Gut health and microbiota during the life cycle of Atlantic salmon (Salmo salar) produced under commercial Arctic conditions : modulation by functional feed ingredients investigated in vivo and in vitro

Abstract

The goal of this thesis was to fill some of the knowledge gaps regarding gut health and microbiota of Atlantic salmon in the life period from late freshwater to seawater in the Arctic region for which two strategies were chosen: Firstly, to observe biomarkers of importance, health-related gut functions in fish in a commercial size experimental farm fed diets without and with functional feed ingredients over a year from the last weeks before seawater transfer, and secondly, to use an in vitro approach to study effects of functional feed ingredients in a gut cell line model to reveal mechanisms underlying possible in vivo effects of functional feed ingredients. Atlantic salmon were observed at four sampling time points from late freshwater stage until about one year in seawater: in May, two weeks before seawater transfer (i.e. FW); in June, four weeks after seawater transfer (SW1); in November (SW2), and in April the following year (SW3) (Paper Ⅰ and Ⅱ). Two series of diets were fed, varying throughout the observation time in nutrient composition according to the requirements of fish, one with functional feed ingredients (Test diet), and the other without (Ref diet). The functional feed ingredients, i.e. nucleotides, yeast cell walls, prebiotic and essential fatty acids, were supplemented to the diets either as a single ingredient or as a mixture based on a strategy developed by the feed producer for commercial production in the Arctic region. Overall, the growth performance of the fish, gut health and gut microbiota varied greatly between the sampling time points, whereas the inclusion of functional feed ingredients in the diet affected the fish only marginally. Compared to fish observed at FW, fish at SW1, i.e. four weeks after seawater transfer, showed substantially lower plasma cholesterol and triglycerides levels. The same was the case for gene expression levels related to immune and barrier functions, i.e. cytokines, T-cell markers and tight junction proteins, indicating the suppressed status of some key physiological functions after seawater transfer. This suppression is most likely a cause of the increased vulnerability to diseases observed in Atlantic salmon just after seawater transfer. During the period SW1 to SW3 the plasma nutrients and most gene expression biomarkers returned to the levels observed at FW. Hyper-vacuolization was observed in the pyloric caeca enterocytes, particularly the fish at SW1 and SW2, indicating a situation of gut mucosa lipid malabsorption. The increasing symptoms of lipid malabsorption corresponded with the up-regulated perilipin-2 (plin2) expression levels. Microbiota in digesta from the distal intestine at FW was dominated by phylum Firmicutes (e.g. genera Lactobacillus, Weissella, Peptostreptococcus and family Ruminococcaceae), Proteobacteria (e.g. genera Photobacterium, Deefgea and Pseudomonas) and Bacteroidetes (mainly genus Flavobacterium). The gut microbiota also showed slight alteration just after seawater transfer, i.e. at SW1. Specifically, it was strongly dominated by the phylum Firmicutes (mainly genus Lactobacillus) and Proteobacteria (mainly genus Photobacterium). As fish progressed towards SW2 and SW3, the genera Lactobacillus and Mycoplasma became more prominent with a corresponding decline in genus Photobacterium. Multivariate association analysis identified the expression levels of gut barrier function genes to be negatively correlated with 26 taxa including genera Megasphaera, Photobacterium and certain lactic acid bacteria (LAB). Also, the relative abundance of Megasphaera was positively correlated with the levels of gut immune gene expressions and plasma nutrients. Mostly, the functional feed ingredients did not significantly affect the observed indicators of production or gut functions of the fish. However, the mix of functional feed ingredients used at SW2, nucleotides, yeast cell walls and essential fatty acids, seemed to represent a metabolic cost for the fish, as indicated by a tendency to slower growth, and a reduction in condition factor. Plasma triglyceride levels showed a corresponding significant decrease. At the same sampling time point, decreased microbial richness and diversity and low relative abundance of LAB were observed in Test-fed fish. The nucleotides, mannan oligosaccharides (MOS) and β-glucans, were selected for evaluation of effects on intestinal epithelial functions at the cellular level using the rainbow trout intestinal epithelial cell line, RTgutGC (Paper ⅠⅡ). The MOS treatment seemed to be the most potent modulator of immune and barrier functions in RTgutGC, and strongly suppressed reactive oxygen species (ROS) production and cell proliferation. Treatment with β-glucans induced high gene expression levels of immune and barrier functions, as well as possibly enhanced barrier functions via increasing transepithelial electrical resistance (TEER) levels and F-actin content. Compared to MOS and β-glucans, nucleotide treatment-induced minor effects, i.e. only on expression levels of a few genes, i.e. interleukin 1 β (il1b), interleukin 8 (il8) and E-cadherin (cdh1). Altogether, this thesis work fills knowledge gaps regarding the dynamics of gut health and microbiota in Atlantic salmon from the late freshwater stage until about one year in seawater in the Arctic region of Norway, effects of strategic use of selected functional feed ingredients throughout the production cycle, as well as mechanisms underlying effects of these functional feed ingredients.

En konsekvens av den forventede økningen i etterspørselen etter atlantisk laks (Salmo salar) er søk etter nye lokaliteter for produksjon. I arktiske områder i Norge finnes muligheter for økt produksjon. Men, ettersom de fleste studier av effekter av variasjon i miljø og fôr på laksens produksjonsbiologi og helse er utført i mer sydlige områder, mangler vi kunnskaper om slike forhold for fisk produsert under arktiske forhold så som virkninger av ekstreme variasjoner i fotoperiode, lav gjennomsnittstemperatur og spesifikke patogener på fiskevelferd og helse. Arbeidet som presenteres i denne avhandlingen hadde som må å fylle kunnskapshull som gjelder tarmfunksjon og helse gjennom kritiske perioder av produksjonssyklusen hos fisk produsert under arktiske forhold. Funksjonelle ingredienser brukes i fiskefôr for å forbedre fiskehelse og sykdomsresistens, spesielt under stressende oppdrettsforhold. Bruken begrunnes med at de kan ha positive effekter på tarmfunksjon og helse, via direkte virkning på tarmslimhinnen og/eller via modulering av tarmmikrobiota. Dokumentasjonen for slike virkninger er imidlertid, svært begrenset, særling under kommersielle forhold. Målet med dette doktorgradsarbeidet var å fylle noen av kunnskapshullene som gjelder produksjon av laks under arktiske strøk. To strategier ble valgt. I den første ble forhold i en kommersiell produksjon indersøkt. I den andre ble effekter av funksjonelle ingredienser på tarmceller studert under in vitro betingelser.