Dietary fatty acids and their ratio: impact on Atlantic salmon health, mineral status and intestinal lipid transport

The rapid growth of the aquaculture industry and the sharp decline of capture fisheries necessitates finding alternative sources for fish oil and fish meal in aquafeeds. However, the inclusion of VOs in aquafeed alters the dietary fatty acid composition, significantly reducing the amount of essential n-3 LC-PUFA, inducing the n-3/n-6 ratio, and increasing the MUFA contents. In the last few decades, numerous studies have been conducted to demonstrate the possibility of partial or complete replacement of FO with vegetable oils (VOs) without any adverse effects on the growth and welfare of the fish, provided sufficient n-3 LC-PUFA from other dietary sources. However, most feeding trials are run in controlled, stable environmental conditions, where there is minimal stress on fish. In contrast, under demanding environmental conditions in sea cages, fish are exposed to various stressors, including fluctuating water temperatures, handling, parasitic pressure, delousing, etc. Therefore, we need increased knowledge on how optimal FA nutrition can be used to maintain a healthy and robust fish that can cope with stressful situations, such as fluctuating environmental conditions and disease pressure. Besides the change in dietary FA profile, increased inclusion of plant ingredients also reduces the supply and availability of dietary minerals to fish. Further, little is known about how this change in the FA profile affects the intracellular fate of these fatty acids in intestinal cells. Therefore, this Ph.D. project investigates how stressful conditions combined with the change in dietary FA level affect the absorption and intracellular fate of dietary fatty acids, stress and immune responses, and the utilization of minerals in the fish. In the present thesis, the three trials were conducted i) short-term challenge experiment, ii) long-term seawater trial, and iii) in vitro trial in RTgutGC cells. In the short-term challenge trial (Paper I), Atlantic salmon were fed diets containing different ratios of n-6/n-3 FA (at 1.3, 2.4, and 6.0 and one diet with a ratio of 1.3 combined with a higher level of n-3 FA and n-6 FA) and challenged with amoebic gill disease (AGD). In the long-term seawater trial (Paper II), Atlantic salmon were fed diets containing graded levels of EPA+DHA (10, 13, 16, and 35 g/kg of feed, and one diet with 13g/ kg of EPA+DHA with reduced total fat content) and fish were subjected to physical stress during delousing. In both trials (Paper I and Paper II), diet did not affect growth under optimal conditions, while a high n-6/n-3 ratio (6:1) (Paper I) and low EPA+DHA (10-16 g/kg of feed) (Paper II) diet had negative impact on growth under AGD challenge and delousing stress, respectively. Furthermore, despite AGD challenge and delousing stress altering the mRNA expression inflammatory and immune marker and oxidative stress markers, both dietary n-6/n-3 ratios and EPA+DHA levels did not alter the expression of any of these genes. In the challenge trial, disease progression, gross gill score, and associated gill pathology were much lower compared to previously reported studies, suggesting the possible genetic resistance of the experimental fish against AGD. In the long-term trial (Paper II), despite increased plasma cortisol level after delousing, dietary EPA+DHA levels had no effect on plasma cortisol, vertebrae deformities, or any other external welfare indicators. Interaction of dietary EPA+DHA on tissue trace mineral status was assessed at delousing (~2.5 kg) and at harvest stage (~ 5 kg). The liver Se, Zn, Fe, Cu, and Mn and plasma Se levels were increased in fish fed a diet high in EPA+DHA (35 g/kg of feed) upon delousing stress. Further, a high dietary EPA+DHA also significantly increased the whole-body Zn, Se, and Mn levels at harvest size fish. This thesis is one of first attempt to use fish intestinal cells (RTgutGC cells) as an in vitro model to study the intracellular fate of FA upon uptake and transport (Paper III), following the 3R principles. The RTgutGC cells offered oleic acid accumulated higher amounts of TAG in the cells, and lead to higher (x6 times) cytosolic lipid droplets (CLDs) accumulation. The accumulation of TAG in CLDs were lower for arachidonic acid (ARA) and palmitic acid (PA) compared to oleic acid (OA). The lower uptake of OA from apical compartment than other FAs (PA and ARA) might indicate the negative consequence of excessive CLDs accumulation and physical barrier to uptake of this FA. A significant amount of ARA was transported as TAG to basolateral compartment and suggesting the better regulated transport for LC-PUFA. Based on the results of this Ph.D. work, it could be concluded that low dietary EPA+DHA (10-16 g/kg of feed) and a high n-6 /n-3 FA ratio (6:1) can have negative impact on growth under challenging environmental conditions. Thus, the optimal dietary n-3 FA needs to be revisited under different challenging situations, and the balance of n-6/n-3 FA in the diet is an important factor that needs to be considered while formulating the diet for Atlantic salmon. Higher inclusion of EPA+DHA (35 g/ kg) in the diet increased the trace mineral levels in plasma, liver, and whole body. Knowledge on the interaction between dietary fatty acids on body mineral stores is crucial for the aquaculture industry to improve the bioavailability and status of these minerals in fish and limit their discharge into the environment. Further, results from this thesis increased the Knowledge on how uptake and transport of FA are affected by their chain length and saturation level. The lipid fraction in current commercial diets for Atlantic salmon contains higher inclusion of rapeseed oil, resulting in a major reduction in SFA and LC-PUFA and increased MUFA levels in diets. This thesis demonstrated that higher inclusion of VOs rich in MUFA results in excessive accumulation of large lipid droplets in the enterocytes, reducing fatty acid absorption efficiency.Doktorgradsavhandlin

Impact of dietary level and ratio of n-6 and n-3 fatty acids on disease progression and mRNA expression of immune and inflammatory markers in Atlantic salmon (Salmo salar) challenged with Paramoeba perurans

The aim of the study was to investigate the influence of dietary level and ratio of n-6/n-3 fatty acids (FA) on growth, disease progression and expression of immune and inflammatory markers in Atlantic salmon (Salmo salar) following challenge with Paramoeba perurans. Fish (80 g) were fed four different diets with different ratios of n-6/n-3 FA; at 1.3, 2.4 and 6.0 and one diet with ratio of 1.3 combined with a higher level of n-3 FA and n-6 FA. The diet with the n-6/n-3 FA ratio of 6.0 was included to ensure potential n-6 FA effects were revealed, while the three other diets were more commercially relevant n-6/n-3 FA ratios and levels. After a pre-feeding period of 3 months, fish from each diet regime were challenged with a standardized laboratory challenge using a clonal culture of P. perurans at the concentration of 1,000 cells L−1. The subsequent development of the disease was monitored (by gross gill score), and sampling conducted before challenge and at weekly sampling points for 5 weeks post-challenge. Challenge with P. perurans did not have a significant impact on the growth of the fish during the challenge period, but fish given the feed with the highest n-6/n-3 FA ratio had reduced growth compared to the other groups. Total gill score for all surfaces showed a significant increase with time, reaching a maximum at 21 days post-challenge and declined thereafter, irrespective of diet groups. Challenge with P. perurans influenced the mRNA expression of examined genes involved in immune and inflammatory response (TNF-α, iNOS, IL4-13b, GATA-3, IL-1β, p53, COX2 and PGE2-EP4), but diet did not influence the gene expression. In conclusion, an increase in dietary n-6/n-3 FA ratio influenced the growth of Atlantic salmon challenged with P. perurans; however, it did not alter the mRNA expression of immune genes or progression of the disease.publishedVersio

Intracellular trafficking of fatty acids in the fish intestinal epithelial cell line RTgutGC

The shift towards higher inclusion of vegetable oils (VOs) in aquafeeds has resulted in major changes in dietary fatty acid composition, especially increased amounts of monounsaturated fatty acids (MUFAs) and decreased polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs). However, little is known about how this change in fatty acid (FA) profile affects the intracellular fate of these fatty acids in the intestinal cells. To investigate this topic, we used the rainbow trout intestinal epithelial cell line (RTgutGC) as an in vitro model. The cells were incubated with either palmitic acid (16:0, PA), oleic acid (18:1n-9, OA), or arachidonic acid (20:4n-6, ARA), to represent the SFA, MUFA, and PUFA, respectively. In all experiments, the RTgutGC were incubated with either non-labeled or radiolabeled FA (PA, OA, or ARA) for 16 h at 190C. The cells were then analyzed for the occurrence of cytosolic lipid droplets (CLD) with confocal microscopy, transcriptomic analysis (non-labeled FA experiments) and lipid class composition in the cells and serosal media from the basolateral side of the cells (radiolabeled FA experiments). CLD accumulation was higher in RTgutGC exposed to OA compared to cells given PA or ARA. This was coupled with increased volume, diameter, and surface area of CLDs in OA treated cells than with other FAs (PA, ARA). The results from radiolabeled FAs performed on permeable transwell inserts showed that OA increased the triacylglycerides (TAG) synthesis and was primarily stored in the cells in CLDs; whereas a significant amount of ARA was transported as TAG to the basolateral compartment. A significant proportion of free FAs was found to be excreted to the serosal basolateral side by the cells, which was significantly higher for PA and OA than ARA. Although there were clear clusters in differentially expressed genes (DEGs) for each treatment group, results from transcriptomics did not correlate to lipid transport and CLD analysis. Overall, the accumulation of TAG in CLDs was higher for oleic acid (OA) compared to arachidonic acid (ARA) and palmitic acid (PA). To conclude, carbon chain length and saturation level of FA differently regulate their intracellular fate during fatty acid absorption.publishedVersio

Long-term feeding of Atlantic salmon with varying levels of dietary EPA+DHA alters the mineral status, but does not affect the stress responses after mechanical delousing stress

publishedVersio

Dietary electrolyte balance of Atlantic salmon (Salmo salar) freshwater feeds: Impact on osmoregulation, mineral metabolism and performance in seawater

Dietary electrolyte balance is the equilibrium of monovalent cations and anions that influence the acid-base balance of the feed (dEB = Na + K − Cl, mEq kg−1). Dietary electrolytes/minerals can influence the physiological changes during smoltification in Atlantic salmon. In this context, we aimed to study if the dEB of the freshwater feeds can be used to pre-adapt the hypoosmotic functionality and the associated effects on mineral metabolism. The dEB of commercial freshwater Atlantic salmon feeds in Norway varied from −9 to 400 mEq kg−1 feed. Three experimental feeds were formulated to study incremental levels of dEB reflecting the low (L-dEB, −50 to 0), median (M-dEB, 200–250) and high (H-dEB, 350–400). Triplicate groups of Atlantic salmon parr (36 g) were fed one of the three feeds for 8 weeks in freshwater at 12 °C. The fish were transferred to full strength seawater in indoor tanks and fed a commercial diet for 6 weeks. Growth was not differentially affected by dEB levels, neither in the freshwater phase nor in the seawater. Plasma electrolytes (Na+ and Cl−) and gill mRNA expression of sodium potassium ATPase (NKA a1b, seawater isoform) were significantly lower in L-dEB fed fish. In the intestine, carbonate precipitates 24 h after seawater transfer was higher in fish fed both L-dEB and H-dEB feeds compared to the M-dEB fed fish. Whole body and plasma mineral levels were significantly affected by dEB levels in freshwater feeds. Interestingly, the carryover effect of dEB in freshwater feeds was significant after 6 weeks in seawater for plasma and whole-body Zn status, with the H-dEB fed fish showing significantly increased body Zn status compared to L-dEB and M-dEB fed fish. The study revealed that mineral metabolism and intestinal response to seawater transfer can be pre-adapted by modulating the electrolyte and/or mineral balance in freshwater feeds in Atlantic salmon. Further, dEB did not affect long term development of cataract or vertebral deformities.publishedVersio

Dietary fatty acids and their ratio: impact on Atlantic salmon health, mineral status and intestinal lipid transport

The rapid growth of the aquaculture industry and the sharp decline of capture fisheries necessitates finding alternative sources for fish oil and fish meal in aquafeeds. However, the inclusion of VOs in aquafeed alters the dietary fatty acid composition, significantly reducing the amount of essential n-3 LC-PUFA, inducing the n-3/n-6 ratio, and increasing the MUFA contents. In the last few decades, numerous studies have been conducted to demonstrate the possibility of partial or complete replacement of FO with vegetable oils (VOs) without any adverse effects on the growth and welfare of the fish, provided sufficient n-3 LC-PUFA from other dietary sources. However, most feeding trials are run in controlled, stable environmental conditions, where there is minimal stress on fish. In contrast, under demanding environmental conditions in sea cages, fish are exposed to various stressors, including fluctuating water temperatures, handling, parasitic pressure, delousing, etc. Therefore, we need increased knowledge on how optimal FA nutrition can be used to maintain a healthy and robust fish that can cope with stressful situations, such as fluctuating environmental conditions and disease pressure. Besides the change in dietary FA profile, increased inclusion of plant ingredients also reduces the supply and availability of dietary minerals to fish. Further, little is known about how this change in the FA profile affects the intracellular fate of these fatty acids in intestinal cells. Therefore, this Ph.D. project investigates how stressful conditions combined with the change in dietary FA level affect the absorption and intracellular fate of dietary fatty acids, stress and immune responses, and the utilization of minerals in the fish. In the present thesis, the three trials were conducted i) short-term challenge experiment, ii) long-term seawater trial, and iii) in vitro trial in RTgutGC cells. In the short-term challenge trial (Paper I), Atlantic salmon were fed diets containing different ratios of n-6/n-3 FA (at 1.3, 2.4, and 6.0 and one diet with a ratio of 1.3 combined with a higher level of n-3 FA and n-6 FA) and challenged with amoebic gill disease (AGD). In the long-term seawater trial (Paper II), Atlantic salmon were fed diets containing graded levels of EPA+DHA (10, 13, 16, and 35 g/kg of feed, and one diet with 13g/ kg of EPA+DHA with reduced total fat content) and fish were subjected to physical stress during delousing. In both trials (Paper I and Paper II), diet did not affect growth under optimal conditions, while a high n-6/n-3 ratio (6:1) (Paper I) and low EPA+DHA (10-16 g/kg of feed) (Paper II) diet had negative impact on growth under AGD challenge and delousing stress, respectively. Furthermore, despite AGD challenge and delousing stress altering the mRNA expression inflammatory and immune marker and oxidative stress markers, both dietary n-6/n-3 ratios and EPA+DHA levels did not alter the expression of any of these genes. In the challenge trial, disease progression, gross gill score, and associated gill pathology were much lower compared to previously reported studies, suggesting the possible genetic resistance of the experimental fish against AGD. In the long-term trial (Paper II), despite increased plasma cortisol level after delousing, dietary EPA+DHA levels had no effect on plasma cortisol, vertebrae deformities, or any other external welfare indicators. Interaction of dietary EPA+DHA on tissue trace mineral status was assessed at delousing (~2.5 kg) and at harvest stage (~ 5 kg). The liver Se, Zn, Fe, Cu, and Mn and plasma Se levels were increased in fish fed a diet high in EPA+DHA (35 g/kg of feed) upon delousing stress. Further, a high dietary EPA+DHA also significantly increased the whole-body Zn, Se, and Mn levels at harvest size fish. This thesis is one of first attempt to use fish intestinal cells (RTgutGC cells) as an in vitro model to study the intracellular fate of FA upon uptake and transport (Paper III), following the 3R principles. The RTgutGC cells offered oleic acid accumulated higher amounts of TAG in the cells, and lead to higher (x6 times) cytosolic lipid droplets (CLDs) accumulation. The accumulation of TAG in CLDs were lower for arachidonic acid (ARA) and palmitic acid (PA) compared to oleic acid (OA). The lower uptake of OA from apical compartment than other FAs (PA and ARA) might indicate the negative consequence of excessive CLDs accumulation and physical barrier to uptake of this FA. A significant amount of ARA was transported as TAG to basolateral compartment and suggesting the better regulated transport for LC-PUFA. Based on the results of this Ph.D. work, it could be concluded that low dietary EPA+DHA (10-16 g/kg of feed) and a high n-6 /n-3 FA ratio (6:1) can have negative impact on growth under challenging environmental conditions. Thus, the optimal dietary n-3 FA needs to be revisited under different challenging situations, and the balance of n-6/n-3 FA in the diet is an important factor that needs to be considered while formulating the diet for Atlantic salmon. Higher inclusion of EPA+DHA (35 g/ kg) in the diet increased the trace mineral levels in plasma, liver, and whole body. Knowledge on the interaction between dietary fatty acids on body mineral stores is crucial for the aquaculture industry to improve the bioavailability and status of these minerals in fish and limit their discharge into the environment. Further, results from this thesis increased the Knowledge on how uptake and transport of FA are affected by their chain length and saturation level. The lipid fraction in current commercial diets for Atlantic salmon contains higher inclusion of rapeseed oil, resulting in a major reduction in SFA and LC-PUFA and increased MUFA levels in diets. This thesis demonstrated that higher inclusion of VOs rich in MUFA results in excessive accumulation of large lipid droplets in the enterocytes, reducing fatty acid absorption efficiency

Intracellular trafficking of fatty acids in the fish intestinal epithelial cell line RTgutGC

The shift towards higher inclusion of vegetable oils (VOs) in aquafeeds has resulted in major changes in dietary fatty acid composition, especially increased amounts of monounsaturated fatty acids (MUFAs) and decreased polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs). However, little is known about how this change in fatty acid (FA) profile affects the intracellular fate of these fatty acids in the intestinal cells. To investigate this topic, we used the rainbow trout intestinal epithelial cell line (RTgutGC) as an in vitro model. The cells were incubated with either palmitic acid (16:0, PA), oleic acid (18:1n-9, OA), or arachidonic acid (20:4n-6, ARA), to represent the SFA, MUFA, and PUFA, respectively. In all experiments, the RTgutGC were incubated with either non-labeled or radiolabeled FA (PA, OA, or ARA) for 16 h at 190C. The cells were then analyzed for the occurrence of cytosolic lipid droplets (CLD) with confocal microscopy, transcriptomic analysis (non-labeled FA experiments) and lipid class composition in the cells and serosal media from the basolateral side of the cells (radiolabeled FA experiments). CLD accumulation was higher in RTgutGC exposed to OA compared to cells given PA or ARA. This was coupled with increased volume, diameter, and surface area of CLDs in OA treated cells than with other FAs (PA, ARA). The results from radiolabeled FAs performed on permeable transwell inserts showed that OA increased the triacylglycerides (TAG) synthesis and was primarily stored in the cells in CLDs; whereas a significant amount of ARA was transported as TAG to the basolateral compartment. A significant proportion of free FAs was found to be excreted to the serosal basolateral side by the cells, which was significantly higher for PA and OA than ARA. Although there were clear clusters in differentially expressed genes (DEGs) for each treatment group, results from transcriptomics did not correlate to lipid transport and CLD analysis. Overall, the accumulation of TAG in CLDs was higher for oleic acid (OA) compared to arachidonic acid (ARA) and palmitic acid (PA). To conclude, carbon chain length and saturation level of FA differently regulate their intracellular fate during fatty acid absorption

Intracellular trafficking of fatty acids in the fish intestinal epithelial cell line RTgutGC

The shift towards higher inclusion of vegetable oils (VOs) in aquafeeds has resulted in major changes in dietary fatty acid composition, especially increased amounts of monounsaturated fatty acids (MUFAs) and decreased polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs). However, little is known about how this change in fatty acid (FA) profile affects the intracellular fate of these fatty acids in the intestinal cells. To investigate this topic, we used the rainbow trout intestinal epithelial cell line (RTgutGC) as an in vitro model. The cells were incubated with either palmitic acid (16:0, PA), oleic acid (18:1n-9, OA), or arachidonic acid (20:4n-6, ARA), to represent the SFA, MUFA, and PUFA, respectively. In all experiments, the RTgutGC were incubated with either non-labeled or radiolabeled FA (PA, OA, or ARA) for 16 h at 190C. The cells were then analyzed for the occurrence of cytosolic lipid droplets (CLD) with confocal microscopy, transcriptomic analysis (non-labeled FA experiments) and lipid class composition in the cells and serosal media from the basolateral side of the cells (radiolabeled FA experiments). CLD accumulation was higher in RTgutGC exposed to OA compared to cells given PA or ARA. This was coupled with increased volume, diameter, and surface area of CLDs in OA treated cells than with other FAs (PA, ARA). The results from radiolabeled FAs performed on permeable transwell inserts showed that OA increased the triacylglycerides (TAG) synthesis and was primarily stored in the cells in CLDs; whereas a significant amount of ARA was transported as TAG to the basolateral compartment. A significant proportion of free FAs was found to be excreted to the serosal basolateral side by the cells, which was significantly higher for PA and OA than ARA. Although there were clear clusters in differentially expressed genes (DEGs) for each treatment group, results from transcriptomics did not correlate to lipid transport and CLD analysis. Overall, the accumulation of TAG in CLDs was higher for oleic acid (OA) compared to arachidonic acid (ARA) and palmitic acid (PA). To conclude, carbon chain length and saturation level of FA differently regulate their intracellular fate during fatty acid absorption

DataSheet_2_Intracellular trafficking of fatty acids in the fish intestinal epithelial cell line RTgutGC.pdf

The shift towards higher inclusion of vegetable oils (VOs) in aquafeeds has resulted in major changes in dietary fatty acid composition, especially increased amounts of monounsaturated fatty acids (MUFAs) and decreased polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs). However, little is known about how this change in fatty acid (FA) profile affects the intracellular fate of these fatty acids in the intestinal cells. To investigate this topic, we used the rainbow trout intestinal epithelial cell line (RTgutGC) as an in vitro model. The cells were incubated with either palmitic acid (16:0, PA), oleic acid (18:1n-9, OA), or arachidonic acid (20:4n-6, ARA), to represent the SFA, MUFA, and PUFA, respectively. In all experiments, the RTgutGC were incubated with either non-labeled or radiolabeled FA (PA, OA, or ARA) for 16 h at 190C. The cells were then analyzed for the occurrence of cytosolic lipid droplets (CLD) with confocal microscopy, transcriptomic analysis (non-labeled FA experiments) and lipid class composition in the cells and serosal media from the basolateral side of the cells (radiolabeled FA experiments). CLD accumulation was higher in RTgutGC exposed to OA compared to cells given PA or ARA. This was coupled with increased volume, diameter, and surface area of CLDs in OA treated cells than with other FAs (PA, ARA). The results from radiolabeled FAs performed on permeable transwell inserts showed that OA increased the triacylglycerides (TAG) synthesis and was primarily stored in the cells in CLDs; whereas a significant amount of ARA was transported as TAG to the basolateral compartment. A significant proportion of free FAs was found to be excreted to the serosal basolateral side by the cells, which was significantly higher for PA and OA than ARA. Although there were clear clusters in differentially expressed genes (DEGs) for each treatment group, results from transcriptomics did not correlate to lipid transport and CLD analysis. Overall, the accumulation of TAG in CLDs was higher for oleic acid (OA) compared to arachidonic acid (ARA) and palmitic acid (PA). To conclude, carbon chain length and saturation level of FA differently regulate their intracellular fate during fatty acid absorption.</p