Project summary
Project summary of NutrEpi WP1 (Gonad).
Objective
The primary goal of the project is to elucidate potential inter-generational effects induced by micronutrients (vitamins and minerals) through the male germ line of Atlantic salmon.
Feeding trial
Our analyses were based on the male gonad 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 male gonad samples at the final harvest stage for gene expression and DNA methylation analysis.
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
| 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
Gonadosomatic index (GSI)
- Harvest: Fish with overgrown gonad (GSI >0.2%) were excluded
GSI = (gonad weight (g) / body weight (g)) * 100
Main findings of NutrEpi WP1 (ganad)
1. Gene expression
- Overall diet effect: both medium (L2) and high (L3) doses of micronutrient supplementation affect gene expression in male gonad. The L3 diet influences more genes than the L2 diet.
- Male liver samples: both L2 and L3 diets affect more genes and biological pathways in male liver compared to male gonad.
2. DNA methylation
- Overall diet effect: clustering analysis shows a clear separation of L2 and L3 from L1 in the regions around transcription start sites (TSSs), specifically between 1000 bp upstream in promoters and 150 bp downstream in exons.
- Affected pathways: micronutrient supplements significantly affect biological pathways related with signalling.
3. Inheritance of DNA methylation in grin3a-like
- Micronutrient supplements significantly hypo-methylate CpG sites in the promoter region of glutamate receptor ionotropic, NMDA 3A-like (grin3a-like).
- This differentially methylated region of grin3a-like is targeted by <histone deacetylase 2 (HDAC2) and a zinc finger protein.
- HDAC2 is an enzyme responsible for the removal of acetyl groups from the core histones.
- DNA methylation in grin3a-like together with HDAC2 is potentially involved in intergenerational epigenetic inheritance that affects early cell development stages in offspring.
To summarise, this project suggests that DNA methylation of grin3A-like together with hdac2 could be a strong candidate for studying intergenerational epigenetic inheritance that affects early cell development stages in offspring. The project also provides extensive data sources for future work.
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