The LeuSENSE project explored possible effects of L-leucine-enriched diets on Atlantic salmon robustness, welfare and growth reared under normal and stressed conditions. Based on pilot studies, it was hypothesized that the essential branched chain amino acid leucine, when added above recommended levels to the salmon diet, would have beneficial effects on fish appetite, muscle growth, fat deposition and resilience to stress.
To test our hypothesis, we first established an effective and standardized stressor protocol. Three different stressors were tested (chasing, hypoxia and a combination of chasing and hypoxia) by repeated exposure for 8 days. All the three stressors induced suppressed weight gain and growth rate, and changes in cortisol, lactate, glucose and creatinine levels in blood in Atlantic salmon post smolts. Additionally, the mRNA levels of corticotrophin-releasing factor (crf), well known for its role in the stress regulation through the Hypothalamic-Pituitary-Interrenal (HPI) axis, in the telencephalon and hypothalamus showed a similar pattern stress-response to cortisol, i.e., higher levels in the stressed groups compared to the non-stress group. Based on all parameters analyzed, chasing was shown to be the most effective method, since it induced the higher levels of stress-response. During the project, we also established new quantitative PCR (qPCR) assays to evaluate the mRNA expression changes of factors involved in the stress-response and appetite control signaling pathway. Specifically, assays for four crf genes, and the neuropeptides agouti-related peptide (agrp), neuropeptide Y (npy), proopiomelanocortin (pomc) and cocaine and amphetamine regulated transcript (cart), as well as the receptor integrating these signals, the melancortin-4 receptor (mc4r) were validated. We also established and validated an in vitro assay to describe the potential of L-leucine to stimulate nutrient sensing pathways that might affect both digestion and appetite control. Results indicate that the L-leucine stimulates the calcium sensing receptor response through the ERK signalling pathway in the presence of Ca2+. L-leucine also seems to stimulate release of the gut hormone cholecystokinin in the salmon hindgut. Additionally, we have also identified and established qPCR assays of amino acid carriers of the SLC7 and SLC38 families, which are known to be key regulators of intracellular leucine.
To address if supplementation of dietary L-leucine attenuates the effects of chronic stress, four L-leucine enriched diets were used to feed Atlantic salmon reared under normal (optimal) or stress (suboptimal) conditions. Feed intake, growth, and mRNA expression levels of key genes involved in appetite control, growth, energy allocation and HPI- stress axis was analysed. The results showed that stressed fish consumed less feed and grew less than unstressed fish, irrespective of diet. Additionally, leucine inclusion resulted only in minor changes in leucine retention and in the expression of genes involved in the regulation of lipid homeostasis (ppara), muscle remodeling (murf), stress-axis (crf), appetite control (pomca) and amino acid carriers of the SLC family (slc7a5, and slc38a). In conclusion, L-leucine did not improve salmon resilience to stress, and neither protein accretion nor growth was affected by the diets. However, it remains to be investigated if leucine supplementation has a positive effect on lipid accumulation and metabolism or oxidation in high fat Atlantic salmon, as shown for other species. Also, the temporal and dynamic effects of leucine administration in the postprandial phase following feeding should be further explored.
The LeuSENSE project investigated the effects of increased levels of dietary L-leucine in Atlantic salmon subject to stress. We have, therefore, established an effective protocol to induce stress in salmon and validated several stress-response markers, notably the identification of 4 corticotrophin-releasing factor genes and validation of gene-specific mRNA expression analyses (qPCR). Several qPCR assays were also established for key genes involved in the appetite control and are now routinely in use in our lab. Overall, the project results suggest that L-leucine supplementation did not improve salmon resilience to stress, which opens new research questions related to the role of leucine in salmon. The project also contributed to establish an in-vitro method to screen for functional response of amino acids and feed ingredients. The LeuSENSE was important in the academic education of two master students, and 3 PhD students that were also partially involved in the project.
LeuSENSE aims to assess if dietary leucine boosts robustness, welfare and growth in Atlantic salmon under stressful conditions. The project builds on our preliminary data and literature, indicating that certain amino acids, particularly leucine, have important positive effects above their nutritional requirement levels under allostatic conditions. During the normal production cycle salmon may encounter episodes of stress related to disease, changes in environmental conditions (temperature, salinity), handling, etc. We propose that a diet rich in leucine will reduce the physiological strain and result in more robust fish with improved lean growth (fillet) with less allocation of fat, and reduced time of production. Therefore, we will investigate how dietary leucine regulates daily feed intake and affects the overall metabolism including muscle growth and fat deposition in salmon reared under normal and stressed conditions. To this end we will combine in vivo, ex vivo and in vitro experiments, and analyse the gene expression profiling in candidate tissues to uncover which processes are vital in the metabolic processes from feed to muscle growth under allostatic conditions. This project will describe how leucine activates specific nutrient sensing receptors in the gastrointestinal tract and triggers gut-brain signalling pathways related to appetite, growth and health. Signals emerging in the gut receive a lot of attention in human nutrition, but virtually nothing is known in fish. LeuSENSE will provide novel information about leucine's role in these signalling pathways and in the metabolism of a teleost and apply the project results into tailoring improved feed for Atlantic salmon under suboptimal conditions.