COMPARATIVE EVALUATION OF 1H NMR SPECTROSCOPY AS A NOVEL TOOL FOR ASSESSING STRESS WITH TRADITIONAL METHODS OF BLOOD CORTICOSTERONE LEVELS AND HETEROPHIL LYMPHOCYTE RATIO IN BROILER CHICKENS (GALLUS DOMESTICUS)
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Stress can be defined as any event that alters physiological homeostasis. Any event that elicits a response (either behavioral or physiological) can be considered a “stressor”. Proper response to a stressor can be beneficial in life threatening situations; however, chronic or repeated exposure can have marked deleterious effects on reproduction, behavior, metabolism and immunocompetence. Identification of stress in poultry research has been done using various physiological endpoints with the two most accepted indicators of stress being elevated corticosterone (CORT) and increased circulating heterophil/lymphocyte (H/L) ratios. The lack of consistency between studies measuring CORT and H/L ratios results in some skepticism when using them to quantify stress and animal welfare in the poultry industry. 1H Nuclear Magnetic Resonance spectroscopy (NMR) may provide insightful mechanistic information on effects of stressors by simultaneously measuring a large number of low molecular weight endogenous metabolites that may potentially be used as biomarkers for stress in chickens (Gallus domesticus). The objectives of this thesis are to identify metabolites associated with chronic stress in broilers fed on a corticosterone containing diet using NMR spectroscopy, evaluate and compare the use of NMR in the identification of stress metabolites to more traditional methods of stress (CORT and H/L ratio), compare the use of CORT, H/L ratio, and metabolic profiles in broilers reared under 4 different lighting photoperiods to evaluate stress and welfare, and compare the use of CORT, H/L ratio and metabolic profiles in broilers reared under 4 different light intensities to evaluate stress and welfare. Chronically stressed broilers fed a CORT (30 mg/kg feed) diet for four weeks had a significant increase in both H/L ratio (P≤0.002) and serum CORT levels (P≤0.001) at all time points of collection (days 14, 21, 28 and 35) in comparison to the control birds. Both H/L ratio and serum CORT levels failed to identify or differentiate any stressed groups in both photoperiod (14L:10D, 17L:7D, 20L:4D and 23L:1D) and lighting intensity (1, 10, 20 and 40 lux) experiments. Behavioral, welfare and production data reported by the sister studies however showed the 23L:1D group (photoperiod) and 1Lux (light intensity) to have compromised welfare. Both H/L ratio and serum CORT levels were highly suggestive of age related changes in the lighting experiments. H/L ratio showed significant difference between the days of sampling in both photoperiod (P≤0.001) and light intensity (P≤0.001) experiments, while serum CORT showed a significant difference between days of sampling in the light intensity experiment (P≤0.001). Metabolomics using Proton Nuclear Magnetic Resonance (NMR) clearly differentiated the chronically stressed birds fed on a CORT diet (30 mg/kg diet) from the control birds. It helped in identifying metabolites like isoleucine, lysine, valine and tryptophan that are highly indicative of stress induced anxiety, depression and behavioral changes. Metabolites like methionine, betaine, histidine suggested compromised methylation process and oxidative stress response. Metabolites threonine, glutamine and histidine suggested reduced immune response while metabolites like carnitine, lactate, glucose and β hydroxybutyrate suggested the undergoing process of energy mobilization. Metabolomic analysis of the serum samples of the photoperiod groups showed the presence of amino acids valine, threonine, lysine, proline, histidine, methionine and other metabolites like creatine, carnitine, choline, glycerol, betaine and lactate. Out of the top common metabolites identified between this study and that of the chronically stressed birds fed CORT (30 mg/kg diet), 5 metabolites (valine, lysine, methionine, histidine and glucose) showed similar relative abundance and trends in lighting groups 20L:4D and 23L:1D. Metabolomic analysis of the lighting intensity groups showed the presence of amino acids isoleucine, threonine, histidine, methionine, tyrosine, serine and other metabolites including carnitine, choline, betaine, lactate, glucose and fructose. Out of the top common metabolites identified between this study and that of the chronically stressed birds fed CORT (30 mg/kg diet), 5 metabolites (isoleucine, histidine, carnitine, glucose and betaine) in lighting groups 1 lux and 40 lux showed similar relative abundance. Findings of this thesis suggest that metabolomics was able to successfully differentiate the chronically stressed birds from the control birds with valuable insights about the physiological process involved in the stress response. In the lighting experiments metabolomics was able to identify metabolites suggestive of the behavioral changes, energy homeostasis, immune response, oxidative stress and osmoregulation. Metabolomics supported evidence of compromised welfare in photoperiod (23L:1D) and lighting intensity (1 lux) while H/L ratio and serum CORT levels failed to do so.
DegreeMaster of Science (M.Sc.)
DepartmentVeterinary Biomedical Sciences
ProgramVeterinary Biomedical Sciences
SupervisorMachin, Karen L.
CommitteeClassen, Henry L.; Singh, Jaswant; Stookey, Joseph M.
Copyright DateApril 2012
Stress, Heterophil, Lymphocyte, Corticosterone, Nuclear Magnetic Resonance, Photoperiod, Light Intensity.