Introduction
Substantial replacement of fishmeal and fish oil with plant ingredients in the diets has caused certain challenges in health and welfare of the Atlantic salmon. Excessive lipid accumulation or steatosis in the pyloric caeca and mid intestine is such a gut health challenge predominant in farmed salmon. Severe steatosis can cause lipid malabsorption resulting floating faeces in surrounding waters also called lipid malabsorption syndrome (LMS). Through a series of studies, i t has been recently established that supplementation of choline, which is deficient in plant-based diets, can prevent the steatosis in Atlantic salmon
(Hansen et al., 2020a; Hansen et al., 2020b; Krogdahl et al., 2020)
. Choline is an essential nutrient and a precursor to produce phosphatidylcholine, the major phospholipid in the lipoproteins that facilitates the transport of lipids from the enterocytes. Moreover, choline can influence epigenetic regulation by being the key methyl donor for DNA or histone methylation.
Epigenetic mechanisms, such as histone modifications, DNA methylation, noncoding RNA and chromatin structure rearrangements can produce heritable changes in gene expression without changing actual genomic sequence . Alterations in DNA methylation in different genomic region s differentially influence gene activities producing diverse physiological and phenotypic changes . Considering the role of choline as methyl donor in DNA methylation, t his study was conducted to evaluate the epigenetic changes in pyloric intestine of Atlantic salmon fed diets containing increasing levels of lipid and choline.
Materials and Methods
Four diets were formulated to have lipid levels of 16% (L16), 21% (L21), 26% (L26) and 31% (L31 ). The first 3 diets were choline deficient and had levels of 1542-1619 mg/kg while the L31 diet had somewhat higher, but still suboptimal choline leve l of 2310 mg/kg. A f eeding trial was conducted with Atlantic sa lmon parr with initial weight of 25g in duplicate tanks per diet group, at 8℃, for 8 weeks.
A genome-wide analysis of DNA methylation patterns was performed by reduced representation bisulfite sequencing (RRBS) to examine the global epigenetic alterations in the pyloric intestine . Library preparation for RRBS was performed with the NuGen ovation RRBS methyl-seq system (Tecan Genomics). Briefly, DNA from 6 replicate samples per diet group were digested with MspI followed by adapter ligation, final repair, bisulfite conversion, amplification and subsequently sequenced on the NextSeq 500. Bisulfite-specific DNA mapping to Atlantic salmon genome and methylation information was extracted u sing Bismark aligner. Differentially methylated cytosines ( DMCs, q value ≤0.1 and methylation differences ≥10%) in CpG context between the genomes of Atlantic salmon fed different diets were analyzed using methylKit package. Genomic feature annotation of DMCs was performed using the HOMER package. Functional annotation of the genes associated with DMCs was performed using g:Profiler online tool and manually inspecting the Ensembl and NCBI data bases.
Results and Discussion
This study revealed that increasing levels of lipid and choline in the diet induced genome-wide epigenetic changes in the pyloric intestine of Atlantic salmon. The h ighest number of differential methylations w ere observed between the fish fed diets with largest choline differences (L31 vs L21) and the lowest number of differential methylations were observed between the fish fed diets with similarly low choline levels and highest lipid differences (L26 vs L16). The observations indicated that choline and/ or high lipid levels epigenetically modulated several genes of membrane components and transporters, adipose lipolysis and lipogenesis, and microRNAs important for lipid homeostasis. Our observations support the vital role of choline in epigenetic regulation also in Atlantic salmon similar to that reported in higher vertebrates (Zeisel, 2017; Korsmo et al., 2019). The knowledge could be valuable for further optimization of the feed formulations for Atlantic salmon.
References
Hansen, A.K.G., Kortner, T.M., Denstadli, V., Måsøval, K., Björkhem, I., Grav, H.J., et al. (2020a). Dose–response relationship between dietary choline and lipid accumulation in pyloric enterocytes of Atlantic salmon (Salmo salar L.) in seawater. British Journal of Nutrition 123(10), 1081-1093. doi: 10.1017/S0007114520000434.
Hansen, A.K.G., Kortner, T.M., Krasnov, A., Björkhem, I., Penn, M., and Krogdahl, Å. (2020b). Choline supplementation prevents diet induced gut mucosa lipid accumulation in post-smolt Atlantic salmon (Salmo salar L.). BMC Veterinary Research 16(1), 32. doi: 10.1186/s12917-020-2252-7.
Korsmo, H.W., Jiang, X., and Caudill, M.A. (2019). Choline: Exploring the Growing Science on Its Benefits for Moms and Babies. Nutrients 11(8), 1823.
Krogdahl, Å., Hansen, A.K.G., Kortner, T.M., Bjӧrkhem, I., Krasnov, A., Berge, G.M., et al. (2020). Choline and phosphatidylcholine, but not methionine, cysteine, taurine and taurocholate, eliminate excessive gut mucosal lipid accumulation in Atlantic salmon (Salmo salar L). Aquaculture 528, 735552. doi: https://doi.org/10.1016/j.aquaculture.2020.735552.
Zeisel, S. (2017). Choline, Other Methyl-Donors and Epigenetics. Nutrients 9(5). doi: 10.3390/nu9050445.
Acknowledgements
The study was one of the series of studies conducted under the GutMatters project funded by The Norwegian Seafood Fund (FHF, project 901435). The authors like to acknowledge researchers and technicians at the NOFIMA’s research station at Sunndalsøra and Nutrition and health group at the Faculty of Veterinary Medicine, Norwegian University of Life Sciences (NMBU).