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Studies had shown that leucine supplementation increases insulin sensitivity and it has been studied that n-3 PUFA may have an anti-inflammatory effect in adipocytes. However, the extent to which dietary sources such as leucine and/or n-3 PUFA act through PFKFB3/iPFK2 to suppress adipocyte inflammatory response has not been studied; PFKFB3/iPFK2 is a regulator that links adipocyte metabolism and inflammatory responses. In this study, the involvement of PFKFB3/iPFK2 in the effects of insulin sensitizing and anti-inflammatory effect of leucine and/or n-3 PUFA are explored using cultured 3T3-L1 adipocytes including wild-type cells, PFKFB3-control cells (iPFK2-Ctrl) and PFKFB3-knockdown cells (iPFK2-KD). In iPFK2-Ctrl cells, leucine supplementation appears to have insulin-sensitizing effects through improving p-Akt/Akt insulin signaling, but have no effect on adiponectin expression, and appear to have limited anti-inflammatory effects. n-3 PUFA supplementation appears to have limited effects on both insulin sensitizing and anti-inflammatory effects in iPFK2-Ctrl. In contrast, n-3 PUFA exhibit pro-inflammatory expression in iPFK2-KD. The results of this study support the hypothesis that PFKFB3/iPFK2 is critically involved in insulin-sensitizing effects of leucine. This role of PFKFB3/iPFK2, however, appears to be independent of anti-inflammatory responses. Given this, it is likely that PFKFB3/iPFK2 only account, in part, for the beneficial effects of leucine. n-3 PUFA stimulate PFKFB3/iPFK2 activity in wild-type adipocytes. However, PUFA do not exhibit anti-inflammatory and insulin-sensitizing effects in controls. In contrast, n3-PUFA exhibit proinflammatory effects in iPFK2-KD cells. Taken together, PFKFB3/iPFK2 is involved, at least in part, in the effects of insulin sensitization of leucine and appears to protect adipocytes from inflammatory responses, which could be exacerbated by n-3 PUFA when PFKFB3/iPFK2 is disrupted.

The aim of this thesis was to investigate how dietary fatty acids affect the risk for cardiometabolic disease, i.e. cardiovascular disease (CVD), type 2 diabetes and obesity. The overall hypothesis was that unsaturated fatty acids and especially the predominant polyunsaturated fatty acid (PUFA) linoleic acid (LA), 18:2n-6, would decrease cardiometabolic risk compared with saturated fatty acids (SFAs), in line with current recommendations to partly replace dietary SFA with PUFA. Papers I and V were observational studies based on the community-based cohort Uppsala Longitudinal Study of Adult Men (ULSAM). Adipose tissue fatty acid composition was determined as biomarker for dietary fat intake. Studies II, III and IV were randomised short-term interventions on human volunteers, in which different dietary fats were provided to the participants. In 71-year-old men, adipose tissue LA and α-linolenic acid (18:3n-3) were associated with insulin sensitivity (euglycaemic clamp), although this association was diminished for LA after adjusting for lifestyle variables. Different SFA displayed divergent associations; only palmitic acid (16:0) was inversely associated with insulin sensitivity (Paper I). In Cox regression analyses, LA was modestly associated with decreased all-cause mortality, but not CVD mortality during 15 years follow-up (Paper V). In a 3+3-week cross-over study on 20 weight-stable volunteers with dyslipidaemia, all foods were provided. A rapeseed oil-based diet distinctly lowered low-density lipoprotein cholesterol and triglycerides compared with a dairy-fat based diet (butter, cream and fatty cheese). Insulin sensitivity or coagulation factors were not affected (Paper II). In a 10-week randomised trial on 67 abdominally obese participants, PUFA (mostly sunflower oil) decreased liver fat compared with SFA (mostly butter) under isocaloric conditions. In individuals considered highly compliant to study diets, lipoproteins were also decreased during the PUFA diet (Paper III). In a 7-week double-blind randomised trial on 41 healthy volunteers, PUFA (sunflower oil) decreased the total:HDL cholesterol ratio compared with SFA (palm oil) during moderate weight gain (1.5 kg) (Paper IV). In conclusion, LA (PUFA) intake is associated with decreased cardiometabolic risk compared with higher SFA intake, overall supporting a beneficial role of non-tropical vegetable oils in place of solid fats in preventing fatty liver and cardiometabolic disorders.

The aim of my thesis was to assess the influence of polyunsaturated fatty acids n 3 (DHA and EPA) on gene expression, mainly PPARy, in model organism. The expression of PPARy gene is increased by these polyunsaturated fatty acids and thus they decrease the risk of development of inflammation and atherosclerosis. This hypothesis was tested on 40 test rats (Wistar albino). The first 7 weeks the rats were fed with a diet containing beef tallow and sweet condensed milk until they manifested mild obesity and inflammation. During other 7 weeks they were divided into 4 groups, each containing 10 individuals. One of the groups was further fed with diet containing beef tallow (control group), other groups received diet with 6% content of oil from safflower added, 6% content of fish oil (high content of EPA) or 6% content of oil from Schizochytrium alga (high content of DHA). The expression of PPARy was measured by using molecular-biology methods at the end of the trial. It was proven that the diet rich on DHA increases PPARy gene expression (P<0,05). The increase of PPARy gene expression in a diet containing fish oil was inconclusive (P>0,05). With these results we confirmed the hypothesis that diet rich on PUFA n3 decreases the risk of inflammation development and atherosclerosis and that DHA is significantly more effective.

ANDREW R. COURSEY, for the Master of Science degree in Zoology, presented 24 June 2011 at Southern Illinois University Carbondale TITLE: ALTERNATIVE FEEDING STRATEGIES TO MAXIMIZE MARINE FEEDSTUFF REPLACEMENT IN LARGEMOUTH BASS Micropterus salmoides CULTURE WHILE MAINTAINING PRODUCTION PERFORMANCE Advisors: Chris Kohler and Jesse Trushenski Harvest of marine resources for feedstuff production is at its maximum sustainable yield and alternative feedstuffs are needed to supply livestock production for an expanding human population having a growing desire for seafood. Aquaculture is currently the fastest growing sector of agriculture, but the high cost and limited supplies of marine feedstuffs for aquafeeds could ultimately limit production of foodfish. Replacement of marine feedstuffs poses a dilemma, however, as replacement of fish meal can affect animal growth, and replacement of fish oil in aquafeeds leads to altered fillet fatty acid profiles without the maximum benefit of high omega-3 (n-3) and long-chain polyunsaturated fatty acids (LC-PUFA) content. Two feeding trials were conducted to determine if poultry byproducts can replace fish meal and fish oil in diets for largemouth bass Micropterus salmoides without affecting production performance or fillet fatty acid composition. Poultry byproducts were able to replace fish meal and fish oil while maintaining production performance of largemouth bass, however, the altered fatty acid profiles of the feeds were reflected in the fillets. Finishing diets containing high levels of fish oil were implemented to restore n-3 and LC PUFA concentrations in the fillet. Twelve weeks of finishing proved suitable to partially restore n-3 and LC-PUFA concentrations while limiting the overall use of marine resources in the largemouth bass production cycle.

Cobia (Rachycentron canadum) aquaculture is poised for expansion, due in part to rapid growth rate, tolerance of culture conditions, and high market value of this species. Similar to other carnivorous marine fishes, the high monetary cost and long-range unsustainability of reliance on fish oil as the principle lipid source for cobia feeds necessitates evaluation of alternative lipid sources to spare or replace fish oil. Unfortunately, alternative lipid-based feeds may affect production performance, and typically yield fillets with reduced levels of long-chain polyunsaturated fatty acids (LC-PUFAs) such as docosahexaenoic acid (DHA, 22:6n-3). Recent research has indicated that diets rich in saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), coupled with the application of so-called "finishing feeds" (feeds containing elevated levels of fish oil relative to grow-out feeds) at the end of the production cycle, may limit or attenuate these effects of fish oil sparing. Accordingly, I conducted two trials to evaluate the usefulness of alternative lipids and finishing in cobia culture. In the first trial, I assessed growth performance and tissue fatty acid composition of juvenile cobia fed diets (~11% lipid, ~48% protein) containing fish oil (control), or 50/50 blends of fish oil and alternative lipids (beef tallow, pork lard, partially and fully hydrogenated soy oils, and traditional soy oil) supplemented with an algal meal (to meet the 22:6n-3 requirement of cobia) for 8 weeks. Although minor differences were observed in feed intake, growth performance was otherwise equivalent among the dietary groups. Tissue fatty acid composition varied significantly among treatments, however, with alternative lipids containing higher levels of saturated (SFAs) and monounsaturated fatty acids (MUFAs) yielding tissue profiles that were most similar to those associated with the fish oil-based control feed. Although beef tallow and hydrogenated soybean oil were largely equivalent in terms of growth performance and fatty acid profile change, beef tallow was selected for further evaluation because of its low cost. In the second trial, beef tallow replaced fish oil in feeds at one of four substitution levels: 0% (100% fish oil), 33%, 67%, or 100% tallow (with algal 22:6n-3 meal included in all feeds as before). Juvenile cobia were raised on these feeds for 8 weeks, then switched to the 100% fish oil-based control feed for an 8-week finishing period. During finishing, subsamples of fish were collected every 2 weeks to quantify changes in tissue fatty acid profile over time as a result of finishing. In the second trial, the overall similarity of tissue fatty profiles to those in the 100% fish oil control treatment increased over the course of finishing, mostly as a result of declines in SFAs and MUFAs. These results suggest that SFA and MUFA-rich lipids, such as fully hydrogenated soy oil and beef tallow, are effective and strategically valuable as partial substitutes for fish oil in cobia feeds, and that finishing is at least partially effective in restoring cobia tissue fatty acid composition to a state approximating that of cobia fed only fish oil as dietary lipid.

Aquaculture is currently the fastest growing sector of protein production, and is expected to overtake the harvest of wild fisheries. Limitations in nutrition, specifically fatty acid nutrition, are preventing even more dramatic growth of many species of commercial importance. Currently, much of the research involving fatty acids examines requirements as being correlated to thermal guilds (warm vs. cool water) or salinity tolerance (marine vs. freshwater). However, recent studies have revealed the potential for trophic level to be as much, if not more, influential in determining fatty acid requirements of a species. As such, two feeding trials were conducted to determine the requirements of two species of different trophic levels (Oreochromis niloticus and Trachinotus carolinus) based on C18 PUFA vs. LC-PUFA. Nile Tilapia, O. niloticus, exhibited similar growth regardless of the inclusion of C18 PUFA or LC-PUFA, however, tissue fatty acid profiles were influenced per the diet provided. As such, it was concluded that Nile Tilapia exhibit the capacity to effectively synthesize LC-PUFA from C18 PUFA as is seen in many species that occupy low trophic levels. Florida Pompano, T. carolinus, did not exhibit any significant differences in growth regardless of the diet provided, but numerical differences indicated benefits towards inclusion of dietary LC-PUFAs. Similar to O. niloticus, tissue fatty acid profiles were significantly affected by dietary treatment. Based on numerical differences in growth performance and significant differences in tissue fatty acids, it was concluded that Florida Pompano show a typical carnivorous requirement for LC-PUFA.

The aim of the study was to determine the impact of diet (natural and formulated feed) on the chemical composition and fatty acids profile of the harvested fish. The content of dry matter in fish flesh resulting from the formulated diet was higher vs. the natural diet (23.94?1.24 % vs. 19.66?0.82 %) with nitrogenous compounds (60.24?2.82 % vs. 72.12?1.75 %), total fat content (24.81?4.51 % vs. 6.14?2.85 %) and ash (7.55?1.28 % vs. 10.54?1.53 %) respectively. The spectrum of fatty acids was determined by gas chromatography using Varian 3800 equipment. Tench fed on a formulated diet in the recirculating system had a significantly higher content (P< 0.05) of monounsaturated fatty acid (MUFA = 43.04?1.68 %) and n-6 polyunsaturated fatty acids (PUFA = 15.47?1.07 %) in their flesh compared to the flesh of fish reared in earth ponds on a natural diet - MUFA (32?5.29 %) and n-6 PUFA (13.6?1.66 %). Tench fed on a natural diet in earth ponds proved to have a significantly higher content (P< 0.05) of n-3 PUFA (16.8?4.38 %) and ? PUFA (30.3 ? 5.3 %) than tench reared in the recirculating system - PUFA n-3 (10.05?0.85 %) and ? PUFA (25.52?1.07%). The ratio n-3/n-6 for fish from earth ponds was 1.2; for fish from the recirculating system the ratio was 0.65. The results show a significantly higher composition of n-3 PUFA in flesh of tench from earth pond with natural food compared to fish on an intensive feeding diet in the recirculating system which showed a higher content of n-6 PUFA.

Tese(doutorado) - Universidade Federal do Rio Grande, Programa de Pós–Graduação em Oceanografia Biológica, Instituto de Oceanografia, 2011. / Submitted by Cristiane Gomides (cristiane_gomides@hotmail.com) on 2013-12-15T17:48:26Z No. of bitstreams: 1 LUCELIA.pdf: 1063843 bytes, checksum: d9a3275320eb091ac6c93c7e1b9d854d (MD5) / Rejected by cristiane soares (krikasoares@live.com), reason: faltam dados na citação on 2013-12-18T19:46:30Z (GMT) / Submitted by Cristiane Gomides (cristiane_gomides@hotmail.com) on 2013-12-19T09:23:08Z No. of bitstreams: 1 LUCELIA.pdf: 1063843 bytes, checksum: d9a3275320eb091ac6c93c7e1b9d854d (MD5) / Approved for entry into archive by cristiane soares (krikasoares@live.com) on 2014-02-23T02:01:54Z (GMT) No. of bitstreams: 1 LUCELIA.pdf: 1063843 bytes, checksum: d9a3275320eb091ac6c93c7e1b9d854d (MD5) / Made available in DSpace on 2014-02-23T02:01:54Z (GMT). No. of bitstreams: 1 LUCELIA.pdf: 1063843 bytes, checksum: d9a3275320eb091ac6c93c7e1b9d854d (MD5) Previous issue date: 2011 / Microalgas marinhas são organismos ricos em lipídios e ácidos graxos, de grande interesse devido ao seu potencial biotecnológico. O objetivo desta tese foi determinar possíveis efeitos sinergéticos da intensidade luminosa (40 e 400mol fótons m-2s-1), temperatura (15 e 30°C), fonte de nitrogênio (NO3- e NH4+) e fase de crescimento (logarítmica e estacionária), sobre o conteúdo e a produtividade de lipídios e ácidos graxos de duas microalgas marinhas Nannochloropsis oculata e Thalassiosira weissflogii. Além disso, foi avaliado, também, como o uso de floculantes aniônicos e catiônicos podem influenciar a extração de lipídio e o perfil de ácidos graxos produzidos por estas microalgas. Os maiores conteúdos e produtividade dos lipídios para ambas as espécies ocorreram em tratamentos com maior intensidade luminosa (400 mol fótons m-2 s-1) e utilizando nitrato como fonte de Nitrogênio. N. oculata acumulou mais lipídios na fase estacionária, enquanto que em T. weissflogii maiores concentrações de lipídios só ocorreram na fase estacionária de tratamentos com menor temperatura (15º C). As duas espécies apresentaram maior produtividade de lipídios em tratamentos com maior produção de biomassa de microalgas. Entretanto, células de N. oculata que cresceram no tratamento com amônio e elevada luminosidade apresentaram grande produtividade de lipídio devido a elevados níveis de concentração deste elemento nas células e não devido ao maior crescimento celular. Portanto uma maior produtividade pode ser obtida empregando estratégias de cultivo que beneficiam, simultaneamente, o crescimento celular e/ou o acúmulo de lipídios nas células. Os ácidos graxos com maior conteúdo em ambas as espécies foram C14:0, C16:0, C16:1, C18:1 e C20:5. Para as duas espécies, maiores concentrações de ácidos graxos saturados e monoinsaturados foram obtidos em tratamentos com maior temperatura (30º C). Ácidos graxos poli-insaturados (PUFAS), especialmente o ácido eicosapentaenóico (C20:5), foi produzido em maior quantidade por N. oculata em tratamentos com baixa luminosidade e Nitrato. O uso de floculantes não alterou a quantidade de lipídios extraídos de ambas as espécies. Entretanto, o floculante aniônico diminuiu o conteúdo de C20:5 em N.oculata, enquanto que em T. weissflogii, os floculantes aniônico e catiônico reduziram os conteúdos de C18:0 e C18:1n9c / Marine microalgae are rich in lipids and fatty acids. These microorganisms are of great interest, mainly due to their biotechnological potential. The aim of this thesis was to determine possible synergistic effects of light intensity (40 and 400 mol photons m-2s-1), temperature (15 and 30° C), nitrogen source (NO3- and NH4+), and growth phase (logarithmic and stationary) in the productivity, content of lipids and fatty acids in two marine microalgae: Nannochloropsis oculata and Thalassiosira weissflogii. Moreover, it was evaluated if the use of anionic and cationic flocculants can influence the extraction of lipids and fatty acid profiles produced by these species. The higher lipid content and productivity for both microalgae occurred in treatments with higher light intensity (400 mol photons m-2 s-1), and with nitrate as nitrogen source. N. oculata accumulated more lipids in the stationary phase, while higher concentrations of lipid in T. weissflogii occurred only in stationary phase from treatment with a lower temperature (15° C). The two microalgae species showed higher lipid yields in treatments with higher biomass production. However, during N. oculata cells grown, the treatment with ammonium and high light showed large lipid productivity due to high concentrations of this element in the cells, and not due to increased cell growth. Therefore highest lipid productivity can be achieved using cultivation strategies that benefit both cell growth and accumulation of lipids in cells. The fatty acids with greater content of both species were C14:0, C16:0, C16:1, C18:1 and C20:5. Higher concentrations of saturated and monounsaturated fatty acids were obtained in treatments with higher temperature (30° C) in both species. Polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (C20:5), was produced in greater amounts by N. oculata in treatments with low light and nitrate. The use of flocculants did not alter the amount of lipids extracted from both species. However, the anionic flocculant decreased content of C20:5 in N. oculata, while for T. weissflogii, the cationic and anionic flocculants reduced content of C18:0 and C18:1n9c.

The thesis contains a theoretical overview, which has focused on classification of lipids, fatty acids and their metabolisms, with emphasis on polyunsaturated fatty acids inclusive of their conversion to eicosanoids, functions and effects of eicosanoids on human health. Furthermore, the theoretical part of the thesis has focused on a food’s enrichment of polysaturated fatty acids n-3 and several possibilities of analytical determination of fatty acids. The practical part of the thesis has aimed attention at the determination of fatty acids in the liver, fat and muscles’ tissue of the experimental rats. The animals were fed with a standard feed mixture, which contains 5 % of fish oil, 5 % of palm oil, 5 % of safflower oil and 5 % of Schizochytrium algae extract. The obtained fat was extracted from the lyophilized samples followed by derivatization and analytical determination of the fatty acid derivatives by gas chromatography. From the results of the experimental part could be determined, that the addition of 5 % of fish oil to the feed mixture of the experimental animals increased the deposition of EPA and DHA in all examined tissues (p < 0.05). The addition of 5 % of the Schizochytrium extract to the animal’s feed mixture had an impact on the effective deposition of DHA and DTA in the examined tissues of all used oils (p < 0.05). Both of the previously mentioned oils can be considered as the most effective nutraceuticals for reducing the ratio of PUFA n-6/n-3 in the food. Unlike the addition of 5 % of safflower or 5 % of palm oil (as a control), ratios of PUFA n-6/n-3 were many times higher. Therefore, from the healthy point of view, this can be concluded as inappropriate.