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Pontifícia Universidade Católica do Rio Grande do Sul
Faculdade de Biociências
PPG- Biologia Celular e Molecular
Aspectos do Metabolismo Energético e da Reprodução de
Hyalella castroi González, Bond-Buckup & Araujo (Crustacea,
Amphipoda, Dogielinotidae) Mantidos em Cultivo
Experimental sob Diferentes Dietas
Fernanda Severo Gering
Professor orientador
Dra. Guendalina Turcato Oliveira
Dissertação de Mestrado
Porto Alegre/RS - 2007
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Pontifícia Universidade Católica do Rio Grande do Sul
Faculdade de Biociências
Programa de Pós-Graduação em Biologia Celular e Molecular
Aspectos do Metabolismo Energético e da Reprodução de Hyalella castroi González, Bond-
Buckup & Araujo (Crustacea, Amphipoda, Dogielinotidae) Mantidos em Cultivo
Experimental sob Diferentes Dietas
Fernanda Severo Gering
Professor orientador
Dra. Guendalina Turcato Oliveira
Dissertação de Mestrado
Porto Alegre – RS – Brasil
2007
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3
DEDICATÓRIA
Dedico o presente trabalho à memória de meu Pai,
Almindo Dias Gering por ter dedicado sua vida a
mim como exemplo de pessoa de caráter e sabedoria;
e a minha Mãe Lenoara Severo Gering que com garra
e determinação se doou, me dando amor, carinho,
afeto e estímulo para superar todas as dificuldades. A
vocês, pelas angústias, preocupações e abdicações
que passaram por minha causa, dedico-lhes esta
conquista como gratidão.
4
AGRADECIMENTOS
Em primeiro lugar, agradeço a Deus pelo dom da vida e pela maravilhosa família a qual
Ele me ofereceu.
Agradeço a uma mulher excepcional em cujo ventre fui abrigada, minha mãe Lenoara
Severo Gering, pelo amor incondicional, carinho, dedicação, pelas palavras de conforto e muitas
vezes de chamada de atenção. Agradeço a esta mulher maravilhosa por ter abdicado de muitas
coisas em prol da minha vida.
Aos meus irmãos que muitas vezes renunciaram às suas vontades por mim.
Da mesma forma, agradeço ao meu marido, Juliano Kreuz Fontoura pela paciência, apoio
e carinho a mim dedicados.
À minha família, que mesmo distante, torce e vibra comigo a cada vitória. Em especial
meu agradecimento para o tio Jauri e a tia Adélia, os primos Deise, Denise e Jadenir que me
ofereceram sua residência, apoio, carinho e acima de tudo força para que este trabalho se
concretizasse.
Em especial minha orientadora Dra. Guendalina Turcato Oliveira pela oportunidade que
me foi dada em fazer parte do seu grupo, sem mesmo me conhecer. Agradeço pelo empenho
desprendido para que este trabalho pudesse ser realizado e também por dedicar parte do seu
tempo a me orientar, com sua competência e sabedoria que sempre me acolheu.
Aos meus grandes amigos Bibiana Kaiser Dutra e Felipe Amorim Fernandes que sempre
ouviram as minhas queixas e reclamações. Obrigada pela convivência, força, palavras de
conforto, auxílio nos experimentos e acima de tudo pela amizade.
À minha amiga Laura que foi peça fundamental para que eu não desistisse do curso.
Aos acadêmicos Luiz e Arielle que me auxiliaram no cultivo dos animais.
Aos colegas de laboratório e professores do Departamento de Ciências Morfofisiológicas
pela convivência e amizade durante este período.
A secretária do Curso de Pós-graduação Kátia Ticiane de Oliveira Bonacine que sempre
se empenhou em resolver meus problemas com muita eficiência.
Ao professor Dr. Antônio Vanderlei dos Santos pela oportunidade e colaboração de
realizar o estágio docente em sua disciplina na Universidade Regional Integrada do Alto Uruguai
e das Missões.
Aos animais que foram sacrificados em prol da ciência.
5
Por fim, agradeço a todos que, de uma maneira ou de outra, contribuíram para que este
trabalho fosse executado com êxito.
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ÍNDICE
Dedicatória........................................................................................................................................3
Agradecimentos................................................................................................................................4
Índice................................................................................................................................................6
Resumo.............................................................................................................................................7
Revisão do Tema..............................................................................................................................8
1. Introdução.....................................................................................................................................8
2. Justificativa.................................................................................................................................14
3.Referências Bibliográficas...........................................................................................................16
ORIGINAL ARTICLE: Aspects of the Energetic Metabolism and Reproduction of the Hyalella
castroi González, Bond-Buckup & Araujo (Crustacea, Amphipoda, Dogielinotidae) Mantained in
Experimental Culture with Different Diets ………………………………………...…………….19
Considerações Finais......................................................................................................................63
ANEXO I: Análises das Composições Centesimais da macrófita Callitriche rimosa e das rações
utilizadas .......................................................................................................................................64
ANEXO II: Comprovante de submissão do artigo.........................................................................68
ANEXO III: Regras do Periódico ao qual o artigo foi submetido..................................................70
7
Resumo
Foi comparado o efeito de diferentes dietas no metabolismo energético, níveis de lipoperoxidação
e atividade da enzima Na
+
/K
+
ATPase de Hyalella castroi assim como sob aspectos reprodutivos.
Este crustáceo vive em ambiente límnico no planalto do Rio Grande do Sul, Brasil. Os animais
foram coletados durante os meses de outono de 2006 em São José dos Ausentes. Em laboratório,
os animais foram mantidos separados por sexo em aquários sob condições controladas e
alimentados ad libitum por 21 dias com diferentes dietas, sendo estas isocalóricas. No final do
período experimental, os animais foram imediatamente congelados para determinação dos
diferentes parâmetros bioquímicos. Parte dos animais foram mantidos nas mesmas condições já
citadas, porém em aquários que permitiam o contato entre machos e fêmeas. Para análise de
alguns parâmetros reprodutivos (número de pareamentos, número de fêmeas ovígeras e número
de juvenis eclodidos). A análise estatística revelou diferença significativa na composição
bioquímica ente os sexos e as dietas ao longo do cultivo experimental. As dietas foram capazes
de alterar o padrão bioquímico dos animais trazidos de campo e determinaram um alto percentual
de sobrevivência ao longo do período de cultivo; contudo, não foram adequadas para permitirem
o pleno sucesso reprodutivo principalmente em relação ao número de fêmeas ovígeras, a
fertilidade e a qualidade dos ovos. Além disso, ambos os sexos mostraram respostas metabólicas
e reprodutivas melhores quando alimentados com a dieta 1, a qual possui maior teor de
carboidratos (43.19g/100g) e menor de proteínas (30.88g/100g).
8
REVISÃO DA LITERATURA
1. Introdução
Diferentes adaptações ao meio ambiente têm sido caracterizadas em todos os níveis de
organização biológica nos mais diversos organismos. Estas adaptações, tanto estruturais como
funcionais, permitiram aos seres vivos à colonização de diferentes habitats. Ao longo dos últimos
anos, as adaptações bioquímicas e metabólicas ao ambiente têm sido bastante estudadas em
moluscos intertidais, peixes, tartarugas aquáticas e em alguns mamíferos, contudo poucos
trabalhos têm abordado a influência de parâmetros ambientais, tais como a hipóxia, a anoxia, a
temperatura, o fotoperíodo, a disponibilidade e qualidade de alimento sobre as adaptações do
metabolismo intermediário em crustáceos. Tanto pelo número de espécies existentes, como pela
diversidade de habitats nos quais vivem, os crustáceos estão entre os animais com o maior êxito
durante sua história evolutiva. Esta diversidade é resultado de seus padrões de vida e de
estratégias reprodutivas (Sastry, 1983).
Grande parte dos Malacostraca é representada pelos Peracarida que juntamente com os
Decapoda são a maioria dos crustáceos, correspondendo a 30% do total. Cerca de 12000 espécies
pertencem à classe Peracarida e representam sete ordens, das quais a Ordem Amphipoda é o
grupo mais representativo dos ecossistemas aquáticos, aproximadamente 6000 espécies, sendo
caracterizado por apresentarem o corpo lateralmente comprimido, olhos compostos e sésseis, e os
primeiro e segundo pares de pereópodos chamados de gnatópodos, são geralmente maiores e
subquelados, servindo para agarrar (Ruppert e Barnes, 2005).
O táxon Amphipoda é bastante diversificado, incluindo Gammaridea, Hiperiidea,
Caprellidea e Ingofiellidea (Ruppert e Barnes, 2005). Os Gammaridea formam um grupo muito
grande, predominando espécies marinhas, distribuídas em 69 famílias. Existem ainda
representantes na água doce e uma única família (Talitridae) que reúne espécies terrestres. No
Rio Grande do Sul, entre ambientes límnicos e marinhos são encontrados representantes de oito
famílias de Gammaridea, das quais se destacam Corophidae, Stenothoidae, Hyalidae,
Ischyriceridae, Gammaridae, Talitridae, Dogielinotidae (Bento e Buckup, 1999) e Dogielinotidae
(González et al.,2005).
O gênero Hyalella, pertencente à família Dogielinotidae, é encontrado em uma série de
habitats de água doce, como reservatórios permanentes, lagos, tanques e riachos estando muitas
vezes aderidas à vegetação, nadando nas colunas d’água ou em buracos cavados no sedimento,
9
sendo importantes membros da fauna bentônica (Kruschwitz, 1978; Wellborn, 1995; Grosso e
Peralta, 1999). No Rio Grande do Sul, há registros de ocorrência de seis espécies deste gênero,
sendo uma delas a Hyalella castroi González et al., (2007) encontrada no município de São José
dos Ausentes, na localidade do Vale das Trutas (González et al., 2005.).
O dimorfismo sexual das espécies de Hyalella é caracterizado pela presença do segundo
par de gnatópodos alargados nos machos. O segundo par de gnatópodos dos machos são usados
para o manuseio das fêmeas durante o comportamento de cópula e os primeiros pares pequenos
são utilizados para carregá-las. As fêmeas maduras de espécies de Amphipoda são facilmente
identificadas pelos seus ovários desenvolvidos, os quais são externamente visíveis, pela presença
de um marsúpio e pela presença de ovos dentro do marsúpio (Kruschwitz, 1978).
Alguns aspectos dos crustáceos, principalmente as estratégias reprodutivas, podem ser
importantes para a interpretação de dados sobre estudos de bioindicação e para o
desenvolvimento de estudos ecotoxicológicos, assim como para programas de conservação. Não
somente os aspectos reprodutivos, mas também outras respostas comportamentais dos crustáceos,
como as mudanças na alimentação, na locomoção ou no comportamento de pré-cópula podem
providenciar respostas preditivas com respeito à bioindicação de toxicidade em determinado
ambiente (Rinderhagen et al., 2000).
A reprodução é um período crítico no ciclo de vida dos animais e está intimamente
relacionada com a capacidade reprodutiva, definida como uma porção das energias corporais
direcionadas para esse propósito. Em crustáceos, a fecundidade é caracterizada como o número
total de ovos no ovário, marsúpio ou externamente presos aos pleópodos das fêmeas (Somers,
1991). Kinne (1961), no entanto, considera ovos, como todas as fases entre a liberação dos
ovócitos e todos os estágios de desenvolvimento subseqüentes anteriores a formação do olho no
embrião. A fecundidade de uma espécie pode ser relacionada ao tamanho ou peso do animal
(Ogawa e Rocha, 1976; Du Preez e McLachlan, 1984; Powell, 1992), a fatores ambientais
(Jensen, 1958), a variações latitudinais (Jones e Simons, 1983), a taxa de sobrevivência das larvas
e/ou juvenis (Branco et al., 1992) e, possivelmente, as reservas metabólicas ou energéticas do
animal.
A biologia e a ecologia das espécies de Amphipoda do Rio Grande do Sul, ainda são
muito pouco conhecidas. Informações sobre a biologia de anfípodos são restritas a Hyalella
azteca, espécie que ocorre na América do Norte e México. Muitas espécies de Amphipoda, por
outro lado, por serem na sua maioria organismos bentônicos, são muito utilizados em testes de
10
toxicidade e bioensaios para avaliação da qualidade do sedimento dos ecossistemas aquáticos.
Este sedimento serve ao mesmo tempo, como depósito e fonte de matéria orgânica e inorgânica,
pelo fato de sua camada superficial ser mais permanente que a coluna de água, servindo,
portanto, como melhor testemunho das atividades ocorridas recentemente na bacia hidrográfica
(Wetzel, 1983; Burton, 1991). Estudos recentes demonstraram que a espécie Corophium
volutator (Crustacea, Amphipoda), também típica do sedimento está sendo usada na expressão da
toxicidade do sedimento em ambientes límnicos (Gerhardt et al., 2005). Contudo, o número de
espécies padronizadas em testes de toxicidade permanece limitado e na sua maioria são utilizados
organismos alóctones, especialmente a espécie Hyalella azteca, contrastando com a riqueza
taxonômica da maioria dos ecossistemas naturais de nosso País (Brendonck e Persoone, 1993).
O estudo do metabolismo intermediário em crustáceos tem demonstrado grande
variabilidade inter e intra-espécies, o que torna difícil a determinação de um perfil metabólico
padrão. Desconsiderando as diferenças entre os métodos bioquímicos empregados pelos diversos
autores esta variabilidade pode ser atribuída a fatores múltiplos, tais como seu habitat (terrestre,
marinho, estuarino ou de água doce), estágio do ciclo de muda, maturidade sexual (especialmente
em fêmeas), estado alimentar, dieta oferecida e sazonalidade, visto que estes fatores determinam
um padrão diferencial de resposta metabólica (Oliveira et al., 2003).
Dados da literatura sobre o metabolismo de carboidratos em crustáceos confirmam a
presença das vias de glicogênese, de glicogenólise e de glicólise em diferentes tecidos (Meenaski
e Scheer, 1968; Chang e O’Connor, 1983). As brânquias, os hemócitos, o músculo e o
hepatopâncreas têm sido propostos como sítios para ocorrência da via gliconeogênica (Johnston e
Davies, 1973; Thabrew et al., 1971; Oliveira e Da Silva, 1997).
Os principais tecidos de reserva de glicogênio em crustáceos são os músculos, o
hepatopâncreas, as brânquias e os hemócitos, porém o local de armazenamento deste
polissacarídeo vária de acordo com a espécie (Parvathy, 1971; Johnston e Davies, 1972; Herreid
e Full, 1988). O glicogênio armazenado é utilizado nos processos de muda, hipóxia e/ou anoxia,
osmorregulação, crescimento, diferentes estágios de reprodução e durante períodos de jejum
(Chang e O’Connor, 1983; Kucharski e Da Silva, 1991a; Kucharski e Da Silva, 1991b; Oliveira
et al., 2001 e 2004).
Segundo Chang e O’Connor (1983) a glicose é o principal monossacarídeo presente na
hemolinfa de crustáceos, tendo seis destinos principais: a síntese de mucopolissacarídeos, a
síntese de quitina, a síntese de ribose e nicotinamida adenina dinucleotídeo fosfato reduzido
11
(NADPH), a formação de piruvato e a síntese de glicogênio (Hochachka et al., 1970; Herreid e
Full, 1988).
Em crustáceos as concentrações de lipídios são bastante elevadas, apesar de não existir
um tecido adiposo diferenciado, os principais locais de armazenamento de lipídios são o músculo
e o hepatopâncreas (O’Connor e Gilbert, 1968; Chang e O’Connor, 1983; Herreid e Full, 1988;
Kucharski e Da Silva, 1991
a; Oliveira et al., 2006). Herreid e Full (1988) verificaram que os
níveis de lipídios no hepatopâncreas excediam em dez vezes os níveis de glicogênio. Nos
crustáceos, as sínteses de ácidos graxos, de diacilglicerol e de triacilglicerol são semelhantes
àquela dos mamíferos. Diversos estudos têm demonstrado que durante períodos de grande
demanda energética, como a muda e a gametogênese, ocorre uma marcante mobilização de
lipídios, principalmente aqueles presentes no hepatopâncreas (Kucharski e Da Silva, 1991b; Rosa
e Nunes, 2003a; Oliveira et al., 2006).
O músculo parece ser a principal fonte de proteínas nos crustáceos e, em decápodos os
níveis de aminoácidos livres nos tecidos atingem valores dez vezes superiores àqueles
encontrados em vertebrados. Diversos trabalhos sugerem que estes aminoácidos estariam
envolvidos nos processos de osmorregulação, estando principalmente ligados ao controle do
volume celular (Huggins e Munday, 1968; Gilles, 1982; Chang e O’Connor, 1983). As proteínas
são constituintes estruturais, funcionais e energéticos dos tecidos e tem um importante papel na
postura, fertilização e desenvolvimento normal dos embriões de crustáceos (Garcia-Guerrero et
al., 2003; Rodriguez-González et al., 2006). Outros estudos têm demonstrado variações no
conteúdo protéico durante o desenvolvimento ovariano de crustáceos, estas variações podem ser
resultado do aumento da biosíntese de muitas proteínas, incluindo enzimas, hormônios e
lipoproteínas envolvidas com a maturação gonadal (Yehezkel et al., 2000; Rosa e Nunes, 2003a e
b; Oliveira et al., 2006; Dutra et al., 2007a e b).
Muitos estudos têm descrito a influencia do jejum no metabolismo de proteínas, gorduras
e carboidratos em crustáceos mostrando uma grande variabilidade interespecífica (Marsden et al.,
1973; Vinagre e Da Silva, 1992; Hervant et al., 1999; Hardy et al., 2000; Hervant e Renault,
2002; Vinagre e Da Silva, 2002; Oliveira et al., 2004). Apesar das diferenças ente os métodos
analíticos utilizados pelos diferentes autores e o período de jejum, múltiplos fatores unidos a
peculiaridades biológicas e ecológicas das diferentes espécies provavelmente contribuem para a
diversidade observada. Um fator, que parece não ser levado em conta nas pesquisas publicadas
12
sobre jejum em crustáceos, é o conteúdo de proteínas ou carboidratos da dieta que estes recebem
antes do período de deprivação alimentar (Oliveira et al., 2004).
Variações sazonais determinam um efeito profundo na composição bioquímica dos
organismos, com modificações observadas na dinâmica e nos níveis de lipídios totais durante o
ciclo reprodutivo principalmente, no tecido gonadal e hepatopancreático tendo sido analisadas em
algumas espécies de Brachyura (Pillay e Nair, 1973) e em outros decápodos (Read e Caulton,
1980; Castille e Lawrence, 1989; Rosa e Nunes, 2003a). Trabalhos desenvolvidos, em nosso
laboratório, com Hyalella curvispina, Hyalella pleoacuta e Hyalella castroi, anfípodos
característicos da região de planície (Hyalella curvispina) e de planalto (Hyalella pleoacuta e
Hyalella castroi) do Rio Grande do Sul, nos permitem verificar, até este momento, um perfil de
resposta sazonal do metabolismo de carboidratos, de proteínas e de lipídios; sendo que estes
resultados parecem estar correlacionados às condições ambientais, a atividade dos animais e ao
período reprodutivo. Observou-se, ainda, que machos e fêmeas diferem quanto ao perfil de
resposta anual dos níveis de lipoperoxidação nas diferentes espécies; onde a lipoperoxidação
parece estar fortemente associada a comportamentos reprodutivos (Dutra et al., 2007a, b).
Os radicais livres são continuamente produzidos pela fosforilação oxidativa e por outros
sistemas biológicos reagindo rapidamente com a maioria das moléculas orgânicas. Estes radicais
podem reagir com lipídios de membrana, proteínas, DNA e também glicídios (Meerson et al.,
1981). Quando reagem com os lipídios de membrana, causam a lipoperoxidação destes, através
de uma série de reações, com conseqüente formação de malondialdeído e outras substâncias que
quando, aquecidas na presença de ácido tiobarbitúrico, formam um composto rosado, medido
espectrofotometricamente (Buege e Aust, 1978; Ohkawa et al., 1979; Llesuy et al. 1985;
Halliwell e Gutteridge, 1995). Sabe-se da literatura especializada que o aumento do estresse
oxidativo, principalmente, em períodos da alta demanda energética, pode aumentar
proporcionalmente, a formação de espécies reativas ao oxigênio e com isto, aumentar a
ocorrência do dano oxidativo (Viarengo et al., 1991; Correia, 2002; Correia et al., 2003;
Timofeyev et al., 2006). Portanto, a utilização e a padronização de uma medida de dano oxidativo
(TBARS) em crustáceos podem refletir alterações biológicas, como as que ocorrem no período
reprodutivo e ou por alterações no meio ambiente.
Díaz-Muñoz et al., (1985) mostraram no córtex cerebral de ratos que a atividade
enzimática da glutationa diminui durante a noite quando o córtex cerebral sofre um aumento na
lipoperoxidação por causa do ritmo de atividade motora e alimentar. Já Fanjul-Moles et al.,
13
(2003) verificaram que a lipoperoxidação no hepatopâncreas do lagostim Procambarus clarkii
não é determinada desta forma, apesar deste animal ser noturno e o ritmo respiratório produzir
radicais no período da noite, coincidindo neste animal com um aumento dos níveis da glutationa
oxidase e da atividade de outros protetores.
A enzima Na
+
K
+
ATPase utiliza a energia derivada da hidrólise do ATP para bombear
para fora da células 3Na
+
transferindo 2K
+
da parte externa para o citosol (Shepherd, 1994);
funcionando assim como um antiporter, sendo um importante instrumento para restaurar o
gradiente iônico nas células nervosas seguindo períodos de atividade elétrica como impulsos
nervosos e potenciais sinápticos (Shepherd, 1994). Esta enzima dimérica existe em diversas
isoformas no cérebro e consome grande parte do ATP disponível (Shepherd, 1994; Bertorello et
al., 1991). Em crustáceos a Na
+
K
+
ATPase exerce um importante papel na manutenção de
gradientes iônicos entre o meio interno (animal) e o habitat; sendo por isto fundamental para a
sobrevivência de animais osmorreguladores. Contudo, poucos estudos sobre esta enzima são
encontrados em crustáceos dulce-aquícolas (Gilles, 1982; Castilho et al., 2001). Esta enzima de
membrana requer fosfolipídios para sua atividade e é altamente vulnerável ao dano oxidativo
visto que sob tais circunstâncias observa-se uma inativação que pode envolver o rompimento dos
fosfolipídios do microambiente da enzima ou danos diretos à proteína causados por radicais do
oxigênio ou por produtos gerados na lipoperoxidação (Fleuranceau-Fleuranceau-Morel et al.,
1999; Lehtosky et al., 1999).
Sabe-se que fatores abióticos como a temperatura e a dureza da água podem influenciar
no cultivo dos organismos (Lewis e Marki, 1981; Persoone et al., 1989), entretanto, dentre todas
as variáveis a dieta a qual os organismos estão submetidos tem se mostrado como fator
determinante no seu desenvolvimento (Kersting e Van der Leeuw, 1976; Lewis e Marki, 1981;
Vijverberg, 1989; Lei et al. 1990; Kawbata e Urabe, 1998; Beatrici, 2000). Beatrici (2000), ao
comparar a resposta de Daphnia similis a três diferentes dietas constatou que os indivíduos
mantidos com uma dieta combinada de alga (Selenastrum caprcornutum) com um complemento
alimentar a base de artêmia reproduziam significativamente mais do que quando cultivados
apenas com algas. Platte (1993) objetivando alcançar uma forma de aumentar a produtividade dos
cultivos de Ceriodaphnia dubia obteve resultados semelhantes ao testar o complemento alimentar
a base de artêmia como uma forma de incrementar a dieta a base de algas dos organismos.
A importância na quantidade e na qualidade do alimento fornecido pode ser avaliada
através do número de filhotes produzidos nos cultivos, uma vez que a dieta pode influenciar
14
diretamente na capacidade reprodutiva dos indivíduos. Herbert (1978) ao estudar o gênero
Daphnia, constatou que o número de neonatos produzidos por fêmeas ovígeras depende
diretamente de sua alimentação. O número de filhotes, juntamente com a sensibilidade de um
organismo a uma substância de referência e o teor de lipídios acumulados são critérios que
podem ser adotados para a avaliação da qualidade do cultivo de organismos utilizados em ensaios
ecotoxicológicos (Zagatto, 1988).
Em um caranguejo estuarino muito estudado, Chasmagnathus granulata, diferentes dietas
alteram significativamente as concentrações de glicose, glicogênio e lipídios nos tecidos e na
hemolinfa deste crustáceo sendo seus níveis correlacionados positivamente com a concentração
de carboidratos da dieta (Kucharski e Da Silva, 1991a). Hernandez-Vergara et al., (2003)
avaliaram o efeito de diferentes concentrações de lipídios em dietas artificiais oferecidas para o
parastacídeo Cherax quadricarinatus, e concluíram que os machos investem suas reservas
metabólicas para o crescimento, enquanto que as fêmeas, com um alto índice hepatossomático,
investem no desenvolvimento gonadal e vitelogênese.
Dutra et al. (2007c) estudando o lagostim de água doce, Parastacus brasiliensis,
verificaram que independente da dieta oferecida (rica em carboidratos ou rica em proteínas)
mantidos em condições controladas de laboratório (temperatura e fotoperíodo) por 15 dias as
marcas metabólicas trazidas de campo pelos animais não são perdidas estando os níveis dos
diferentes metabólitos relacionados principalmente ao período reprodutivo. Padrão semelhante
foi encontrado por Ferreira et al. (2005) com as mesmas dietas, porém estudando o anomura de
água doce Aegla platensis.
2. Justificativa
Os crustáceos são freqüentemente utilizados como bioindicadores e biomonitores em
vários sistemas aquáticos, alguns aspectos deste grupo como as estratégias reprodutivas e as
respostas comportamentais (mudanças na alimentação, na locomoção ou no comportamento de
pré-cópula) podem ser importantes para a interpretação de dados sobre estudos da bioindicação e
para o desenvolvimento de estudos ecotoxicológicos (Rinderhagen et. al., 2000). Alguns estudos
desenvolvidos com crustáceos, especialmente anfípodos, têm demonstrado a importância da
determinação de parâmetros bioquímicos como biomarcas em estudos de ecotoxicologia e
monitoramento ambiental (Brendonck & Persoone, 1993; Hebel et al., 1997; Dutra et al., 2007d).
Tendo em vista a crescente preocupação com as alterações provocadas no ambiente
aquático resultante das diversas atividades humanas cresce também, a utilização de
15
bioindicadores e de testes de toxicidade ou bioensaios para avaliação destes impactos. Vários
organismos são utilizados nestes testes, como algas, microcrustáceos, poliquetos, oligoquetos,
larvas de insetos e peixes (Plate 1993). Entre os critérios para a seleção destes organismos,
destaca-se um amplo conhecimento da distribuição da espécie, localização dentro da estrutura
trófica, conhecimento da biologia, hábitos nutricionais e fisiologia, manutenção e cultivo em
laboratório (Environmetal Protection Agency-EPA, 1989).
Em Amphipoda, a rápida adaptação às condições de laboratório, com eventos
reprodutivos em um mesmo período, desenvolvimento embrionário rápido, elevadas densidades
sob as quais são encontrados em seu habitat, a fácil determinação do sexo e o tamanho dos
espécimes, facilitam observações de seu ciclo de vida (Krushwitz, 1978; Pennak, 1953; Cooper,
1965; Borowsky, 1991; Duan et al., 1997). Neste sentido, esta pesquisa visa contribuir para o
conhecimento de aspectos do metabolismo energético e da reprodução de Hyalella castroi em
laboratório, estabelecendo uma dieta adequada para a manutenção desta espécie em cultivo;
fornecendo assim, subsídios de cunho bio-ecológico e fisiológico que permitam o uso destes
animais autóctones como modelo experimental em estudos futuros de toxicologia e de
monitoramento ambiental.
16
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19
Aspects of the Energetic Metabolism and Reproduction of the Hyalella castroi González,
Bond-Buckup & Araujo (Crustacea, Amphipoda, Dogielinotidae) Mantained in
Experimental Culture with Different Diets
Fernanda Severo Gering
1
, Luiz Ferrúa Farias de Oliveira
1
, Bibiana Kaiser Dutra
1
, Guendalina
Turcato Oliveira
1#
1
Laboratório de Fisiologia da Conservação, Faculdade de Biociências, Programa de Pós-
Graduação em Biologia Celular e Molecular, PUCRS
Correspondence to:
#
Dra. Guendalina Turcato Oliveira
Pontifícia Universidade Católica do Rio Grande do Sul
Faculdade de Biociências
Depto. de Ciências Morfofisiológicas
Laboratório de Fisiologia da Conservação
Avenida Ipiranga, 6681 Pd. 12, Bloco C, Sala 266
CP. 1429, Zip Code: 90619-900
Porto Alegre, RS, Brazil
Telephone: 33203545 (4342)
Fax:3320-3612
E-mail: [email protected]
Keyword: Crustacea, Amphipod, Energy Metabolism, Hyalella castroi, Diets, Lipoperoxidation,
Na
+
/K
+
ATPase activity
20
Abstract
Was compared the effect of different diets in the energy metabolism (total lipids,
cholesterol, proteins and glycogen), in the levels of lipoperoxidation and activity of
Na
+
/K
+
ATPase of Hyalella castroi. We also investigated some patterns of the life cycle like
survival, formation of reproductive precopulatory mating pairs and number of ovigerous females
after 21 days of cultivation with different diets. These crustaceans live in limnetic environments
of the plateau (1200m a.s.l) of the state of Rio Grande do Sul, in southern Brazil. The animals
were collected in the autumn of 2006 in São José dos Ausentes. In the laboratory, the animals
were kept submerged in aquariums, separated by sex, under controlled conditions and were fed
ad libitum, for 21 days with different diets. At the end of this period, the animals were
immediately frozen for determination of biochemical parameters and enzyme. Statistical analysis
(ANOVA) revealed significant differences in biochemical composition between the sexes and
diets. These diets changed the biochemical patterns of the animals taken from the natural
environment, determine a high survival rate, and not improve the reproduction (fecundity and
egg quality), these points may be more investigate. In both sexes showed metabolic and
reproductive response more adequate when cultivated with diet 1, which was have more
carbohydrate (43.19g/100g) and less protein (30.88g/100g) that the diet 2 (carbohydrate =
28.99g/100g and protein = 39-78g/100g).
21
Introduction
Members of the genus Hyalella are common in the Nearctic and Neotropical regions, with
51 described species (González & Watling, 2001). They are found in a variety of freshwater
habitats, such as permanent reservoirs, lakes, impoundments, and streams, and often cling to the
vegetation, swim in the water, or burrow in the sediment, where they are important members of
the benthic fauna (Kruschwitz, 1978; Wellborn, 1995; Grosso & Peralta, 1999). In the Rio
Grande do Sul, Brazil, occurred six species of this genus, one this is Hyalella castroi González,
Bond-Buckup and Araujo (2007) found in the municipally of São José dos Ausentes (1200m
a.s.l., in the region of Aparados da Serra), in Vale das Trutas, Rio Grande do Sul (González et al.,
2005.)
Aquatic organisms exist in a constantly fluctuating habit, with changes in photoperiod,
temperature, pH, dissolved organic matter, dissolved oxygen and quality of the food and food
supply (Reid and Wood 1976). Organisms must alter of their physiological and biochemical
processes in order to cope with these changes.
Carbohydrates are often included in crustacean artificial diets for their protein-sparing
effect. By supplying energy to support routine metabolism, a greater quantity of protein is
directed towards somatic growth (Shiau and Peng, 1992; Rosas et al., 2000). However, although
glucose is the main sugar circulating in the haemolymph of crustaceans, inclusion of this
monosaccharide in the diet of penaeid shrimps was associated with low growth rates, increased
mortality and poor protein conversion efficiencies (Abdel-Rahman et al., 1979; Rosas et al.,
2001; Cuzon et al., 2001).
In crustaceans, glycogen is stored mainly in the muscles, hepatopancreas, gills, and
hemocytes; however, the storage locations vary among different species (Parvathy, 1971;
Johnston & Davies, 1972; Herreid & Full, 1988). The stored glycogen is used in the processes of
22
change, hypoxia and/or anoxia, osmoregulation, growth, different periods during reproduction,
and during periods of starvation (Chang & O’Connor, 1983; Kucharski & Da Silva, 1991a;
Kucharski & Da Silva, 1991b; Rosa & Nunes, 2003a; Oliveira et al., 2001 and 2004).
Several studies have evaluated maturation, eye ablation (Sagi et al., 1997; Wongprasert et
al., 2006), use of hormones (Abdu et al., 2001), fecundity (King, 1993), and reproductive cycle
(Villarreal et al., 1999; Serrano-Pinto et al., 2004) of the redclaw crayfish under laboratory
conditions. Different diets have been used in these studies. Diet plays an important role in
crayfish broodstock condition (Holdich, 2002). Broodstock nutrition is important for reproductive
success, because egg and larval production are strongly dependent on the diets offered (Bromage,
1995; Harrison, 1997; García-Ulloa, 2000). Protein is the most critical ingredient in practical
diets, because it is expensive and growth responses are affected (Cortés-Jacinto et al., 2003;
Thompson et al., 2005). According to Harrison (1997), the amount of protein required in
broodstock diets for maturation and production of eggs is higher than the level required for
growth, because gonad maturation is a process of intense protein synthesis, mainly during
vitellogenesis (Abdu et al., 2000).
The muscle is apparently the main protein-storage location in crustaceans. In decapods,
free amino acids in the tissues reach levels ten times higher than those observed in vertebrates.
Several studies suggest that these amino acids may participate in osmoregulation, and in the
control of cellular volume (Gilles, 1982; Chang and O’Connor, 1983; Schein et al., 2004). Other
studies have demonstrated a variation in protein content during ovarian development in
crustaceans. These variations may result in increased synthesis of several proteins, including
enzymes, hormones, and lipoproteins involved in gonad maturation (Yehezkel et al., 2000; Rosa
and Nunes, 2003b).
23
Lipid concentrations are high in crustaceans, although they have no differentiated adipose
tissue but store lipids mainly in muscle tissue and in the hepatopancreas (O’Connor and Gilbert,
1968; Chang and O’Connor, 1983; Herreid and Full, 1988; Kucharski and Da Silva, 1991a;
Oliveira et al., 2006). During periods of high energy demand, such as molting and
gametogenesis, large amounts of lipids are mobilized, especially from the hepatopancreas
(Kucharski and Da Silva, 1991a; Rosa and Nunes, 2003a; Oliveira et al., 2006). Herreid and Full
(1988) observed that lipid levels in the hepatopancreas were higher than the levels of glycogen.
Malondialdehyde, a breakdown product of lipid endoperoxides, is an expression of lipid
peroxidation and has been used with success in aquatic invertebrates as a general indicator of
toxicant stress derived from various types of contamination (Zwart et al., 1999; Livingstone,
2001; Wilhelm Filho et al., 2001; Timofeyev et al., 2006). Neuparth et al., 2005 described that in
Gammarus locusta maintained with sediments have high levels of organic matter content present
higher levels of lipoperoxidation. Effectively, some authors agree that endogenous variables like
nutritional status, age, sex, growth and reproduction influence the peroxidation status of
organisms (Viarengo et al., 1991; Correia, 2002; Correia et al., 2003).
Some studies have reported that the peroxidation of membrane phospholipids induced by
reactive oxygen species and/or free radicals leads to alterations in the membrane structure and
functions (Halliwell and Gutteridge, 1986; Vercesi et al., 1997). These degenerative changes can
affect dynamic properties of the membranes such as fluidity and permeability, and consequently
the activity of various membrane-associated enzymes (Meccoci et al., 1997). Several
investigators have reported that lipid peroxidation products disrupt neuronal ion homeostasis by
impairing the function of membrane-bound ion-motive ATPases such as Na
+
/K
+
ATPase (Keller
et al., 1997; Mark et al., 1997).
24
Dutra et al. (2007b), working with H. curvispina (Shoemaker 1942), suggest that the lipid
reserves seem to be an important source of energy used during reproduction, in both males and
females; whereas glycogen and proteins may be used during periods of intense activity or intense
variation in environmental conditions. This correlation was found too by Chang & O’Connor
(1983), Kucharski and Da Silva (1991a), Rosa and Nunes (2003b) and Oliveira et al. (2006) in
their works with other crustaceans. Dutra et al. (2007b) showed in H. curvispina that levels of
lipoperoxidation may be related to reproductive behavior, motor and feeding activity, and
variation of the photoperiod.
The amphipods species are benthonic organisms, for this they are utilized for toxicity tests
and bioassays for evaluation of the water quality of the sediment of the aquatic ecosystem.
Recently studies demonstrated that the specie Corophium volutator (Crustacea, Amphipoda),
tipic of the sediment, is adjusted to expressed of toxicity of the sediment in liminic environments
(Gerhardt et al. 2005). However, the number of the species patronized for the toxicity tests are
limited and the major are alloctone organisms, specially the specie Hyalella azteca, in contrast
with the taxonomic richness of the natural ecosystems in Brazil (Brendonck and Persoone, 1993).
The aim of the present work was to characterize the response of the intermediate
metabolism (total lipids, cholesterol, proteins, and glycogen), of the levels of lipoperoxidation
(TBARS) and of the activity of Na
+
K
+
ATPase in Hyalella castroi maintained in experimental
culture with two different diets. We also investigated some patterns of the life cycle like survival,
formation of reproductive couples and number of ovigerous females and juveniles eclosion for
patronization of this specie for future use to toxicity tests.
MATERIAL AND METHODS
The animals were cared for in accordance with guidelines such as the Guide for the Care
and Use of Laboratory Animal (1996, published by National Academy Press, 2101 Constitution
25
Ave. NW, Washington, DC 20055, USA) and Brazilian laws. The animal were used with the
permission of the Ethic Committee of the Pontifícia Universidade Católica do Rio Grande do Sul
(License 06/03423).
Description of the collection place:
The animals were collected between April to June of 2006 (autumn) in a stream in the São
José dos Ausentes, RS (28°47’00”S – 49°50’53”W), this place is characterized by little
anthropogenic influence. Three periods of collect (April, May and June) permit that were use
animals of the same population, but not the same generation, this form we have sure that results
are significantly for all population. Animals and macrophytes (Callitriche rimosa) were collected
by means of fish traps and bottom grabs in same hour of the day.
The animals were transported in cold water (5°C) in insulated containers to the
Laboratory of Conservation Physiology of PUCRS (Pontifícia Universidade Católica do Rio
Grande do Sul), where they were separated by sex and placed in aerated aquariums for 24 hours
without food.
In order to characterize the collect place the following abiotic parameters were measured
during months of collect: pH, water temperature and hardness of the water. pH was determined
with a portable pHmeter (Quimis/400H), and water temperature with a thermometer of internal
scale. The hardness of the water was determined using a classic method of volumetric
complexation (Adad, 1982).
Experimental procedure:
After this 24-hour period, the animals were kept submerged in aerated aquariums, in
density of 1 animal per liter of water, with an average temperature of 23±1ºC and a photoperiod
of 14:10 hours light/dark. The amphipods were divided into two groups, which were fed ad
libitum in late afternoon, when most of the animals were active, for a period of 21 days. Males
26
and females stay in the aquariums during 21 days separated with a nylon fabric, but stay in
chemical contact, because the water passed by the two parts of aquarium. They were fed one of
two diets, the first group (Diet 1) received macrophytes and ration for fishes (ALCOM: fresh
shrimp, fish flour, soy protein hydrolizated, corn cream, wheat flour, marine algae flour,
dehydrated carrot, leavenings, soy oil, vitamin C, mineral vitaminic supplement, inorganic
minerals, additives to pigment and antioxidant BHT), and the second group (Diet 2) received
macrophytes and commercial ration for fishes with add spiruline algae (ALCOM: fish flour, soy
protein hydrolizated, corn cream, wheat flour, marine algae flour, leavenings, soy oil, vitamin C,
mineral vitaminic supplement, inorganic minerals, dehydrated spinach, antioxidant BHT spiruline
and prebiotic additive).
The centesimal composition of these rations was determined by ICTA (Instituto de
Ciência e Tecnologia dos Alimentos) of the UFRGS (Universidade Federal do Rio Grande do
Sul) and showed in Table 1, diet 1 and diet 2 are isocaloric. After 7, 14 and 21 days of
experimental culture a group of each diet of each sex was cryoanesthetized, weighed on an
electronic balance (± 0.001), and then stored frozen at -80°C until they were used to determine
the biochemical parameters.
Reproductive parameters
After of the period of 24 hours, 10 males and 10 females were distributed in each
aquarium of the 20 liters in a total of the six aquarium and 60 animals (three aquariums for the
diet 1 and three for the diet 2); in this experiment we were permit the physical contact between
male and female. The animals were observed every day, during 21 days, and the number of the
couples and ovigerous females was quantified (Plaistow, 2003).
Survival and Mortality
27
The survival and the mortality of the animals during the experimental cultures were
registered.
Biochemical Analyses
Metabolites
Metabolic determination for H. castroi was done in total homogenates of three pools of
twelve males and twelve females each. One pool was used for determination of glycogen and
proteins, the second pool for quantification of lipids and cholesterol, and the third pool for
quantification of lipoperoxidation levels. Metabolic parameters were determined in quintuplicate
by used spectrophotometric methods.
a. Glycogen was extracted from tissues following the method described by Van Handel 1965, and
glycogen levels in the animals were determined as glucose equivalent, after acidic hydrolysis
(HCl) and neutralization (Na
2
CO
3
), following the method of Geary et al. 1981. Glucose was
quantified using a Biodiagnostic kit (glucose-oxidase). Results are presented as in mmol/g of
animal.
b. Proteins were quantified as described by Lowry et al. 1951, with bovine albumin (Sigma Co.)
as the standard. Results are expressed mg/ml of homogenate.
c. Lipids were extracted from tissue homogenized with an Omni Mixer Homogenizer in a 2:1
(v/v) chloroform-methanol solution, according to Folch et al. 1957. Total lipids in this
homogenate were determined by the sulfophosphovanillin method (Meyer and Walter 1980).
This method consists of oxidizing cellular lipids to small fragments after chemical digestion with
hot concentrated sulfuric acid. After the addition of a solution of vanillin and phosphoric acid, a
red complex is formed witch is measured with spectrophometer (530nm). The levels of total
cholesterol were measured by the reactions of cholesterol esterase, cholesterol oxidase and
peroxidase enzymes (Labtest Kit/Liquiform). Results are expressed as mg/g of animals.
28
d. Lipoperoxidation levels were quantified by the method of Buege and Aust (1978) by
measuring reactive substances to Thiobarbituric Acid (TBA-RS), using the extraction method of
Llesuy et al. (1985). Results are expressed in nmol of TBARS/mg of protein.
Activity of Na
+
/K
+
-ATPase
The membrane was extracted from five animals, according to Barnes (1993). The pool
were homogenized (10% W/V) in cold buffer Tris (40mM) and phenylmethylsulfonyl fluoride (1
mM; from Sigma, St. Louis, MO) with pH adjusted to 7.40. The homogenate was centrifuged at
10000Xg at 4°C, and the supernatant was collected and centrifuged at 40.000Xg (4°C). The pellet
was resuspended in the same buffer and centrifuged again at 40.000Xg (4°C). This last
supernatant was then used as the source of Na
+
/K
+
ATPase. Na
+
/K
+
ATPase activity was measured
according to the method described by Esmann (1988) adjustment by Dutra et al. (2007d).
Incubation medium A contained ATP (5 mM; from Sigma), NaCl (60 mM), KCl (10 mM) and
MgCl (40mM), with the pH adjusted to 7.40. In the incubation medium B, KCl was replaced by
ouabain (1 mM; from Sigma). Aliquots of homogenate were incubated at 30°C in both mediums
A and B, for 30 min with the equivalent of 10 mg of the proteins. The enzymatic reaction was
stopped by addition of 10% trichloroacetic acid. The inorganic phosphate released was
determined using the method of Chan (1986), in a spectrophotometer at 630 nm. Any difference
in phosphate concentration between medium A and B was attributed to Na
+
/K
+
ATPase activity.
All determinations were done in quadruplicate. Results are expressed in µmol of the Pi.mg of
protein
-1
.min
-1
.
Statistical Analysis
The results are expressed as mean ± standard error. For statistical analysis of the different
periods of experimental culture, a one-way ANOVA test was used, followed by a Bonferroni test.
For comparisons between different diets and sexes, a two-way ANOVA was used. The
29
comparisons of experimental culture with dates of the natural environmental and the number of
ovigerous females between different diets were did with Student’s T test. All the metabolic
parameters were homogeneous (Levene test), and were normally distributed (Kolmogorov-
Smirnov test). The significance level adopted was 5%. All the tests were done with the program
Statistical Package for the Social Sciences (SPSS- 11.5) for Windows.
Results
Abiotic Conditions Analyses
The environmental and experimental culture abiotic factors are present in Table 2. When
compared the temperature of the environmental with the experimental culture occurred a
significant difference, because the temperature of the collected local was lowest in relation of the
experimental culture temperature. The pH and hardness of water were constant in both situations,
in environment pH was 7.09 ± 0.21 and in experimental culture 7.00 ± 0.40, already the hardness
of the water in environment was 1.12 ± 0.52 ppm of CaCO
3
and in experimental culture 0.98 ±
0.46 ppm of CaCO
3.
Reproductive parameters
The number of the couples (paired formed) and the ovigerous females feeding with diet 1
and diet 2 are present in Table 3. The amphipods feeding with diet 1 paired 22 % more than the
animal maintained with the diet 2. The females feeding with diet 1 showed a higher indice of
ovigerous females than the females maintained with the diet 2, however in both cultures, the
maximum period of the females showed eggs was 4 days, after this period not was observed
juvenis eclosion in aquariums. Although, the number of ovigerous females was low in relation
the number of the couples formed in both diets.
Survival rate
30
The survival of the males and females feeding with diet 1 and with diet 2 are present in
Table 4. We verified that during period of experimental culture males and females feeding with
diet 1 showed survival rate 11.20% and 13.40%, respectively, higher than the animal feeding
with diet 2. However, there was no significant difference between the sexes feeding with the
same diet in none of type of diet. The survival rate in diet 1 to vary to 94.5%, until 98.20% and in
diet 2 the rate to vary of 83.64% until 89.10%, in both sexes considering the three experiments.
Metabolic parameters
Glycogen:
Figure 1A shows the glycogen concentration in males and females in the natural
environmental and cultivation with diet 1. Males of H. castroi cultivated by 7 days present a
glycogen levels higher (1.3 times) than the animals collected in natural environment, this levels
continued increasing until 14 days of culture, and after 21 days this polysaccharide was lower
than 14 days and environment. Already, in females was observed an increase in glycogen in 7
days of experiment, and this polysaccharide remain high until finish of the experiment (21 days).
There was a significant difference in the behavior of glycogen levels between males and females
feeding with diet 1 (p<0.05).
Glycogen content in males and females of H. castroi in the natural environmental and
maintained with diet 2 showed in the Figure 1B. Males feeding with diet 2 showed a peak (1.7
times) of glycogen in 7 days of experiment, and these levels decreased gradually until 21 days of
cultivation. In females was observed in 7 days a decrease of 20% in the content of glycogen, after
14 days of culture this polysaccharide increase (30%), and in the finish experiment the glycogen
levels returned to the values of 7 days. There was a significant difference in the behavior of
glycogen levels between males and females feeding with diet 2 (p<0.05).
31
There was a significant difference in the levels of glycogen during 21 days of cultivation
between males submitted to the different diets (p<0.05); the same pattern was found to females
feeding with different diets (p<0.05).
Proteins:
Protein concentrations in males and females in the environment and feeding with diet 1
are showed in Figure 2A. Males showed after seven days of cultivation an increase of 2 times in
levels of proteins, in 14 days was found a decrease of 40% in this values, and in the end of the
experiment (21 days) the content of proteins returned the levels of 7 days. Already, in females
the proteins were higher after 7 days of cultivation, although gradually decreased until 21 days
reaching levels lower than the environment and 7 days of culture. There was a significant
difference between total protein content in females and males maintained with the diet 1(p<0.05).
The levels of total protein in males and females in the environment and feeding with diet
2 are showed in Figure 2B. Males feeding with the diet 2 by 7 days showed values of total
proteins 2.2 times highest than the males of environment; these levels remained constant until 14
days and decrease after 21 days of experiment returned the values of the animals in natural
environment. In females, the levels of total proteins after 7 days of the diet 2 were similar to the
amphipods collected in natural environment, although these levels decreased significantly during
the time of culture (14 and 21 days). There was a significant difference between total proteins
content between males and females maintained with the diet 2 (p<0.05).
When was compared different diets we observed a significant difference of the behavior
of the total proteins during 21 days of the experiment, the same response was observed in females
(p<0.05).
Total Lipids:
32
The concentrations of total lipids in males and females in the environment and feeding
with diet 1 are shown in Figure 3A. Levels of this metabolite in males were lowest (p<0.05) after
seven days of culture and maintained stable until 14 days of experiment, after 21 days of feeding
this levels showed a decrease of approximately 2.2 times. Females present values of lipids lowest
than the animals of environment after 7 days, although after 14 days this levels was increase, and
these animals shows a new decreasing in their lipidic reserve in the end of the experiment (21
days).There was a significant difference between total lipid content of males and females
maintained in laboratory feeding with diet 1.
Total lipids content in males and females of H. castroi in the environmental and
maintained with diet 2 showed in the Figure 3B. The males collected in natural environmental
present a total lipids levels approximately 2.6 times highest (p<0.05) than the animals fed with
the diet 2 by seven days, while the females maintained by the same period no shows a
significantly difference in this metabolite (p>0.05). We observed that in males the level was
decreasing gradually until minimum values in 21 days of culture. Already, the females shows a
peak after 14 days of experiment, their levels are 3.7 times highest in relation the females
cultivate by 7 days, the levels of total lipids decreasing (2.2 times) in the end of the experimental
period (21 days), although these levels were highest. There was a significant difference in the
behavior of total lipids levels between males and females feeding with diet 2 (p<0.05). There was
no significant difference in the levels of total lipids in males submitted to the different diets
(p>0.05); but the females showed a significant difference between the group that received diet 1
and diet 2 (p<0.05).
Cholesterol:
The concentrations of cholesterol in males and females in the environment and feeding
with diet 1 are shown in Figure 4A. The males and females of the environment present a content
33
of total cholesterol 12.7 and 3.7 times, respectively, higher than the animals maintained in
laboratory with diet 1 by 7 days. When the levels of this metabolite are compared between the
experimental periods, we observed that in males the level was highest after 14 days of
experiment, but with 21 days of feeding they shows a turn back to the levels showed after seven
days of culture. The same response was observed in females during the experiment cultivation.
There was no significant difference between total cholesterol content of males and females
maintained in laboratory feeding with diet 1 (p>0.05).
The cholesterol content in males and females of H. castroi in the environmental and
maintained with diet 2 showed in the Figure 4B. The males and females collected in natural
environmental present a cholesterol levels approximately 13.3 and 1.8 times highest than the
animals fed with the diet 2 by seven days (p<0.05). When the levels of this metabolite are
compared during the periods of cultivation we verified that males no shows significant difference
between the levels of cholesterol (p>0.05). Already, in females after 14 days in experimental
culture was observed levels 1.8 times highest than of seven days, and cholesterol decreasing (7.0
times) in the end of the period (21 days). There was a significant difference in the behavior of
cholesterol levels between males and females feeding with diet 2 (p<0.05).
There was no significant difference in the levels of cholesterol in males submitted to the
different diets (p>0.05); but the females showed a significant difference between the group that
received diet 1 and diet 2 (p<0.05).
Levels of Lipoperoxidation
Figure 5A shows the levels of lipoperoxidation in males and females in the environmental
and maintained with diet 1. Males and females feeding to 7 days were present values of
lipoperoxidation lower that animals of the natural environment. After 14 days of experiment the
levels of lipoperoxidation decrease in males and increase in females. Already, in the 21 days of
34
culture this response was inverted, when in males the levels increased and in females the levels
decreased. There was a significant difference in the behavior of lipoperoxidation levels between
males and females feeding with diet 1 (p<0.05).
The levels of lipoperoxidation in males and females of H. castroi in the environmental
and maintained with diet 2 showed in the Figure 5B. The males and females collected in natural
environmental present a lipoperoxidation levels approximately 5.5 and 2.3 times higher,
respectively, than the animals fed with the diet 2 by seven days. When the levels of TBARS are
compared between the experimental periods, we observed after 14 days, that in males and
females the level was increase 2.4 and 2.0 times, respectively, and increase again after 21 days in
both sexes. There was no significant difference in the levels of lipoperoxidation between males
and females submitted to the diet 2 in the different times of culture (p>0.05). There was a
significant difference in the levels of lipoperoxidation in males submitted to the different diets
(p<0.05); the same pattern showed by males was found when was made the comparison between
females feeding with diet 1 and diet 2 (p<0.05).
Na
+
/K
+
ATPase activity
Na
+
/K
+
ATPase activity in males and females in the environment and feeding with diet 1
was showed in Figure 6A. The males after 7 days of diet 1 present value of Na
+
/K
+
ATPase
activity lowest (1.7 times) that the animals collected in natural environment, in the 14 days the
levels showed an increase and returned the initial values in 21 days of culture. Already in females
was observed an increase after seven days of culture with diet 1, the values were similar in 14
days, and this activity increased in the finish of the experiment (21 days).
Figure 6B shows the levels of activity of Na
+
/K
+
ATPase in males and females in the
environment and feeding with diet 2. The males collected in the environment showed values of
activity of Na
+
/K
+
ATPase 1.3 times highest than the males feeding with the diet 2 by 7 days, after
35
14 days of experimental culture was observed an increase of the 2.4 times in this activity, in 21
days the Na
+
/K
+
ATPase remained elevated in relation of 7 days. Already, in females were
verified an increase of 8.1 times in seven days of culture, the levels showed a gradually decrease
until 21 days of experiment, however this activity was higher in relation 7 days and environment.
There was a significant difference between levels of activity of Na
+
/K
+
ATPase of males and
females maintained with the diet 2 (p<0.05). There was a significant difference of the
Na
+
/K
+
ATPase activities values between males maintained with different diets; the same pattern
showed by males was found when was made the comparison between females feeding with diet 1
and diet 2 (p<0.05).
Discussion
Integral to the development of a diet for any species included the identification of their
requirements of protein, lipid and energy. Protein is required to provide the fundamental units
amino acids for growth, while dietary lipid provides both essential fatty acids and other some
forms of the energy needed for the metabolic processes of growth (D’Abramo et al., 1997).
Further energy can also be derived from the metabolism of protein and some dietary
carbohydrates. Most studies on nutrition of freshwater crustaceans are focused on proteins
(Cortés-Jacinto et al. 2003, 2004; Thompson et al.. 2004) and scarce information is available on
carbohydrates and lipids (Hernandez-Vergara et al. 2003). These two nutrients have important
roles not only as energy sources but also in development and reproduction of crustaceans. The
accumulation of energy reserves in species of crustaceans dependent upon unstable food
resources has been reported by several authors (Lee et al., 1971; Griffiths, 1977; Oliveira et al.,
2003; Rosa and Nunes, 2003b).
36
In this work diets (1 or 2) determined different responses to glycogen, total proteins,
lipoperoxidation levels and Na
+
/K
+
ATPase activity in both sexes these amphipods, and total
lipids and cholesterol levels only in females. The levels of glycogen and total lipids verified in
these amphipods, in both diets were similar to levels observed in other crustaceans feeding diet
rich in proteins (Kucharski and Da Silva, 1991; Ferreira et al., 2005). The results may suggest
that amphipods present a major content of proteins in natural diet.
Dutra et al. (2007c) reported that H. castroi explore the sediment predominantly, where it
finds more organic matter of animal origin, in which it can burrow, these activities require
different levels of energy consumption, as well as allowing consumption of organic substances,
although her feeding habits is unknown. For H. azteca, Hargrave (1970) reported that it is an
omnivorous deposit feeder, primarily feeding on algae and bacteria associated with the sediments
and aquatic macrophytes. It has been recorded feeding on dead animal and plant matter (Cooper
1965). Byrén et al. (2002) showed in two species of the amphipods Monoporeia affinis and
Pontoporeia femorata that the settled phytoplankton and detrital organic matter are considered
their main found source but bacteria, meiofauna and temporary meiofauna are also included in
the diet. Casset et al. (2001), studying Hyalella curvispina in a river in Argentina, suggest that
this amphipod is herbivorous, feeding mainly on the phytobenthos and occasionally on sediment.
In the present work we verified that animals that received diet 1 that have less protein
(30.88%) and more carbohydrate (43.19%) than diet 2 (protein 39.78% and carbohydrate
28.99%) present a higher rate of survival and number of couples. Although, we was observed in
both diets a lower number of ovigerous females and fertility (number of juveniles liberated by
females). In present work was not verified juveniles liberated by ovigerous females. These facts
can be related with a significant decrease in proteins reserves verified in females in both diets
and/or quantity of the proteins in diets (1 and 2) and /or higher temperature of the water in
37
aquariums. Castiglioni and Buckup (submitted) studying the reproductive strategies in H. castroi
in laboratory conditions (19°C and 12/12hours light/dark) showed a fecundity of the 7 to 42 eggs
and a fertility of the 16 to 36 juveniles per females. In this study developed to Castiglioni and
Bond-Buckup (2007) the animals were feed with macrophyte more fish food with 43% of the
protein.
The importance of the quantity and quality of the food provided can be evaluated through
the offspring produced in the cultures, because the diet can directly influence the reproductive
capacity of individuals. Herbert (1978), studying the genus Daphnia, observed that the number of
neonates produced by ovigerous females depends directly on the food ingestion. The number of
juveniles, together with the sensitivity of the organism to some reference substance and the
course of accumulation of lipids, can be adopted as criteria to evaluate the quality of the cultures
of organisms used in ecotoxicological bioassays (Zagatto, 1988).
According Goimier et al. (2006) studying Litopenaeus setiferus verified that in diet that
have protein in excess could produce a stress provoking loss of immune control, melanization and
loss of sperm quality change reproductive success. Other studies have been demonstrated that
protein in excess (45% of protein) provokes a reduction in growth rate in several shrimp species
(García et al., 1998; Sheen and Huang, 1998; Kureshy and Davis, 2002; Pascual et al., 2004). A
difference in protein catabolism and the stress provoked by changes in internal ammonia
concentration was related to the reduction in growth rate of shrimp fed protein in excess (Rosas et
al., 1995, 1996, 2001; Taboada et al., 1998).
In general, the lowest levels of glycogen in females and males feeding with diet 2 in
relation the animals feeding with diet 1 can be explain by lower percentage of carbohydrates
contained in this diet (diet 2). Where diet 1 present 43.19g/100g of carbohydrates and diet 2
38
present 28.99g/100g. Studies developed with other crustaceans, like C. granulata (Kucharski and
Da Silva, 1991), A. platensis (Ferreira et al., 2005) and P. brasiliensis (Dutra et al., 2007c) shows
that increase of the quantity of carbohydrate in diet change the homeostasis of glycogen when
authors compared with the animals that received a high-protein diet, determining an increase in
levels of this polysaccharide.
In crustaceans as in other animals, the regular functioning of organs such as the brain and
39
indicated that such a cost is more likely to result from pair formation than from the cost of
carrying the female, as had previously been assumed. Males benefit from precopulatory mate
guarding by maximizing their chances of fertilizing females’ eggs once they become receptive.
However, the optimal time a male spends guarding each female will depend upon the costs
associated with precopulatory mate guarding. Finally, precopulatory mate guarding may be
energetically costly (Robinson and Doyle 1985; Elwood and Dick 1990; Sparkes et al. 1996;
Jormalainen et al. 2001). Robinson and Doyle (1985) showed that in Gammarus lawrencianus
the feeding rates of males were reduced while they were in precopula, but female feeding rates
were unaffected. However, Sparkes et al.. (1996) showed that in the isopod Lirceus fontinalis
(Rafinesque-Schmaltz 1820), precopulatory mate guarding was associated with only a short-term
(<36 h) reduction in glycogen reserves, and there was no reduction when pairs were given access
to food.
In this work, male feeding with diet 1 and diet 2 maintained their proteins reserves similar
to natural environment after 21 days of the cultivation. Although, female feeding with both diets
showed a decrease in these reserves, however females that received diet 1 showed higher
decrease in protein levels. A major cost of energy to reproduction activity, principally, synthesis
of vittelin in females can be explained the lower quantity of protein, especially in diet 1.
Protein is an essential for tissue growth and maintenance, is an expensive component of
formulated diets (Cortés-Jacinto et al., 2003; Thompson et al., 2005). When insufficient energy is
available in a diet from non-protein sources, protein may be catabolized to meet the energy
requirements at the cost of nutrient supply somatic growth (Capuzzo and Lancaster, 1979;
Sedgwick, 1979). The most efficient diets contain sufficient non-protein energy sources (lipid
and carbohydrate) that are metabolized preferentially to protein to meet general energy
requirements, leaving an organism to direct the maximum level of available dietary protein into
40
growth (Sedgwick, 1979; Bautista, 1986). Barclay et al. (1983), working with Penaeus
esculentus, showed that during the period of starvation the abdominal muscle makes the largest
41
Dutra et al. (2007a) described that in H. castroi, that proteins, lipids and cholesterol were
depleted during precopula and copula (winter) because H. castroi, like other crustaceans, produce
large eggs. Egg size is related to maternal investment, mainly the lipid metabolism (Rosa and
Nunes 2003 a and b). Lipids are the main source of energy throughout embryonic development,
and the amount of lipids is generally correlated with the size of eggs and the time interval
between spawning and hatching (Petersen and Anger 1997; Rainuzzo et al. 1997). Cholesterol is
a vital component of cell membranes and is the precursor of bile acids, steroids, and molting
hormones. It is reported to be an essential nutrient for growth and survival of all crustacean
species (Abdel-Rahman et al., 1979).
The two diets determined different response in levels of the Na
+
/K
+
ATPase activities in
males and females and after 7 days of experimental culture already were possibly to observe
change in the environmental pattern for this enzyme In both diets, the females always showed
values of the Na
+
/K
+
ATPase activity higher that males, this fate can be related with more intense
degradation of the glycogen and total lipids. The animals feeding with diet 2 were present levels
of activity of Na
+
/K
+
ATPase higher in relation that animals feeding with diet 1 during the period
of the study (21 days).
The fact of the levels of Na
+
/K
+
ATPase activity was lower in animals feeding with diet 1
maybe can explain by the higher levels of lipoperoxidation. Some works reported that the
peroxidation of membrane phospholipids induced by reactive oxygen species and/or free radicals
leads to alterations in the membrane structure and functions (Halliwell and Gutteridge, 1986;
Vercesi et al. 1997). These degenerative changes can affect dynamic properties of the membranes
such as fluidity and permeability and consequently the activity of various membrane-associated
enzymes (Meccoci et al. 1997). Several investigators have reported that lipid peroxidation
42
products disrupt neuronal ion homeostasis by impairing the function of membrane-bound ion-
motive ATPases such as Na
+
/K
+
ATPase (Keller et al. 1997; Mark et al. 1997).
Dutra et al. (2007b) showed in females of H. castroi a peak of lipoperoxidation in
autumn. This season, antecede the peak of reproduction that occurred in winter (Araujo et al.
2005), the males showed too a peak of lipoperoxidation, during the period of precopulation and
copulation (autumn) because they consume more energy for precopulatory mate guarding and
carrying females during this period. However, these animals not pared in laboratory, but females
showed mature ovaries, we can be suggesting that they maintained the preparation for the
reproductive period.
In conclusion, our results showed that these diets changed the biochemical patterns of the
animals taken from the natural environment and but not can be improve the reproduction (fertility
and egg quality), these points may be more investigate. In both sexes showed metabolic response
more adequate when cultivated with diet 1, which was have more carbohydrate, less protein and
major quantity of protein of animal origin.
43
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50
Captions to Tables:
Table 1: Centesimal composition of the diets 1 and diet 2.
Table 2: Water temperature (°C), pH, and Hardness of water (ppm of CaCO
3
) in natural
environmental and experimental conditions. The results are expressed as mean ± standard error of
the mean.
Table 3: Number of the couples and ovigerous females of the Hyalella castroi maintained in
experimental culture with different diets.
Table 4: Indices of the survival of the males and females of the Hyalella castroi maintained in
experimental culture with different diets.
51
Captions to Figures:
Figure 1: Concentration of glycogen of Hyalella castroi in environment and maintained in
experimental culture. (A) Males and females feeding with diet 1; (B) Males and females feeding
with diet 2. Columns represent the mean, and bars represent the standard error of the mean.
Results are expressed in mmol/g. The same letter represents a significant difference between the
days of culture or environment. # a significant difference between sexes maintained with the
same diets. & represents significant difference between males feeding with different diets. *
represents significant difference between females feeding with different diets.
Figure 2: Concentration of total proteins of Hyalella castroi in environment and maintained in
experimental culture. (A) Males and females feeding with diet 1; (B) Males and females feeding
with diet 2.Columns represent the mean, and bars represent the standard error of the mean.
Results are expressed in mg/ml. The same letter represents a significant difference between the
days of culture or environment. # a significant difference between sexes maintained with the
same diets. & represents significant difference between males feeding with different diets. *
represents significant difference between females feeding with different diets.
Figure 3: Concentration of total lipids of Hyalella castroi in environment and maintained in
experimental culture. (A) Males and females feeding with diet 1; (B) Males and females feeding
with diet 2. Columns represent the mean, and bars represent the standard error of the mean.
Results are expressed in mg/g. The same letter represents a significant difference between the
days of culture or environment. # a significant difference between sexes maintained with the
same diets. * represents significant difference between females feeding with different diets.
Figure 4: Concentration of cholesterol of Hyalella castroi in environment and maintained in
experimental culture. (A) Males and females feeding with diet 1; (B) Males and females feeding
with diet 2. Columns represent the mean, and bars represent the standard error of the mean.
Results are expressed in mg/g. The same letter represents a significant difference between the
days of culture or environment. # a significant difference between sexes maintained with the
same diets. * represents significant difference between females feeding with different diets.
Figure 5: Levels of lipoperoxidation of Hyalella castroi in environment and maintained in
experimental culture. (A) Males and females feeding with diet 1; (B) Males and females feeding
with diet 2. Columns represent the mean, and bars represent the standard error of the mean.
Results are expressed in nmol of TBARS/mg of protein. The same letter represents a significant
difference between the days of culture or environment. # a significant difference between sexes
maintained with the same diets. & represents significant difference between males feeding with
different diets. * represents significant difference between females feeding with different diets.
Figure 6: Activity of Na
+
/K
+
ATPase of Hyalella castroi in environment and maintained in
experimental culture. (A) Males and females feeding with diet 1; (B) Males and females feeding
with diet 2. Columns represent the mean, and bars represent the standard error of the mean.
Results are expressed in µmol of Pi/min. mg of protein. The same letter represents a significant
difference between the days of culture or environment. # a significant difference between sexes
maintained with the same diets. & represents significant difference between males feeding with
different diets. * represents significant difference between females feeding with different diets.
52
Table 1
Compound Diet 1
Macrophyte and Ration 1
Diet 2
Macrophyte and Ration 2
Water content (g/100g) 5.30 7.26
Ashes (g/100g) 11.02 14.15
Protein (g/100g) 30.88 39.78
Fat (g/100g) 6.19 4.99
Fiber (g/100g) 3.59 4.83
Carbohydrates (g/100g) 43.19 28.99
Total Caloric Value (Kcal/100g) 351.59 319.99
53
Table 2
Abiotic Factors Environmental Experimental Culture
Temperature
16.40± 0.35 23.00 ± 1.00
pH
7.09 ± 0.21 7.00 ± 1.00
Hardness of Water
1.12 ± 0.52 0.98 ± 0.65
54
Table 3
Days
Couples
Diet 1
Couples
Diet 2
Ovigerous Females
Diet 1
Ovigerous Females
Diet 2
1
8 10 0 0
2
10 9 0 0
3
10 9 0 0
4
2 2 0 1
5
2 2 0 1
6
3 2 0 1
7
3 2 0 1
8
2 0 2 0
9
2 1 2 0
10
2 1 2 0
11
2 1 1 0
12
3 1 0 0
13
2 1 0 0
14
2 3 0 0
15
4 3 0 0
16
5 3 0 0
17
3 4 0 0
18
3 1 0 0
19
3 2 0
0
20
3 3 0 0
21
3 3 0 0
Total
77 63 2 1
55
Table 4
1º Culture 2º Culture 3º Culture Mean
Males –Diet 1
98.20% 94.55% 96.36% 96.37%
Females –Diet 1
98.20% 96.36% 98.20% 97.59%
Males –Diet 2
83.64% 89.10% 85.45% 86.06%
Females –Diet 2
87.27% 85.45% 85.45% 86.06%
56
Glycogen - Diet 1
0
1
2
3
4
5
6
7
8
9
10
Environment 7 days 14 days 21 days
Time of Experimental Culture
mmol/g
Male
Female
Glycogen - Diet 2
0
1
2
3
4
5
6
7
8
9
10
Environment 7 days 14 days 21 days
Time of Experimental Culture
mmol/g
Male
Female
ade
bdf
cef
abc
abc
ade
bdf
cef
# & *
abc
ade
bdf
cef
abc
ce
ad
bde
#
B
A
Figure 1
57
Total Proteíns - Diet 1
0
0,5
1
1,5
2
2,5
Environment 7 days 14 days 21 days
Time of Experimental Culture
mg of protein/ml of homogenate
Male
Female
Total Proteins - Diet 2
0
0,5
1
1,5
2
2,5
Environment 7 days 14 days 21 days
Time of Experimental Culture
mg of protein/ml of homogenate
Male
Female
ab
acd
bce
de
ab
ac
bc
#
abc
ad
bde
ce
a
acd
bc
bd
# & *
A
B
Figure 2
58
Total Lipids - Diet 1
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
Environment 7 days 14 days 21 days
Time of Experimental Culture
mg of lipids/g of animal
Male
Female
Total Lipids - Diet 2
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
Environment 7 days 14 days 21 days
Time of Experimental Culture
mg of lipids/g of animal
Male
Female
abc
ab ade
cd
bd
ace
ce
bde
abc
ad
be
cde
abc
ade
bdf
cef
# *
A
Figure 3
59
Total Cholesterol - Diet 1
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
Environment 7 days 14 days 21 days
Time of Experimental Culture
mg of cholesterol/g of animal
Male
Female
Total Cholesterol - Diet 2
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
Environment 7 days 14 days 21 days
Time of Experimental Culture
mg of cholesterol/g of animal
Male
Female
abc
ab
ad
ac
bde
cd
ce
bd
*
A
abc
a
b
c
ab
acd
bde
ce
#
B
Figure 4
60
Lipoperoxidação - Diet 1
0
5
10
15
20
25
30
35
Environment 7 days 14 days 21 days
Time of Experimental Culture
nmol of TBARS/mg of protein
Male
Female
Lipoperoxidation - Diet 2
0
5
10
15
20
25
30
35
Environment 7 days 14 days 21 days
Time of Experimental Culture
nmol of TBARS/mg of protein
Male
Female
abc
abc
ade
ade
bdf
bdf
cef
cef
abc
ab
ade
bdf
cef
ace
bcf
ef
# & *
A
B
Figure 5
61
Na+/K+ATPase - Diet 1
0
10
20
30
40
50
Environment 7 days 14 days 21 days
Time of Experimental Culture
moles Pi/min.mg of protein
Male
Female
Na+/K+ATPase - Diet 2
0
10
20
30
40
50
Environment 7 days 14 days 21 days
Time of Experimental Culture
moles Pi/min.mg of protein
Male
Female
abc
abc
ade
bde
bdf
ce
cef
abc
abc
ade
ade
bdf
bdf
cef
cef
# & *
#
A
B
ad
Figure 6
62
CONSIDERAÇÕES FINAIS
O presente estudo visou padronizar uma espécie de Hyalella, Hyalella castroi, em cultivo
experimental em laboratório para sua possível utilização posteriormente em testes de toxicidade.
De acordo com o objetivo proposto e os resultados obtidos neste trabalho, podemos concluir que:
63
ANEXO I
64
INSTITUTO DE CIÊNCIA E TECNOLOGIA DE ALIMENTOS
DEPARTAMENTO DE CIÊNCIAS DOS ALIMENTOS
RELATÓRIO DE ENSAIO
NÚMERO: 171B/2004 DATA ENTRADA: 10/10/2005
DATA SAÍDA: 27/10/2005
SOLICITANTE: Bibiana Kaiser Dutra
Fone: 9191.1954
AMOSTRA: Macrófita Callitriche rimosa
ENSAIOS:
Umidade.........................................................................................................93,84g/100g
Cinzas...............................................................................................................1,26g/100g
Proteina ...........................................................................................................1,24g/100g
Lípidios ............................................................................................................0,28g/100g
Fibra Bruta......................................................................................................1,34g/100g
Carboidratos ...................................................................................................2,04g/100g
VCT ...........................................................................................................9,52Kcal/100g
Prof. Adriano Brandelli Heloisa H. Chaves Carvalho
Coordenador Prestação de Serviços Nutricionista
CRN 1484
Referências: Ministério da Agricultura. Laboratório Nacional de Referência Animal. Métodos analíticos para
análise e seus ingredientes. Brasília, 1981. V.2: Métodos físicos e químicos.
Official methods of the Association of Official Analytical Chemists. AOAC, 1995. Cap. 4, seção
4.5.0.1.
Portaria n. 108 de 04 e 11 de setembro de 1991, Método n. 04. Diário Oficial da União, Brasília, p.
11813-19819, 17 setembro de 1991. Seção 1.
Normas Analíticas do Instituto Adolfo Lutz. 3 Ed. São Paulo, 1985. V. 1: Métodos Químicos e Físicos
para análise de alimentos.
Válido somente para a(s) amostra(s) fornecida(s) pelo interessado.
UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL -INSTITUTO DE CIÊNCIA E TECNOLOGIA DE
ALIMENTOS
Av. Bento Gonçalves, 9.500 - Campus do Vale - Prédio 43 212 - Porto Alegre - RS - C. Postal 15 090 CEP 91.501- 970
Prestação de serviços - telefone: (051) 3316-6248 - Fax: (051) 316-7048
E MAIL BROMO.ICTA @UFRGS.BR
65
INSTITUTO DE CIÊNCIA E TECNOLOGIA DE ALIMENTOS
DEPARTAMENTO DE CIÊNCIAS DOS ALIMENTOS
RELATÓRIO DE ENSAIO
NÚMERO: 170B/2004 DATA ENTRADA: 10/10/2005
DATA SAÍDA: 27/10/2005
SOLICITANTE: Bibiana Kaiser Dutra
Fone: 9191.1954
AMOSTRA: Ração em Flocos para Peixes – Ração 1
ENSAIOS:
Umidade...........................................................................................................5,13g/100g
Cinzas.............................................................................................................11,02g/100g
Proteina .........................................................................................................30,88g/100g
Lípidios ............................................................................................................6,19g/100g
Fibra Bruta......................................................................................................3,59g/100g
Carboidratos .................................................................................................43,19g/100g
VCT ........................................................................................................351,99Kcal/100g
Prof. Adriano Brandelli Heloisa H. Chaves Carvalho
Coordenador Prestação de Serviços Nutricionista
CRN 1484
Referências: Ministério da Agricultura. Laboratório Nacional de Referência Animal. Métodos analíticos para
análise e seus ingredientes. Brasília, 1981. V.2: Métodos físicos e químicos.
Official methods of the Association of Official Analytical Chemists. AOAC, 1995. Cap. 4, seção
4.5.0.1.
Portaria n. 108 de 04 e 11 de setembro de 1991, Método n. 04. Diário Oficial da União, Brasília, p.
11813-19819, 17 setembro de 1991. Seção 1.
Normas Analíticas do Instituto Adolfo Lutz. 3 Ed. São Paulo, 1985. V. 1: Métodos Químicos e Físicos
para análise de alimentos.
Válido somente para a(s) amostra(s) fornecida(s) pelo interessado.
UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL -INSTITUTO DE CIÊNCIA E TECNOLOGIA DE
ALIMENTOS
Av. Bento Gonçalves, 9.500 - Campus do Vale - Prédio 43 212 - Porto Alegre - RS - C. Postal 15 090 CEP 91.501- 970
Prestação de serviços - telefone: (051) 3316-6248 - Fax: (051) 316-7048
E MAIL BROMO.ICTA @UFRGS.BR
66
INSTITUTO DE CIÊNCIA E TECNOLOGIA DE ALIMENTOS
DEPARTAMENTO DE CIÊNCIAS DOS ALIMENTOS
RELATÓRIO DE ENSAIO
NÚMERO: 136B/2006 DATA ENTRADA: 28/11/2006
DATA SAÍDA: 11/12/2006
SOLICITANTE: Bibiana Kaiser Dutra
Fone: 9191.1954
AMOSTRA: Ração em Flocos para Peixes - Ração 2
ENSAIOS:
Umidade...........................................................................................................7,26g/100g
Cinzas.............................................................................................................14,15g/100g
Proteina .........................................................................................................39,78g/100g
Lípidios ............................................................................................................4,99g/100g
Fibra Bruta......................................................................................................4,83g/100g
Carboidratos .................................................................................................28,99g/100g
VCT ........................................................................................................319,99Kcal/100g
Prof. Adriano Brandelli Heloisa H. Chaves Carvalho
Coordenador Prestação de Serviços Nutricionista
CRN 1484
Referências: Ministério da Agricultura. Laboratório Nacional de Referência Animal. Métodos analíticos para
análise e seus ingredientes. Brasília, 1981. V.2: Métodos físicos e químicos.
Official methods of the Association of Official Analytical Chemists. AOAC, 1995. Cap. 4, seção
4.5.0.1.
Portaria n. 108 de 04 e 11 de setembro de 1991, Método n. 04. Diário Oficial da União, Brasília, p.
11813-19819, 17 setembro de 1991. Seção 1.
Normas Analíticas do Instituto Adolfo Lutz. 3 Ed. São Paulo, 1985. V. 1: Métodos Químicos e Físicos
para análise de alimentos.
Válido somente para a(s) amostra(s) fornecida(s) pelo interessado.
UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL -INSTITUTO DE CIÊNCIA E TECNOLOGIA DE
ALIMENTOS
Av. Bento Gonçalves, 9.500 - Campus do Vale - Prédio 43 212 - Porto Alegre - RS - C. Postal 15 090 CEP 91.501- 970
Prestação de serviços - telefone: (051) 3316-6248 - Fax: (051) 316-7048
E MAIL BROMO.ICTA @UFRGS.BR
67
ANEXO II
68
69
ANEXO III
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