Determinar o conteúdo dos lipídeos presentes nos tecidos animais nem sempre resulta em uma resposta com precisão, devido á pouca disponibilidade de métodos adequados para a extração e determinação quantitativa destes lipídeos. A extração de lipídeos de matrizes complexas, como o pescado, por meio dos métodos tradicionais de Bligh & Dyer e Folch normalmente utilizam solventes com elevada toxicidade, cujos resíduos podem gerar um elevado impacto ambiental. A chamada segurança química está diretamente ligada à qualidade de vida e alerta para as questões de controle e prevenção dos efeitos adversos dos solventes ao ser humano e ao ambiente, compreendendo desde a extração, uso e descarte desses e seus resíduos. Portanto, o objetivo deste trabalho foi estudar as condições de extração de lipídeos totais de resíduos do processamento de tilápias, visando à proposição de um método alternativo que garanta uma eficiência tão satisfatória quanto as obtidas pelos métodos tradicionais de extração. Ademais, foi desenvolvido um método de extração mais seguro ao ambiente utilizando solventes menos tóxicos que os propostos pelo método de Bligh &Dyer, por meio da aplicação experimental de um diagrama de fases hexano- etanol- água. Após a identificação da melhor combinação de solventes, os extratos obtidos com hexano- etanol apresentaram rendimento eficiente em lipídeos totais e capacidade de extrair os componentes lipídicos polares e apolares das amostras analisadas. O melhor aproveitamento dos lipídeos a serem extraídos da matriz resíduos de peixes na proporção em massa de solvente hexano: etanol: água, foram da solução 1 (1: 7: 1,375) para a carcaça, da solução 2 (1: 2,5: 1,1875) para a cabeça e da solução 4 (4: 1: 4,375) para as vísceras totalizando 14,13%, 14,69 e 47,63% de teor de lipídeos, respectivamente. Logo, ao buscar sistemas de solventes alternativos ao clorofórmio - metanol, para extrair lipídeos de resíduos do processamento de tilápias, foi possível identificar as melhores proporções de hexano: etanol: água como substituição do método tradicional proposto por Bligh & Dyer (1959). / The total lipid content of animal tissues is not always accurately measured due to the lack of adequate methods for lipid extraction and quantitative determination of tissue fat. The extraction of lipids from complex matrices, such as fish, using the traditional methods proposed by Bligh &Dyer and Folch usually, employs solvents with high toxicity, whose residues can generate a high environmental impact. The chemical safety concern nowadays is directly linked to quality of life involving control and prevention of adverse effects of solvents to humans and the environment, ranging from the extraction, use and disposal of these and their residues. Moreover, the procedures of sample preparation, the sample: solvent ratio, the proportion of solvents applied, and the order of addition of solvents during the experiment are not standardized in these methods. Therefore, the present study aimed to assess the extraction conditions of total lipids from fish processing residues, and thus propose a standardized method which ensures a maximum efficiency in lipid extraction. Furthermore, a more environmental-friendly oil extraction method using less toxic solvents than those proposed by the Bligh & Dyer method (i.e. using the hexane-ethanol-water phase diagram) was developed. After identifying the best combination of solvents, the extracts obtained with hexane- ethanol showed effective yield on total lipids and the ability to extract the polar lipid components and nonpolar of the samples. The best use of lipids to be extracted from fish waste matrix, were the solution 1 (1: 7: 1,375) for the carcass of solution 2 (1: 2,5: 1,1875) for the head and the solution 4 (4: 1: 4,375) for the viscera totaling 14,13%, 14,69% and 47,63%(4: 1: 4,375) respectively. Therefore, to seek alternative solvent systems chloroform - methanol to extract lipids of tilapia processing waste, it was possible to identify the best proportions of hexane: ethanol: water as replacing the traditional method proposed by Bligh & Dyer (1959).

Um terço da captura mundial de pescado não é empregada para o consumo direto na alimentação humana, seguindo para elaboração de rações ou é desperdiçada como resíduo. O ideal seria utilizar a matéria-prima em toda a sua extensão e recuperar os subprodutos, evitando a própria formação do resíduo. Com os objetivos de aumentar a receita e a eficiência de produção da indústria e, conseqüentemente, minimizar os problemas ambientais e de sanidade, provenientes do resíduo de pescado, procedeu-se à elaboração da silagem química do resíduo de beneficiamento da tilápia. A tilápia do Nilo (Oreochromis niloticus) é hoje a espécie mais popular no Brasil. A tilápia é cultivada em 22 estados brasileiros e a sua produção anual está entre 30 e 40 mil t. A tilápia foi uma das primeiras espécies oriundas da aqüicultura a ser beneficiada, sendo atualmente comercializada na forma de filés congelados, tecnologia emergente, porém com rendimento de cerca de 30%. Com isso, há necessidade de se transformar o resíduo gerado com o beneficiamento do filé em subprodutos, diminuindo o impacto ambiental. A silagem foi elaborada após homogeneização em cutter e acidificação da biomassa com 3% de ácido fórmico: propiônico, 1:1, adição do antioxidante BHT e manutenção do pH ao redor de 4,0. Foram realizadas análises para determinação da umidade, proteína, lipídios e cinza. Os aminoácidos foram examinados em auto analisador após hidrólise ácida, à exceção do triptofano determinado por colorimetria. A silagem de tilápias apresentou teores semelhantes ou maiores aos preconizados pelo padrão da FAO para todos os aminoácidos essenciais, com exceção do triptofano. Os valores mais elevados encontrados foram para o ácido glutâmico, leucina e lisina. Os resultados indicam uma possível utilização da silagem, preparada a partir do resíduo de processamento da Tilápia-do-Nilo, como fonte proteíca na formulação de ração para peixes. Entretanto, para o aproveitamento da silagem é necessário que se estabeleçam técnicas para remoção do óleo presente com a finalidade de aumentar a estabilidade do produto e seu valor comercial. A oxidação dos lipídios presentes pode causar a formação de peróxidos que podem complexar as proteínas com conseqüente destruição dos aminoácidos. O presente trabalho buscou, também, estabelecer a melhor via de extração do óleo presente na silagem ácida de tilápia do Nilo (Oreochromis niloticus), bem como caracterizar o óleo obtido. A fração lipídica foi extraída por três métodos de extração, a saber, centrifugação (3500 × G/30 min), Soxhlet (AOAC,1990) e Bligh & Dyer (1969). Os resultados para rendimento e índice de peróxido foram, respectivamente, 65,53% e 0,00 mEq/1000g de O2, 44,27% e 25,00 mEq/1000g de O2, 46,87% e 0,00 mEq/1000g de O2. O óleo de tilápia contém, em mg/100g, 28,60; 16,30 e 3,10 dos ácidos oléico, linoléico e linolênico, respectivamente. A centrifugação demonstrou ser a melhor metodologia de extração, por preservar as características físico-químicas do óleo e propiciar maior rendimento. O conteúdo de lipídios na silagem de tilápias, com base na matéria úmida é de 3,99 g/100g, com a retirada da fração lipídica através da centrifugação, restou na amostra apenas 1,54 g/100g de lipídios, o que é considerado um nível aceitável para inclusão em rações para animais. / One third of the world fishing produce is not directly applied in human consumption. Instead, it is used in the elaboration of feed or wasted as residue. It would be ideal to use the raw material thoroughly and to recover by-products, preventing the generation of residues. With the objectives of increasing the income and the production of the industry, as well as minimizing environmental and health problems from fish residues, chemical silage from Tilapia processing residues was produced. The Nile tilapia (Oreochromis niloticus) is the most popular species in Brazil today. Tilapia is cultivated in 22 Brazilian states and its annual production is between 30 and 40 thousand tons. Tilapia was one of the first aquaculture species to be processed after harvesting, being currently commercialized in the form of frozen fillets, however, with a yield of about 30%. Thus, there is a need for transforming the residues generated by the fillet processing into by-products, minimizing the environmental impact. The silage was elaborated after cutter-homogenization and acidification of the biomass with 3% formic acid: propionic, 1:1, addition of antioxidant BHT and maintenance of pH at about 4.0. Analyses for the determination of the moisture, protein, lipids and ash were carried out. The amino acids were examined in an auto analyzer after acid hydrolysis, except for the triptophane which was determined through colorimetry. The tilapia silage presented contents that were similar to or higher than the FAO standards for all essential amino acids, except for the triptophane. The highest values found were for lysine and leucine. The results indicate a possible use of the silage prepared from the Nile tilapia processing residue as a proteic source in the manufacturing of fish feed. However For the use of the silage, oil-removing techniques are necessary in order to increase the stability of the product as well as its commercial value. Lipid oxidation may cause peroxide formation, which can produce complex proteins with a consequent destruction of amino acids. The present work tried to determine the best form of extracting the oil from the acid silage of Nile tilapia (Oreochromis niloticus) and to caracterize the silage oil. The lipid fraction was extracted by three extraction methods: centrifugation (3500 × G·30 min-1), Soxhlet (AOAC, 1990) and Bligh & Dyer (1969). The results for yield and peroxide value were, respectively, 65.53% and 0.00 mEq·1000g-1 of O2, 44.27% and 25.00 mEq·1000g-1of O2, 46.87% and 0.00 mEq·1000g-1 of O2. The lipid fraction showed 28,60; 16,30 and 3,10mg·1000g-1 of oleic, linoleic and linolenic acids, respectively. Centrifugation proved to be the best extraction methodology due to the preservation of the physical-chemical characteristics of the oil and greater yield. The lipid content of the tilapia silage, on a fresh matter basis, was 3.99g·100g-1. With the removal of the lipid fraction by centrifugation, only 1.54 g·100g-1of lipids could be found in the sample, which is considered an acceptable level for its inclusion in animal feed.

Submitted by Robert Guaracy Aparecido Cardoso Araujo null (roberty_kadu@hotmail.com) on 2017-02-04T14:33:13Z No. of bitstreams: 1 Dissertação.pdf: 1123603 bytes, checksum: 04494f1c8cf34365a9d7ad9cb0ffd4de (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-02-07T13:05:58Z (GMT) No. of bitstreams: 1 araujo_rgac_me_dra.pdf: 1123603 bytes, checksum: 04494f1c8cf34365a9d7ad9cb0ffd4de (MD5) / Made available in DSpace on 2017-02-07T13:05:58Z (GMT). No. of bitstreams: 1 araujo_rgac_me_dra.pdf: 1123603 bytes, checksum: 04494f1c8cf34365a9d7ad9cb0ffd4de (MD5) Previous issue date: 2017-01-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / É importante a determinação atualizada dos valores nutricionais dos alimentos para a prática de formulação de rações para aves. O objetivo desta pesquisa foi avaliar o perfil de ácidos graxos e energia metabolizável aparente corrigida pelo balanço de nitrogênio (EMAn) de diferentes fontes de óleos (soja, girassol, linhaça e peixe) para galinhas poedeiras da linhagem Hysex White, com 24 semanas de idade. As dietas experimentais consistiram de: T1 – ração basal, T2 – ração basal + 10% de inclusão de óleo de soja, T3 – ração basal + 10% de inclusão de óleo de girassol, T4 – ração basal + 10% de óleo de linhaça e T5 – ração basal + 10% de óleo de peixe, distribuídas num delineamento inteiramente casualizado com sete repetições e oito aves por unidade experimental. O perfil de ácidos graxos das fontes lipídicas foi analisado por cromatografia gasosa. O ensaio de digestibilidade foi realizado pelo método de coleta total de excretas, utilizando-se 280 poedeiras em 35 gaiolas experimentais, incluindo aves do grupo controle. Os resultados foram submetidos à análise de variância e as médias dos tratamentos foram comparadas pelo teste de tukey à 5% de probabilidade. Os maiores níveis de ácidos graxos insaturados foram encontrados nas fontes de origem vegetal, com mais de 80% enquanto o óleo de peixe apresentou 58,38%. Os valores de EMAn na matéria natural foram: 9.334 kcal/kg para o óleo de soja; 10.533 kcal/kg para o óleo de girassol; 10.928 kcal/kg para o óleo de linhaça e 9.005 kcal/kg para o óleo de peixe. Os valores de EMAn entre as fontes lipídicas foram diferentes entre si, ressaltando a importância de haver uma matriz nutricional individual para cada óleo nos livros e tabelas sobre informações nutricionais dos ingredientes. / It is important to determine the current nutritional values of ingredients for the practice of feed formulations for birds. The objective of this research was to determine the fatty acid profile and evaluate the apparent metabolizable energy corrected for nitrogen balance (AMEn) of different lipid sources (soybean, sunflower, linseed and fish) for laying hens of Hysex White at 24 weeks of age. The experimental diets were: T1 – basal diet, T2 - basal diet + 10% soybean oil addition, T3 - basal diet + 10% sunflower oil addition, T4 - basal diet + 10% linseed oil and T5 - basal diet + 10% fish oil, in a completely randomized design with seven replicates with eight birds per experimental unit. The fatty acid profile of lipid sources was analyzed by gas chromatography. The digestibility trial was held by the total excreta collection method, using 280 birds in 35 experimental cages, including the birds of the control group. The results were submitted to analysis of variance and the means of treatments were compared by the tukey test at 5% probability. The highest levels of unsaturated fatty acids among the four lipid sources were found in sources of vegetable origin, with more than 80% while fish oil had 58.38%. The AMEn in natural matter were: 9.334 kcal / kg for soybean oil; 10.533 kcal / kg for sunflower oil; 10.928 kcal / kg for linseed oil and 9.005 kcal / kg for fish oil. The AMEn values among the lipid sources were different, emphasizing the importance of having an individual nutritional matrix for each oil in the books and tables on nutritional information of the ingredients. / FAPESP: 2015/14423-6

Os efeitos tóxicos decorrente do consumo de peixes contaminados com metilmercúrio (MeHg) tem sido muito discutido no Brasil, especialmente na Região Amazônica, onde estudos a esse respeito têm mostrado resultados conflitantes. Fatores nutricionais associados à exposição ao MeHg ou a forma na qual o MeHg se encontra ligada no peixe poderiam estar alterando sua toxicidade. Diante destas controvérsias, ratos foram subcronicamente expostos à solução de MeHg, selênio (Se) e óleo de peixe, ou foram alimentados com ração contendo peixes contaminados com MeHg. Biomarcadores de estresse oxidativo, presença de processo inflamatório, genotoxicidade, pressão sistólica, concentração de óxido nítrico (NO), de mercúrio (Hg) total e de Se em diferentes tecidos foram avaliados. Ratos expostos à solução de MeHg, mostraram significante diminuição de antioxidantes endógenos, aumento na peroxidação lipídica, hipertensão, inflamação de tecidos, dano ao DNA e diminuição de NO. A associação MeHg+Se mostrou significativa proteção antioxidante e antigenotóxica, porém não foi capaz de proteger a inflamação induzida pelo MeHg e, surpreendentemente, ratos tratados somente com Se apresentaram aumento significativo na pressão sistólica. Na associação MeHg+óleo de peixe observou-se significativa ação anti-inflamatória, antigenotóxica e anti-hipertensiva, porém não se observou ação antioxidante do óleo de peixe. A absorção do Hg nos diferentes tecidos não foi modificada pela presença de Se ou óleo de peixe. Já em ratos que receberam ração contendo peixe contaminado com MeHg, poucas alterações foram observadas. Houve aumento do biomarcador de peroxidação lipídica malondialdeído em ratos que receberam a ração com o peixe contaminado, bem como a indução de dano no DNA. A partir da 11ª semana, hipertensão também foi observada e o NO não se alterou. Assim, observou-se que o MeHg em solução induziu efeitos oxidantes, inflamação, genotoxicidade e hipertensão, e o MeHg presente no peixe induziu efeitos consideráveis, porém menos severos. A forma química com que o MeHg está presente no peixe, combinado com os nutrientes do peixe como o Se e os ácidos graxos poliinsaturados poderiam estar contribuindo para reduzir os efeitos tóxicos desse elemento. / The toxic effects due the consumption of fish contaminated with methylmercury (MeHg) have been widely discussed in Brazil, especially in Amazon region, where studies have shown confound results. Nutritional factors associated with MeHg exposure or the form of MeHg in fish could be changing MeHg toxicity. Thus, rats were subchronically exposed to MeHg in solution, selenium (Se) and fish oil, or rats were fed with a diet containing MeHg-contaminated fish. Oxidative stress biomarkers, presence of inflammation, genotoxicity, systolic blood pressure, nitric oxide (NO) levels and total mercury (Hg) and Se in different tissues were determined. Rats exposed to MeHg in solution showed endogenous antioxidants significantly reduced, increase of lipid peroxidation, hypertension, tissue inflammation, DNA damage and decrease of NO levels. MeHg+Se association presented antioxidant protection and antigenotoxic effetc, but it was not able to protect the inflammation induced by MeHg. Surprisingly, rats treated only with Se also showed increase in systolic blood pressure. MeHg+fish oil association presented a significant antiinflammatory and antigenotoxic effects and antihypertensive action; however it was not observed antioxidant capacity of fish oil. Hg absorption in different tissues was not modified by Se or fish oil treatments. On the other hand, rats fed with diet containing MeHg-contaminated fish presented few alterations in biomarkers. A lipid peroxidation biomarker showed significantly increased in rats fed with MeHgcontaminated fish as well as induction of DNA damage. From the 11th week, a hypertension was observed and NO levels did not change. In conclusion, it was observed that MeHg solution was able to induce oxidative effects, inflammation, genotoxicity and hypertension, and MeHg linked in fish induced considerable but mild effects. The chemical form that MeHg is present in fish combined with nutrients from fish, as Se and polyunsaturated fatty acids could contribute to reducing toxic effects of MeHg.

From animal experimentation, and studies using in vitro models, there was evidence in the literature to suggest that dietary fibre may influence contractility and motility of the gastrointestinal tract and long chain (LC) n-3 polyunsaturated fatty acids (PUFAs) from marine sources may influence contractility of smooth muscle cells in blood vessels. The hypothesis of this thesis was that dietary fish oil and/or fibre influence the contractility of isolated intact sections of gut smooth muscle tissue from small animal models. Methodology was established to measure in vitro contractility of intact pieces of guinea pig ileum with the serosal side isolated from the lumen. It was demonstrated that four amino acid peptides from κ-casein (casoxins) applied to the lumen overcame morphine-induced inhibition of contraction. Using this established technology, the guinea pig was used to investigate the effects of dietary fibre and fish oil supplementation on gut in vitro contractility. In separate experiments, changes in sensitivity to electrically-driven and 8-iso-prostanglandin (PG)E₂-induced contractility were demonstrated for dietary fibre and fish oil. A modified, isolated gut super-perfusion system was then established for the rat to validate these findings. It was subsequently shown that LC n-3 PUFA from dietary fish oil significantly increased maximal contraction in response to the G-protein coupled receptor modulators, acetylcholine and the eicosanoids PGE₂, PGF₂α, 8-iso-PGE₂ and U-46619 in ileum but not colon, without changes in sensitivity (EC₅₀), when n-3 PUFA as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) had been incorporated to a similar degree into the gut total phospholipid membrane pool. It was further established that the spontaneously hypertensive rat (SHR) had a depressed prostanoid (PGE₂and PGF₂α) response in the gut that could be restored by dietary fish oil supplementation (5% w/w of total diet) in the ileum but not the colon. Importantly, the muscarinic response in the colon of the SHR was increased by fish oil supplementation with DHA likely to be the active agent. Dietary fish oil dose experiments deduced differential increases in response occurred at fish oil concentrations of 1% for muscarinic and 2.5% (w/w) for prostanoid stimulators of the ileum with no difference in receptor-independent KCl-induced depolarization-driven contractility. Studies combining high amylose resistant starch (HAMS, 10% w/w) and fish oil (10% w/w) fed to young rats demonstrated a low prostanoid response that was enhanced by dietary fish oil but not resistant starch. There was however, an interactive effect of the HAMS and fish oil noted for the muscarinic-mimetic, carbachol. Generally, resistant starch increased the large bowel short chain fatty acid pool with a subsequent lower pH. Binding studies determined that while the total muscarinic receptor binding properties of an isolated ileal membrane fraction were not affected in mature rats by dietary fish oil, young rats had a different order of muscarinic receptor subtype response with a rank order potency of M₃ > M₁ > M₂ compared to mature animals of M₃ > M₂ > M₁ with fish oil altering the sensitivity of the M₁ receptor subtype in isolated carbachol-precontracted ileal tissue. In conclusion, experiments using the guinea pig and rat gut models demonstrated that dietary fish oil supplementation, and to a lesser degree fibre, increased receptor-driven contractility in normal and compromised SHR ileum and colon. Further, changes in responsiveness were demonstrated in the developing rat gut prostanoid and muscarinic receptor populations that could be altered by dietary fish oil. Preliminary evidence suggested that fish oil as DHA may alter receptor-driven gut contractility by mechanisms involving smooth muscle calcium modulation. Defining the role that dietary fibre and fish oil, and other nutrients, play in normal and diseased states of bowel health such as inflammatory bowel disease (IBD), where contractility is compromised, are among the ongoing challenges. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1316907 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

A whole-cell DHA-rich algal product (A-DHA, provided by Evonik Industries) that is rich in DHA (125 mg DHA/g dry matter) is a possible replacement for fish oil in salmonid diets. The nutrient digestibilities of the algal product were measured in rainbow trout in freshwater and in Atlantic salmon in saltwater (32-33 ppm). In experiment 1, rainbow trout (initial weight ~ 300g) were randomly assigned to 12 x 120 L tanks (n = 10 per tank). A reference diet containing 1% Celite as an indigestible marker and three test diets with increasing percentage of A-DHA substitution (6.67%, 13.33% and 20%) were fed. Feces were collected using a settling column and feed and feces analyzed for digestible dry matter (DM), gross energy (GE), ash, crude protein (CP), essential amino acids and total lipid. The digestibility of six long-chain fatty acids including 18:1n-9 (OA), 18:2n-6 (LA), 18:3n-3 (ALA), 20:4n-6 (ARA), 20:5n-3 (EPA) and 22:6n-3 (DHA) was measured. In experiment 2, Atlantic salmon (~170g) were randomly distributed to 12 fiberglass tanks (600L) with 106 fish per tank. The fish were assigned to four diets with the same levels of A-DHA inclusion as for rainbow trout and yttrium oxide (Y2O3) was used as an inert marker. Feces were collected by stripping and the digestibilities of DM, CP and lipid as well as OA, LA, ALA, ARA, EPA and DHA were determined. In experiment 1, the apparent digestibility of dietary DM, GE and lipid in rainbow trout declined significantly with increasing inclusion of A-DHA (P < 0.01). The inclusion of A-DHA had no effect on the digestibility of CP and ash as well as the availability of essential amino acids (P > 0.05). Furthermore, increased inclusion of A-DHA resulted in significantly lower digestibility of ARA, EPA and DHA (P < 0.05). A similar pattern was seen in the digestibility of OA, LA and ALA, although the effect of A-DHA inclusion was not statistically significant. Regression analysis revealed that nutrient contribution from A-DHA had significantly negative linear and quadratic effects on the apparent digestibility of DM, GE, and lipid. The inclusion levels of A-DHA had both significantly negative linear and quadratic effects on digestibility of LA and ALA, whereas only significantly negative linear effect was found on OA. Significantly negative linear and quadratic regressions were observed for the digestibility of ARA, EPA and DHA. The linear regression for CP was significantly negative and the regressions for the individual amino acids were not significant (P > 0.05). In experiment 2, dietary inclusion of A-DHA had a significantly negative effect on lipid digestibility in Atlantic salmon, at all inclusion rates whereas the significant negative effect on digestibilities of DM and CP was only observed in fish fed 20% A-DHA. The digestibilities of OA, LA, ALA and EPA were greater than 91%. In contrast, the apparent digestibilities of ARA and DHA decreased significantly with increasing substitution of A-DHA (P < 0.01). Significantly negative linear and quadratic regressions were found between nutrient contribution from A-DHA to the diets and apparent digestibility of DM, CP and lipid, so were LA, EPA and DHA. However, there were only significant quadratic regressions for OA, ALA and ARA, but not significant linear effects. Subsequently, a twelve-week feeding trial in rainbow trout was conducted to investigate the impact of replacing fish oil with A-DHA in canola-oil-based diets on the growth performance and fatty acid composition and retention. Four experimental diets containing only canola oil (CO; 13.5%), fish oil (FO; 13.5%), canola oil and fish oil (C+F; 7.4% and 6.1%, respectively) or canola oil and A-DHA (C+A; 15.5% and 6%, respectively) were formulated to contain 386.2 g/kg digestible crude protein and 17.58 MJ/kg digestible energy. In addition, the C+A diet was formulated to have the same DHA concentration as in the C+F diet. Each diet was fed to three tanks of rainbow trout (average initial weight of 70g; n = 17/tank) and the fish were fed to apparent satiation 2 times daily. At the end of the growth trial, all fish approximately tripled their weight. No significant differences were noted between the dietary treatments in growth performance as measured by final weight, average weight gain, feed intake, specific growth rate (SGR) and feed conversion ratio (FCR). Although FO and C+A fed fish tended to accumulate more lipids, final whole body lipid content did not differ significantly between dietary treatments (P = 0.11). The concentrations of EPA, DHA as well as total n-3 fatty acid were significantly higher in fish fed the FO diet than fish fed the other 3 diets. The C+A fed fish had lower EPA and higher DHA concentrations compared with the CO and C+F fed fish; however, the differences were not significant. Apparent retention of total lipid in the trout was not significantly influenced by treatments (P > 0.05). Similarly, dietary treatments had no significant effect on the apparent retention of total saturated fatty acids, total mono-unsaturated fatty acids, n-3 polyunsaturated fatty acids and n-6 polyunsaturated fatty acids. The retention of 18:4n-3 (SDA) was significantly higher (> 100%) in fish fed CO and C+A compared with fish fed FO and C+F (< 51%), indicating greater bioconversion of ALA to SDA in the CO and C+A fed fish than in FO and C+F fed fish. The retention of EPA in the CO and C+A fed fish was over 100%, suggesting a net synthesis of EPA in these treatment groups. In contrast, the EPA retention in the FO and C+F fed fish was 55 and 21%, respectively, which showed a tendency to be significantly lower than that in the other two groups (P = 0.09). The CO fed fish had significantly higher DHA retention than fish fed the other 3 diets. The DHA retention in the FO fed fish (112%) was numerically but not significantly higher than in the C+F (66%) and C+A fed fish (73%). Thus, feeding the C+A to rainbow trout resulted in DHA retention equal to feeding the C+F.

Die Abdeckung des Energiebedarfs ist insbesondere bei Sport- und Zuchtpferden mit einer Aufnahme hoher Stärkemengen gekoppelt. Daraus können jedoch sowohl fermentative Störungen, bedingt durch das Abfluten von Stärke in den Dickdarm, als auch metabolische Veränderungen resultieren, wobei letztere insbesondere in Form von gesteigerten Glucose- und Insulinreaktionen zum Ausdruck kommen und nicht zuletzt in der Entstehung einer Insulinresistenz münden können. In der vorliegenden Arbeit sollte der Einfluss verschiedener Nährstoffe bzw. Futtermittel in Kombination mit der Aufnahme einer stärkereichen Mahlzeit auf die Glucose- und Insulinreaktion bei gesunden Pferden überprüft werden. Für die Versuche standen insgesamt zwölf Wallache (Alter: 6 ± 4 Jahre, Gewicht: 552 ± 84 kg) zur Verfügung. Die Untersuchungen unterteilten sich in drei Fragestellungen, bei denen die jeweiligen Futtermittel randomisiert oder blockweise angeboten wurden. Als stärkereiche Mahlzeit wurde einmal täglich morgens Bruchmais in einer Dosierung von 2 g Stärke / kg KM verfüttert. Im ersten Versuchsabschnitt erhielten die Pferde verschiedene Heuzuteilungsformen (ad libitum oder restriktiv (0,6 kg / 100 kg KM)) in der Nacht vor Bruchmaisaufnahme sowie nach der Fütterung des Bruchmaises. Im zweiten Versuch wurde der Bruchmais in Kombination mit extrahierten Rohfaserquellen (0,2 g / kg KM Cellulose / Hemicellulose oder 0,1 g / kg KM Apfelpektin) oder 0,2 g Rohprotein / kg KM (in Form von Maiskleber) verfüttert. Im dritten Versuchsabschnitt erfolgte die Zulage von 0,2 ml Soja- oder Fischöl / kg KM zum Bruchmais. Je nach Fragestellung durchliefen die Pferde eine acht- bis elftägige Adaptationsphase, nach welcher sich ein- bis zweitägige Blutprobenentnahmetage anschlossen. Hierbei wurde den Pferden über einen Venenverweilkatheter bis 510 Minuten ppr. jeweils in halbstündigen Abständen Blut entnommen. Im Plasma wurden die Blutparameter Glucose (GOD-Methode) und Insulin (Radioimmunoassay) bestimmt. Es konnte gezeigt werden, dass die Zufuhr von Rohfaser in Form von Heu vor und nach Aufnahme einer stärkereichen Ration keinen wesentlichen Einfluss auf die Glucosereaktion hatte. Hierbei wurden die Glucosekonzentrationen nach zwölfstündiger Nüchterung durch die Heufütterung während der Blutentnahme nicht beeinflusst (AUCHeu restriktiv nachts, Heu restriktiv tags 132 ± 72,5 mmol x min / l, AUCHeu restriktiv nachts, Heu ad libitum tags 144 ± 114 mmol x min / l, Behandlung p > 0,05). Die Heuaufnahme während der Blutentnahme führte zu signifikant höheren Glucosekonzentrationen (Behandlung p = 0,02) wenn die Pferde vor der Bruchmaisaufnahme ad libitum Zugang zu Heu hatten (Heu ad libitum nachts, Heu ad libitum tags). Die Flächen unter den Glucosekurven wiesen jedoch keine signifikanten Unterschiede zwischen den Behandlungen auf (AUCHeu ad libitum nachts, Heu restriktiv tags 89,5 ± 50,5 mmol x min / l, AUCHeu ad libitum nachts, Heu ad libitum tags 113 ± 62,7 mmol x min / l, Behandlung p > 0,05). Die Heufütterung während der Blutentnahme ließ nach zwölfstündiger Nüchterung signifikant höhere Insulinkonzentrationen (AUCHeu restriktiv nachts, Heu ad libitum tags 5996 ± 4460 µU x min / ml) erkennen als ohne Heufütterung (AUCHeu restriktiv nachts, Heu restriktiv tags 1626 ± 1040 µU x min / ml, Behandlung p = 0,02). Die Pferde zeigten weiterhin signifikant höhere Insulinwerte, wenn nach der Fütterung des Bruchmaises Heu ad libitum aufgenommen werden konnte (AUCHeu ad libitum nachts, Heu restriktiv tags 1275 ± 845 µU x min / ml, AUCHeu ad libitum nachts, Heu ad libitum tags 3701 ± 2163 µU x min / ml, Behandlung p = 0,04). Die Zugabe extrahierter Rohfaserquellen wie Cellulose / Hemicellulose sowie Pektin zu einer definierten Maisaufnahme zeigte keinen signifikanten Einfluss auf die Glucose- (AUCBruchmais 230 ± 163 mmol x min / l, AUCCellulose / Hemicellulose 259 ± 215 mmol x min / l, AUCPektin 274 ± 106 mmol x min / l, Behandlung p > 0,05) und Insulinreaktion (AUCBruchmais 8885 ± 4024 µU x min / ml, AUCCellulose / Hemicellulose 8767 µU x min / ml, AUCPektin 10657 µU x min / ml, Behandlung p > 0,05). Durch die additive Supplementierung von Sojaöl- und Fischöl zu einer stärkehaltigen Mahlzeit ließen sich keine signifikanten Unterschiede in der Glucose- (AUCSojaöl 268 mmol x min / l, AUCFischöl 234 mmol x min / l, AUCBruchmais 257 ± 113 mmol x min / l) und Insulinreaktion (AUCSojaöl 4640 µU x min / ml, AUCFischöl 3653 µU x min / ml, AUCBruchmais 4703 ± 3365 µU x min / ml) im Vergleich zur isolierten Bruchmaisaufnahme erkennen. Die Ergänzung von Protein zu einer stärkehaltigen Mahlzeit zeigte sehr variable Reaktionen innerhalb der Einzelpferde, wobei zwei Pferde keine Reaktion und zwei Pferde erhöhte Insulinreaktionen zeigten. Unterschiede in den Glucosereaktionen wurden nicht beobachtet. Zusammenfassend ist hervorzuheben, dass die gewählten Fütterungskonzepte (Zulage extrahierter Faserquellen, Protein oder Fett) bei einer Stärkezufuhr von 2 g Stärke / kg KM keinen signifikanten Einfluss auf die Glucose- und Insulinreaktionen hatten. Dagegen führte die Fütterung von Heu ad libitum nach Aufnahme einer stärkereichen Ration zu erhöhten Insulinkonzentrationen. Um die Glucose- und Insulinreaktion nachhaltig abschwächen zu können, ist als effiziente Maßnahme eine Reduktion der aufgenommenen Stärkemenge pro Mahlzeit zu empfehlen, wohingegen die Zulage von Rohfaser und Fett bei standardisierter Stärkeaufnahme keine Abschwächung der postprandialen Glucose- und Insulinreaktion bewirkt. / In order to meet horses’ requirements of work and exercise feeding high amounts of starch is common practice. However, a high intake of starch can increase the risk of either fermentative disorders due to an overload of starch in the hindgut, or of metabolic disorders leading to exaggerated blood glucose and insulin concentrations which possibly could induce insulin resistance. The aim of this study was to investigate the effects of different nutrients in combination with the intake of a starchy meal on blood glucose and insulin concentrations in healthy horses. Twelve geldings (age: 6 ± 4 years, body weight (BW): 552 ± 84 kg) were fed the respective diets either in a randomized order or in a block design. The experiment was divided into three feeding trials. In each trial, horses received cracked corn (2 g starch / kg BW) in the morning (0800 h). In the first trial horses were fed cracked corn and grass hay in four different orders (hay ad libitum or hay restrictive (0.6 kg / 100 kg BW)), in the night before and after the intake of cracked corn. In the second trial cracked corn was mixed with extracted diatary fibers (cellulose / hemicellulose (0.2 g / kg BW) or apple pectin (0.1 g / kg BW)) or with corn gluten (2 g crude protein / kg BW). In the third trial cracked corn was fed either in combination with soybean oil (0.2 ml / kg BW) or in combination with fish oil (0.2 ml / kg BW). Each feeding period consisted of eight to eleven days of acclimatization to the different diets followed by one or two blood collection days. Blood samples were taken via a venous catheter in half-hour intervals up to 510 minutes ppr. Plasma glucose concentrations were measured by a glucose oxidase assay, and insulin levels were determined using a radioimmunoassay. There was no influence of hay feeding during blood collection after a 12 h overnight fast on plasma glucose concentrations (AUChay restriktive night, hay restriktive day 132 ± 72.5 mmol x min / l, AUChay restriktive night, hay ad libitum day 144 ± 114 mmol x min / l, treatment p > 0.05). In contrast, hay feeding during the blood collection period led to significant higher plasma glucose concentrations (treatment p = 0.02) when horses had ad libitum access to hay in the night before the intake of cracked corn. There was no significant difference in the AUC (AUChay ad libitum night, hay restriktive day 89.5 ± 50.5 mmol x min / l, AUChay ad libitum night, hay ad libitum day 113 ± 62.7 mmol x min / l, treatment p > 0.05). Furthermore, horses which were fed hay ad libitum after the intake of cracked corn demonstrated significant higher insulin concentrations (AUChay restriktive night, hay ad libitum day 5996 ± 4460 µU x min / ml) than horses without hay feeding (AUChay restriktive night, hay restriktive day 1626 ± 1040 µU x min / ml, treatment p = 0.02). In case of ad libitum hay feeding during the night significant higher insulin concentrations were found in horses which had ad libitum access to hay after intake of cracked corn when compared to horses without hay feeding during blood collection (AUChay ad libitum night, hay restriktive day 1275 ± 845 µU x min / ml, AUChay ad libitum night, hay ad libitum day 3701 ± 2163 µU x min / ml, treatment p = 0.04). The addition of extracted cellulose / hemicellulose and extracted apple pectin did not change the glycaemic (AUCcracked corn 230 ± 163 mmol x min / l, AUCcellulose / hemicellulose 259 ± 215 mmol x min / l, AUCpectin 274 ± 106 mmol x min / l, treatment p > 0,05) and insulinaemic responses (AUCcracked corn 8885 ± 4024 µU x min / ml, AUCcellulose / hemicellulose 8767 µU x min / ml, AUCpectin 10657 µU x min / ml, treatment p > 0.05) in comparison to isolated intake of cracked corn. There were no differences in postprandial glucose (AUCsoybean oil 268 mmol x min / l, AUCfish oil 234 mmol x min / l, AUCcracked corn 257 ± 113 mmol x min / l, treatment p > 0,05) and insulin responses (AUCsoybean oil 4640 µU x min / ml, AUCfish oil 3653 µU x min / ml, AUCcracked corn 4703 ± 3365 µU x min / ml, treatment p > 0.05) between feeding either cracked corn alone or in combination with soybean oil or fish oil. Protein supplementation of a starchy meal resulted in highly individual insulin responses. Two horses showed higher insulin concentrations after addition of protein to the corn meal, whereas two horses showed no reaction to the respective diet. There were no differences in the glycaemic responses. In conclusion, the results demonstrated no influence of the applied feeding strategies including supplementation of a starchy meal (2 g / kg BW) by extracted dietary fibers, protein or fat on postprandial glucose and insulin responses. In contrast, hay feeding increased the plasma insulin concentrations after the intake of cracked corn. To attenuate the glycaemic and insulinaemic responses in the horse efficiently, a reduction of starch intake per meal to quantities below 1 g starch per kg BW and meal is recommended, whereas feeding strategies like the addition of fiber or fat to a standardised starch intake do not improve glucose metabolism in healthy horses.

From animal experimentation, and studies using in vitro models, there was evidence in the literature to suggest that dietary fibre may influence contractility and motility of the gastrointestinal tract and long chain (LC) n-3 polyunsaturated fatty acids (PUFAs) from marine sources may influence contractility of smooth muscle cells in blood vessels. The hypothesis of this thesis was that dietary fish oil and/or fibre influence the contractility of isolated intact sections of gut smooth muscle tissue from small animal models. Methodology was established to measure in vitro contractility of intact pieces of guinea pig ileum with the serosal side isolated from the lumen. It was demonstrated that four amino acid peptides from κ-casein (casoxins) applied to the lumen overcame morphine-induced inhibition of contraction. Using this established technology, the guinea pig was used to investigate the effects of dietary fibre and fish oil supplementation on gut in vitro contractility. In separate experiments, changes in sensitivity to electrically-driven and 8-iso-prostanglandin (PG)E₂-induced contractility were demonstrated for dietary fibre and fish oil. A modified, isolated gut super-perfusion system was then established for the rat to validate these findings. It was subsequently shown that LC n-3 PUFA from dietary fish oil significantly increased maximal contraction in response to the G-protein coupled receptor modulators, acetylcholine and the eicosanoids PGE₂, PGF₂α, 8-iso-PGE₂ and U-46619 in ileum but not colon, without changes in sensitivity (EC₅₀), when n-3 PUFA as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) had been incorporated to a similar degree into the gut total phospholipid membrane pool. It was further established that the spontaneously hypertensive rat (SHR) had a depressed prostanoid (PGE₂and PGF₂α) response in the gut that could be restored by dietary fish oil supplementation (5% w/w of total diet) in the ileum but not the colon. Importantly, the muscarinic response in the colon of the SHR was increased by fish oil supplementation with DHA likely to be the active agent. Dietary fish oil dose experiments deduced differential increases in response occurred at fish oil concentrations of 1% for muscarinic and 2.5% (w/w) for prostanoid stimulators of the ileum with no difference in receptor-independent KCl-induced depolarization-driven contractility. Studies combining high amylose resistant starch (HAMS, 10% w/w) and fish oil (10% w/w) fed to young rats demonstrated a low prostanoid response that was enhanced by dietary fish oil but not resistant starch. There was however, an interactive effect of the HAMS and fish oil noted for the muscarinic-mimetic, carbachol. Generally, resistant starch increased the large bowel short chain fatty acid pool with a subsequent lower pH. Binding studies determined that while the total muscarinic receptor binding properties of an isolated ileal membrane fraction were not affected in mature rats by dietary fish oil, young rats had a different order of muscarinic receptor subtype response with a rank order potency of M₃ > M₁ > M₂ compared to mature animals of M₃ > M₂ > M₁ with fish oil altering the sensitivity of the M₁ receptor subtype in isolated carbachol-precontracted ileal tissue. In conclusion, experiments using the guinea pig and rat gut models demonstrated that dietary fish oil supplementation, and to a lesser degree fibre, increased receptor-driven contractility in normal and compromised SHR ileum and colon. Further, changes in responsiveness were demonstrated in the developing rat gut prostanoid and muscarinic receptor populations that could be altered by dietary fish oil. Preliminary evidence suggested that fish oil as DHA may alter receptor-driven gut contractility by mechanisms involving smooth muscle calcium modulation. Defining the role that dietary fibre and fish oil, and other nutrients, play in normal and diseased states of bowel health such as inflammatory bowel disease (IBD), where contractility is compromised, are among the ongoing challenges. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1316907 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Methylmalonic acidemias are inherited metabolic disorders characterized by methylmalonate (MMA) accumulation and neurological dysfunction, including seizures. Dietary fatty acids are known as an important energy source and reduce seizure activity in selected acute animal models. This study investigates whether the chronic treatment with fish oil or with oleic acid attenuates MMA-induced seizures. Adult male Wistar rats were treated with fish oil (85 mg/kg), oleic acid (85 mg/kg) or vehicle (0.42 % aqueous Cremophor EL , 4 mL/kg/body weight/day), p.o., for 75 days. In the 73th day were implanted a cannula in the right lateral ventricle with electrodes over the parietal cortex for EEG recording. In the 76th day half the animals from each group were injected with NaCl (2.5 μmol/2.5 μL, i.c.v.), and the other half with MMA (2.5 μmol/2.5 μL, i.c.v.), and seizure activity was measured by EEG recording with concomitant behavior monitoring. The effect of prostaglandin E2 (PGE2) on Na+,K+-ATPase activity of slices of cerebral cortex from NaCl-injected (control) animals was determined. Fish oil administration increased the latency for MMA-induced tonic-clonic seizures and reduced the mean amplitude of ictal EEG recordings. Oleic acid decreased mean amplitude of ictal EEG recordings. Treatment with fish oil prevented PGE2-induced decrease of Na+,K+-ATPase activity in cortical slices in vitro. The results support a major anticonvulsant role for fish oil against MMA-induced seizures. The decreased sensitivity of Na+,K+-ATPase from fish oil-treated animals to the inhibitory effect of PGE2 may be related to its currently reported anticonvulsant activity. / A acidemia metilmalônica é um erro inato do metabolismo caracterizado pelo acúmulo tecidual de ácido metilmalônico (MMA) e disfunção neurológica, incluindo convulsões. Os ácidos graxos dietéticos são conhecidos como fonte de energia e reduzem a atividade convulsivante em determinados modelos experimentais agudos de convulsão. Este estudo investiga se o tratamento crônico com óleo de peixe ou com ácido oléico atenua as convulsões induzidas por MMA. Ratos Wistar machos adultos foram tratados com óleo de peixe (85 mg / kg), ácido oléico (85 mg / kg) ou veículo (solução aquosa de Cremophor EL® 0,42%, 4 mL / kg de peso corporal / dia), via oral, por 75 dias. No 73º dia foi implantada uma cânula no ventrículo lateral direito e dois eletrodos sobre o córtex parietal para o registro eletroencefalográfico. No 76º dia metade dos animais de cada grupo foi injetada com NaCl (2,5 Smol / 2,5 SL, i.c.v.), e outra metade com MMA (2,5 Smol / 2,5 SL, i.c.v.), e a atividade convulsiva foi medida por EEG e monitoramento comportamental concomitantemente. O efeito da prostaglandina E2 (PGE2) na atividade Na+, K+-ATPase foi determinada em fatias de córtex cerebral dos animais injetados com NaCl (controle). A administração de óleo de peixe aumentou a latência para as convulsões tônico-clônicas induzidas por MMA e reduziu a amplitude média dos registros ictais de EEG. O ácido oléico diminuiu a amplitude média dos registros ictais de EEG. O tratamento com óleo de peixe preveniu a diminuição da atividade da Na+, K+-ATPase induzida por PGE2 em fatias corticais in vitro. Os resultados suportam um papel importante anticonvulsivante do óleo de peixe sobre as convulsões induzidas por MMA. A prevenção da redução da Na+, K+-ATPase induzida por PGE2 nos animais tratados com óleo de peixe pode estar relacionada à sua atividade anticonvulsivante atualmente relatada.

Orientador: Valquíria Cação Cruz-Polycarpo / Resumo: É importante a determinação atualizada dos valores nutricionais dos alimentos para a prática de formulação de rações para aves. O objetivo desta pesquisa foi avaliar o perfil de ácidos graxos e energia metabolizável aparente corrigida pelo balanço de nitrogênio (EMAn) de diferentes fontes de óleos (soja, girassol, linhaça e peixe) para galinhas poedeiras da linhagem Hysex White, com 24 semanas de idade. As dietas experimentais consistiram de: T1 – ração basal, T2 – ração basal + 10% de inclusão de óleo de soja, T3 – ração basal + 10% de inclusão de óleo de girassol, T4 – ração basal + 10% de óleo de linhaça e T5 – ração basal + 10% de óleo de peixe, distribuídas num delineamento inteiramente casualizado com sete repetições e oito aves por unidade experimental. O perfil de ácidos graxos das fontes lipídicas foi analisado por cromatografia gasosa. O ensaio de digestibilidade foi realizado pelo método de coleta total de excretas, utilizando-se 280 poedeiras em 35 gaiolas experimentais, incluindo aves do grupo controle. Os resultados foram submetidos à análise de variância e as médias dos tratamentos foram comparadas pelo teste de tukey à 5% de probabilidade. Os maiores níveis de ácidos graxos insaturados foram encontrados nas fontes de origem vegetal, com mais de 80% enquanto o óleo de peixe apresentou 58,38%. Os valores de EMAn na matéria natural foram: 9.334 kcal/kg para o óleo de soja; 10.533 kcal/kg para o óleo de girassol; 10.928 kcal/kg para o óleo de linhaça e 9.005 kcal/... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre