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

Abstract

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.