Genetics of one-carbon metabolism in sheep in relation to productivity, fertility and health

This project is focussed on the health, productivity and welfare of sheep. We will concentrate on aspects of metabolism that affect lifelong health and wellbeing. Specifically, we will study a key aspect of metabolism referred to as one-carbon (1C) metabolism. This is important because it affects many key processes in the cell, including DNA synthesis, DNA methylation and cell proliferation. It does this by delivering methyl groups, which are central to these biochemical reactions. Deficiencies in metabolites involved in these pathways, such as choline, methionine, folate and vitamin B12, have adverse effects on animal development and wellbeing. For example, deficiencies in vitamin B12 and/or folate can affect fertility and fetal development, and lead to poor growth, vascular disease and metabolic syndrome in adult animals and humans. 1C metabolism pathways are complex and are affected by many genes. We hypothesise that mutations in these genes will affect 1C metabolism and the vulnerability of animals to micronutrient deficiencies. In this study we will identify such mutations, determine their functional significance (i.e. what they do and how important they are), test their impact in animals fed different diets, and find ways to use this information to improve the welfare of farm animals.

Firstly, we will identify mutations (i.e. single nucleotide polymorphisms or SNPs) in around 40 genes directly affecting 1C metabolism, and in very closely related pathways involved in energy metabolism, cell proliferation, DNA synthesis and DNA methylation. To achieve this in sheep we will create our own sequence data. We estimate that we will identify 2000-4000 SNPs by these methods.

We will then conduct a large-scale study with liver samples collected from around 300 sheep slaughtered at local abattoirs. We will genotype each sample (to determine which SNPs are present), measure gene expression and conduct a comprehensive analysis of all metabolites involved in 1C metabolism. Interpretation of these data will inform us on SNP function and how these SNPs affect pathways involved in 1C metabolism. To achieve this we will collate the required information and, using our knowledge of these genomes combined with complex bioinformatic analyses, determine the subset of SNPs that have the greatest impact on 1C metabolism. We expect to identify an estimated 100 or so functionally significant SNPs in these genes. We will then construct a 'SNP chip'; i.e. a tool to genotype sheep simultaneously and cheaply for many SNPs.

This chip will be used to screen several flocks of sheep to identify 24 'Low-risk' and 24 'High-risk' weaned lambs, and 24 'Low-risk' and 24 'High-risk' breeding ewes. We will then monitor these animals in separate studies involving Control and Methyl-Deficient diets (i.e. two genotypes by two diets). For weaned lambs we will focus on effects on growth, animal health and liver metabolism, and carcass yields and composition. For breeding ewes we will also focus on animal health and liver metabolism, but extend studies to consider effects on chemical modifications to DNA (i.e. DNA methylation) in early (Day 16) male and female embryos.

This research will provide novel insights into nutrient x gene interactions for many components of 1C metabolism and how they affect (a) lamb production efficiency, health and welfare, and (b) early development of mammalian embryos influencing fertility and the long-term health and wellbeing of offspring. We will be able to use this information to help breed animals with better functioning 1C metabolism (leading to permanent improvements in welfare and productivity) and/or to improve animal diets. As 1C metabolism also influences human development and health our results will also have biomedical research benefits, and increase the utility of sheep as a model species for this type of research.

One-carbon (1C) metabolism delivers methyl groups for use in a plethora of key cellular reactions including DNA synthesis and methylation. Dietary deficiencies in 1C-related metabolites impair lifelong health and wellbeing in humans and farm animals, and reduce farm-animal productivity. We hypothesise that SNPs in genes associated with 1C metabolism alter the sensitivity of animals to micronutrient deficiencies. This project will identify SNPs in 1C metabolism in sheep, determine their functional significance, and confirm their practical relevance in two selection-based nutritional studies involving weaned lambs and breeding ewes. By re-sequencing and data mining, we expect to identify between 2000-4000 SNPs in or near 40 genes encoding enzymes involved in 1C metabolism and closely related pathways related to energy metabolism, cell proliferation, DNA synthesis and DNA/histone methylation. A comprehensive analysis of SNPs, transcripts and all metabolites involved in 1C metabolism will follow using liver samples collected from ca. 300 sheep. Outputs from these experiments will be the source of data for a 'SNP selection' pipeline, to identify an estimated 100 or so functionally significant SNPs, associated with these genes, for chip construction. This chip will be used to screen several sheep flocks to identify 24 'Low-risk' and 24 'High-risk' breeding ewes, and similar groups of lambs, to participate in two separate prospective 2 x 2 factorial studies involving Control and Methyl-Deficient diets. Measureable endpoints will be growth, immune status, liver metabolism and carcass quality (for lambs), liver metabolism and DNA methylation (MBD-Seq) in Day 16 embryonic cells (for breeding ewes). This research will provide novel insights into nutrient x gene interactions for the many components of 1C metabolism with benefits for animal productivity, health and welfare, improving our understanding of how diet can influence long-term epigenetic programming via the germline.

This BBSRC supported Industrial Partnership Award (with three Levy Boards: EBLEX, HCC and AgriSearch) will advance both fundamental and applied knowledge, with primary non-academic beneficiaries being livestock breeders (producers), companies involved in genetic improvement of sheep, the animal feed industry, veterinarians and the animal health-care pharmaceutical industry. However, we anticipate that the development of a validated software pipeline for associating SNPs with specific changes in metabolism will also be of value to human health-care professionals and the human health-care pharmaceutical industry.

Outputs from this programme (e.g. functional SNP chips, specific dietary advice) may require further industry-sponsored refinement. However, it is probable that specific SNPs in just a few genes will have the greatest influence on 1C metabolism and metabolic outcomes. As this becomes evident so this knowledge can be integrated into practical programmes quickly via the routes outlined below. It is likely that the initial impact of this study will be realised within 3-5 years of project completion, perhaps following further Levy-Board sponsored field-scale studies within the national flock. This is likely to involve a small number of monitor farms where, using Electronic Identification Systems (EID) and following specific pedigrees, we would validate the merits of our approach by (i) quantifying variant-allele frequencies for 1C-metabolism genes and (ii) relating these to measures of animal performance, health and wellbeing. We can also establish a selected resident population of ewes, with contrasting SNP profiles, for future industry-supported studies that could, for example, test the efficacy of pertinent trace-element supplementation strategies at key stages of the life-course (or annual production cycle). This would help identify, for susceptible animals, the most suitable stages of development to mitigate trace-element deficiencies; the efficacy of which would be related to the status of animals identified as being genetically tolerant to such deficiencies.

Differences in digestive metabolism between ruminants and non-ruminants are recognised and, as we have previously demonstrated, can be suitably accommodated when formulating diets. These studies reported similar metabolic responses and developmental effects on insulin resistance in both sheep and rat offspring. Furthermore, we have shown that ewes and women undergoing controlled ovarian stimulation during IVF elicit similar ovarian responses to folate status. Following the successful conclusion of the current proposal we would be able to conduct direct interventional studies (whole animal, cell, embryo culture) in sheep to further address the effects of various SNP combinations on metabolic, developmental and epigenetic responses to varied 1C-metabolite status and relate these observations to parallel human studies. In this regard, we can also make use of the validated software pipeline for associating 1C SNPs with specified changes in metabolism in human studies.

At project completion, and in collaboration with the Levy Boards, we will host seminars/workshops with breed societies from across the UK, to discuss trace-element deficiencies in sheep and the use of contemporary genomic tools to facilitate selection (e.g. via the National Sheep Association (NSA) or via the biennial 'Sheep Breeders Roundtable'). Project completion coincides with the BBSRC Animal Science Forum held in conjunction with the British Society of Animal Science (~500 delegates from industry, the veterinary profession, various public bodies and media). Nottingham also hosts a successful annual meeting (~120 delegates) directed specifically at the animal feed industry. Papers at this meeting are published in the acclaimed series 'Recent Advances in Animal Nutrition', Nottingham University Press. The PI and Co-I Bishop will coordinate these activities.