Deliverables Dietary vegetable lipids and intestinal integrity - effects on fatty acid composition, barrier functions, pathogen translocation, microbiota and disease resistance Lipid composition of TAG changes rapidly in liver and anterior intestine when fish were fed diets enriched in one particular fatty acid; 18:1, 18:2 or 18:3. TAG fatty acid composition rapidly approached the level of the selected fatty acid in the diet. In the posterior intestine and muscle the response was slower, indicating that these tissues do not use neutral lipids directly from the diet. The polar lipids also changed in accordance to the diet but only to about one third of the effect seen for non-polar lipids, suggesting that the fish are able to keep the fatty acid composition of the cell membranes at a fairly stable level irrespective of the dietary fatty acid supply. Mixed vegetable diets, all containing a small amount of marine oil resident in the un-extracted fishmeal, showed less pronounced changes in tissue fatty acid composition. When fed the extreme diets, the fish encountered problems in maintaining the levels of 20:5n-3 and 22:6n-3 in their polar lipids, a pattern that was not obvious in the fish fed mixed diets. Even fish fed only the small amounts of marine lipids that were retained in the fish meal (2% of total lipids) were relatively successful in maintaining their levels of 20:5n-3 and 22:6n-3 in a similar range as the fish receiving 40% and even 100% fish oil (FO). This suggests that the fatty acids, 20:5n-3 and 22:6n-3, are dietary requirements for the fish but that even small amounts of marine fatty acids are almost sufficient to meet this requirement. Fish fed sunflower oil (SO) were distinguished from the other dietary groups in that these fish were not fully capable of maintaining their levels of 20:5n-3, instead they seem to increase their levels of 20:4n-6 in the membranes which probably is due to the ample supply of 18:2n-6 from the SO and may lead to altered membrane characteristics. Fish fed the extreme diets showed severe accumulation of lipid droplets in the enterocytes. This situation sometimes led to damaged cells and cell junctions. In the fish fed mixed diets, there was a substantial accumulation of lipid droplets but not to a damaging degree. A higher amount of the fatty acid 16:0 in the mixed diets might be a reason for lower amount of droplets. Another possibility is that he higher amounts of PC in the mixed diets diminish the formation of lipid droplets. Even though the lipid droplet formation and the cell damage was observed in all dietary groups, only SO and rapeseed oil (RO) physiologically affected the intestinal barrier functions as judged by paracellular permeability and epithelial resistance. SO affected the barrier functions of Atlantic salmon, whereas RO was the main effector in rainbow trout. Exposure of the intestinal epithelium to the pathogen bacteria Aeromonas salmonicida created ultra structural changes with loosened junctional complexes and loosened whole enterocytes. This was mainly apparent in the anterior intestine of Atlantic salmon and in the posterior intestine of rainbow trout. Exposure of the intestinal epithelium is also followed by a decrease in intestinal permeability, suggesting that the pathogen bacteria are able to affect the barrier properties of the epithelium. A similar effect is seen when exposing the epithelium to the bacterial endotoxin, LPS, as well as to culture medium from live A. salmonicida. This clearly indicates that the bacteria itself can secrete virulent factors of which a probable candidate is LPS. Thus, if fish fed a diet that negatively affects the barrier functions, i.e. one containing SO and are orally infected with a pathogen bacteria, this may lead to an increased risk for systemic infections. However, in the present project no effect of different mixed dietary vegetable oils on the actual translocation rate of A. salmonicida could be demonstrated. Rather, Atlantic salmon parr fed SO showed a lower translocation rate of pathogen bacteria then corresponding groups fed FO. This can be due to several possibilities: The change in composition of membrane lipids may affect the transcellular translocation routes, decreasing those even though the paracellular routes are increased. The increase in the paracellular pathway may increase the transfer of bacterial components and virulent factor that act as antigens and stimulate the immune system, which might give a decreased actual translocation rate. Also, the present study reveals that the only two diets demonstrated the presence of Carnobacterium mobile-like strains, which are endogenous bacteria with antagonistic activity against A. salmonicida, was SO and RO. Thus, even though some of the extreme and mixed vegetable diets changed the fatty acid composition, affected the ultra structure of the epithelium and the intestinal barrier properties, the overall growth, performance and intestinal bacterial translocation of the fish was unaffected in the short term perspective, but it may lead to health effects in the long term perspective. Dietary vegetable lipids and nutrient uptake Although optimal use of high energy diets can increase protein efficiency and despite the adaptive response of rainbow trout to high-fat diets through: - Maintenance of energy intake until values of 20.5 kJ DE/g diet, - Increased utilization of lipids for energy purposes and - Reduction of hepatic lipogenesis, trout were still depositing to maintain the chemical composition of their whole-body and tissues. Simply stated, feeding trout high fat diets produces fatter fish, lipid deposition being primary in the visceral cavity and to a lesser extent in muscle. Since the development with age of adipose tissue is correlated with a recruitment of pre-adipocytes, increased storage capacity by dietary manipulations results from hyperplasic and hypertrophic processes in trout. Besides, the use of high fat diet depresses de novo fatty acid synthesis and increases lipid storage of dietary origin. Therefore, a low quality dietary lipid profile may impair the nutritional value of fish flesh as human food. A change in dietary lipids clearly effects the fatty acid composition of the polar lipids constituting the enterocyte membranes. The effect was pronounced in fish fed only vegetable oils, whereas fish fed small amounts of marine lipids retained in the fishmeal (2% of total lipids), were relatively successful in maintaining their levels of 20:5n-3 and 22:6n-3 in a similar range as the fish receiving 40% and even 100% fish oil (FO). Fish fed sunflower oil (SO) were distinguished from the other dietary groups in that these fish were not fully capable of maintaining their levels of 20:5n-3, they seem to increase their levels of 20:4n-6 in the membranes which probably is due to the ample supply of 18:2n-6 from the SO and may lead to altered membrane characteristics. Intra-membrane proteins, like the active transporters of water soluble nutrients, glucose and amino acids, can be affected by changes the surrounding membrane lipid composition. The intestinal uptake of the non-toxic, non-essential amino acid proline was not affected by dietary vegetable oils when fed in a mixture. Using a diet containing only SO, both the proline and the essential fatty acid leucine uptake were negatively affected. Mixtures of dietary vegetable oils instead increased the uptake rate of the two essential amino acids leucine and histidine. In summary, the uptake rate of essential amino acids seems to be more sensitive to changes in dietary replacements then the non essential amino acids and a mixture of vegetable oils seems to affect the active uptake mechanisms to a lesser extend then diets containing only SO. The same pattern seems to be present for intestinal glucose uptake. A very interesting and for aquaculture important life stage of the Atlantic salmon is the parr-smolt transformation, during which cortisol and GH, among other hormones have regulatory functions. Apart from being a developmental hormone, cortisol is also a stress related hormone and the consistently elevated plasma levels of cortisol in fish fed SO indicates that the substitution of fish oil by SO creates a situation of chronic stress. These elevated cortisol levels also affects the intestinal uptake of amino acids revealing a higher uptake rate coincident with the peak in plasma cortisol levels that is most pronounced in the fish fed SO. A substitution of FO with vegetable oils changes the composition of fatty acids in the diets and this will probably affect the intestinal uptake rate of specific fatty acids. The uptake of selected fatty acids was indeed dependent and regulated by dietary vegetable oils. Fish fed a SO based diet, rich in the fatty acid 18:2n-3, showed a down regulation of intestinal fatty acid uptake of both 18:2n-3 and the fatty acid 16:0 whereas fish fed a linseed oil-based diet, rich in 18:3n-3, instead showed an up regulation of the intestinal uptake of 18:3n-3 and 16:0. These findings open up a new and important field of research. A better understanding of the physiological mechanisms and regulatory events of this specific intestinal uptake of fatty acids would be of great importance for the design of diets containing mixed vegetable oils. The apparent co-transport of unsaturated fatty acids with 16:0 opens up interesting question as to whether the transport of unsaturated fatty acids, including their incorporation into TAG and/or phospholipids, is dependent on a co-uptake of the saturated fatty acid 16:0. Thus, even though the substitution of fish oils with vegetable oils does not affect the overall growth, performance or digestibility, this substitution, especially when using vegetable oils of just one source, may lead to changes in intestinal uptake mechanisms for both water and lipid soluble nutrients. Furthermore, an increased knowledge about the specificity and regulation of intestinal fatty acid uptake mechanisms may be valuable when designing the proportions of fatty acids constituting vegetable oil mixtures to be used for diets. Effects of lipids on gastro-intestinal function in rainbow trout and regulation of feed intake by peptides and hormones The data generated from the present project suggest that rainbow trout are able to regulate their feed intake in relation to energy content, and that their decreased food intake at high lipid diets may also reflect a compromise between reduced protein intake and increased lipid intake. Vegetable oils may accelerate the gastric emptying rate, with linseed oil having the strongest effect, which suggests that the regulation of gastric emptying is not functioning optimally. In addition, gastric emptying time increased with increasing dietary lipid levels, but at higher levels the effect levelled off. Thus, it appears that the regulation of gastric emptying may somehow break down at extremely high lipid levels, indicating a higher energy throughput and lower energy utilization. The postprandial regulatory mechanisms behind these effects need to be addressed in future studies. Among CCK, GRP and ghrelin, only GRP was effective in regulating food intake, potently suppressing food intake. Thus, GRP is a candidate satiety factor in rainbow trout. As plasma GRP levels did not change after feeding, the most likely pathway for GRP action on food intake would be a local action at afferent nerves, being effective at low concentrations to terminate a meal. The RIA-analysis also revealed that the circulating GRP levels are generally very low. As GRP accelerated gastric emptying, the suggestion that GRP decreases feed intake at the gastrointestinal level by slowing the gastric emptying rate is not supported (Himick and Peter 1994b). From our data, it is hypothesized that CCK could be related to gastrointestinal function regulating the proper processing of food in the intestine at different diet composition rather that having a role in the feedback inhibition of food intake. Alternatively, CCK may have a transient acute effect on food intake, or regulates the time between meals, that could not be detected in the present experimental set-up. It can be speculated that CCK delays gastric emptying time and possibly also mediates the decreased food intake at high lipid levels, which is supported by the CCK levels being elevated at high dietary lipid levels. The physiological role of CCK needs to be clarified and further studies should be designed to take into account rapid effects of CCK on food intake and its effect on interfeeding period duration as well as its functional relation to dietary lipids. Although a role for ghrelin in food intake regulation in rainbow trout is not supported by the obtained data, it may well be linked to nutritional status. Finally, this part of the GUTINTEGRITY project has established a number of new techniques to study voluntary food intake and gastrointestinal passage of food and the regulation of these processes by different substances, along with the methods to measure CCK and GRP in plasma. This has generated important basic knowledge that significantly contributes to our understanding of gastrointestinal physiology in relation to food intake and appetite regulation, and also raises a number of interesting scientific issues that may be addressed in future studies. The gained information will help for a better design of diet compositions by increasing the usefulness of lipids of vegetable origin. Feed waste and release to the environment will be reduced by a better matching between feed distribution and fish appetite. These data will be published in the scientific literature. The developed methodologies of RIA assessment of CCK and GRP levels in fih, as well as the method of intraperitoneal cannulation, will be used in further research applications and projects. Dietary vegetable lipids and diet discrimination capacities The present study confirms the possibility to use self-feeders for determining feed preferences in fish. The rainbow trout clearly detected the presence of vegetable oils and expressed a preference for the fish oil diet. Among the three vegetable oils tested, linseed oil was the most avoided, followed by sunflower oil, while rapeseed oil was better accepted by the fish. The avoidance of the vegetable oil diets was compensated by increased demands for the FO diet. As a consequence, despite the important differences in relative feed preference, no differences were observed in total feed intake or in final body weights of fish from the different dietary treatment combinations. Longer term feeding studies with similarly high-fat diets and full fish oil replacement are necessary to distinguish if present preferences were steered by the flavour or by the nutritional value of the oils. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
