Effects of dietary co-exposure to fungal and herbal functional feed additives on immune parameters and microbial intestinal diversity in rainbow trout (Oncorhynchus mykiss).

Misuse and overuse of antibiotics in aquaculture has proven to be an unsustainable practice leading to increased bacterial resistance. An alternative strategy involves the inclusion of immunostimulants in fish diets, especially fungal and herbal compounds already authorized for human consumption, hence without environmental or public health concerns. In this study, we used a holistic and cross-disciplinary pipeline to assess the immunostimulatory properties of two fungi: Trametes versicolor and Ganoderma lucidum; one herbal supplement, capsaicin in the form of Espelette pepper (Capsicum annuum), and a combination of these fungal and herbal additives on rainbow trout (Oncorhynchus mykiss). We investigated the impact of diet supplementation for 7 weeks on survival, growth performance, cellular, humoral, and molecular immune parameters, as well as the intestinal microbial composition of the fish. Uptake of herbal and fungal compounds influenced the expression of immune related genes, without generating an inflammatory response. Significant differences were detected in the spleen-tlr2 gene expression. Supplementation with herbal additives correlated with structural changes in the fish intestinal microbiota and enhanced overall intestinal microbial diversity. Results demonstrated that the different treatments had no adverse effect on growth performance and survival, suggesting the safety of the different feed additives at the tested concentrations. While the mechanisms and multifactorial interactions remain unclear, this study provides insights not only in regard to nutrition and safety of these compounds, but also how a combined immune and gut microbiota approach can shed light on efficacy of immunostimulant compounds for potential commercial inclusion as feed supplements.

Interaction between genetics and inulin affects host metabolism in rainbow trout fed a sustainable all plant-based diet.

Inulin affects nutrition and metabolism in many animals. Although inulin is widely used in the diet of teleosts, its mechanism of action is unknown. Here, we investigated the effect of inulin (2 %) on the intestinal microbiome and metabolism in rainbow trout (Oncorhynchus mykiss) selected for growth and survival when fed a 100 % plant-based diet (suave) and a control line (temoin). Metabolic responses to the two factors (line and inulin) in liver, intestine, muscle and adipose were tissue-specific, with line and interaction between the two factors influencing overall expression in liver. In the intestine, inulin and line and in muscle, line influenced the expression of metabolic genes. Microbiota between the mucus and digestive contents was significantly different, with genera from Proteobacteria being more abundant in the mucus, whereas genera from the Firmicutes and Planctomycetes being more abundant in contents. Effect of inulin and interaction between factors on the microbiome was evident in contents. The significant taxa of control and inulin-fed groups differed greatly with Streptococcus and Weissella being significantly abundant in the inulin-fed group. There was a general trend showing higher levels of all SCFA in temoin group with propionic acid levels being significantly higher. An operational taxonomic unit (OTU) belonging to the Ruminococcaceae was significantly abundant in suave. The tissue-specific correlations between OTU and gene expression may indicate the link between microbiome and metabolism. Together, these results suggest that line and inulin impact the gene expression in a tissue-specific manner, possibly driven by specific OTUs enriched in inulin-fed groups and suave.

Positive effects of bubbles as a feeding predictor on behaviour of farmed rainbow trout.

Occupational enrichment emerges as a promising strategy for improving the welfare of farmed animals. This form of enrichment aims to stimulate cognitive abilities of animals by providing them with more opportunities to interact with and control their environment. Predictability of salient daily events, and in particular predictability of feeding, is currently one of the most studied occupational enrichment strategies and can take several forms. In fish, while temporal predictability of feeding has been widely investigated, signalled predictability (based on a signal, such as light or sound) has received little attention. Depending on the type of predictability used and the ecology of the species, the effects on fish welfare often differ. The present study aimed to determine which feeding predictability would be most appropriate for rainbow trout, the main continental farmed fish in Europe, and what the consequences might be for their welfare. We tested four feeding predictability conditions: temporal (based on time of day), signalled (based on bubble diffusion), temporal + signalled (based on time and bubble diffusion), and unpredictable (random feeding times). Behavioural and zootechnical outcomes recorded were swimming activity, aggressive behaviours, burst of accelerations, and jumps, emotional reactivity, and growth. Our results showed that rainbow trout can predict daily feedings relying on time and/or bubbles as predictors as early as two weeks of conditioning, as evidenced by their increased swimming activity before feeding or during feed omission tests, which allowed to reinforce their conditioned response. Temporal predictability alone resulted in an increase in pre-feeding aggressive behaviours, burst of accelerations, and jumps, suggesting that the use of time as the sole predictor of feedings in husbandry practices may be detrimental to fish welfare. Signalled predictability with bubbles alone resulted in fewer pre-feeding agonistic behaviours, burst of accelerations, and jumps than in the temporal predictability condition. The combination of temporal and signalled predictability elicited the highest conditioned response and the level of pre-feeding aggression behaviours, burst of accelerations and jumps tended to be lower than for temporal predictability alone. Interestingly, fish swimming activity during bubble diffusion also revealed that bubbles were highly attractive regardless of the condition. Rainbow trout growth and emotional reactivity were not affected by the predictability condition. We conclude, therefore, that the use of bubbles as a feeding predictor could represent an interesting approach to improve rainbow trout welfare in farms, by acting as both an occupational and physical enrichment.

Sources of variation of DNA methylation in rainbow trout: combined effects of temperature and genetic background.

Phenotypic plasticity is a key component of the ability of organisms to respond to changing environmental conditions. In this study, we aimed to study the establishment of DNA methylation marks in response to an environmental stress in rainbow trout and to assess whether these marks depend on the genetic background. The environmental stress chosen here was temperature, a known induction factor of epigenetic marks in fish. To disentangle the role of epigenetic mechanisms such as DNA methylation in generating phenotypic variations, nine rainbow trout isogenic lines with no genetic variability within a line were used. For each line, half of the eggs were incubated at standard temperature (11°C) and the other half at high temperature (16°C), from eyed-stage to hatching. In order to gain a first insight into the establishment of DNA methylation marks in response to an early temperature regime (control 11°C vs. heated 16°C), we have studied the expression of 8 dnmt3 (DNA methyltransferase) genes, potentially involved in de novo methylation, and analysed global DNA methylation in the different rainbow trout isogenic lines using LUMA (LUminometric Methylation Assay). Finally, finer investigation of genome-wide methylation patterns was performed using EpiRADseq, a reduced-representation library approach based on the ddRADseq (Double Digest Restriction Associated DNA) protocol, for six rainbow trout isogenic lines. We have demonstrated that thermal history during embryonic development alters patterns of DNA methylation, but to a greater or lesser extent depending on the genetic background.

Regulation of the corticosteroid signalling system in rainbow trout HPI axis during confinement stress.

This study aims to shed light on corticosteroid regulation of stress in teleost fish with focus on the corticosteroid signalling system. The role of the mineralocorticoid-like hormone 11-deoxycorticosterone (DOC) in fish is still enigmatic, as is the function of the mineralocorticoid receptor, MR. Low plasma DOC levels and ubiquitous tissue distribution of MR question the physiological relevance of the mineralocorticoid-axis. Furthermore, the particular purpose of each of the three corticosteroid receptors in fish, the glucocorticoid receptors, GR1 and GR2, and the MR, is still largely unknown. Therefore we investigate the regulation of cortisol and DOC in plasma and mRNA levels of MR, GR1 and GR2 in the HPI-axis tissues (hypothalamus, pituitary and interrenal gland) during a detailed confinement stress time-course. Here we show a sustained up-regulation of plasma DOC levels during a confinement stress time-course. However, the low DOC levels compared to cortisol measured in the plasma do not favour an activity of DOC through MR receptors. Furthermore, we show differential contribution of the CRs in regulation and control of HPI axis activity following confinement stress. Judged by the variation of mRNA levels negative feedback regulation of cortisol release occurs on the level of the pituitary via MR and on the level of the interrenal gland via GR2. Finally, asa significant effect of confinement stress on CR expressions was observed in the pituitary gland, we completed this experiment by demonstrating that corticosteroid receptors (GR1, GR2 and MR) are co-expressed in the ACTH cells located in the adenohypophysis. Overall, these data suggest the involvement of these receptors in the regulation of the HPI axis activity by cortisol.

Selection for adaptation to dietary shifts: towards sustainable breeding of carnivorous fish.

Genetic adaptation to dietary environments is a key process in the evolution of natural populations and is of great interest in animal breeding. In fish farming, the use of fish meal and fish oil has been widely challenged, leading to the rapidly increasing use of plant-based products in feed. However, high substitution rates impair fish health and growth in carnivorous species. We demonstrated that survival rate, mean body weight and biomass can be improved in rainbow trout (Oncorhynchus mykiss) after a single generation of selection for the ability to adapt to a totally plant-based diet (15.1%, 35.3% and 54.4%, respectively). Individual variability in the ability to adapt to major diet changes can be effectively used to promote fish welfare and a more sustainable aquaculture.