Introduction
Changing feed raw materials from marine to plant-based alters the requirement levels of certain micronutrients for Atlantic salmon (Salmo salar). Recommendations have been re-evaluated through the EU-funded ARRAINA project (Vera et al., 2020, Hemre et al., 2016) and feeding plant-based diets through smoltification of Atlantic salmon needs verification of optimal levels of one-carbon (1C) nutrients. Recent research points to those 1C nutrients, which are methionine as a key micronutrient along with folic acid, vitamin B6 (pyridoxine) and vitamin B12 (cobalamin), for improved and healthy growth through smoltification (Espe et al., 2020). NRC* funded projects explore the 1C nutrient-responsive mechanisms in muscle, which are of metabolic, molecular and epigenetic nature and that can help to explain the improved growth observed. The availability of certain nutrients can affect histone tail modifications and DNA methylation that together regulate mRNA expression and thereby control metabolism, which is one underlying explanation for nutritional programming. Epigenetic changes during early life stages such as early impact in pre-smolts can program life-long consequences on physiology, robustness and growth. Understanding how growth is controlled by those non-genetic mechanisms becomes important for a rapidly growing aquaculture industry whose concerns are to optimize production, sustainability and quality.
Material and methods
Two experimental diets provided by Skretting ARC were fed to Atlantic salmon 6 weeks prior to smoltification until 3 months after saltwater transfer in triplicate tanks at Skretting’s research station. A Control and a moderate 1C+ diet contained varying levels of methionine (6.7 and 9.5 g/kg), folate (2.6 and 4.8 mg/kg), vitamin B12 (0.15 and 0.18 mg/kg) and vitamin B6 (6.75 and 9.31 mg/kg), respectively. The formulation of the Control diet included 1C nutrients on requirement and recommended levels (NRC, 2011, Espe et al., 2014, Hemre et al., 2016), whereas 1C+ diet contained a moderate 1C nutrient surplus package to support maximal performance. Both diets contained 240 g/kg soy protein and 150 g/kg pea concentrates as main protein source, and smaller amounts of fishmeal (120 g/kg) and krill meal (20 g/kg). Lipid source was a mixture of rapeseed oil (81 g/kg) and fish oil (127 g/kg). Muscle samples from both dietary groups were taken in the end of the freshwater and in the on-growing saltwater period. Global metabolic profiling was performed on pooled muscle samples (five individuals per tank, n=3). RNA and DNA were extracted from single muscle samples (n=9) for gene expression (RNA-seq, n=9), DNA methylation (RRBS, n=9) and analysis of a histone tail methylation (H3K4me3) and acetylation (H3K27ac) mark (CUT&RUN, n=6).
Results
Salmon fed the 1C+ diet throughout smoltification significantly increased body weight and decreased liver weight in the seawater period, which was reflected in a higher condition factor and specific growth rate compared to the Control group (Espe et al., 2020). There were no differences in growth performance during the freshwater period. Feed conversion ratio and protein utilization were not different during either the freshwater or the saltwater period. Pre-liminary results from both metabolic and gene expression signatures in muscle revealed significant 1C nutrient-dependent changes already at pre-smolt, but differences intensified when analyzing post-smolt muscle (Adam et al., under review). 1C+ fed salmon showed less free amino acid and putrescine levels, and higher methionine and glutathione amounts in post-smolt muscle. Transcriptional differences between both dietary treatments revealed lower expression of genes related to translation, growth, and amino acid metabolization in post-smolt muscle when fed additional 1C nutrients. DNA methylation and histone tail modification data are under analysis and key results will be presented.
Discussion and conclusion
Increased 1C nutrient levels given in the feed over the challenging smoltification period resulted in best growth performance in the saltwater period. The overall metabolic profile in muscle of salmon fed the Control diet suggests a lower amino acid utilization for protein synthesis, and increased methionine metabolization in polyamine and redox homeostasis, whereas gene expression profiles are indicative of compensatory growth regulation at local muscle tissue level. Linking both metabolic and gene expression profiles, our findings point to fine-tuned nutrient-gene-interactions fundamental for improved growth capacity through better amino acid utilization for protein accretion when salmon was fed additional 1C nutrients throughout smoltification. Following a multi-omics approach, integration of metabolic and gene expression with DNA methylation and histone tail modification profiles will reveal possible epigenetic mechanisms involved in improved growth. These results also provide documentation for adjustment of dietary nutrient levels in aquaculture and highlight the employment of nutritional programming strategies on healthy and robust growth.
* The Research Council of Norway (NFR-295118, NFR-267787).
References
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