Project summary

Project summary of NutrEpi WP1 (Liver).

Objective

The primary goal of the project is to elucidate the effect of micronutrient (vitamins and minerals) on metabolic pathways from a genetic and epigenetic point of view. Specifically, our aim is to analyse and understand the influences of micronutrients in Atlantic salmon feed through a whole life cycle feeding trial (over 54 weeks for salmon) conducted under the EU funded ARRAINA (advanced research initiatives for nutrition and aquaculture) project.

Background

With the rapid expansion of the aquaculture industry in the last two decades, we can hardly sustain traditional practices as feeding fish to grow other fish. Hence, aquaculture feeds have been shifted to containing more plant-based materials. Consequently, the optimised levels of nutrition, including micronutrient, are altered and need to be updated to produce healthier and more nutritious fish.

New knowledge regarding genetic and epigenetic regulations acquired through this project may contribute to the global effort on solving a word-wide problem of micronutrient deficiencies called “hidden hunger”. Ending all forms of malnutrition including micronutrient deficiencies is one of the SDGs (sustainable development goals) proposed by the UN.

Feeding trial

Our analyses were based on the liver samples of Atlantic salmon fed with three graded levels of micronutrient supplementation. The main outcome of the feeding trail was summarised in Vera et al., 2020.

Experimental design

We collected liver samples at the final harvest stage for gene expression and DNA methylation analysis.

Experimental design
Three groups of Atlantic salmon were fed with different levels of micronutrients through the trial.

Experimental feed

Through the trial, Atlantic salmon in triplicate groups - L1, L2, L3 - were fed with graded levels of micronutrient supplements, formulated with nutrition package (NP).

  • L1: 100% NP (recommended composition of micronutrients)
  • L2: 200% NP
  • L3: 400% NP
Added micronutrient concentrations (mg/kg) within the NP.
Nutrient group Micronutrient L1 L2 L3
Vitamin Vitamin A 3.79 7.58 15.16
Vitamin D3 0.05 0.1 0.2
Vitamin E 102.4 204.9 409.8
Vitamin K3 9.82 19.64 39.28
Thiamine (B1) 2.67 5.34 10.68
Riboflavin (B2) 8.3 16.6 33.2
Vitamin B6 4.77 9.54 19.08
Vitamin B12 0.25 0.5 1
Niacin (vitamin B3) 24.8 49.6 99.2
Pantothenic Acid (vitamin B5) 17.15 34.3 68.6
Folic Acid (vitamin B9) 2.82 5.64 11.28
Biotin (vitamin B7) 0.14 0.28 0.56
Vitamin C 80 160 320
Micro-mineral Cobalt (Co) 0.94 1.88 3.76
Iodine 0.67 1.34 2.68
Selenium 0.23 0.46 0.92
Iron 32.64 65.28 130.6
Manganese 12.03 24.06 48.12
Copper 3.24 6.48 12.96
Zinc 66.92 133.8 267.7
Macro-mineral Calcium (Ca) 0.4 0.8 1.6
Amino acid Taurine 2450 4900 9800
Histidine 1400 2800 5600
Cholesterol Cholesterol 1100 2200 4400

Growth performance

Body weights

  • Smolt: L2 showed the best growth followed by L3
  • Harvest: Both L2 and L3 showed significantly better growth

Hepatosomatic index (HSI)

  • Smolt: L1 had a significantly higher HSI than L2 and L3
  • Harvest: No significant difference among L1, L2 and L3
  • HSI = (Liver weight (g) / Fish weight (g)) × 100
Barplot of Atlantic salmon body weights Barplot of Atlantic salmon hepatosomatic index values
Body weights and HSI at smolt and final harvest stages.

Main findings of NutrEpi WP1 (liver)

1. Gene expression

  • Overall diet effect: clustering analysis clearly separates our 12 liver samples into three groups by diet.
  • Dose dependent effect: micronutrient supplements significantly affect several biological pathways, especially those related with lipid metabolism, in a dose dependant manner (L3 < L2 < L1).

2. DNA methylation

  • Overall diet effect: clustering analysis shows no obvious separations by diet.
  • Affected pathways: micronutrient supplements significantly affect biological pathways related with cell-adhesion and cell-signalling.

3. Epigenetic regulations of acetyl-CoA carboxylase alpha (acaca)

  • Micronutrient supplements significantly down-regulates gene expression with L3 having the lowest expression level (L3 < L2 < L1).
  • Micronutrient supplements significantly hyper-methylate CpG sites in its promoter region (L3 > L2 > L1).
  • acaca is involved in the upstream regulation of the lipid biosynthesis pathway.

To summarise, our study shows that micronutrient supplementation suppresses liver gene expression in the pathways related to lipid metabolism and increases the methylation rates in the acetyl-CoA carboxylase alpha (acaca) gene, which is involved in the upstream regulation of the lipid biosynthesis pathway.

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