Long -term feeding with high plant protein based diets in gilthead seabream (Sparus aurata, L.) leads to changes in the inflammatory and immune related gene expression at intestinal level

[EN] Background: In order to ensure sustainability of aquaculture production of carnivourous fish species such as the gilthead seabream (Sparus aurata, L.), the impact of the inclusion of alternative protein sources to fishmeal, including plants, has been assessed. With the aim of evaluating long-term effects of vegetable diets on growth and intestinal status of the on-growing gilthead seabream (initial weight = 129 g), three experimental diets were tested: a strict plant protein-based diet (VM), a fishmeal based diet (FM) and a plant protein-based diet with 15% of marine ingredients (squid and krill meal) alternative to fishmeal (VM+). Intestines were sampled after 154 days. Besides studying growth parameters and survival, the gene expression related to inflammatory response, immune system, epithelia integrity and digestive process was analysed in the foregut and hindgut sections, as well as different histological parameters in the foregut. Results: There were no differences in growth performance (p = 0.2703) and feed utilization (p = 0.1536), although a greater fish mortality was recorded in the VM group (p = 0.0141). In addition, this group reported a lower expression in genes related to pro-inflammatory response, as Interleukine-1 beta (il1 beta, p = 0.0415), Interleukine-6 (il6, p = 0.0347) and cyclooxigenase-2 (cox2, p = 0.0014), immune-related genes as immunoglobulin M (igm, p = 0.0002) or bacterial defence genes as alkaline phosphatase (alp, p = 0.0069). In contrast, the VM+ group yielded similar survival rate to FM (p = 0.0141) and the gene expression patterns indicated a greater induction of the inflammatory and immune markers (il1 beta, cox2 and igm). However, major histological changes in gut were not detected. Conclusions: Using plants as the unique source of protein on a long term basis, replacing fishmeal in aqua feeds for gilthead seabream, may have been the reason of a decrease in the level of different pro-inflammatory mediators (il1 beta, il6 and cox2) and immune-related molecules (igm and alp), which reflects a possible lack of local immune response at the intestinal mucosa, explaining the higher mortality observed. Krill and squid meal inclusion in vegetable diets, even at low concentrations, provided an improvement in nutrition and survival parameters compared to strictly plant protein based diets as VM, maybe explained by the maintenance of an effective immune response throughout the assay.The research has been partially funded by Vicerrectorat d'Investigacio, Innovacio i Transferencia of the Universitat Politecnica de Valencia, which belongs to the project Aquaculture feed without fishmeal (SP20120603). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.Estruch-Cucarella, G.; Collado, MC.; Monge-Ortiz, R.; Tomas-Vidal, A.; Jover Cerdá, M.; Peñaranda, D.; Perez Martinez, G.... (2018). Long -term feeding with high plant protein based diets in gilthead seabream (Sparus aurata, L.) leads to changes in the inflammatory and immune related gene expression at intestinal level. BMC Veterinary Research. 14. https://doi.org/10.1186/s12917-018-1626-6S14Hardy RW. Utilization of plant proteins in fish diets: effects of global demand and supplies of fishmeal. Aquac Res. 2010;41:770–6.Martínez-Llorens S, Moñino AV, Vidal AT, Salvador VJM, Pla Torres M, Jover Cerdá M, et al. Soybean meal as a protein source in gilthead sea bream (Sparus aurata L.) diets: effects on growth and nutrient utilization. Aquac Res. 2007;38(1):82–90.Tacon AGJ, Metian M. 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Cloning, Functional Characterization and Nutritional Regulation of Delta 6 Fatty Acyl Desaturase in the Herbivorous Euryhaline Teleost Scatophagus Argus

Marine fish are generally unable or have low ability for the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, with some notable exceptions including the herbivorous marine teleost Siganus canaliculatus in which such a capability was recently demonstrated. To determine whether this is a unique feature of S. canaliculatus or whether it is common to the herbivorous marine teleosts, LC-PUFA biosynthetic pathways were investigated in the herbivorous euryhaline Scatophagus argus. A putative desaturase gene was cloned and functionally characterized, and tissue expression and nutritional regulation were investigated. The full-length cDNA was 1972 bp, containing a 1338 bp open-reading frame encoding a polypeptide of 445 amino acids, which possessed all the characteristic features of fatty acyl desaturase (Fad). Functional characterization by heterologous expression in yeast showed the protein product of the cDNA efficiently converted 18:3n-3 and 18:2n-6 to 18:4n-3 and 18:3n-6, respectively, indicating D6 desaturation activity. Quantitative real-time PCR showed that highest D6 fad mRNA expression was detected in liver followed by brain, with lower expression in other tissues including intestine, eye, muscle, adipose, heart kidney and gill, and lowest expression in stomach and spleen. The expression of D6 fad was significantly affected by dietary lipid and, especially, fatty acid composition, with highest expression of mRNA in liver of fish fed a diet with a ratio of 18:3n-3/18:2n-6 of 1.72:1. The results indicated that S. argus may have a different LC-PUFA biosynthetic system from S. canaliculatus despite possessing similar habitats and feeding habits suggesting that LC-PUFA biosynthesis may not be common to all marine herbivorous teleosts

Characterization and modulation of gene expression and enzymatic activity of delta-6 desaturase in teleosts : a review

International audienceThere is currently considerable interest in understanding how the biosynthetic pathways of highly unsaturated fatty acids (HUFA) are regulated in fish. The aim is to know if it is possible to replace fish oils (FO), rich in HUFA, by vegetable oils (VO), poor in HUFA and rich in their 18 carbon fatty acid precursors, in the feed of cultured fish species of commercial importance. Thus many studies have focussed on delta-6 desaturase (Delta 6D) since it is the rate-limiting enzyme involved in HUFA biosynthesis from precursors. The aims of this paper were (i) to review and compare the structure, function, and tissue distribution of the Delta 6D gene in teleosts and (ii) to review the effect of nutrition and environment on the modulation of Delta 6D gene expression and on the activity of this enzyme in teleosts. Most existing studies have clearly shown that Delta 6D is modulated by nutrition and environment in freshwater fish. This modulation allows the control of lipid metabolism and the maintenance of cell membrane functionality. Delta 6D gene expression and enzymatic activity were higher in fish fed VO diets than in those fed FO diets, irrespective of their life cycle in seawater or freshwater; this concurs with expectations regarding the compensation for HUFA deficiency in VO. However, the magnitude of these increases was not great enough to maintain HUFA tissue content. Such a decrease in tissue content may result either from competition between substrates for Delta 6D or from the inhibition of subsequent steps in HUFA biosynthesis, such as elongation or delta-5 desaturation activity. Other studies showed that Delta 6D enzyme activity is stimulated at low temperatures and low salinities, which keep cell membranes fluid. In salmonids, Delta 6D would then play an important role in the adaptation to salinity changes at parr-smolt transformation. In marine fish, similar nutritional and environmental modulations may occur, although conflicting data exist. A low expression of the Delta 6D gene or the involvement of genetic, environmental, or hormonal factors could explain why Delta 6D appears to be barely functional in marine fish. This review shows that, despite the large number of studies investigating the regulation of Delta 6D, little is known about the molecular mechanisms involved. Furthermore, the nutritional and environmental regulation of other enzymes involved in HUFA biosynthesis still need to be investigated to obtain a better understanding of the regulation of HUFA biosynthetic pathways in teleosts, ultimately leading to improvements in fish production

Plant oils' inclusion in high fish meal‐substituted diets: effect on digestion and nutrient absorption in gilthead sea bream (Sparus aurata L.)

Here, we performed an 11-week trial to study the effects of four experimental diets on the digestion, nutrient absorption and intestinal histology of gilthead sea bream. The diets were formulated with a low fish meal content (25%) and were rich (75%) in plant proteins. Fish oil (FO) was replaced at 0%, 33%, 66% and 100% by graded levels of a blend of vegetable oils (VO) (diets FO, 33VO, 66VO and 100VO respectively). Protease activity increased in the pyloric caeca (PC) and decreased in the proximal intestine (PI) of the 66 and 100VO groups, while lipase and alpha-amylase activities were not modified by VO. The capacity of brush-border membrane vesicles obtained from PC, PI and distal intestine to absorb amino acids was not modified, but D-glucose and linoleic acid absorption diminished when VO was included in the diets.In summary, these results show that when 75% of protein is provided by vegetable sources in the diet of gilthead sea bream, up to 66% of FO can be replaced by a blend of VO without compromising digestive processes. Only the total replacement of VO results in an impaired digestion, with reduced total protease activity and accumulation of lipid droplets in the enterocytes of the PI

Modifications of intestinal nutrient absorption in response to dietary fish meal replacement by plant protein sources in sea bream (Sparus aurata) and rainbow trout (Onchorynchus mykiss)

The effects of partial (75%; diet PP75) and total (100%; diet PP100) replacement of fish meal by plant protein sources on the intestinal nutrient absorption of gilthead sea bream and rainbow trout were examined over a 12-week growth trial. The diets comprised a mixture of plant ingredients (corn gluten meal, wheat gluten, extruded peas and rapeseed meal) and met the amino acid requirements of the fish. A third group of fish on a fish meal diet was used as a control for comparative purposes. Diets were tested in triplicate and fish were fed twice a day until visual satiation. At the end of the trial, we measured amino acid (L-leucine, L-lysine, L-phenylalanine, Lalanine and L-proline) and D-glucose absorption at short term (6 h) and long-term (36 h and 48 h in rainbow trout and sea bream respectively) post-feeding by means of brush border membrane vesicles obtained from pyloric caeca, proximal intestine and distal intestine. The absorption pattern at 6 h post-feeding was modified in both species in response to fish meal replacement. In PP75-fed trout absorption was delayed from pyloric caeca to proximal and/or distal intestinal segments, thus total absorption capacity was maintained in this group. On the contrary, total uptake was significantly decreased in trout fed the PP100 diet and in sea bream on both PP75 and PP100 diets. Glucose transport capacity was increased in both experimental sea bream groups and in PP75 trout. Long-term transport capacity was up-regulated for both species. Our results show that intestinal nutrient absorption is modified in response to the use of high levels of plant protein sources and that these changes are species-specific

Mar Biotechnol

Fish are the main source of the n-3 highly unsaturated fatty acids, which are crucial for human health. Their synthesis from C18 precursors is mediated by desaturases and elongases, but the activity of these enzymes has not been conclusively established in marine fish species. This study reports the cloning, tissue expression, and functional characterization of a sea bass (Dicentrarchus labrax L.) Δ6-desaturase and one of its splicing variants. Two cDNAs with open reading frames of 1,346 and 1,354 bp were cloned and named D6D and D6D-V, respectively. Both deduced protein sequences (445 and 387 amino acids, respectively) contained two transmembrane regions and the N-terminal cytochrome b5 domain with the HPGG motif characteristic of microsomal desaturases. D6D presents three histidine-rich regions, whereas in D6D-V, an insertion of eight nucleotides in the boundaries of exons 10 and 11 modified the third histidine-rich domain and led to insertion of a premature STOP codon, resulting in a shorter predicted protein. Quantitative real-time polymerase chain reaction assay of gene expression showed that D6D was highly expressed in the brain and intestine, and to a lesser extent, in muscle and liver; meanwhile, D6D-V was expressed in all tissues tested, but at level at least 200-fold lower than D6D. Functional analysis in yeast showed that sea bass D6D encodes a fully functional Δ6-desaturase with no residual Δ5-desaturase activity. This desaturase does not exhibit a clear preference for n-3 versus n-6 C18 substrates. Interestingly, D6D-V is a nonfunctional protein, suggesting that the C-terminal end is indispensable for protein activity

Effects of a vegetable diet deprived in HUFA on the regulation of FADS2 activity in the European sea bass (Dicentrarchus labrax): a potential limiting step in HUFA biosynthesis

International audienc