Dietary Fish Meal Level and a Package of Choline, β-Glucan, and Nucleotides Modulate Gut Function, Microbiota, and Health in Atlantic Salmon (Salmo salar, L.)

Abstract

Steatosis and inflammation have been common gut symptoms in Atlantic salmon fed plant rich diets. Choline has recently been identified as essential for salmon in seawater, and β-glucan and nucleotides are frequently used to prevent inflammation. The study is aimed at documenting whether increased fishmeal (FM) levels (8 levels from 0 to 40%) and supplementation (Suppl) with a mixture of choline (3.0 g/kg), β-glucan (0.5 g/kg), and nucleotides (0.5 g/kg) might reduce the symptoms. Salmon (186 g) were fed for 62 days in 16 saltwater tanks before samples were taken from 12 fish per tank for observation of biochemical, molecular, metabolome, and microbiome indicators of function and health. Steatosis but no inflammation was observed. Lipid digestibility increased and steatosis decreased with increasing FM levels and supplementation, seemingly related to choline level. Blood metabolites confirmed this picture. Genes in intestinal tissue affected by FM levels are mainly involved in metabolic and structural functions. Only a few are immune genes. The supplement reduced these FM effects. In gut digesta, increasing FM levels increased microbial richness and diversity, and changed the composition, but only for unsupplemented diets. An average choline requirement of 3.5 g/kg was indicated for Atlantic salmon at the present life stage and under the present condition.

Figure 1

Growth rate as indicated by thermal growth coefficient (TGC) and feed conversion ratio (FCR) in fish fed two series of diets varying in fishmeal level, one series without additional supplement (No suppl) and the other supplemented (Suppl) with a mixture of choline chloride (0.3%), β-glucan (0.05%) and nucleotides (0.05%). For statistics see Table 2.

Figure 2

Pyloric intestine somatic index (PISI) and hepatosomatic index (HIS) in fish fed two series of diets varying in fishmeal level, one series without supplements (No suppl) and the other supplemented (Suppl) with a mixture of choline chloride (0.3%), and β-glucan (0.05%) and nucleotides (0.05%). For statistics see Table 3.

Figure 3

Digestibility of groups of fatty acid which showed a significant effect of dietary fishmeal level as well as a significant effect of the dietary supplement (No Suppl and Suppl), i.e., a mixture of 0.3% choline chloride, 0.05% β-glucan and 0.05% nucleotides. Note the expanded scale on the y-axis. For statistics, see Table 4.

Figure 4

Estimated digestibility of choline in FM and in the choline supplement (estimated by difference). Effect of dietary fishmeal level and supplementation (No suppl vs. Suppl) with a mixture of 0.3% choline chloride, 0.05% β-glucan, and 0.05% nucleotides on digestibility of choline. The statistical evaluation showed no significant effect of dietary fishmeal levels. For the unsupplemented diet series, the results were as follows: p (model) = 0.2359; R² = 0.22; Intercept = 91.3; Reg coeff = 0.037, and for the supplemented diet series: p (model) = 0.118; R² = 0.36; Intercept = 95.9; Reg coeff = −0.012; Effect of Supplementation: p < 0.0001. Note the expanded scale on the y-axis.

Figure 5

Effects of increasing fishmeal level on bile salt concentration in chyme from the distal half of the pyloric intestine (PI2) in fish fed two series of diets, one without (No suppl) and the other with (Suppl) mixture of supplements, i.e., 0.3% choline chloride, 0.05% β-glucan, and 0.05% nucleotides. For statistics, see Table 5.

Figure 6

Effects of increasing fishmeal level on trypsin activity in chyme from the two halves of the pyloric intestine (PI1 and PI2) in fish fed diet series with the mixture of supplements, i.e. 0.3% choline chloride, 0.05% β-glucan, and 0.05% nucleotides. For both sections, the regression model was highly significant. For the fish fed the unsupplemented diet series, there was no significant effect of fishmeal level. For statistics, see Table 5.

Figure 7

Effects of increasing fishmeal level on the activity of leucine-aminopeptidase (LAP) activity in chyme from the pyloric intestine (PI) in fish fed two series of diets, one without (No suppl) and the other with (Suppl) mixture of supplements, i.e., 0.3% choline chloride, 0.05% β-glucan, and 0.05% nucleotides. For statistics, see Table 6.

Figure 8

Results of analyses of plasma biomarkers which showed significant effects of dietary fishmeal level and of supplementation (No suppl vs. Suppl). For statistics, see Table 7.

Figure 9

Effects of increasing dietary level of fishmeal given without (No suppl) and with (Suppl) a mixture of 0.3% choline chloride, 0.05% β-glucan and 0.05% nucleotides (Suppl) on plasma levels of biomarkers current for the present study and with an important relationship with choline metabolism and function.

Figure 10

Representative histology images of the pyloric caeca mucosa showing: (a) tissue with normal morphology of pyloric caeca enterocytes with no vacuolization changes; (b) Pyloric caeca tissue with enterocyte steatosis changes graded as moderate and characterized by multifocal areas (blue asterisks) with the lipid vacuolization; and (c) Pyloric caeca with enterocyte steatosis (blue asterisks) affecting almost all the enterocytes and scored as severe in the histology assessment.

Figure 11

Representative images of the distal intestine mucosa showing the histology changes observed. (a) DI tissue with normal and healthy morphology, alongside; (b) DI mucosa with mild enteritis characterized by infiltration of the submucosal compartment (blue arrows) and some mild loss of enterocyte vacuolization (blue asterisks); and (c) DI mucosa with moderate enteritis with a higher level of infiltration of the submucosal compartment (blue arrows) and mild infiltration of the lamina propria (black arrows) but with no reduction in mucosal fold height or loss on enterocyte vacuolization.

Figure 12

Results of histological examination of (a) pyloric caeca regarding the degree of enterocyte vacuolization (steatosis; numbers at the bottom of the columns indicate fishmeal level), and (b–e) distal intestine inflammation as represented by the degree of increase in immune cell infiltration/cellularity in submucosa and lamina propria, mucosal fold height, and supranuclear vacuolization in fish fed diets with increasing levels of FM, without (No supplement) or with (Suppl) a mixture of 0.3% choline chloride, 0.05% β-glucan, and 0.05% nucleotides.

Figure 13

Results of histological examination of liver tissue for (a) vacuolation indicated by H&E staining and (b) vacuolation as indicated by PAS staining indicating carbohydrate accumulation in fish fed diets with increasing levels of FM, without (No supplement) or with (Supplemented) a mixture of 0.3% choline chloride, 0.05% β-glucan, and 0.05% nucleotides. Numbers at the bottom of the columns indicate the fishmeal levels of the diet.

Figure 14

Correlation between FM levels and expression of genes encoding proteins with metabolic functions in the distal intestine. (a) lipids, retinol, steroids, iron, and heme. (b) metabolism of reactive oxygen species and biotransformation. Data are correlation (Pearson) with FM in feeds with and without supplements (Supp− and Supp+). Genes downregulated in the distal intestine with dietary enteritis (Kortner et al. [49]) are marked with ^∗.

Figure 15

Effect of supplement on gene expression in distal intestine. Data are expression ratios (folds) of diets with and without supplement; differential expression is marked with underlined bold italics.

Figure 16

The alpha diversity indices for digesta-associated microbiota in the intestine of Atlantic salmon fed with diets containing diverse levels of fishmeal and their respective counterparts with functional ingredients. (a) Observed species and (b) Shannon index. Different letters indicate significant differences among the dietary groups without supplementation. ^∗ indicates significant difference between the dietary groups without and with supplementation.

Figure 17

Results for the ten most abundant genera present in the treatments selected for these analyses, i.e., FM0, FM11, FM17, FM40, FM0f, FM11f, FM17f, and FM40f.

Figure 18

Relationship between diet choline level and relative weight (%) of PI (PISI), average sum of histological scores (Normal =1, Mild =2, Moderate =3, Marked =4, Severe =5), and total plasma choline level (mM) as indicated by the polynomes best fitting the observations. Visual examination of the trend line for the histoscores indicates an average choline requirement of about 3200 mg/kg, based on the PISI and histological scores, and about 3000 mg/kg based on the plasma choline levels.