| The physical, endocrine and metabolic responses of livestock to road transport have been evaluated by conventional haematological and biochemistry parameters for more than twenty years. However, these measures are relatively insensitive to subtle metabolic adaptations. In this thesis, we applied NMR-based metabonomics to assess system-wide metabolic responses as expressed in urine and serum of a large cohort of animals (n=80) subjected to 12 hour and 48 hour road transport (without food and water), with two replicates. Sera and urine samples were taken at pre-transport, and at 0 h,24 h,48 h, and 72 h post-transport. Our results revealed clear metabolic responses to road transport. The amplification of the response and recovery trajectory in the cohort subjected to the longer duration transport could be observed in both blood serum and urine samples using Multivariate Statistical Analysis (MVSA), which demonstrated the animals transported for 48 hours exhibited a deeper metabolic response to the transport event, and a complex and expanded metabolic trajectory over the 72 h recovery period. Betaine, phenylacetylglycine, creatine/creatinine, hippurate, azelate, and a number of acyl glycines were characterized as key metabolites in urine study. For blood serum study, glucose, lipoprotein, betaine, valine, isoleucine and leucine are responsible for the metabolic changes. Therefore, the profiling of 1H NMR spectra revealed that the transported animals experienced altered gut and energy metabolism, muscle catabolism and possibly a renal response. Intriguingly, the excretion of acyl glycines and a dicarboxylic acid was observed after transport and during recovery, implicating peroxisomal fatty acid oxidation as a metabolic response to transport-induced stress.We also report the identification of crotonyl glycine, a key metabolite in the metabolic response to long duration road transport. Urine, one of the most important biological samples, has been widely investigated in NMR-based metabonomics studies. Thousands of metabolites are excreted into urine, causing extensive overlap of signals in NMR spectra. Currently, less than one thousand metabolites can be identified through data in publicly available sources, whereas numerous chemical compounds which are potential biomarkers for the clinical application still need to be identified. We found an unknown constituent was important in separating the urine from animals undergoing different treatments. To identify this metabolite, a pool of urine was separated by reversed phase HPLC. The complexity of 1H NMR spectra of the collected fractions was reduced, greatly improving the resolution, and revealing that the unknown metabolite had separated into a single fraction. This fraction was spiked by synthesized crotonyl glycine which proved the identification. Mass determination of parent ion and its fragments by nano ESI-TOF-MS also helped to confirm the identity of crotonyl glycine. Our results demonstrated the combination of analytical methods has a great application in the discovery of potential biomarkers.To eliminate the influences of pH and ionic strength on chemical shifts variations, we optimized buffers for sheep urine and serum samples. Ten healthy sheep were involved in the analysis. After comparing the effects of saline,0.075 M,0.1 M, 0.25 M, and 0.5 M phosphate buffer on minimizing variations of chemical shifts, we determined the 0.25 M phosphate buffer as the buffer for sheep serum with the consideration of matching and tunning issue of probe. The same buffer was also selected for urine samples.In conclusion, NMR-based metabonomics combined with statistical analysis can offer fresh insights into livestock research.
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