The role of stress and dietary micronutrients in fish health
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The overall theme of this thesis has been the study of the effects of well known and potentially novel stressors on fish health and how some of these may be modulated by dietary supplements of micronutrients, using both in vitro and in vivo analysis. In vitro experiments with cultured fish cells (EPC-A1, GFSk-S1) evaluated the potential of niacin and selenium to reduce cytotoxicity and genomic instability (DNA damage) induced by ultraviolet radiation exposure, following assay validation with reference toxins. Whilst cytotoxicity was determined by the neutral red retention (NRR) assay, genomic stability was evaluated by either a standard or modified version of the single cell gel electrophoresis (SCGE) or 'Comet' assay. Niacin as nicotinamide (NAM) significantly reduced levels of UVB induced DNA damage (single strand breaks). Selenium supplements, as sodium selenite or seleno-L-methionine also showed a protective effect against H202, UVA and UVB induced cytotoxicity and oxidative DNA damage. An additional in vitro study was carried out to identify environmental nanoparticles as a potential novel source of stress for fish. Titanium dioxide (Ti02) nanoparticle exposure to GFSk-81 cells caused dose-dependent increases in cytotoxicity (NRR assay) and oxidative DNA damage (Comet assay). These effects were exacerbated by combined exposures of Ti02 with UVA. Electron spin resonance (ESR) and spin trapping suggested that Ti02 induced oxidative stress may be primarily due to the production of hydroxyl radicals (OH). Two in vivo experiments were carried out in order to evaluate the effects of husbandry stress (netting and confinement stress) on antioxidant capacity, immune function and genomic stability in trout (Oncorhynchus mykiss) and carp (Cyprinus carpio) , In both studies on trout and carp, certain health parameters were shown to be sensitive to husbandry stress, and may be useful biomarkers of stress in future studies. Total antioxidant capacity (TAC) was reduced in both carp and trout after stress. Respiratory burst capacity of blood leukocytes was also affected by stress but differently in carp than in trout. In carp, husbandry stress apparently stimulated the production of free radicals by leukocytes whereas in trout it was suppressed. In trout, stress was also shown to increase oxidative DNA damage, as measured by the Comet assay. Health parameters that appeared not to be significantly affected by husbandry stress in this study include SOD activity, ALP activity, red blood cell fragility and complement activity (for carp only). In the second experiment on carp, a 10 week feeding trial was also conducted prior to stress induction to evaluate the potential modulatory effects of selenium, zinc and vitamin E on any husbandry induced stress effects observed. Health parameters were also evaluated pre stress in order to elicit any effects of micronutrient supplementation on health parameters in unstressed fish. Selenium supplementation significantly increased glutathione peroxidase activity post stress, but not pre stress, indicating an increased requirement of selenium in stressed fish. No other differences were observed between dietary treatments for any parameter measured either pre stress or post stress, indicating that levels of selenium, zinc and vitamin E were probably sufficient in the control diet. Further work is needed to enhance our understanding on the interaction between, and the role of, stress and dietary micronutrients in fish health using a suite of assays to monitor all aspects of fish health, both in vitro and in vivo.
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