NMR-metabolomics profiles of two goat breeds with different level of tolerance to seasonal weight loss Mariana Palma Lorenzo E. Hernández-Castellano Noemí Castro Anastasio Arguëllo Juan Capote Manolis Matzapetakis André Martinho de Almeida mpalma@itqb.unl.pt 3 rd DairyCare Conference. Zadar, Croatia. 5 6 October, 2015
Goats historic, economic and nutritional importance valuable nutritional supply in developing countries important sources of animal protein recent increase of interest for their milk and dairy products benefits in health maintenance good nutritional properties lower allergenic risk Haenlei, 2001. J Dairy Sci. 84:2097:2115; Boyazoglu et al., 2005. Small Rum Res. 60:13-23; Blummel et al., 2010 CRIDA. 18-20
Goats Understanding physiology of small ruminants Identification of physiological markers Genetic improvement (breed selection)
Goats Understanding physiology of small ruminants Identification of physiological markers Genetic improvement (breed selection) Improvement Production yields and quality milk meat wool Disease management Animal welfare Economic importance
Seasonal Weight Loss Tropical, Sub-Tropical and Mediterranean Climate: Rainy season - abundant pastures Dry season - poor and scarce pastures Animals may lose up to 30% of their live weight: Seasonal Weight Loss (SWL) Major limitation in milk and production in drought-prone regions. Herrera et al..2001. J Climate. 14: 3889-3903 ; Almeida et al.. 2006. Trop Anim Health Prod. 38: 443-449.
Seasonal Weight Loss Some breeds show higher adaptation to dry environments and resistance to SWL effects. Comparative study of breeds naturally tolerant to SWL with susceptible ones Differences at gene, protein or metabolite level??? Metabolic response could help defining physiological pathways relating genetic consequences of selection to increased production. Distinctive characteristics could be used as probes for breed selection. Almeida et al.. 2006. Trop Anim Health Prod. 38: 443-449.
Aim Study the effect of feed-restriction in two goat breeds with different levels of tolerance to SWL (mammary gland and milk) Identify physiological markers candidate indicative of SWL tolerance Characterization of the goat mammary gland and milk metabolomes using NMR
Canaries Archipelago - West of North Africa, in Atlantic Ocean - Subtropical Climate Zone GoogleEarth. 2013 rainfall arid climate NASA. 2013 Herrera et al..2001. J Climate. 14: 3889-3903
Canary Breeds common ancestry Palmera Breed - adapted to rainy climate - low tolerance to pasture scarcity Majorera Breed - adapted to arid climate - high tolerance to pasture scarcity La Palma Fuerteventura NASA. 2013 Lérias et al..2013. Trop Anim Health Prod. 45: 1731-1736
Methodology Sample collection Palmera breed lactation N = 10 CTRL Group (N = 6) Restricted Feeding Group: 15-20% initial live weight (N = 4) Majorera breed lactation N = 9 CTRL Group (N = 4) Restricted Feeding Group: 15-20% initial live weight (N = 5) Regular: - weight measurement - milking - milk yield recording 23 days Mammary gland biopsy Milk
Methodology I. Mammary gland tissue fraction extraction - Bligh & Dyer Method: methanol-water/chloroform extraction aqueous fraction II. NMR Experiments 800 MHz 1D-NOESY, J-Resolved 20min 60 min /sample III. Metabolite Profiling I. Milk ultracentrifugation: separation of fat, proteins and serum 3 KDa filtration serum Chenomx NMR Suite IV. Statistical Analysis SIMCA Software Multivariate analysis
Mammary Gland: metabolite profiling
Mammary Gland: metabolite profiling (mmol/g tissue) Amino Acids Metabolism Carbohydrates Metabolism Nucleotide Metabolism Energy Metabolism TCA Cycle Cofactors and Vitamins Metabolism
Mammary Gland: metabolite profiling (mmol/g tissue) Amino Acids Metabolism Carbohydrates Metabolism Nucleotide Metabolism Energy Metabolism TCA Cycle Cofactors and Vitamins Metabolism 47 metabolites: all common constituents of mammary gland and meat
Mammary Gland: metabolite profiling (mmol/g tissue) Amino Acids Metabolism Carbohydrates Metabolism Nucleotide Metabolism Energy Metabolism TCA Cycle Cofactors and Vitamins Metabolism Lactose ~2x10-2 mmol/g tissue
Mammary Gland: metabolite profiling (mmol/g tissue) Amino Acids Metabolism Carbohydrates Metabolism Nucleotide Metabolism Energy Metabolism TCA Cycle Cofactors and Vitamins Metabolism Lactose ~2x10-2 mmol/g tissue NADP + ~4x10-5 mmol/g tissue
Mammary Gland: metabolite profiling (mmol/g tissue) Amino Acids Metabolism Carbohydrates Metabolism Nucleotide Metabolism Energy Metabolism TCA Cycle Cofactors and Vitamins Metabolism
Mammary Gland: metabolite profiling (mmol/g tissue) Amino Acids Metabolism Creatinine (mmol/g tissue) Creatinine Only significant difference between breeds in control groups (p < 0.05) Could be related to different body weights between breeds (Majorera is bigger)
Mammary Gland: multivariate analysis Principal Component Analysis (PCA) PCA: - unsupervised analysis - cluster the samples (scores) - identify metabolites responsible for the group clustering (loadings) [NC=3] PCA is not capable of separating the 4 groups.
Mammary Gland: multivariate analysis Partial Least Squares Discriminant Analysis (PLS) & Orthogonal Partial Least Squares Discriminant Analysis (OPLS) PLS Majorera breed OPLS Palmera breed [NC=2; Q 2 =0.81; ρ CV-ANOVA =0.91] [NC=1+3+0; Q 2 =0.66; ρ CV-ANOVA =1.01] PLS/OPLS analysis was necessary to differentiate between control and restricted groups. PLS / OPLS: - supervised analysis - evaluate repeated variables between groups - highlight differences between groups
In the end of the experiment: Majorera breed Palmera breed Acetate * IMP Phosphocholine IMP ATP/ADP/AMP Phosphocholine (Significant differences p < 0.05)
In the end of the experiment: Majorera breed Palmera breed Acetate * IMP Phosphocholine IMP ATP/ADP/AMP Phosphocholine (Significant differences p < 0.05)
In the end of the experiment: Majorera breed Palmera breed Acetate * Variations are consistent between breeds IMP Phosphocholine Differences could be related to: metabolism adaptation to the low-energy diet microflora composition * IMP ATP/ADP/AMP Phosphocholine (Significant differences p < 0.05)
Milk serum: metabolite profiling
Milk serum: metabolite profiling (mm) Amino Acids Metabolism Cofactors and Vitamins Metabolism Carbohydrates Metabolism Other compounds: TCA Cycle Nucleotide Metabolism
Milk serum: metabolite profiling (mm) Amino Acids Metabolism Cofactors and Vitamins Metabolism Carbohydrates Metabolism Other compounds: TCA Cycle Nucleotide Metabolism 50 metabolites: all common constituents of milk
Milk serum: metabolite profiling (mm) Amino Acids Metabolism Cofactors and Vitamins Metabolism Carbohydrates Metabolism Other compounds: TCA Cycle Nucleotide Metabolism
Milk serum: metabolite profiling (mm) Amino Acids Metabolism Cofactors and Vitamins Metabolism Carbohydrates Metabolism Other compounds: TCA Cycle Nucleotide Metabolism Lactose ~120 mm Isoleucine ~2x10-4 mm
Milk serum: multivariate analysis Principal Component Analysis (PCA) [NC=3] PCA roughly separate control and restricted groups.
Milk serum: multivariate analysis Partial Least Squares Discriminant Analysis (PLS) PLS Majorera breed PLS Palmera breed [NC=2; Q 2 =0.91; ρ CV-ANOVA =0.46] [NC=2; Q 2 =0.82; ρ CV-ANOVA =0.62] PLS differentiate between control and restricted groups in both breeds.
In the end of the experiment: Majorera breed Palmera breed Acetyl-L-carnitine Creatine Fucose * Hippurate * Acetyl-L-carnitine Creatine Fucose * Lactate Methylmalonate * Adenosine Carnitine Citrate Dimethylglycine Fumarate * N-acetylglucosamine * Succinate * TMAO * UDP-galactose UDP-glucose Alanine Carnitine Citrate Fumarate N-acetylglucosamine * Succinate UDP-galactose (Significant differences p < 0.05)
In the end of the experiment: Majorera breed Palmera breed Acetyl-L-carnitine Creatine Fucose * Hippurate * Acetyl-L-carnitine Creatine Fucose * Lactate Methylmalonate * Adenosine Carnitine Citrate Dimethylglycine Fumarate * N-acetylglucosamine * Succinate * TMAO * UDP-galactose UDP-glucose Alanine Carnitine Citrate Fumarate N-acetylglucosamine * Succinate UDP-galactose (Significant differences p < 0.05)
In the end of the experiment : Majorera breed Palmera breed Acetyl-L-carnitine Creatine Fucose * Hippurate * Adenosine Carnitine Citrate Dimethylglycine Fumarate * N-acetylglucosamine * Succinate * TMAO * UDP-galactose UDP-glucose Variations are consistent between breeds Differences could be related to: changes in carbohydrates and aminoacids-related metabolites changes in microflora * Acetyl-L-carnitine Creatine Fucose * Lactate Methylmalonate * Alanine Carnitine Citrate Fumarate N-acetylglucosamine * Succinate UDP-galactose (Significant differences p < 0.05)
Main outcomes First metabolite profiling of mammary gland and milk serum from goats by NMR Significant differences between control and restricted groups in both breeds. Effects of nutritional restriction (SWL): adaptation to the low-energy diet (variations in carbohydrates and aminoacidsrelated metabolites) variations in and microflora composition
Main outcomes Milk exhibits more metabolites with significant differences between control and restricted groups, than the mammary gland better sample to study SWL tolerance effects. Majorera breed shown more variations in milk serum than Palmera indicative of a faster and prompt adaptation to SWL. Microflora profile and milk composition should be considered in future studies about SWL.
Acknowledgments Working teams & Participant Institutions : Manolis Matzapetakis André Martinho de Almeida Joana Lérias José Salvado BioMolecular NMR Group Juan Capote Lorenzo Hernandez-Castellano Noemi Castro Anastacio Arguello Funding: PhD Grant SFRH/BD/85391/2012 Project PTDC/CVT/116499/2010 NMR Spectrometers are part of The National NMR Facility (RECI/BBB-BQB/0230/2012) 36
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