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

Interaction between Dietary Vitamin D3 and Vitamin K3 in Gilthead Seabream Larvae (Sparus aurata) in Relation to Growth and Expression of Bone Development-Related Genes

Vitamins D and K are essential fat-soluble nutrients that intervene in bone development processes among other biological functions. The present study is aimed at investigating the potential combined effect of dietary supplementation with vitamin D3 (cholecalciferol) and vitamin K3 (menadione) in gilthead seabream (Sparus aurata) larvae. For that purpose, seabream diets were supplemented with different combinations of vitamin D3/vitamin K3 (mg/kg diet) as follows: 0.00/0, 0.06/70, 0.06/170, 0.13/70, 0.13/170, 0.40/70, and 0.40/170. Feeding gilthead seabream larvae (22 days post hatch) for 21 days with the diets supplemented with 0.06-0.13 mg/kg vitamin D3 and 70 mg/kg vitamin K3 (diets 0.06/70 and 0.13/70) led to the highest larval growth and survival and the highest expression of important biomarkers of both bone development and health, such as bmp2, osx, and mgp, and calcium homeostasis, such as pthrp and casr. However, the increased supplementation with both vitamins at 0.40 mg/kg vitamin D3 and 170 mg/kg vitamin K3 (diet 0.40/170) reduced larval growth and survival, downregulated bmp2 and pthrp expressions, and upregulated osx and mgp, causing an unbalance in the relative expression of these genes. The results of the present study have shown the interaction between vitamin D3 supplementation and vitamin K3 supplementation in larval performance and gene expression related to bone development and calcium homeostasis, denoting the significance of a correct balance between both vitamins in larval diets.publishedVersio

Effect of dietary electrolyte balance on the interplay between water fluxes and digestive functioning along the gastrointestinal tract of freshwater rainbow trout (Oncorhynchus mykiss)

Aquaculture feed formulation is shifting from fish meal and fish oil toward other ingredients, such as plant-based ingredients, which lead to different levels and forms of minerals in diets. Dietary minerals are essential not only for growth, but also for acid-base balance and the homeostasis of fish body fluids. The gastrointestinal tract (GIT) is involved in the process of osmoregulation of salt and water during digestion, but this process can be hampered when the dietary electrolyte balance (dEB = Na - K + Cl, mEq/kg) is altered. To better understand the interaction between feeding and osmoregulation along the GIT, two diets with low (−100 mEq/kg) and high (+600 mEq/kg) dEB were designed. Freshwater rainbow trout (average initial weight, 306 g) were fed for 6 weeks. Faeces were collected during the last week for digestibility analyses. The final sampling took place over three days, with fish being sampled at 2 time points after feeding (3 and 7 h) and dissected to collect chyme from 4 GIT segments: stomach, proximal, middle and distal intestine. Chyme was analysed for dry matter, pH, osmolality, crude protein (CP) and mineral content. Yttrium oxide (Y2O3) was used as an inert marker to measure water fluxes, mineral fluxes, and nutrient digestibility from different gut segments. Both dEB and time after feeding altered (p 0.05). dEB also affected (p < 0.01) chyme pH and sodium (Na) fluxes in the stomach. Faecal digestibility of dry matter (DM) and CP was higher (p < 0.001) when fish were fed a high dEB diet compared to a low dEB diet. The opposite was observed for Ca availability, which was lower at the high dEB diet than at the low dEB diet (p < 0.001). In contrast to faecal digestibility, CP and mineral digestibility measured in the different gut segments were different predominantly in the middle and distal intestine. Our findings suggest that the GIT plays a role in regulating the alterations caused by the contrasting dEB, and that this regulation is stronger in the middle and distal intestine. Furthermore, this regulation affects nutrient and mineral digestibility in the middle and distal segments of the GIT.publishedVersio

Effects of vitamin D3 supplementation in gilthead seabream (Sparus aurata) juveniles fed diets high in plant based feedstuffs

Modern aquaculture feeds tend to contain lower levels of fish based ingredients, while increasing the content of plant ingredients. However, this may alter the vitamin profile of the feeds, leading to unbalanced vitamin supply. Requirements for several vitamins have been established for species such as carps and salmonids, but adequate levels for gilthead sea bream are yet unknown.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

Long-term effect of parental selenium supplementation on the one-carbon metabolism in rainbow trout (Oncorhynchus mykiss) fry exposed to hypoxic stress

This study evaluated how different forms of selenium (Se) supplementation into rainbow trout broodstock diets modified the one-carbon metabolism of the progeny after the beginning of exogenous feeding and followed by hypoxia challenge. The progeny of three groups of rainbow trout broodstock fed either a control diet (Se level: 0·3 µg/g) or a diet supplemented with inorganic sodium selenite (Se level: 0·6 µg/g) or organic hydroxy-selenomethionine (Se level: 0·6 µg/g) was cross-fed with diets of similar Se composition for 11 weeks. Offspring were sampled either before or after being subjected to an acute hypoxic stress (1·7 mg/l dissolved oxygen) for 30 min. In normoxic fry, parental Se supplementation allowed higher glutathione levels compared with fry originating from parents fed the control diet. Parental hydroxy-selenomethionine treatment also increased cysteine and cysteinyl–glycine concentrations in fry. Dietary Se supplementation decreased glutamate–cysteine ligase (cgl) mRNA levels. Hydroxy-selenomethionine feeding also lowered the levels of some essential free amino acids in muscle tissue. Supplementation of organic Se to parents and fry reduced betaine-homocysteine S-methyltransferase (bhmt) expression in fry. The hypoxic stress decreased whole-body homocysteine, cysteine, cysteinyl-glycine and glutathione levels. Together with the higher mRNA levels of cystathionine beta-synthase (cbs), a transsulphuration enzyme, this suggests that under hypoxia, glutathione synthesis through transsulphuration might have been impaired by depletion of a glutathione precursor. In stressed fry, S-adenosylmethionine levels were significantly decreased, but S-adenosylhomocysteine remained stable. Decreased bhmt and adenosylmethionine decarboxylase 1a (amd1a) mRNA levels in stressed fry suggest a nutritional programming by parental Se also on methionine metabolism of rainbow trout.publishedVersio

A meta-analysis on the nutritional value of insects in aquafeeds

A major challenge for development of sustainable aquafeeds is its dependence on fish meal and fish oil. Similarly, it is unwanted to include more plant ingredients which adds more pressure on resources like arable land, freshwater and fertilisers. New ingredients that do not require these resources but rather refine and valorise organic side streams, like insects, are being developed. Increasing evidence indicates that using insect ingredients in aquafeeds are a sustainable alternative and considerable progress has been made on this topic in the past years. The aim of this chapter is to present a comprehensive and systematic analysis of the data available on the impact of insects in aquafeeds. Systematic search, collection and selection of relevant literature from databases such as Web of Science and NCBI was performed. The literature search enabled 91 scientific papers from peer-reviewed journals, comprising a dataset of 415 experimental diets, including 35 different aquatic species and 14 insect species to be included in this meta-analysis, covering what we consider a close to complete representation of credible publications on this topic. Information on aquatic species, insect species, dietary composition (amino acids, fatty acids, proximate composition) and performance outputs (growth performance indicators and nutrient digestibility) were included in the construction of the dataset. Regression models and principal component analyses were performed on the meta-data. The results from the meta-analysis revealed a great degree of variation in the maximum threshold for insect inclusion in aquafeeds (from 4 to 37%) based on subgroups of trophic level of aquatic species, insect species used, statistical method and the output parameter. Overall, a maximum threshold of 25-30% inclusion of insects in aquafeeds for uncompromised performance is suggested. Reduction in protein digestibility, imbalanced amino acid profile and increasing levels of saturated fatty acid were identified as major factors limiting higher inclusion of insects in aquafeeds.publishedVersio

Dietary selenium required to achieve body homeostasis and attenuate pro-inflammatory responses in Atlantic salmon post-smolt exceeds the present EU legal limit

Selenium (Se) supplementation either as inorganic or organic form was evaluated in Atlantic salmon post-smolt in vivo and in vitro. The basal diet was formulated to be low in fish meal and contain 0.24 mg Se kg−1; six other diets with Se inclusion of 0.15, 0.4, 0.7 or 1.1 mg kg−1 as sodium selenite (SS) and 0.15 or 0.4 mg kg−1 as L-selenomethionine (SM) were formulated from the basal diet. The diets were fed to Atlantic salmon post-smolt (mean initial weight, 216 ± 27 g) in triplicate groups (35 fish tank−1) and reared in flow-through seawater (33 ppt) at 10–12 °C for 9 weeks. At the end of the feeding trial, whole fish and tissues were sampled for in vivo assessment; whereas, liver cells and head kidney leukocytes (HKL) were isolated and their primary cultures used for in vitro assessment following exposure to hydrogen peroxide (H2O2), lipopolysaccharide (LPS) or poly I:C (PIC). Growth, feed intake, feed conversion ratio, specific growth rate, hepato-somatic index, proximate composition and mineral concentration of the whole fish (except for Se) were unaffected by dietary Se (p > .05). Hematocrit was significantly higher in fish fed the 0.4 mg Se supplemented feeds, irrespective of the Se source (p = .02). The Se concentration in whole body, liver, muscle, plasma, kidney and liver/kidney Se ratio increased with increasing dietary Se concentration (p < .0001). Level of oxidised glutathione (GSSG) in liver and head kidney followed a quadratic function (p < .05) indicating that the concentrations were lower at intermediate SS supplementation of 0.4 and 0.65 mg Se kg−1 (total Se, 0.65 and 0.87 mg Se kg−1). Impact of Se sources on glutathione redox status was similar. Slope-ratio analysis revealed SM to be more efficient than SS in improving apparent availability, whole body or tissues Se status, Se retention and reducing Se loss to the environment. In vitro, the mRNA expression of p38mapk and aif, in liver cells were affected by the impact of dietary Se, but not by the treatment of H2O2 (p < .05). In the HKL, the LPS and PIC induced pro-inflamatory action of il-1β, cox2, nfkβ and viperin were attenuated by SM supplementation, but not by SS (p < .001). Dietary Se supplementation required to the basal diet containing 0.24 mg Se kg−1 was 0.41 mg Se as SS (total 0.65 mg kg−1) or 0.17 mg Se as SM (total 0.41 mg kg−1) based on body Se homeostasis or tissue Se status. SM inclusion at 0.4 mg kg−1 diet (total, 0.65 mg kg−1) attenuated LPS or PIC induced pro-inflammatory responses in vitro. Overall, Se requirement of Atlantic salmon post smolt was 0.27 mg kg−1 diet, on availale basis. Dietary Se level required to maintain body Se homeostasis and improved health status of Atlantic salmon fed plant-based diets (0.65 mg kg−1 diet) exceed the existing EU maximum limit of 0.5 mg Se kg−1 diet. SM as the Se source in salmon feeds has the potential to improve salmon health and reduce Se emissions from Norwegan salmon farming by 60 to 70%.publishedVersio

Parental selenium nutrition affects the one-carbon metabolism and the hepatic dna methylation pattern of rainbow trout (Oncorhynchus mykiss) in the progeny

Selenium is an essential micronutrient and its metabolism is closely linked to the methionine cycle and transsulfuration pathway. The present study evaluated the effect of two different selenium supplements in the diet of rainbow trout (Onchorhynchus mykiss) broodstock on the one-carbon metabolism and the hepatic DNA methylation pattern in the progeny. Offspring of three parental groups of rainbow trout, fed either a control diet (NC, basal Se level: 0.3 mg/kg) or a diet supplemented with sodium selenite (SS, 0.8 mg Se/kg) or hydroxy-selenomethionine (SO, 0.7 mg Se/kg), were collected at swim-up fry stage. Our findings suggest that parental selenium nutrition impacted the methionine cycle with lower free methionine and S-adenosylmethionine (SAM) and higher methionine synthase (mtr) mRNA levels in both selenium-supplemented treatments. DNA methylation profiling by reduced representation bisulfite sequencing (RRBS) identified differentially methylated cytosines (DMCs) in offspring livers. These DMCs were related to 6535 differentially methylated genes in SS:NC, 6890 in SO:NC and 7428 in SO:SS, respectively. Genes with the highest methylation difference relate, among others, to the neuronal or signal transmitting and immune system which represent potential targets for future studies.publishedVersio