Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (ARA, 20:4ù6), eicosapentaenoic acid (EPA, 20:5ù3) and docosahexaenoic acid (DHA, 22:6ù3) have been shown to have many health benefits, some of which include lowering blood pressure, providing protection against cardiovascular diseases and improving brain and eye functions. Entomopathogenic fungi, a group of fungal pathogens able to infect insects, were previously reported to produce substantial amounts of VLCPUFAs, however the genes involved in the biosynthesis of these fatty acids have yet to be identified. This research started with fatty acid analysis of five entomopathagenic fungi, of which Conidiobolus obscurus and Conidiobolus thromboides were found to produce high levels of VLCPUFAs such as ARA and EPA. Thus, these two fungal species were selected as potential gene sources for the enzymes involved in the biosynthesis of VLCPUFAs. Using degenerate reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE) methods; we cloned two full-length putative ∆6 desaturase cDNAs (CoD6 and CtD6) from the two fungi.<p> Functional expression of CoD6 in Saccharomyces cerevisiae showed it codes for a functional Ä6 desaturase, which can introduce a Ä6 double bond into linoleic acid and á-linolenic acid, respectively. However, expression of CtD6 in S. cerevisiae showed it does not have any Ä6 desaturase activity. Using degenerate RT-PCR and RACE, we also cloned two full-length ∆6 elongase cDNAs (CoE6 and CtE6) from the C. obscurus and C. thromboides species. Functional expression of these genes in S. cerevisiae showed CoE6 and CtE6 code for functional ∆6 elongase. Substrate specificity analysis indicated that both preferentially elongate 18-carbon Ä6 desaturated fatty acids, such as ã-linolenic acid and stearidonic acid. In addition, CtE6 can also elongate 20-carbon VLCPUFAs, such as ARA and EPA. The entire eicosatetraenoic acid (ETA, 20:4ù3) biosynthetic pathway was reconstituted in yeast using four genes, CoD6 (a ∆6 desaturase) and CoE6 (a ∆6 elongase) from Conidiobolus obscurus, CpDes12 (a Ä12 desaturase) and CpDesX (a ù3 desaturase) from Claviceps purpurea. Yeast transformant expressing the four genes produced several new fatty acids. Among them, eicosatetraenoic acid (ETA) accounts for approximately 0.1% of the total fatty acids. Although the level of ETA in the transformant is low, this represents the first report describing the reconstitution of the entire ETA pathway in yeast without exogenous supplementation of any fatty acids.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成23年3月25日 村松崇氏の博士論文として提出された

A study was performed to investigate the effect of diet modifications on skin and hair coat condition in canines. The study included 24 normal adult dogs fed a baseline diet (Ol'Roy[trademark]), during an acclimation period of 12 wk (Phase I). Nine female Beagles and 15 male Hound mix-breed dogs were used. For the next 12 wk (Phase II) the dogs were divided into three groups and fed one of three specially formulated diets. They contained similar ingredients and had similar nutrient profiles except for the following differences: Diet A contained lower but adequate amounts of dietary zinc and linoleic acid than diet B. Diet C was similar to B with respect to zinc and linoleic acid but contained more α-linolenic acid. An evaluation panel conducted skin and hair coat condition scoring on wk 0, 4, 7, and 12 (Phase I) and wk 14, 16, 19, and 24 (Phase II). The panel evaluated the dogs for glossiness, softness, scale, greasiness, and overall condition. Transepidermal water loss (TEWL) and skin hydration (HYDR) assessments were determined on wk 3, 7, and 11 (Phase I) and wk 11, 12, 15, 19, and 23 (Phase II) using a Tewameter[trademark] and Corneometer[trademark] respectively. Blood samples were collected on d 0, 5, 8, 16, 28, 56, and 84. Profiles of plasma phospholipid fatty acids were determined at each collection period. Serum zinc concentrations were analyzed on wk 12, 14, and 24. The hypothesis was that a diet containing increased LA, ALA, and zinc concentrations (diet C) would show improvements of skin and hair coat condition in dogs compared to the other diets. All three test diets caused significant improvements compared to Ol'Roy[trademark]. Diet B caused more improvement than diet A in both subjective and objective assessments of skin and hair coat. Based on mean values diet B is better to be fed to dogs that need to improve skin hydration and diet C should be fed to dogs that need to decrease TEWL. Diet C not only led to improvements in skin and hair coat condition, but also provided additional benefit by producing less pro-inflammatory conditions in the skin.

Long-chain polyunsaturated fatty acids (LCPUFA) are essential for proper neural and retinal development in many mammalian species. One objective of this research was to investigate the effects of dietary &#945;-linolenic acid (ALA) and LCPUFA on the fatty acid composition of canine plasma phospholipids (PL) and milk during the gestation and lactation periods. The fatty acid composition of plasma PL and the retinal development of puppies reared on the same experimental diets as their mothers were also investigated. Enriching the canine gestation/lactation diet with ALA (6.8% DM) does not result in enrichment of docosahexaenoic acid (DHA) in the milk. From this data it can be inferred that peroxisomal elongation and desaturation of LCPUFA does not occur in canine mammary tissue. Dose responses of linoleic acid (LA), ALA and DHA were observed in the plasma of adult dogs during gestation and lactation and in puppies during both the suckling and post-weaning periods. Plasma PL fatty acid data from puppies indicate that canine neonates are capable of synthesizing LCPUFA from ALA, but that plasma enrichment of the newly-synthesized DHA does not compare with that obtained from preformed DHA in the diet. Visual function was assessed via electroretinography (ERG) in 12-wk old canines. One-way ANOVA revealed significantly better visual performance in dogs fed the highest amounts of n-3 LCPUFA. Puppies in this group demonstrated the greatest rod response as measured by the amplitude and implicit time of the a-wave. Neonates reared on the lowest dietary levels of both ALA and n-3 LCPUFA exhibited the poorest visual function. A novel parameter devised in this study was the threshold intensity, which was the initial intensity at which the a-wave was detectable. Again, puppies consuming the greatest concentrations of n-3 LCPUFA responded significantly sooner, i.e. exhibited greater rod sensitivity, than other diet groups. The findings of this research underscore the importance of preformed n-3 LCPUFA in the diet, rather than ALA, as a means of enriching neural tissues in DHA during the developmental period. Moreover, dietary DHA appears to be related to improved visual performance in developing canines.

No description available.

ABCD2 (D2) is a peroxisomal ATP binding cassette (ABC) transporter that is expressed in brain, adrenal and liver. D2 is transcriptionally regulated by key transcriptional factors that control lipid and glucose metabolism. Therefore, we examined its role in adipose tissue. These studies revealed that D2 is highly abundant in adipose tissue and upregulated during adipogenesis. However, D2 deficiency does not affect either adipogenesis or lipid accumulation. An examination of the lipid profile of adipose tissue revealed the accumulation of C20 and C22 fatty acids in D2 deficient (D2‐/‐) mice. When challenged with a diet enriched in erucic acid (C22:1, 10% kcal), this lipid accumulated in both liver and adipose tissue. Following 8 weeks of diet, D2‐/‐ mice showed increased adiposity, glucose intolerance, dyslipidemia and steatosis. Analysis of the hepatic lipid profile showed significant changes away from poly unsaturated fatty acids (PUFAs) and toward C18‐22 mono‐unsaturated fatty acids (MUFA). RT‐PCR of the mRNA from the adipose tissue and liver revealed significant changes in lipogenic (ACC, SCD1 & 2) and PUFA synthesis (Δ5 & 6‐desaturase) genes in D2‐/‐ mice. The molecular mechanisms by which D2 regulates lipid metabolism in adipose tissue remains unclear. To explore potential mechanisms, the subcellular localization of D2 in adipose tissue was determined. Our results demonstrated that D2 resides in a distinct subclass of peroxisomes that does not containing classical peroxisomal markers such as pex19 or PMP70, but are positive for pex14. In conclusion, our studies reveal a novel role of D2 and peroxisomes in the protection from disruptions of lipid metabolism induced by dietary erucic acid and that D2 resides in a unique compartment within adipocytes that plays a yet to be elucidated role in the regulation of lipid metabolism.

The role of dietary fatty acids and their subsequent effects on metabolism has received considerable attention in mammalian species. It is becoming increasingly clear that fatty acids have metabolic roles over and above their influence on energy density of the diet. Recent studies have linked changes in the fatty acyl composition of the plasma membrane, induced by the dietary fat profile, to alterations in both lipid and glucose metabolism. These dietary induced changes have profound effects on insulin action, glucose transport and enzyme activity that regulate triglyceride and fatty acid synthesis, factors that ultimately influence protein and lipid deposition of animals. Because of their high growth rate, broiler chickens have a high requirement for energy and the use of triglycerides as a major energy source has resulted in a fat carcass. A change in the glucose-insulin balance has been suggested as being the main reason for differences in adiposity between broilers selected for fatness or leanness. The hypotheses of this thesis is based on the finding that dietary polyunsaturated fatty acids (PUFA�s) increase the sensitivity of muscle tissue to insulin and this would presumably augment insulin-stimulated glucose uptake into muscle cells. Therefore, increasing the capacity of broiler muscle tissue to utilise glucose as its principal energy substrate would reduce the bird�s reliance on triglycerides and this inturn would result in a leaner carcass. The aims of this study are firstly to explore the role that dietary PUFA�s from the n-3 and n-6 series have on the growth and body composition of broiler chickens and secondly to determine the relationships between dietary fatty acid profile, tissue insulin sensitivity and lipid deposition. Because dietary fatty acids have been implicated in the modulation of hormones important for the growth and development of animals, a third aim of this thesis is to determine the effects of dietary n-3 and n-6 polyunsaturated fatty acids on pituitary and adrenal sensitivity. Since the modulation of metabolism by dietary fatty acids has been attributed to changes in the fatty acyl composition of the plasma membrane, the final aim of this study will be to investigate and characterise the molecular species of the breast muscle choline and ethanolamine phospholipids. Feeding either fish oil a source of n-3 PUFA�s or sunflower oil a source of n-6 PUFA�s fatty acids to broiler chickens resulted in a significant (P<0.01) reduction in the abdominal fat pad mass and a modest increase in breast muscle mass when compared to broilers fed edible tallow. Associated with the changes in carcass composition was an alteration in energy substrate utilisation. This was reflected by lower respiratory quotients and reduced triglyceride and insulin concentrations for the chickens fed the two PUFA diets. Coupled to the shift in energy metabolism was a significant (P<0.05) increase in the proportion of PUFA�s incorporated into the abdominal fat pad and breast muscle. The dietary fat supplements resulted in the incorporation of specific fatty acid subtypes. Feeding fish oil significantly increased the proportion of long-chain n-3 PUFA�s whereas feeding sunflower oil significantly increased the proportion of long-chain n-6 PUFA�s compared to tallow feeding whose tissues were dominated by a higher proportion of saturated fatty acids. It was further shown that dietary n-3 and n-6 PUFA�s enhanced glucose /insulin action. Feeding either fish oil or sunflower oil to broiler chickens increased insulin action when examined by an intravenous glucose tolerance test. The maximal insulin release in response to the glucose infusion was higher in the tallow fed group compared to either the sunflower oil or fish oil groups. To estimate the disappearance rate of glucose from the plasma and its incorporation into tissues, 2-deoxy-D-3H glucose was infused into each chicken. There were no significant differences in the clearance rate of 2-deoxy-D-3H glucose from the plasma. However, when measured under steady state conditions, the labelled glucose incorporation into the breast muscle was greater in birds fed fish oil compared to either tallow or sunflower oil feeding. The dietary fatty acid induced increase in insulin action suggests that the sensitivity of muscle cells to insulin was enhanced. This modulation of tissue sensitivity by dietary fatty acids was also shown to occur at the level of the pituitary. To provide an estimate of pituitary sensitivity, bolus GnRH and GHRH infusions were given on different days to chickens fed the three dietary treatments. Feeding sunflower oil (n-6 PUFA�s) increased the level of GH that was released in response to the GHRH infusion when compared to birds fed either tallow or fish oil (n-3 PUFA�s). This dietary fatty acid modulation appears to be specific to certain pituitary cell types as there was no effect on LH secretion following the GnRH infusion. Dietary fatty acid modulation of endocrine gland sensitivity is particular to the gland type. Although the dietary treatments mediated a distinct pattern in pituitary sensitivity to GHRH infusion, these same three diets did not influence adrenal sensitivity, as there was no difference in the corticosterone profile following either ACTH or CRF infusion. The previously observed physiological changes for the three dietary groups was expected to be positively correlated to an alteration of the plasma membrane phospholipids induced by the dietary fatty acids. Supplementation with fish oil (n-3 PUFA�s) significantly increased levels of both eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) into the choline (PC) and ethanolamine (PE) breast muscle phospholipids compared to either sunflower oil (n-6 PUFA�s) or tallow supplementation. The increase in n-3 PUFA incorporation was associated with a corresponding decrease in the proportion of arachidonic acid (AA; 20:4n-6) an event that would presumably alter substrate availability for the 1- and 2-series eicosanoids. However, feeding sunflower oil or tallow gave a molecular species profile that was remarkably similar in both fatty acid subtype and proportion. This suggests that the plasma membrane dynamics would be similar for these two dietary groups. Therefore, it is appears that factors other than a change in the fatty acyl- composition of the plasma membrane may be responsible for modifying the physiology of the broiler.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.

Feeds that maximize reproductive potential are needed to ensure success of captive broodstock. Nutritional requirements for somatic growth of juvenile fishes differ from nutritional requirements of mature fishes and are largely species-specific. Broodstock nutritional research has focused primarily on lipid and fatty acid requirements and their effects on reproductive conditioning due to the importance of these nutrients in providing metabolic energy and structural elements, i.e. for phospholipids for embryonic development. Development of suitable broodstock feeds are limited by not knowing fatty acid requirements for many species. Once requirements are identified, plant, grain, marine, algal, and fungal lipid sources can be blended to develop least-cost diet formulations. The objectives of this dissertation are to (1) evaluate white bass Morone chrysops ovum fatty acid composition and reproductive performance after feeding maternal broodstock graded levels of squid to fish oil; (2) evaluate flax, canola, and corn oils as alternatives to marine oil(s) in white bass maternal broodstock diets; (3) determine extent to which grain oils can replace marine oils in female white bass broodstock diets in order to maintain reproductive performance and quantify fatty acid utilization of larvae with different initial fatty acid profiles; and (4) assess dietary supplementation of 20:4n-6, 20:5n-3, and 22:6n-3 concentrates to boost reproductive performance of female white bass fed primarily plant oil-based lipid sources. Of the marine and plant oils tested, menhaden fish oil provided female white bass broodstock the fatty acids (~3.9% n-3 long-chain polyunsaturated fatty acids; LC-PUFA; dry matter basis) necessary to maximize embryonic survival; however, flax oil, due to its low 18:2n-6 and high 18:3n-3 content, showed promise as a suitable plant oil candidate for partial if not complete marine oil substitution in female white bass broodstock feeds. Differential responses in embryonic and larval survival resulted in comparable total larval yields at 5 days post hatch (DPH) after feeding female broodstock graded levels (0, 33, 67, or 100%) flax to fish oils for 30 weeks prior to spawning. At the end of the endogenous feeding period, fatty acid compositions of flax and fish oil-fed broodstock progeny deviated from initial ova composition. Although n-3 LC-PUFA from menhaden fish oil are essential for embryonic survival, sunshine bass appear to have lower n-3 LC-PUFA requirements after hatch. Larval survival was highly dependent on the presence of C18 PUFA present due to flax oil inclusion in maternal diets. Embryonic survival of progeny produced from broodstock fed dietary saturated fatty acid-rich plant lipids supplemented with intact LC-PUFA concentrates (~3.4% n-3 LC-PUFA; dry matter basis) was similar to that of the broodstock fed the menhaden fish oil control diet containing 4.8% n-3 LC-PUFA. Although the dietary requirement for n-3 LC-PUFA was reduced by feeding these LC-PUFA concentrates in combination with plant lipids, menhaden fish oil is still the most viable option for least cost broodstock diet formulations intended for white bass.

Seafood is the number one source of essential fatty acids, particularly, long-chain polyunsaturated fatty acids (LC-PUFA) in the human diet. As global population growth eventually surpasses what the world's wild commercial stocks can provide, reliance on the aquaculture industry to expand production will continue to increase in order to meet the demands of consumers worldwide. Currently, fluctuations in supply and cost coupled with environmental sustainability and contaminant concerns have motivated the aquaculture industry to research alternative lipid sources and feeding strategies in order to reduce the reliance on marine-derived resources. For most cultured species, replacing fish oil with terrestrial plant-based lipid sources is a minor dietary modification that has little consequence on production performance. However, fish raised on these plant-based lipid alternatives contain considerably higher medium chain polyunsaturated fatty acids (MC-PUFA) and n-6 fatty acids and less beneficial LC-PUFA and n-3 fatty acids within the fillets, thus negatively impacting the nutritional value of cultured seafood to the consumer. In order to alleviate this problem, producers can employ finishing strategies to restore fillet LC-PUFA content prior to harvest. As a complement to this approach, provision of dietary saturated fatty acids (SFA) and/or monounsaturated fatty acids (MUFA) in lieu of MC-PUFA appears to maximize the retention of LC-PUFA deposition during the grow-out period and may increase deposition during finishing. Accordingly, my objectives were to 1) assess whether the SFA, MUFA, and MC-PUFA content of the alternative lipid affected LC-PUFA levels in Nile Tilapia fed reduced fish oil feeds; and 2) using the optimal alternative lipid identified in the first objective, assess increasing fish oil replacement rates in conjunction with finishing to maximize product nutritional value and minimize fish oil usage in Nile Tilapia culture. To address the first objective, I assessed production performance and tissue composition of Nile Tilapia fed diets containing fish oil or blends of fish oil and various soybean-derived alternative lipids. Quadruplicate tanks of juvenile Nile Tilapia were fed diets containing fish oil (FISH, high in LC-PUFA) or a 50:50 blend of fish oil and standard (STD-SO, high in MC-PUFA), saturated fatty acid-enriched (SFA-SO, high in SFA), low α-linolenic (LO-ALA-SO, high in MC-PUFA), or hydrogenated (HYD-SO, high in MUFA) soybean oil for 16 weeks. Partial replacement of fish oil with soybean oils did not significantly affect production performance with the exception of the HYD-SO diet which yielded significantly reduced growth efficiency in comparison with some of the experimental diets, though not the FISH control. Despite distinctly different dietary fatty acid profiles, fillet fatty acid composition was similar among fish fed the FISH, SFA-SO, and HYD-SO diets. However, feeding the STD-SO and LO-ALA-SO diets resulted in significant enrichment of less desirable MC-PUFA and n-6 fatty acids within the fillet. Fillet LC-PUFA levels were equivalent among all groups despite the 50% reduction in dietary LC-PUFA intake among fish fed the soybean oil-based feeds. Based on these results, incorporation of STD-SO, SFA-SO, or LO-ALA-SO could be used as partial replacements for fish oil in Nile Tilapia feeds without impairing production performance, though SFA-rich soybean oils appeared to be the best alternative for maintaining a more "fish oil"-associated fillet fatty acid profile. Accordingly, the SFA-enriched soybean oil was selected for further study in the second objective trial that evaluated the effects of graded levels of fish oil replacement without or without implementation of finishing periods on production performance and fillet fatty acid composition. Nile Tilapia were fed feeds containing 100% fish oil (100-FO), the previously assessed SFA-enriched soybean oil (100-SFA-SO), or blends of fish oil and SFA-enriched soybean oil (50-SFA-SO, 75-SFA-SO). Triplicate groups of fish were fed the aforementioned diets exclusively throughout the feeding trial (100-SFA-SO unfinished, 75-SFA-SO unfinished, 50-SFA-SO unfinished) or in conjunction with 4 or 8 weeks of finishing with the 100-FO feed (100-SFA-SO + 4 wks, 100-SFA-SO + 8 wks, 75-SFA-SO + 4 wks, 75-SFA-SO + 8 wks, 50-SFA-SO + 4 wks, 50-SFA-SO + 8 wks) for a total of 20 weeks. Production performance was unaffected by dietary inclusion of SFA-enriched soybean oil when fed exclusively or in combination with fish oil, though growth performance was lower than observed in the previous trial and likely confounded by behavioral interactions and frequent spawning. After 12 weeks of consuming the SFA-enriched soybean oil grow-out diets, fillet levels of n-3 LC-PUFA were not statistically different from 100-FO control levels despite different levels of dietary inclusion. However, the high dietary levels of SFA in the experimental feeds did not translate into increased fillet SFA content, suggesting selective retention of LC-PUFA at the expense of fillet SFA. Finishing for 4 or 8 weeks increased fillet n-3 LC-PUFA content in all groups, though it appears that the 50- and 75-SFA-SO diets were more successful in maintaining acceptable health promoting n-3:n-6 ratios. Based on these results, SFA-enriched soybean oil-based feeds can be used as a cost-saving measure during grow-out, and the effects of these feeds on fillet fatty acid profile can be reversed to a considerable extent in as little as 4 weeks by implementing a finishing period prior to harvest. This approach is a promising strategy for minimizing fish oil usage while maximizing product value of cultured Nile Tilapia.