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

Abstract

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-1β (il1β, 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 (il1β, 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 β, 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.

Keywords: Gene expression; Gilthead seabream; Histology; Intestine; Krill meal; Squid meal; Vegetable meal.

Fig. 1

Evaluation and scoring system used to assess histological parameters of gilthead seabream foregut. a Measurements performed in a foregut histological section (20×). b Detail of villi with a certain grade of infiltration of the lamina propia and the epithelia. Enterocytes nuclei were displaced in some cases. Epithelial vacuolization can also be observed in a normal grade (40×). c Enterocytes showed aligned nuclei in a basal position. Villi presented a low grade of infiltration of their lamina propia and of the epithelia, and low vacuolization. A certain grade of infiltration in the submucosa layer can be observed (20×). SL, ML, SML, VL, VT and number of GC were measured six times per section, and averages were obtained for each section (six sections per group, n = 6). V, EN, EI, LPI and SMI were assessed in each section (n = 6) using the following scoring system: V, normal (1) to hypervacuolated (4); EN, basal (1) to apical (4); EI, low (1) to markedly increased (4); LPI, low (1) to markedly increased (4); SMI, low (1) to markedly increased (4). SL, serous layer; ML, muscular layer; SML, submucosa layer; VL, villi length; VT, villi thickness; LP, lamina propria; GC, goblet cells; V, supranuclear absorptive vacuoles; EN, enterocytes nuclei; EI, epithelial infiltration; LPI, lamina propria infiltration; SMI, submucosa infiltration

Fig. 2

Average weight (g) and survival rate (%) evolution of gilthead seabream along the assay period. Average weight mean and standard error (bars) and survival rate (line) of each experimental group were displayed in different colours (Black: VM; Grey: FM; White: VM+). Different superscripts on the bars indicate significant statistical differences in the average weight during the growth trial (p < 0.05). Data are means of triplicate groups (n = 20). Asterisks indicate the existence of significant differences in the survival rate along the assay at p < 0.05

Fig. 3

Relative gene expression in the intestine of gilthead seabream fed different experimental diets. a Interleukine-1β (il1β); b Interleukine-6 (il6); c Cyclooxigenase-2 (cox2); d Intestinal Mucin (imuc); e Immunoglobulin M (igm). f Occludin (ocl); g Alkaline Phosphatase (alp); h Trypsin (tryp); i Peptide Transporter 1 (pept1). Bars represent relative gene expression (mean + standard error, n = 9), for each group, in the foregut (FG, black bars) and the hindgut (HG, grey bars). Superscript letters on the bars indicate differences between experimental groups in each section, at p < 0.05. Asterisks indicate differences between intestinal sections in each experimental group, at p < 0.05. Capital letters at the top of the graph indicate differences between experimental groups, regardless the intestinal section (n = 18, p < 0.05), when interaction between factors (diet and section) is not significative (Table 5)

Fig. 4

Histological assessment of foregut sections of gilthead seabream fed different experimental diets, according to Fig. 1. Frequency bar charts showing differences in a supranuclear absorptive vacuolization (V), b enterocytes nuclei (EN), c enterocytes infiltration (EI), d lamina propria infiltration (LPI) and e submucosa infiltration (SMI). f Dispersion graph representing values of the first and second components for each foregut section assessed, obtained from the Principal component analysis of histological foregut scores according to diet. Only sections evaluated in all parameters were included in the Principal component analysis (n = 5 for VM and FM, n = 6 for VM+)