Introduction
The proportion of plant-based ingredients being used to replace marine resources in Atlantic salmon (Salmo salar) feed is in constant growth. Hence, the lipid profile of the diets is changing, what can in turn affect the performance and health of fish. According to Lucas (1991), exposure to specific conditions during critical early life periods can lead to stable and long-lasting changes in the DNA, resulting in potential lifetime consequences during adult life. The application of nutritional programming in freshwater stages of Atlantic salmon has shown benefits in feed efficiency and nutrient retention when using plant ingredients in a stimulus diet compared to marine during first feeding (Clarkson et al., 2017). Moreover, physiological adaptations have been described at gene expression level in vegetable stimulated groups after challenged (Vera et al., 2017). These changes could have improved the use of nutrients as well as enhanced the fish tolerance to vegetable-based feed. However, it is important to determine whether these benefits can be extrapolated to seawater stage and if a “booster” is needed. Methylation of the DNA is one of the most important epigenetic modifications, and it is well characterized that various environmental factors, such as diet or stress, can result in changes in DNA methylation which can persist through life and even become hereditary (Skjærven et al., 2022). The aim of the present study was to validate the long-term effect of nutritional programming and the possible interactions of nutrition with factors such as genotype and epigenetics.
Materials and methods
A trial was performed with Atlantic salmon from six families characterised by either high or low pigmentation genotypes. Fish were distributed into four groups and exposed to two stimulus diets; with either marine or vegetable ingredients, both delivered within the first three weeks of exogenous feeding. After that period, they were maintained with a commercial marine diet until 14 weeks after seawater transfer. All the groups were then challenged with a vegetable-based feed which was delivered from week 66 until the end of the trial when they reached harvest size. Body weight and total fork length were recorded before fish were processed for sample extraction. Samples were taken during freshwater and in four sampling points through seawater. Nutritional analyses included proximate and fatty acid composition of feed, whole fish and tissue samples. Additionally, DNA from liver fragments was extracted and used for the Reduced Representation Bisulphite Sequencing (RRBS) libraries which were sequenced for all the samples.
Results
Genotype had a significant effect on body weight at the start of the marine phase, with high pigment group showing larger weights compared to low pigment, however, these differences disappear at the end of the intermediate phase. The same factor dictated differences for SGR throughout the seawater phase and for nutrient retention. In the last, genotype explained the variations between all the nutrients analysed (protein, lipid, LA, ARA, ALA, EPA, DPA, DHA), yet again, these differences weren’t extended until the end of the challenge. On regards of biochemical composition for whole fish during freshwater, both dry matter and lipid content were affected by the stimulus diet, with vegetable stimulated fish presenting larger values compared to those marine stimulated groups. Lipid content and dry matter also showed differences in seawater during the intermediate phase, although differences were explained by genotype rather than by stimulus diet. All differences disappeared by the mid challenge sampling point. In general, changes observed in growth parameters and nutritional composition were related to genotype rather than to a specific stimulus diet and differences disappeared at the end of the challenge. The exception was anterior intestine from which differences in the proportions of ARA, DHA, n-3 PUFA and total PUFA were explained by the nutritional stimulus and the effects maintained until the end of the challenge.
When looking at the impact of nutritional stimulus in DNA methylation, only a couple of differentially methylated CpG (DMCpG) sites were found after a 3-week exposition to stimulus feed, with 1 showing increased methylation levels (hypermethylation) and 1 presenting decreased levels (hypomethylation) in the marine compared to the vegetable treatment. In seawater, and before the vegetable challenge started, the number of DMCpG sites increased, with 109 hypermethylated and 21 hypomethylated in the marine-stimulated group relative to the vegetable-stimulated. After 14 weeks of challenge in seawater the number of sites were 4-fold above the previous values, with 112 hypermethylated and 508 hypomethylated in the marine stimulated group relative to the vegetable stimulated. Finally, after 36 weeks of vegetable challenge in seawater, the number of DMCpG sites decreased compared to the last sampling with a total of 21 sites identified with 17 hypermethylated and 4 hypomethylated in marine relative to vegetable stimulated groups. The same trend is shown when looking at the differences between low and high pigment retention genotypes. These preliminary results show that nutritional stimulus had a clear impact on the methylome of the fish. Differences between marine and vegetable stimulated fish became larger during the challenge diet phase, suggesting that nutritional programming may alter the response of the fish to a vegetable-based diet via epigenetic mechanisms. The ability of the fish to retain pigment also has an impact on the epigenetic response to the diet, suggesting that different genotypes may be more adaptable to vegetable diets.
References
Clarkson, M., Migaud, H., Metochis, C., Vera, L. M., Leeming, D., Tocher, D. R., & Taylor, J. F (2017). Early nutritional intervention can improve utilisation of vegetable-based diets in diploid and triploid Atlantic salmon (Salmo salar L.). British Journal of Nutrition, 118 (1), 17-29.
Lucas, A (1991). Programming by early nutrition in man. The childhood environment and adult disease, 156, 38-55.
Skjærven, K. H., Adam, A. C., Takaya, S., Waagbø, R., & Espe, M. (2022). Nutritional epigenetics. In Cellular and Molecular Approaches in Fish Biology (pp. 161-192). Academic Press.
Vera, L. M., Metochis, C., Taylor, J. F., Clarkson, M., Skjaerven, K. H., Migaud, H., & Tocher, D. R (2017). Early nutritional programming affects liver transcriptome in diploid and triploid Atlantic salmon, Salmo salar. BMC genomics, 18 (1), 886.
Acknowledgements
This study received funding from the European Union Commission: Horizon 2020 – AquaIMPACT. Additional funding for K. F. was provided by the National Agency for Research and Development (ANID), Chilean Government.