Dietary and genetic regulation of B12 metabolism in sheep

Cobalt (Co) deficiency in sheep can result in poor animal health and performance, ultimately decreasing farm profitability. Development of strategies to improve the health and productivity of Co deficient animals at different stages of the production cycle, but particularly of rapidly growing lambs, is therefore of significant interest and major economic importance to the sheep industry. Cobalt is an essential component of vitamin B12, and therefore the nutritional status of this trace-element can impact on one-carbon (1C) metabolism and associated pathways. A number of factors influence the prevalence and severity of Co/B12 deficiency in sheep including nutritional status, stage of production cycle and geographical location. In addition, human studies have provided clear evidence for genetic predispositions to 1C metabolic imbalances. Currently, however, knowledge of such susceptibilities in farm animals does not exist. In response to these gaps in knowledge, this thesis sought to increase our understanding of nutritional and genetic regulation of B12 metabolism in sheep through supplementation trials and genetic analyses. The first part of this thesis (Chapters 2 and 3) addressed nutritional aspects of B12 deficiency. These studies sought to analyse the biochemical consequences of decreased B12, to formally assess the interaction between Co and Se, and to evaluate the effectiveness of a novel long-acting supplementation strategy. B12 status was determined though biochemical assessment of primary and secondary metabolite concentrations in blood. Live weights and body condition scores were recorded to assess the potential consequences of depleted B12. The efficacy of SMARTShot®, a long-acting injectable microencapsulated source of B12, compared to the commonly used Co bolus on repletion of weaned Co deficient lambs determined its usefulness as a method of repletion. This method of supplementation was found to be a beneficial option for young lambs due to its ease and speed of administration and ability to supplement light weight lambs from 3 weeks of age. Whilst it was not possible to address the primary hypothesis of Chapter 3, that a Se deficiency masked an underlying Co deficiency, this study provided evidence of seasonal and year-to-year variation in B12 status, BCS and live-weight gains highlighting the requirement for strategic ‘risk management’ approaches to supplementation. The second part of this thesis (Chapters 4 and 5) addressed the genetic aspect of B12 deficiency. The aim of Chapter 4 was to address the hypothesis that single nucleotide polymorphisms (SNPs) in genes involved in or directly related to 1C metabolism lead to inter-individual variability in metabolic responses to micronutrient deficiencies in sheep. Furthermore, as every farm has different nutritional challenges and with evidence of geographic and ethnic variation in frequencies of 1C metabolic gene polymorphisms in humans, Chapter 5 sought to characterise 1C genetic diversity between breeds. The objectives of Chapter 4 were addressed in a series of analyses which included depleting the Co status of weaned lambs by feeding a Co and S deficient barley-based diet and confirmation of status as in previous chapters. From these animals, DNA samples were collected for genotyping and liver samples were collected for quantification of metabolite concentrations through GCMS, HPLC and LC-MS/MS. An Illumina Infinium® iSelect® Custom Array with 4,576 probes capturing SNPs in 115 1C metabolism and related genes, and 108 related epigenetic regulators was constructed and used to genotype the collected DNA. Following this, a pipeline of bioinformatic analyses was developed, including a genome-wide association study (GWAS), facilitating identification and prioritisation of functionally significant SNPs. 33 SNPs were found to be significantly associated with concentrations of biochemical parameters related to 1C metabolism in the study population of 360 Texel lambs. Of particular biological interest were two SNPs in CUBN; a gene fundamental in the transport of B12 for provision to the methionine cycle and propionate pathways. Chapter 5 investigated population differentiation of four commercially important breeds in the UK; Texel, Suffolk, Bluefaced Leicester and Swaledale, for SNPs in 1C related genes, with specific focus on the 33 functionally significant SNPs identified in Chapter 4. Of particular significance, Swaledale sheep differed genetically from the other three breeds, predominantly for functionally significant SNPs in CUBN identified in Chapter 4. Furthermore, interbreed variation in allele frequency was also observed for SNPs in important 1C regulatory genes including MTHFR, MTHFD1, MTR, MUT and MAT1. Overall, outcomes of the studies in Chapters 4 and 5 provide an unprecedented understanding of the functional significance of complex relationships between 1C metabolites and variants in 1C metabolic genes, providing a basis for further study into judicious targeted supplementation and selection for genetic tolerance to Co deficiency in breeding programmes. In summary this thesis confirms that B12 deficiency in sheep varies with respect to geographical location, stage of production cycle and nutritional availability partly as a consequence of seasonal and inter-year variability. Furthermore, the studies detailed in this thesis represent the first investigation of genetic influences on 1C metabolism in sheep, providing evidence for a possible genetic predisposition to 1C metabolic imbalances, resulting in inter-individual and inter-breed variation in the regulation of 1C and other B12 associated metabolic pathways

Comprehensive and quantitative profiling of B vitamins and related compounds in the mammalian liver

A method for the simultaneous quantification of B vitamins and related amines in one-carbon (1C) metabolism would benefit the study of diet and genetic/epigenetic regulation of mammalian development and health. We present a validated method for the simultaneous quantitative analysis of 13 B vitamers and four related 1C-pathway amine intermediates in liver using hydrophilic interaction chromatography (HILIC) coupled to electrospray ionization tandem mass spectrometry. Frozen sheep liver samples (50 mg) were homogenized in cold 50% acetonitrile containing 1% acetic acid with the addition of two isotope labelled internal standards. Hot acid hydrolysis was applied to release the protein-bound forms. The separation of 17 analytes was achieved using a pHILIC column with a total run time of 13 min. Detection was achieved in electrospray positive ionisation mode. Limits of detection for the majority of analytes were within the range of 0.4-3.2 pmol/g. The method was applied to 266 sheep liver samples and revealed that adenosylcobalamin, methylcobalamin, pyridoxic acid, flavin adenine dinucleotide and thiamine were the major forms of the B vitamers present with pyridoxal 5’-phosphate and thiamine pyrophosphate being detected at lower concentrations. Trimethylglycine and methylglycine were the predominant 1C-related amines measured. As anticipated, the B vitamin status of individuals varied considerably, reflecting dietary and genetic variation in our chosen outbred model species. This method offers a simple sample extraction procedure and provides comprehensive coverage of B vitamins coupled with good sensitivity and reliability

Investigative power of Genomic Informational Field Theory (GIFT) relative to GWAS for genotype-phenotype mapping

Identifying associations between phenotype and genotype is the fundamental basis of genetic analyses. Inspired by frequentist probability and the work of R.A. Fisher, genome-wide association studies (GWAS) extract information using averages and variances from genotype-phenotype datasets. Averages and variances are legitimated upon creating distribution density functions obtained through the grouping of data into categories. However, as data from within a given category cannot be differentiated, the investigative power of such methodologies is limited. Genomic Informational Field Theory (GIFT) is a method specifically designed to circumvent this issue. The way GIFT proceeds is opposite to that of GWAS. Whilst GWAS determines the extent to which genes are involved in phenotype formation (bottom-up approach), GIFT determines the degree to which the phenotype can select microstates (genes) for its subsistence (top-down approach). Doing so requires dealing with new genetic concepts, a.k.a. genetic paths, upon which significance levels for genotype-phenotype associations can be determined. By using different datasets obtained in ovis aries related to bone growth (Dataset-1) and to a series of linked metabolic and epigenetic pathways (Dataset-2), we demonstrate that removing the informational barrier linked to categories enhances the investigative and discriminative powers of GIFT, namely that GIFT extracts more information than GWAS. We conclude by suggesting that GIFT is an adequate tool to study how phenotypic plasticity and genetic assimilation are linked.</p

Dietary and genetic regulation of B12 metabolism in sheep

Cobalt (Co) deficiency in sheep can result in poor animal health and performance, ultimately decreasing farm profitability. Development of strategies to improve the health and productivity of Co deficient animals at different stages of the production cycle, but particularly of rapidly growing lambs, is therefore of significant interest and major economic importance to the sheep industry. Cobalt is an essential component of vitamin B12, and therefore the nutritional status of this trace-element can impact on one-carbon (1C) metabolism and associated pathways. A number of factors influence the prevalence and severity of Co/B12 deficiency in sheep including nutritional status, stage of production cycle and geographical location. In addition, human studies have provided clear evidence for genetic predispositions to 1C metabolic imbalances. Currently, however, knowledge of such susceptibilities in farm animals does not exist. In response to these gaps in knowledge, this thesis sought to increase our understanding of nutritional and genetic regulation of B12 metabolism in sheep through supplementation trials and genetic analyses. The first part of this thesis (Chapters 2 and 3) addressed nutritional aspects of B12 deficiency. These studies sought to analyse the biochemical consequences of decreased B12, to formally assess the interaction between Co and Se, and to evaluate the effectiveness of a novel long-acting supplementation strategy. B12 status was determined though biochemical assessment of primary and secondary metabolite concentrations in blood. Live weights and body condition scores were recorded to assess the potential consequences of depleted B12. The efficacy of SMARTShot®, a long-acting injectable microencapsulated source of B12, compared to the commonly used Co bolus on repletion of weaned Co deficient lambs determined its usefulness as a method of repletion. This method of supplementation was found to be a beneficial option for young lambs due to its ease and speed of administration and ability to supplement light weight lambs from 3 weeks of age. Whilst it was not possible to address the primary hypothesis of Chapter 3, that a Se deficiency masked an underlying Co deficiency, this study provided evidence of seasonal and year-to-year variation in B12 status, BCS and live-weight gains highlighting the requirement for strategic ‘risk management’ approaches to supplementation. The second part of this thesis (Chapters 4 and 5) addressed the genetic aspect of B12 deficiency. The aim of Chapter 4 was to address the hypothesis that single nucleotide polymorphisms (SNPs) in genes involved in or directly related to 1C metabolism lead to inter-individual variability in metabolic responses to micronutrient deficiencies in sheep. Furthermore, as every farm has different nutritional challenges and with evidence of geographic and ethnic variation in frequencies of 1C metabolic gene polymorphisms in humans, Chapter 5 sought to characterise 1C genetic diversity between breeds. The objectives of Chapter 4 were addressed in a series of analyses which included depleting the Co status of weaned lambs by feeding a Co and S deficient barley-based diet and confirmation of status as in previous chapters. From these animals, DNA samples were collected for genotyping and liver samples were collected for quantification of metabolite concentrations through GCMS, HPLC and LC-MS/MS. An Illumina Infinium® iSelect® Custom Array with 4,576 probes capturing SNPs in 115 1C metabolism and related genes, and 108 related epigenetic regulators was constructed and used to genotype the collected DNA. Following this, a pipeline of bioinformatic analyses was developed, including a genome-wide association study (GWAS), facilitating identification and prioritisation of functionally significant SNPs. 33 SNPs were found to be significantly associated with concentrations of biochemical parameters related to 1C metabolism in the study population of 360 Texel lambs. Of particular biological interest were two SNPs in CUBN; a gene fundamental in the transport of B12 for provision to the methionine cycle and propionate pathways. Chapter 5 investigated population differentiation of four commercially important breeds in the UK; Texel, Suffolk, Bluefaced Leicester and Swaledale, for SNPs in 1C related genes, with specific focus on the 33 functionally significant SNPs identified in Chapter 4. Of particular significance, Swaledale sheep differed genetically from the other three breeds, predominantly for functionally significant SNPs in CUBN identified in Chapter 4. Furthermore, interbreed variation in allele frequency was also observed for SNPs in important 1C regulatory genes including MTHFR, MTHFD1, MTR, MUT and MAT1. Overall, outcomes of the studies in Chapters 4 and 5 provide an unprecedented understanding of the functional significance of complex relationships between 1C metabolites and variants in 1C metabolic genes, providing a basis for further study into judicious targeted supplementation and selection for genetic tolerance to Co deficiency in breeding programmes. In summary this thesis confirms that B12 deficiency in sheep varies with respect to geographical location, stage of production cycle and nutritional availability partly as a consequence of seasonal and inter-year variability. Furthermore, the studies detailed in this thesis represent the first investigation of genetic influences on 1C metabolism in sheep, providing evidence for a possible genetic predisposition to 1C metabolic imbalances, resulting in inter-individual and inter-breed variation in the regulation of 1C and other B12 associated metabolic pathways