Soya saponins and prebiotics alter intestinal functions in Ballan wrasse (Labrus bergylta)

Abstract

A 5-week feeding trial was conducted in the cleaner fish Ballan wrasse (Labrus bergylta) for a better understanding of the basic biology of the intestinal functions and health in this stomach less species. During the trial, Ballan wrasse was fed either a reference diet, the reference diet supplemented with (i) a commercial prebiotic (Aquate™ SG, 0·4 %) expected to have beneficial effects, (ii) soya saponins (0·7 %) expected to induce inflammation or (iii) a combination of the prebiotics and the soya saponins to find a remedy for gut inflammation. Blood, intestinal tissue and gut content from four consecutive intestinal segments (IN1 - IN4) were collected. No significant differences in fish growth were observed between the four dietary groups. Saponin supplementation, both alone and in combination with prebiotics, increased weight index of IN2 and IN3 and decreased blood plasma glucose, cholesterol and total protein. Dry matter of intestinal content and activity of digestive enzymes were not affected by diet. Histomorphological analyses revealed a progressing inflammation with increased infiltration by immune cells particularly into the distal parts of the intestine in fish fed diets with saponins, both alone and in combination with prebiotics. Gene expression profiles obtained by RNA sequencing and quantitative PCR mirrored the histological and biochemical changes induced by the saponin load. The study demonstrated that Ballan wrasse gut health and digestive function may be markedly affected by feed ingredients containing antinutrients.

Keywords: Antinutrients; Ballan wrasse; Cleaner fish; Gut health; Prebiotics.

Fig. 1.

A representative image showing the bifurcation of intestinal mucosal fold.

Fig. 2.

Representative images of the mucosa of Ballan wrasse intestine that were scored as normal, mild, moderate and marked (images a-d, respectively) for infiltration of lamina propria by inflammatory cells. Image d shows a notable increase in width of the lamina propria.

Fig. 3.

Representative images of the mucosal epithelium of the Ballan wrasse intestine that were scored as normal, mild, moderate, or marked for intraepithelial lymphocyte infiltration (images a-d, respectively).

Fig. 4.

Maltase capacities in IN1, IN2 and IN4. Values are means with standard errors as error bars. Mean values with different letters are significantly different. P values derived from one-way ANOVA for diet effect were 0·30, less than 0·01 and less than 0·01 in IN1, IN2 and IN4, respectively. IN1, IN2 and IN4, intestinal regions 1, 2 and 4 in a proximo-distal axis.

Fig. 5.

LAP capacities in IN1, IN2 and IN4. Values are means with standard errors as error bars. Mean values with different letters are significantly different. P values derived from one-way ANOVA for diet effect were 0·11, 0·22 and 0·39 in IN1, IN2 and IN4, respectively. IN1, IN2 and IN4, intestinal regions 1, 2 and 4 in a proximo-distal axis.

Fig. 6.

Digesta bile salt concentration in IN1 to IN4. Values are means with standard errors as error bars. Mean values with different letters are significantly different. P values derived from one-way ANOVA for diet effect were 0·16, 0·01, less than 0·01 and 0·20 from IN1 to IN4, respectively. IN1–4, intestinal regions 1–4 in a proximo-distal axis.

Fig. 7.

Diet effects on the cellularity of the lamina propria in the different regions of the Ballan wrasse intestine. LP, lamina propria. IN1–4, intestinal regions 1–4 in a proximo-distal axis.

Fig. 8.

Diet effects on the intraepithelial lymphocyte infiltration in the different regions of the Ballan wrasse intestine. IEL, intraepithelial lymphocyte. IN1–4, intestinal regions 1–4 in a proximo-distal axis.

Fig. 9.

Diet effects on the numbers of intraepithelial rodlet cells and eosinophilic granular cells (EGCs) in the mucosal epithelium in the different regions of the Ballan wrasse intestine. IN1–4, intestinal regions 1–4 in a proximo-distal axis.

Fig. 10.

Venn diagram showing the number of differentially expressed genes affected by either saponin, prebiotic or their combination. Among them, 308, 2128 and 1078 genes were exclusively affected by saponin, prebiotic and combination treatments, respectively; 133 genes were influenced by both saponin and prebiotic treatments; 341 genes were influenced by both saponin and combination treatments; 283 genes were influenced by both prebiotic and combination treatments and 273 genes were influenced by all treatments. Venn diagram was created by using venny 2·1·0.

Fig. 11.

Comparative ingenuity pathway analysis showing A) significant canonical pathways (Z-score > 2), B) disease and function (Z-score > 2·5) and C) predicted upstream regulators (Z-score > 4) related to immune function in Ballan wrasse (Labrus bergylta) as a result of adding either saponins, prebiotics or saponin + prebiotic in the feed. All analysis were filtered using p < 0·05 in addition to Z-score filtering. Non-significant terms are marked as ‘X’.

Fig. 12.

Comparative ingenuity pathway analysis showing A) significant canonical pathways (Z-score > 2), B) disease and function (Z-score > 2·5) and C) predicted upstream regulators (Z-score > 4) related to lipid metabolism in Ballan wrasse (Labrus bergylta) as a result of adding either saponins, prebiotics or saponin + prebiotic in the feed. All analysis were filtered using p < 0·05 in addition to Z-score filtering. Non-significant terms are marked as ‘X’.

Fig. 13.

Distal intestine gene expression of A: chymotrypsin A (ctra), vitamin C transporter (slc23a1), cluster of differentiation 36 (cd36), fatty acid binding protein (fabp2), aquaporin 8 (aqp8), squalene epoxidase (sqle) and lanosterol 14α-demethylase (cpy51a1). B: interleukin 1 beta (il1b), interleukin 6 (il6), NOD-like receptor family CARD domain containing 3 (nlrc3), lysozyme (lyz), cluster of differentiation 40 (cd40), immunoglobulin M (igm), cluster of differentiation 38 (cd38), mannose receptor C-type 1 (mrc1), proliferating cell nuclear antigen (pcna), catalase (cat), matrix metallopeptidase 13 (mmp13), IgGFc-binding protein (fcgbp). Values are means with standard errors as error bars. Mean values with different letters are significantly different. P values derived from statistical analysis are given.