Application of Gas Chromatography-Mass Spectrometry Metabolite Profiling Techniques to the Analysis of Heathland Plant Diets of Sheep IFAT PARVEEN,* ,² JON M. MOORBY, ² MARIECIA D. FRASER, ² GORDON G. ALLISON, ² AND JOACHIM KOPKA ‡ Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth SY23 3EB, United Kingdom, and Max Planck Institute of Molecular Plant Physiology, Am Mu ¨hlenberg 1, D-14467 Golm, Germany Little is known about how plant biochemistry influences the grazing behavior of animals consuming heterogeneous plant communities. The biochemical profiles of grassland species are mostly restricted to major nutritional characteristics, although recent developments in analytical techniques and data analysis have made possible the detailed analysis of minor components that may influence animal feeding preferences, performance, and health. In the present study, gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF/MS) was used to profile the abundances of metabolites in nine specific heathland plant groups and in three mixed forage diets containing 10, 20, or 30% heather (Calluna vulgaris) and also in plasma and feces from sheep offered one of the three diets. Statistical and chemometric approaches, that is, principal component analysis (PCA) and hierarchical cluster analysis (HCA), were used to discriminate between these diets and between individual animals maintained on these diets. It is shown that GC-TOF/MS analysis of sheep plasma allowed distinction between the very similar diets by PCA and HCA, and, moreover, the plant metabolites responsible for the differences observed have been identified. Furthermore, metabolite markers of herbage mixtures and individual plant groups have been identified, and markers have been detected in sheep plasma and feces. KEYWORDS: Diet composition; GC-TOF/MS; ruminant nutrition; metabolite markers INTRODUCTION Free-ranging ruminants are significant components of the agri- ecosystem, and their health and production performance depend upon the nutritive value of the complex plant communities available for consumption. Choices made by large herbivores regarding the type and quantity of plant material grazed can have a profound effect on species richness and diversity and, consequently, on the structure and function of the agri- ecosystem. Furthermore, the dynamic distribution of nutrients and minerals through trampling and excretion can also affect the ecosystem (1). Therefore, a key element to understanding factors affecting long-term sustainability of ecosystems is an understanding of foraging preferences on heterogeneous swards. This would provide objective guidance for efficient range management through habitat restoration and maintenance and the development of management guidelines for grazing sensitive ecosystems. Many plants consumed by herbivores contain the nutrients needed to meet basic requirements, but they can also contain a diverse and complex array of secondary compounds that provide some degree of defense against predation, disease, competition, and adverse climatic conditions. These compounds are fre- quently antinutritional or toxic, yet little is known about how they influence the choices and aversions of animals in their grazing behavior and the associated impact on health and performance. In addition, the composition of primary metabolites varies greatly among sward components and has significant effects on animal performance and dietary selection. Few detailed studies have been conducted with free-ranging animals grazing complex plant communities. This is primarily due to difficulties in accurately determining animal intake and diet composition in such environments. Earlier investigations in- volved direct observation of the grazing animal (2, 3). However, although the method proved to be simple, problems in species identification and quantification of plants consumed were major disadvantages. More recent approaches include microhistological procedures (1, 3-5), stable C-isotope discrimination (1, 6-8), use of plant wax marker compounds (1, 9-11), and near-infrared spectroscopy (NIRS) (12-15). The advent of the postgenomic era has brought powerful high- throughput analytical methods coupled to advanced chemometric techniques. One area that has benefited directly from these * Corresponding author [telephone +44 (0) 1970 823207; fax +44 (0) 1970 828357; e-mail ifat.parveen@bbsrc.ac.uk]. ² Institute of Grassland and Environmental Research. ‡ Max Planck Institute of Molecular Plant Physiology. J. Agric. Food Chem. 2007, 55, 1129-1138 1129 10.1021/jf062995w CCC: $37.00 © 2007 American Chemical Society Published on Web 01/24/2007