The added value of molecular phenotypes: towards the identification of animal welfare proxies
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1 The added value of molecular phenotypes: towards the identification of animal welfare proxies Luca Fontanesi Department of Agricultural and Food Sciences Division of Animal Sciences University of Bologna Bologna, Italy
2 The added value of molecular phenotypes: towards the identification of animal welfare proxies.. for breeding and selection purposes
3 Animal welfare is a multi-dimensional concept: From a pure biological approach: Behaviour Physiology Immunology Biochemistry
4 Our omics study in pigs It combines: Metabolomics Genomics Hypothesis free approach Hypothesis generating approach
5 Metabolomics measures all endogenous metabolites of a tissue or body fluid under given conditions (i.e.metabolome) From Junot et al. 2014
6 Metabolomics (Genomics free)
7 Animal resources ~1,000 pigs ~300 phenotypes (~200 metabotites) ~15,000 performance tested Italian Large White ~8,000 performance tested Italian Duroc ~6,000 performance tested Italian Landrace ~400 pigs ~300 phenotypes (~200 metabolites)
8 Metabolites Metabotypes Quantitative measure of metabolites Phenotypes Molecular phenotypes
9 Metabolites Metabotypes Quantitative measure of metabolites Phenotypes Molecular phenotypes Targeted metabolomic analysis on blood plasma: ~200 metabolites Measured by LC-MS/MS or FIA-MS/MS with a AB SCIEX 4500 mass spectrometer
10 Metabotypes Metabolite classes No. Biological relevance (selected examples) Acylcarnitines 40 Energy metabolism, fatty acid transport and mitochondrial fatty acid oxidation, ketosis, oxidative stress, mitochondrial membrane damage (apoptosis) Amino acids 21 Amino acid metabolism, urea cycle, activity of gluconeogenesis and glycolysis, insulin sensitivity/resistance, neurotransmitter metabolism, oxidative stress Biogenic amines 19 Neurological disorders, cell proliferation, cell cycle progression, DNA stability, oxidative stress Hexoses 1 Carbohydrate metabolism Glycerophospholipids 90 - lysophosphatidylcholine acyl lysopc a Cx:x Degradation of phospholipids (phospholipase 14 activity), membrane damage, signalling cascades, fatty acid profile - Phosphatidylcholine diacyl PC aa Cx:x 38 Dyslipidemia, membrane composition and damage, - Phosphatidylcholine acyl-alkyl PC ae Cx:x 38 fatty acid profile, activity of desaturases Sphingolipids 15 Signalling cascades, membrane damage (e.g. neurodegeneration)
11 Metabotypes Metabolite classes No. Biological relevance (selected examples) Acylcarnitines 40 Energy metabolism, fatty acid transport and mitochondrial fatty acid oxidation, ketosis, oxidative stress, mitochondrial membrane damage (apoptosis) Amino acids 21 Amino acid metabolism, urea cycle, activity of gluconeogenesis and glycolysis, insulin sensitivity/resistance, neurotransmitter metabolism, oxidative stress Biogenic amines 19 Neurological disorders, cell proliferation, cell cycle progression, DNA stability, oxidative stress Hexoses 1 Carbohydrate metabolism Glycerophospholipids 90 - lysophosphatidylcholine acyl lysopc a Cx:x Degradation of phospholipids (phospholipase 14 activity), membrane damage, signalling cascades, fatty acid profile - Phosphatidylcholine diacyl PC aa Cx:x 38 Dyslipidemia, membrane composition and damage, - Phosphatidylcholine acyl-alkyl PC ae Cx:x 38 fatty acid profile, activity of desaturases Sphingolipids 15 Signalling cascades, membrane damage (e.g. neurodegeneration)
12 Metabotypes Metabolite classes No. Biological relevance (selected examples) Acylcarnitines 40 Energy metabolism, fatty acid transport and mitochondrial fatty acid oxidation, ketosis, oxidative stress, mitochondrial membrane damage (apoptosis) Amino acids Database 1 21 Amino acid metabolism, urea cycle, activity of gluconeogenesis and glycolysis, insulin sensitivity/resistance, neurotransmitter metabolism, oxidative stress Neurological disorders, cell proliferation, cell cycle Biogenic amines 19 Database 2 progression, DNA stability, oxidative stress Hexoses 1 Carbohydrate metabolism Glycerophospholipids 90 Database 3 Degradation of phospholipids (phospholipase - lysophosphatidylcholine acyl lysopc a Cx:x 14 activity), membrane damage, signalling cascades, fatty acid profile - Phosphatidylcholine diacyl PC aa Cx:x 38 Dyslipidemia, membrane composition and damage, Database 4 - Phosphatidylcholine acyl-alkyl PC ae Cx:x 38 fatty acid profile, activity of desaturases Signalling cascades, membrane damage (e.g. Sphingolipids 15 neurodegeneration) Database 5 metabotypes
13 Metabolytes that we analysed Metabolytes present in body fluids
14 Metabotypes
15 Metabotypes Heatmap with phenotypic correlations metabotypes metabotypes
16 Metabotypes 0.8 Heritability Large White Duroc Acylcarnitines Glycerophospholipids Sphingolipids Amino acids Biogenic amines
17 Metabotypes 0.8 Heritability Large White Duroc kynurenine serotonin taurine Acylcarnitines Glycerophospholipids Sphingolipids Amino acids Biogenic amines
18 Serotonin: neurotransmitter, known also as the happiness drug Kynurenine: intermediate metabolite of the tryptophan catabolism
19 Taurine:
20 Metabotypes Gaussian Graphical Model (GGM) Based on partial correlation coefficients (PCC): pairwise Pearson correlation coefficients conditioned against the correlation with all other metabolites
21 Metabotypes Top 8 positive GGM edge weights (i.e. partial correlation coefficients, PCC) in our data set Metabolite Metabolite PCC SM C18:0 SM C18: lysopc a C16:0 lysopc a C18: SM C16:1 SM C18: PC aa C38:6 PC aa C40: PC aa C38:3 lysopc a C20: PC aa C40:4 PC aa C40: lysopc a C18:2 lysopc a C20: Serotonin Taurine desaturation (1) elongation (1) elongation (1) elongation (1) phospholipid association? desaturation (1) elong.+ desat. (2) interaction/regulation?... lysophosphatidylcholine acyl: lysopc Phosphatidylcholine diacyl: PCaa Phosphatidylcholine acyl-alkyl: PCae Sphingolipids: SM
22
23 Metabotypes Top 8 positive GGM edge weights (i.e. partial correlation coefficients, PCC) in our data set Metabolite Metabolite PCC SM C18:0 SM C18: lysopc a C16:0 lysopc a C18: SM C16:1 SM C18: PC aa C38:6 PC aa C40: PC aa C38:3 lysopc a C20: PC aa C40:4 PC aa C40: lysopc a C18:2 lysopc a C20: Serotonin Taurine desaturation (1) elongation (1) elongation (1) elongation (1) phospholipid association? desaturation (1) elong.+ desat. (2) interaction/regulation?... lysophosphatidylcholine acyl: lysopc Phosphatidylcholine diacyl: PCaa Phosphatidylcholine acyl-alkyl: PCae Sphingolipids: SM
24 How can we link potential «welfare bio-markers» with genomics information?
25 Metabolomics + Genomics
26 Running title: Searching the happiness genes in pigs
27 Animal resources ~1,000 pigs ~300 phenotypes (~200 metabotypes) ~15,000 performance tested Italian Large White ~8,000 performance tested Italian Duroc ~6,000 performance tested Italian Landrace ~400 pigs ~300 phenotypes (~200 metabotypes)
28 PorcineSNP60 BeadChip Genotype space Internal phenotypes Metabotypes External phenotypes
29 PorcineSNP60 BeadChip Genotype space Internal phenotypes Metabotypes ~200 metabotypes External phenotypes
30 PorcineSNP60 BeadChip Genotype space Internal phenotypes Metabotypes External phenotypes ~200 metabotypes 200 x 200 = ratios
31 Single Italian Large White Italian Duroc Combined
32 Single Kynurenine Ornithine Arg Citrulline His Pro serotonine Lys Italian Large White Thr Ser His Ala serotonine Kynurenine Citrulline Citrulline Asn Lys Italian Duroc Kynurenine Ornithine Arg Citrulline serotonine Combined Phe Ile Citrulline Lys
33 Single kynurenine Italian Large White Italian Duroc Combined
34 Single kynurenine Italian Large White SNP7 SNP6 SNP5 SNP4 SNP1 SNP2 SNP3 Kynurenine 3-monooxygenase (KMO)
35 Single kynurenine Italian Large White Tryptophan catabolism SNP7 SNP6 SNP5 SNP4 SNP1 SNP2 SNP3 Immunity/ inflammation IDO, TDO Kynurenine 3-monooxygenase (KMO) Behaviour
36 Single serotonin Italian Large White Italian Duroc Combined
37 Single serotonin Italian Large White SLC6A4 Solute carrier family 6 (neurotransmitter transporter, serotonin), member 4
38
39 Arg Arg Ala Asn His His Lys Ser Thr Lys Kynurenine Kynurenine Kynurenine Ile Pro Lys Phe Citrulline Citrulline Ornithine Ornithine Citrulline Citrulline Citrulline Italian Large White Italian Duroc Combined Single serotonine serotonine serotonine SM PCae PCaa PCaa SM PCae PCae PCaa LysoPC PCae PCaa PCaa PCae SM PCaa PCae
40 Our omics study in pigs Hypothesis generating Nutrigenetics Behaviour
41 Conclusions
42 Internal Conclusions Opportunities Strengths S W O T Weaknesses Threats External
43 Internal Conclusions Opportunities Strengths S W O T Weaknesses Threats External
44 Internal Conclusions Opportunities Strengths S W O T External Metabolomics: technology gap Weaknesses Threats
45 Internal Conclusions Opportunities Strengths S W O T Limited number of metabolites External Metabolomics: technology gap Weaknesses Data analysis: New approaches needed Threats
46 Internal Conclusions Opportunities Strengths S W O T Close link to genomics Genomics + metabolomics = basic biological mechanisms Limited number of metabolites External Metabolomics: technology gap Weaknesses Data analysis: New approaches needed Threats
47 Internal Conclusions Opportunities Strengths S W O T Close link to genomics Genomics + metabolomics = basic biological mechanisms Biochemistry Behaviour genetics New breeding opportunities for animal welfare Limited number of metabolites External Metabolomics: technology gap Weaknesses Data analysis: New approaches needed Threats
48 University of Bologna Department of Agricultural and Food Sciences Samuele Bovo Gian Luca Mazzoni Giuseppina Schiavo Anisa Ribani Valerio Joe Utzeri Antonia Bianca Samoré Emilio Scotti Francesca Bertolini Stefania Dall Olio Paolo Trevisi Paolo Bosi Department of Surgical and Medical Sciences Endocrinology Unit Flaminia Fanelli Marco Mezzullo Uberto Pagotto Department of Statistical Sciences Giuliano Galimberti Daniela Giovanna Calò Associazione Nazionale Allevatori Suini Funded by: Innovagen project (MiPAAF) AGER project (Fondazioni Bancarie) FARB project (University of Bologna)
49 Thank you!
50 The added value of molecular phenotypes: towards the identification of animal welfare proxies Luca Fontanesi Department of Agricultural and Food Sciences Division of Animal Sciences University of Bologna Bologna, Italy
Dissecting complex traits in pigs: metabotypes illuminate genomics for practical applications
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