660

microbicide in hydrogen peroxide which passes freely through membranes (Campa-1661

Cordova et al., 2002). The antioxidant catalase (CAT) and glutathione peroxidase (GPx) 1662

remove hydrogen peroxide from the cells (Campa-Córdova et al., 2002). A significant 1663

quantity passes into the extracellular environment and can cause damage to cells 1664

(Warner, 1994; Campa-Cordova et al., 2002). To avoid this damage, cells and organisms 1665

use three defense strategies; one involves low molecular weight antioxidants such as 1666

ascorbic acid, α-tocopherol and glutathione, which can directly interact with ROS and 1667

neutralizing those (Campa-Cordova et al., 2002). The other two involve a variety of 1668

enzymes that metabolize ROS (SOD, catalase, glutathione peroxidase) or 1669

macromolecular damage repair as nucleic acids, proteins and lipids (DNA repair 1670

enzymes, proteases, lipases, etc.) caused by ROS (Warner, 1994; Campa-Cordova et al., 1671

2002). 1672

In NCLA treatments was increased in HH that is explained by the fact in this 1673

study were not exposed to any pathogen so no requiring activation of components 1674

associated with phagocytosis and/or coagulation. Thus, there is an increased percentage 1675

of GH with a greatest amount of immunoefetoras cell components and 1676

immunoregulatory to protect the shrimp against possible pathogenic attacks. There little 1677

research with differential hemocyte count, being more found total hemocyte count, and 1678

other study with compounds supplemented becomes delicate comparisons by the 1679

difference in composition and functionality of the compound. In conclusion, NCLA 1680

treatments improved the immune response by increasing the number of granular 1681

hemocytes. 1682

1683

1684

61

5. CONCLUSION 1685

The GST activity was tissue-specific, with greater activity in gills. However, the NLA 1686

induced increase in GST activity in the hepatopancreas. Likewise that the GST activity 1687

the GSH concentration is tissue-specific with greater performance in the muscle 1688

although not presenting significant difference between treatments had higher 1689

concentration in this tissue has direct antioxidant activity and not as a substrate for 1690

detoxification activity. Antioxidant capacity also showed a standard tissue-specific, with 1691

higher antioxidant capacity hepatopancreas no evident action of the performance of 1692

NLA against peroxyl radicals. Lipid peroxidation levels were lower in muscle, with 1693

obvious performance NLA action. It can say that the groups with NLA there was an 1694

increase in the percentage of granular hemocytes, cells with higher amounts of 1695

components immunocompetents, but cannot say that the NLA is a compound with 1696

immunostimulatory behavior. 1697

1698

Acknowledgments. José Maria Monserrat and Wilson Wasielesky Junior receive 1699

productivity research fellowships from the Brazilian agency CNPq (process numbers 1700

PQ 307880/2013-3 and PQ 310993/2013-0, respectively). 1701

1702

1703

1704

1705

1706

1707

1708

62

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glutathione synthesis, which is reversible with lipoic acid. Proc. Natl. Acad. Sci 1853

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antioxidants but different. Redox Report. 15, 207–216. 1859

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electron paramagnetic resonance spectroscopy. Arch. Biochem. Biophys. 420, 1862

209–216. 1863

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based microtiter plate assay for glutamate-cysteine ligase activity. Anal. 1868

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environmental impact and perspectives. Ocean Coast. Manage. 50, 538–550. 1871

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enzyme activity and body composition of juvenile Litopenaeus vannamei in 1873

zero-water exchange tanks manipulating C/N ratio in feed. Aquaculture. 356–1874

357, 147–152. 1875

Xu, W-J., Pan, L-Q., 2013. Enhancement of immune response and antioxidant status of 1876

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C/N ratio of feed input. Aquaculture. 412–413, 117–124. 1878

Zar, J. H., 1984. Biostatistical analysis. New Jersey: Prentice Hall. 1879

1880

68

Table captions 1881

Table 1 – Parameters physical and chemical water quality on average function (± 1 1882

standard deviation). In saline water (SW) or in bioflocs system (BFS). Animals were fed 1883

with ration suplemented with nanocapsules containing lipoic acid (NCLA) or without 1884

the antioxidant (empty nanocapsules - NC). Asterisk (*) indicate significant differences 1885

(p <0.05) between groups. 1886

Parameter SW-NC SW-NCLA BFT-NC BFT-NCLA

Temperature °C (n=186) 27.01 (± 0,01) 27.01 (± 0,03) 27.03 (± 0,01) 27.01 (± 0,02)

[DO] (mg L-1) (n=186)* 6.45 (± 0.01) 6.51 (± 0.01) 5.56 (± 0.05) 5.61 (± 0.04)

Salinity (n=186) 31.00 (± 0.01) 31.00 (± 0.01) 31.98 (± 0.01) 31.96 (± 0.01)

pH (n=186) 7.89 (± 0.06) 7.94 (± 0.02) 8.00 (± 0.01) 7.82 (± 0.07)

Ammonia (mg L-1) (n=66) 1.72 (± 1.36) 1.71 (± 1.43) 1.84 (± 2.53) 1.90 (± 2.86)

Nitrite (mg L-1) (n=66)* 0.55 (± 0.75) 0.53 (± 0.69) 0.27 (± 0.39) 0.25 (± 0.33)

Nitrate (mg L-1) (n=30)* 3.64 (± 1.88) 0.18 (0.69) 84.60 (± 5.57) 80.90 (± 5.55)

1887

Table 2 – Average (± 1 standard deviation) initial and final weight in shrimp 1888

Litopenaeus vannamei reared in saline water (SW) or in bioflocs system (BFS). Animals 1889

were fed with ration suplemented with nanocapsules containing lipoic acid (NCLA) or 1890

without the antioxidant (white nanocapsules - WNC). Statistical differences between 1891

treatments were represented by different lower case letters (p<0.05) for both initial and 1892

final weight. Number of samples analyzed per treatment: 45. 1893

Treatments Average initial weight (g) Average final weight (g) Survival

SW-NC 5.90 (± 0.08)a 9.72 (± 0.06)c 100%

SW-NCLA

5.91 (± 0.07)a

11.85 (± 0.11)b

100%

BFT-NC

5.91 (± 0.04)a

9.43 (± 0.04)d

100%

BFT-NCLA

5.91 (± 0.01)a

12.34 (± 0,05)a

100%

1894

69

Figure captions 1895

1896

Figure 1. (a) Mean activity (± 1 standard error) of glutathione S-transferase (GST) 1897

(nmol of CDNB-GSH conjugate/min/mg of protein). (b) Mean (± 1 standard error) of 1898

reduced glutathione (GSH) concentration (nmol GSH/mg protein). (c) Mean 1899

concentration (± 1 standard error) of thiobarbituric acid reactive substances (TBARS) 1900

70

(TMP nmol/mg tissue). In all cases, measurements were performed in gills, 1901

hepatopancreas and muscle of L. vannamei reared in saline (SW) or in biofloc system 1902

(BFT) and exposed to empty nanocapsules or nanoencapsulated lipoid acid. Different 1903

letters indicate significant differences (p< 0.05) between the experimental groups. 1904

Similar letters (lowercase in gills; uppercase in hepatopancreas and Roman numerals in 1905

muscle) means absence of statistical differences (p> 0.05). Number of samples analyzed 1906

per treatment: 11 – 15. 1907

1908

1909

Figure 2. Mean (± 1 standard error) of percentage of hyaline and granular hemocytes in 1910

hemolymph from shrimps L. vannamei submitted to different experimental conditions in 1911

the same treatments. Shrimps were reared in saline water (SW) or in bioflocs system 1912

(BFT) and fed with ration supplemented with nanocapsules containing lipoic acid 1913

(NCLA) or empty nanocapsules (NC). Similar letters (lowercase in hyaline hemocytes 1914

and uppercase in granular hemocytes) means absence of statistical differences (p>0.05). 1915

Number of samples analyzed per treatment: 7. 1916

71

CAPÍTULO III 1917

1918

1919

Effects of lipoic acid in the total antioxidant capacity in 1920

biofloc 1921

1922

1923

1924

Átila Clivea da Silva Martins, Wilson Wasielesky Junior, José 1925

Maria Monserrat 1926

1927

1928

1929

Será submetido a revista Aquaculture 1930

1931

1932

1933

1934

1935

1936

72

ABSTRACT 1937

Several studies prove the effectiveness of biofloc in improving growth, survival and 1938

feed conversion to shrimp species Litopenaues vannamei, and the presence of bioactive 1939

compounds that help the immune and antioxidant defense. In this study, was observed 1940

the total antioxidant capacity of biofloc with application of antioxidant lipoic acid in 1941

different concentrations (control, [1,25μM], [2,5μM], [5μM] and [10μM]). Using the 1942

method of total antioxidant capacity against peroxyl radical (ACAP), for which was 1943

found increase in antioxidant capacity with the application of lipoic acid, especially in 1944

the treatment [10μM]. 1945

1946

Keywords: biofloc, bioactive compound, lipoic acid, total antioxidant capacity. 1947

1948

1 INTRODUCTION 1949

The biofloc Technology (BFT) is a production system with minimal water 1950

exchange, which includes the development of microorganisms aggregate belonging to 1951

different taxonomic groups (bacteria, micro-algae, protozoa and other) together with 1952

debris (organic and inorganic particles) that are suspended in the water column with 1953

constant aeration (Avnimelech, 2009; Cardona et al., 2015). The aggregates or biofloc 1954

of this microbial community in this system helps to maintain the water quality by 1955

setting of the recycling of nutrients (nitrogen compounds) and can be used as a source 1956

of proteins and lipids to aquatic organisms including fish and shrimp (Burford et al., 1957

2004; Avnimelech, 2009). 1958

From the ecology point of view, the biofloc can be understood as a 1959

microecosystem constituted by a great diversity of microorganisms (bacteria, 1960

phytoplankton, zooplankton), which develop at the expense of biological processes 1961

occurring in environment of raised. Thus, the nutritional value of biofloc is directly 1962

linked to the composition of the microbial communities and bioactive compounds 1963

present in these organisms. In the studies by Ju et al. (2008), Xu and Pan (2013) and 1964

Cardona et al., (2015), they proposed that animals raised in biofloc system showed an 1965

73

increase in immune response and antioxidant status, probably for the intake of bioactive 1966

compounds present in these organisms. 1967

In an intensive rearing system may increase the action of bioactive compounds 1968

by exogenous addition of other compounds with bioactive properties to assist in the 1969

wellness of animals raised, in this sense lipoic acid (LA) is presented as a multifaceted 1970

bioactive agent. It is known that the physiological level, this compound chelates free 1971

radicals, induces increase in the reduced glutathione concentration and restores the 1972

levels of other antioxidants; in this way also assists in maintaining the intracellular 1973

redox state (Shay et al., 2009). 1974

The quantification of biofloc is monitored through the settleable solids in BTF 1975

system (Samocha et al., 2010; Ray et al., 2010; Schveitzera et al., 2013) and the 1976

presence of these solids over 10–14 mL L-1, may increase consumption of dissolved 1977

oxygen limiting their concentrations and may change other variables such as alkalinity 1978

and pH (Tovar and Erazo, 2009; Samocha et al., 2010; Ray et al., 2010). The 1979

measurement of settleable solids may be a staunchly useful to interpret the interactions 1980

among the AL and biological processes that lead to increased or decreased of the biofloc 1981

quantity. 1982

The application of AL in biofloc can increase the antioxidant capacity of biofloc, 1983

providing a means of reducing the damaging effects of reactive oxygen species (ROS) 1984

(Ghiselli et al., 2000). The ROS such as superoxide anion (O2·-); hydroxyl radical (OH·) 1985

and hydrogen peroxide (H2O2) are produced in cells of the organisms in normal or 1986

pathologic physiological conditions (Wajner et al., 2004), and can also be generated by 1987

cyclically dissolved oxygen and practice use of hydrogen peroxide to establish the 1988

oxygen levels in the water with biofloc by reduction or absence of mechanical aeration 1989

(Cardona et al., 2015). These ROS act on biomolecules that comprise cell membranes, 1990

proteins, DNA and RNA and the negative effects of this process are counteracted or 1991

mitigated by the action of endogenous or exogenous antioxidants (Barreiros et al., 1992

2006). 1993

An antioxidant may be defined as any substance that, when present at low 1994

concentrations compared with an oxidizable substrate, significantly delays or prevents 1995

oxidation of said substrate (Dickinson and Forman, 2002). The sum of the antioxidant 1996

74

activity of an antioxidant compound is known as total antioxidant capacity (Bartosz, 1997

2003). The term antioxidant activity is restricted to characterize the rate constant of a 1998

single antioxidant at a specific oxidant (generally free radical), whereas the antioxidant 1999

capacity should refer to the number of moles of oxidant eliminated by a given sample 2000

(Ghiselli et al., 2000). 2001

Therefore, the objective of this study is to evaluate the effects of lipoic acid in 2002

biofloc over time (8 days), by analyzing the total antioxidant capacity against peroxyl 2003

radicals. 2004

2005

2 MATERIAL AND METHODS 2006

2.1 EXPERIMENTAL DESIGN 2007

It was held at the Green House I (GHI), in Marine Aquaculture Station of the 2008

Federal University of Rio Grande - FURG (Brazil), from 26 October to 2 November of 2009

2014. In 15 tanks plastics (70 L), with a useful volume of 50 L each. Treatments were 2010

set at: Control (0 mg LA L-1 of biofloc), Concentration of 1.25 µM (0.26 mg L-1), 2011

Concentration of 2.5 µM (0.51 mg L-1), Concentration of 5 µM (1.03 mg L-1) and 2012

Concentration of 10 µM (2.06 mg L-1); all in triplicate. The collections of biofloc were 2013

made every 24 hours. After the collection of biofloc, were added the AL concentrations. 2014

2015

2.2 DILUTION LIPOIC ACID IN SODIUM HYDROXIDE 2016

The fixed concentrations of synthetic α-lipoic acid (> 99% purity, Sigma-2017

Aldrich) were dissolved individually for each tank, with sodium hydroxide solution and 2018

distilled water to ensure full dilution of lipoic acid following the methodology Amado et 2019

al. (2011). After dilution, the pH was adjusted to 7.5. The concentrations of LA were 2020

added every 24 hours (Table 1). 2021

2022

2023

2024

75

Table 1: Preparation of lipoic acid solution. NaOH (sodium hydroxide; 2mM), dwH2O 2025

(distilled water), LA (lipoic acid). From the overall calculation of 300 mL of NaOH to 1 2026

g of LA and 1000 mL of dwH2O to 80 g of NaOH. 2027

Treatment NaOH (g) dwH2O (mL) LA (g)

Control

1.25 µM

2.5 µM

5.0 µM

10.0 µM

0

0.62

1.22

2.47

4.94

0

7.8

15.3

30.9

61.8

0

0.26

0.51

1.03

2.06

2028

2.3 WATER QUALITY ANALYSIS 2029

Daily physical and chemical parameters of water were monitored, including 2030

dissolved oxygen (mg L-1) and temperature (°C) with YSI multiparameter, salinity with 2031

optical refractometer (Atago 103, ± 1 ppt), pH with electrode Mettler Toledo FEP20 - 2032

FiveEasy Plus™. Determination of alkalinity (mg CaCO3 L-1) was made by the method 2033

APHA (1985), and calculating the concentration of sodium bicarbonate was performed 2034

according Furtado et al. (2011). The analyzes of total ammonia (NH3 + NH4+ mg L-1) by 2035

method UNESCO (1983) and nitrite (mg L-1) according Benderschneider and Robinson 2036

(1952) (Figure 1). 2037

The settleable solids (organic and inorganic particulate matter) were determined 2038

by sedimentation of solids in Imhoff cones where placed 1 L of water and allowed to 2039

stand for 1 h, time which is made to read the settleable solids mL L-1 (Tovar and Erazo 2040

2009) (Figure 1). 2041

2042

2.4 COLLECTION AND HOMOGENIZATION OF SAMPLES 2043

Samples were collected after reading the Imhoff cone, in which the water was 2044

removed by siphoning and flake placed in falcon (50 mL) and stored on ice, and then 2045

they were transferred to eppendorf (2 mL) and centrifuged at 800 x g being the 4 °C for 2046

10 minutes and stored at -80 °C. For homogenization, the biofloc samples were weighed 2047

76

in eppendorff, added methanol (100%) on relationship 1000 mg 1000 μL-1, 2048

homogenized and stirred for 3 hours, then centrifuged for 10 minutes at 10000 rpm and 2049

4 °C, the supernatant removed and it used to determine the antioxidant capacity against 2050

peroxyl radicals. 2051

2052

2.5 DETERMINATION OF TOTAL ANTIOXIDANT CAPACITY AGAINST 2053

PEROXYL RADICAL (ACAP) 2054

It was conducted in accordance with the protocol Amado et al. (2009), but for 2055

this work was not the fixation of the protein concentration. After white microplate was 2056

added to 127.5 µL of a reaction buffer consisting of 0.3575 g of ethanesulfonic acid, 4.2 2057

hydroxyethyl-piperazine-1 (HEPES), 0.7455 g of potassium chloride (KCl), 0.0102 g 2058

magnesium chloride (MgCl2) dissolved in 50 mL of MilliQ water with pH adjusted to 2059

7.2; 10 µL tissue extract; 7.5 µL of water MilliQ to samples without ABAP (2,2 azobis 2060

2metilpropianoamidina dihidrocloreto) or 7.5 µL ABAP solution for samples with 2061

ABAP (peroxyl radical generator) and 10 µL of H2DCF-DA solution (diacetate '2,7 2062

diclorofluresceín). Reading is performed at time zero and then every 5 minutes to 2063

complete 30 minutes in fluorimeter plates (FilterMax F5, Multi-mode microplate 2064

reader) using a wavelength of 530 nm emission and 485 nm excitation, at 37 °C, the 2065

temperature that favors ABAP thermolysis. This method quantifies the ability of the 2066

tissue to neutralize the ROS generated by the decomposition of ABAP, including 2067

enzymatic antioxidant defenses and/or non-enzymatic, by calculating the relative area 2068

performed according Monserrat et al. (2014). The relative area has an inverse 2069

relationship with the antioxidant capacity, the area where lower values indicate a higher 2070

relative antioxidant capacity and vice-versa. 2071

2072

2.6 STATISTICAL ANALYSIS 2073

Data were expressed in mean ± standard deviation. On analysis by two-factor 2074

ANOVA (different concentrations of lipoic acid and time). Previously, the assumptions 2075

of normality and homogeneity of variance were analyzed. Mean comparisons were 2076

made using the Newman-Keuls test. In all cases, we used a 5% significance level. 2077

77

3 RESULTS 2078

The water quality parameters such as temperature got overall average of 28.48 ± 2079

0.09 °C. The dissolved oxygen was not significantly different (p>0.05) between 2080

treatments or over time, getting between minimum 7.68 ± 0,05 and maximum 7.99 ± 2081

0,02 mg L-1, both in control at time 0 and time 2, respectively. The pH showed changes 2082

in time 4, 5 and 6 in the control treatment, with values of 7.81 ± 0.04, 7.69 ± 0.04 e 7.62 2083

± 0.07 respectively and have minimum of 7.62 and maximum of 8.10 on 3 (time 3) both 2084

in the control treatment. The lowest salinity was measured on time 1 in the treatment 2085

[1.25 µM] with 26.27 ± 2.99 and from time 4 was in 30 for all treatments. For alkalinity, 2086

the minimum value of 126.67 ± 53.64 mg CaCO3 L-1 occurred at time 5 in the treatment 2087

[10 µM], with a maximum of 213.33 ± 38.33 at time 3 for the same treatment. The 2088

ammonia presents change in time 4 in treatment [5 µM], with maximum value of 0.41 ± 2089

0.32 mg L-1 and have minimum of 0.02 ± 0.00 mg L-1 at time 3 in treatment [10 µM]. 2090

The minimum levels of nitrite was among 0.03 ± 0,00 mg L-1 in the control treatment at 2091

times 2, 6 and 7 and maximum levels at time 5, 6 e 7 with value 0.34 ± 0.23, 0.31 ± 0.15 2092

and 0.34 ± 023 mg L-1 respectively in treatment [1.25 µM]. The settleable solids 2093

increased over time and showed significant differences (p>0.05) when compared to 2094

control, with a minimum value of 2.33 ± 0.33 mL L-1 at time 2 in the treatment control 2095

and up to 216.67 ± 16.67 mL L-1 in time 6 in treatment [1.25 µM]. 2096

For total antioxidant capacity, at time 0 was there is significant difference 2097

(p<0.05) between control and of the treatments 1.25 µM (0.26 mg L-1), 2.5 µM (0.51 2098

mg L-1), 5 µM (1.03 mg L-1) and 10 µM (2.06 mg L-1). At time 1, noted increase in total 2099

antioxidant capacity with significant difference (p<0.05) between control and of the 2.5 2100

µM (0.51 mg L-1), 5 µM (1.03 mg L-1) and 10 µM (2.06 mg L-1). At time 2, there was a 2101

significant difference (p<0.05) between control and the other treatments, following this 2102

pattern for the times 3, 4, 5, 6 and 7 (Figure 2). 2103

2104

2105

2106

2107

78

2108

Figure 1 – Mean concentration (±1 standard error) of the physico and chemical 2109

parameters. Number of samples analyzed in each treatment: 3 2110

79

2111

Figure 2 – Mean values (± 1 standard error) of total antioxidant capacity against peroxyl 2112

radicals (expressed in relative area) in biofloc. Different letters indicate significant 2113

80

differences (p<0.05) between experimental groups after performing Newman-Keuls 2114

post hoc test. Number of samples analyzed in each treatment: 9. 2115

2116

4 DISCUSSION 2117

Water quality parameters were monitored to comply with requirements physical 2118

and chemical of the water for species Litopenaues vannamei, following values proposed 2119

by Lin and Chen (2001; 2003), Ray et al. (2010) and Samocha et al. (2010), that 2120

recommended: (i) temperature should be between 28–30 °C, because low temperature 2121

decreases growth and high temperature cause stress; (ii) dissolved oxygen greater than 5 2122

mg L-1, reduces stress on the shrimp; (iii) pH in 7.5, preferably greater than 7; (iv) 2123

salinity greater than 10 with a tolerance of 4–35, high salinity help in buffering capacity 2124

of water; (v) alkalinity greater than 160 mg L L-1, assists in balancing the pH; (vi) 2125

ammonia in 3.35 mg L-1 with salinity of 25 and 3.95 mg L-1 in salinity 35; (vii) nitrite 2126

levels between 5–25 mg L-1, thresholds rise with increased salinity; (viii) settleable 2127

solids 10–14 mg L-1, increase in the floc concentration may result in anaerobic mean. 2128

For this work, on time 1 the salinity had decreased to 26.67 ± 2.99, but is still in the 2129

recommended range for L. vannamei. On time 5, when the alkalinity has reached 2130

minimum 126.67 ± 53.64 mg CaCO3 L-1, was added sodium bicarbonate to increase the 2131

alkalinity levels. On time 4, showed an increase of ammonia remaining with value of 2132

0.41 ± 0.32 mg L-1, below the recommended amount. On time 5, 6 and 7, an increase in 2133

nitrite levels getting the value of 0.34 ± 0.23 mg L-1; 0.31 ± 0.15 mg L-1 and 0.34 ± 0.23 2134

mg L-1, below the recommended values. At the end of the experiment, only settleable 2135

solids were beyond to standard recommended for shrimp L. vannamei, with a significant 2136

increase in treatments with LA, which may suggest that the AL induces increased 2137

amount of floc or the increase was due to the absence of shrimp in the tank, providing 2138

an imbalance between production and consumption floc. 2139

The total antioxidant capacity of biofloc increased since the first day of 2140

application of AL, highlighting to treatments [2.5 µM], [5 µM] and [10 µM]. On the 2141

second day of the experiment, the increase occurred in treatments [1.25 µM] and [10 2142

µM]. On the third day, the increase was observed in treatment [10 µM]. On the fourth 2143

day, increased total antioxidant capacity was observed [10 µM]. On the fifth day, the 2144

81

increase occurred in treatments [1.25 µM] [µM 5] and [10 µM]. Thus, AL induces 2145

increasing the capacity of floc cells to neutralize ROS. On day 6 and 7 was observed 2146

decrease in antioxidant capacity with respect to treatment with AL, suggesting that 2147

under experimental conditions the biofloc has limited capacity to absorb AL or increase 2148

the amount of floc, evidenced by the increase in settleable solids, reduces the 2149

concentration and the LA effects. For the control treatment, no significant change over 2150

time. 2151

2152

5 CONCLUSION 2153

The lipoic acid induces increase in total antioxidant capacity in biofloc, having 2154

the concentration of 10 µM AL per liter of water at most the total antioxidant capacity 2155

over time. 2156

2157

6 FUTURE PERSPECTIVE 2158

Lipoic acid can convey their bioactive properties without causing damage to 2159

biofloc, but these benefits can be apply to the community that makes up the biofloc or 2160

it benefits are community-specific? 2161

Once lipoic acid increases the total antioxidant capacity of the flake, the dose of 2162

10 µM of LA per liter of water can influence the nutritional composition of biofloc? 2163

2164

Acknowledgments. José M. Monserrat, Wilson Wasielesky Junior, are research 2165

fellowships from the Brazilian agency CNPq (process numbers PQ 307880/2013-3, PQ 2166

310993/2013-0, respectively). 2167

2168

2169

2170

2171

82

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capacity against peroxyl radicals in aquatic organisms: Application to evaluate 2175

microcystins toxicity. Science of the Total Environment. 407, 2115–2123. 2176

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2011. Chemoprotection of lipoic acid against microcystin-induced toxicosis in 2178

common carp (Cyprinus carpio, Cyprinidae). Comparative Biochemistry and 2179

Physiology, Part C. 154, 146–153. 2180

Avnimelech, Y., 2009. Biofloc Technology - A practical guide Book. Baton Rouge, 2181

Louisiana, United States, The World Aquaculture Society. 2182

Barreiros, A.L.B.S., David, J.M., David, J.P., 2006. Estresse oxidativo: relação entre 2183

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Burford, M.A., Thompson, P.J., McIntosh, P.R., Bauman, R.H., Pearson, D.C., 2004. 2187

The contribution of flocculated material to shrimp (Litopenaeus vannamei) 2188

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Boone juveniles at diferente salinity levels. Journal of Experimental Marine 2212

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Lin, Y-C., Chen, J-C., 2003. Acute toxicity of nitrite on Litopenaeus vannamei (Boone) 2214

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Miglioranza, K.S., Amé, M.V., Wunderlin, D.A., 2014. Antioxidant, phase II and 2217

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(Anablapidae) and its influence on endolsulfan accumulation and toxicity. 2219

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CONCLUSÃO GERAL 2255

Os camarões criados em sistema de biofloco, em 30 dias de exposição, tiverem 2256

aumento na competência antioxidante, sendo a resposta tecido-específica para camarão 2257

branco do pacífico Litopenaus vannemei. Deste modo, algumas comunidades de 2258

microrganismo do sistema BFT influencia no estado redox de tecidos de camarão 2259

(brânquias, hepatopâncreas e músculo), como por exemplo: diminuição da concentração 2260

de glutaiona reduzida em brânquias e hepatopâncreas e níveis de peroxidação lipídica 2261

menores em hepatopâncreas. 2262

Com suplementação com ácido lipóico nanoencapsulado, aumento atividade 2263

gluationa S-transferase em hetaopâncreas, aumento da concentração de glutationa 2264

reduzida em músculo, maior capacidade antioxidante no hepatopâncreas e níveis de 2265

peroxidação lipídica menos em músculo. E aumento dos hemóctios granulares em 2266

camarões exposto ao ácido lipóico. E, portanto, o ácido lipóico foi capaz de aumentar 2267

capacidade antioxidante e diminuir o dano oxidativo em tecido do camarão branco do 2268

pacífico Litopenaus vannemei com ação antioxidante tecido-específica. 2269

O ácido lipóico foi capaz de aumentar a capacidade antioxidante no biofloco em 2270

concentração de [10 µM] (2,06 mg de AL/L de biofloco), com aumento gradual da 2271

capacidade antioxidante durante 5 dias. 2272

2273