Recently, oxidative stress has been highlighted as a potential mediator underlying life-history trade-offs in animals. However, despite growing interest in the role of oxidative stress as a mechanistic explanation of trade-offs, the importance of oxidative stress in wild populations remain poorly understood. In this thesis, I use four commonly applied markers of oxidative stress. I apply two markers of oxidative damage, protein carbonyls and malondialdehyde, and two markers of antioxidant protection, superoxide dismutase activity and total antioxidant capacity. These were applied to samples collected in 2010-2013 and 2015 from the wild population of Soay sheep (Ovis aries), St Kilda, Scotland. I investigate correlations among different markers of oxidative stress, and their within individual repeatability over time. In addition, I examine the role played by oxidative stress in mediating life-history traits at different life-stages; specifically, I test for associations between oxidative stress, growth and survival in Soay sheep lambs. I also investigate the oxidative costs of reproduction in adult Soay sheep females and I test whether any such costs vary with age. My findings reveal, firstly, that plasma markers of oxidative stress vary dramatically among years, and that the different markers of oxidative damage and antioxidant protection applied throughout this thesis, are uncorrelated with each other (Chapter 2). This indicates that oxidative stress is a multifaceted process, where each oxidative stress marker may reflect different and potentially uncoupled biochemical processes (Chapter 2). Second, faster lamb growth shows a weak, positive association with malondialdehyde. However, growth is not associated with variation in the other three markers (Chapter 3). In addition, lamb survival also shows marker dependent associations; lambs with higher superoxide dismutase activity are more likely to survive their first winter, as are male but not female lambs with lower protein carbonyl content. Survival does not vary with malondialdehyde or antioxidant capacity. Thus, different markers of oxidative stress capture different aspects of the complex relationships between individual oxidative state, physiology and fitness (Chapter 3). Third, protein carbonyl content and superoxide dismutase activity measured at birth and at four month old in the lambs, show no within individual repeatability, although there is a significant difference in mean marker values over time (Chapter 4). This indicates that these markers of oxidative stress might reflect transient, rather than general, physiological states. Finally, I find some evidence for an oxidative cost of reproduction and for age-related variation in oxidative stress (Chapter 5). However, once again, effect are highly marker and year dependent, and I find no consistent patterns of variation across the two oxidative damage markers or across the two antioxidant protection markers, as they all show different responses to both breeding and age (Chapter 5). Together, my results provide some support for the association between oxidative stress and life-histories, but the effects are both marker and year dependent. Furthermore, the lack of correlation among different markers and the lack of within individual repeatability of oxidative stress markers highlight the need for careful selection and interpretation of plasma oxidative stress markers in the wild.

Appendices removed due to copyright restrictions Appendix 1: Beausoleil, N J, Stafford, KJ, Mellor, DJ. 2005. Sheep show more aversion to a dog than to a human in an arena test. Applied Animal Behaviour Science, 91 : 2 1 9-232. Appendix 2: Beausoleil, NJ, Stafford, KJ, MelIor, DJ. Does direct human eye contact function as a warning cue for domestic sheep, Ovis aries? Journal of Comparative Psychology, J 20 (3) : 269-279. Appendix 3:Beausoleil, NJ, Stafford, KJ, MelIor, DJ. 2004. Can we use change in core body temperature to evaluate stress in sheep? Proceedings of the New Zealand Society of Animal Production, 64: 72-76. Appendix 4: Beausoleil, N.J, Mellor, DJ, Stafford, KJ 2004. Methods for marking New Zealand wildlife: amphibians, reptiles and marine mammals. Department of Conservation, Wellington, New Zealand 147p. ISBN 0-478-2263 1-4. Appendix 5: Mellor, DJ, Beausoleil, NJ, Stafford, KJ 2004. Marking amphibians, reptiles and marine mammals: Animal welfare, practicalities and public perceptions in New Zealand. Miscellaneous Publication, Department of Conservation, Wellington, New Zealand 55p. ISBN 0-478-22563-6. / Both humans and dogs are integral in sheep production systems; however, which is more aversive to sheep, or indeed, whether either causes significant stress, has not been shown experimentally. The aim of this thesis was to examine some behavioural and physiological responses of domestic sheep to the presence of humans or dogs. An arena test was used to measure the relative aversion of sheep to the presence of a human or dog, as well as to elucidate differences in the responses of flocks at the University of Western Australia (UWA) which were putatively selected for differences in fearfulness. A Y maze preference test was used to 'ask' sheep whether they preferred a human shaking a rattle or a barking dog. In both tests, adrenocortical responses were measured concurrently to support the interpretation of behaviour. The presence of a human or dog in the arena elicited significantly more avoidance and vigilance behaviour and less exploration than did the presence of a control object. However, the dog elicited significantly more of this fear-related behaviour, and significantly larger adrenocortical responses than did the human. Sheep also expressed a clear preference for a human shaking a rattle over a barking dog in the Y maze test and exhibited larger adrenocortical responses to the dog than to the human in the Y maze facility. The UWA flocks differed in their expression of locomotor and vocal activity; MA sheep were more active/vocal than the other flocks, not only in the presence of the human but also with the box or dog. MA sheep expressed less avoidance and vigilance and more exploration than the other flocks in the presence of the human and exhibited significantly lower plasma cortisol concentrations than LA sheep after exposure to the human (10-min sample). However, there were no inter-flock differences in fear-related behaviour or adrenocortical responses when the flocks were presented with the box or dog. The results do not support the notion that the UWA flocks have been selected for differences in a consistent predisposition to react fearfully. The adrenocortical responses measured in these studies were only moderate in magnitude and duration, with peak plasma cortisol concentrations 2-3 times higher than pre-treatment values, and all concentrations returning to pre-treatment levels within one hour of the start of treatment. If these observations are confirmed in practical situations, the presence of humans and dogs during routine handling should cause little concern on the basis of animal welfare. However, limiting the presence of dogs in certain situations (e.g. before slaughter) may reduce stress in domestic sheep. Significant methodological developments in this research include the use of multivariate statistical techniques to analyze arena behaviour, the concurrent measurement of adrenocortical and behavioural responses in the arena and Y maze tests, and the explicit testing of the effects of individual lateral biases on choice behaviour in a Y maze test. Future studies should measure sheep stress responses to the presence of humans and dogs in practical situations.

Appendices removed due to copyright restrictions Appendix 1: Beausoleil, N J, Stafford, KJ, Mellor, DJ. 2005. Sheep show more aversion to a dog than to a human in an arena test. Applied Animal Behaviour Science, 91 : 2 1 9-232. Appendix 2: Beausoleil, NJ, Stafford, KJ, MelIor, DJ. Does direct human eye contact function as a warning cue for domestic sheep, Ovis aries? Journal of Comparative Psychology, J 20 (3) : 269-279. Appendix 3:Beausoleil, NJ, Stafford, KJ, MelIor, DJ. 2004. Can we use change in core body temperature to evaluate stress in sheep? Proceedings of the New Zealand Society of Animal Production, 64: 72-76. Appendix 4: Beausoleil, N.J, Mellor, DJ, Stafford, KJ 2004. Methods for marking New Zealand wildlife: amphibians, reptiles and marine mammals. Department of Conservation, Wellington, New Zealand 147p. ISBN 0-478-2263 1-4. Appendix 5: Mellor, DJ, Beausoleil, NJ, Stafford, KJ 2004. Marking amphibians, reptiles and marine mammals: Animal welfare, practicalities and public perceptions in New Zealand. Miscellaneous Publication, Department of Conservation, Wellington, New Zealand 55p. ISBN 0-478-22563-6. / Both humans and dogs are integral in sheep production systems; however, which is more aversive to sheep, or indeed, whether either causes significant stress, has not been shown experimentally. The aim of this thesis was to examine some behavioural and physiological responses of domestic sheep to the presence of humans or dogs. An arena test was used to measure the relative aversion of sheep to the presence of a human or dog, as well as to elucidate differences in the responses of flocks at the University of Western Australia (UWA) which were putatively selected for differences in fearfulness. A Y maze preference test was used to 'ask' sheep whether they preferred a human shaking a rattle or a barking dog. In both tests, adrenocortical responses were measured concurrently to support the interpretation of behaviour. The presence of a human or dog in the arena elicited significantly more avoidance and vigilance behaviour and less exploration than did the presence of a control object. However, the dog elicited significantly more of this fear-related behaviour, and significantly larger adrenocortical responses than did the human. Sheep also expressed a clear preference for a human shaking a rattle over a barking dog in the Y maze test and exhibited larger adrenocortical responses to the dog than to the human in the Y maze facility. The UWA flocks differed in their expression of locomotor and vocal activity; MA sheep were more active/vocal than the other flocks, not only in the presence of the human but also with the box or dog. MA sheep expressed less avoidance and vigilance and more exploration than the other flocks in the presence of the human and exhibited significantly lower plasma cortisol concentrations than LA sheep after exposure to the human (10-min sample). However, there were no inter-flock differences in fear-related behaviour or adrenocortical responses when the flocks were presented with the box or dog. The results do not support the notion that the UWA flocks have been selected for differences in a consistent predisposition to react fearfully. The adrenocortical responses measured in these studies were only moderate in magnitude and duration, with peak plasma cortisol concentrations 2-3 times higher than pre-treatment values, and all concentrations returning to pre-treatment levels within one hour of the start of treatment. If these observations are confirmed in practical situations, the presence of humans and dogs during routine handling should cause little concern on the basis of animal welfare. However, limiting the presence of dogs in certain situations (e.g. before slaughter) may reduce stress in domestic sheep. Significant methodological developments in this research include the use of multivariate statistical techniques to analyze arena behaviour, the concurrent measurement of adrenocortical and behavioural responses in the arena and Y maze tests, and the explicit testing of the effects of individual lateral biases on choice behaviour in a Y maze test. Future studies should measure sheep stress responses to the presence of humans and dogs in practical situations.

Appendices removed due to copyright restrictions Appendix 1: Beausoleil, N J, Stafford, KJ, Mellor, DJ. 2005. Sheep show more aversion to a dog than to a human in an arena test. Applied Animal Behaviour Science, 91 : 2 1 9-232. Appendix 2: Beausoleil, NJ, Stafford, KJ, MelIor, DJ. Does direct human eye contact function as a warning cue for domestic sheep, Ovis aries? Journal of Comparative Psychology, J 20 (3) : 269-279. Appendix 3:Beausoleil, NJ, Stafford, KJ, MelIor, DJ. 2004. Can we use change in core body temperature to evaluate stress in sheep? Proceedings of the New Zealand Society of Animal Production, 64: 72-76. Appendix 4: Beausoleil, N.J, Mellor, DJ, Stafford, KJ 2004. Methods for marking New Zealand wildlife: amphibians, reptiles and marine mammals. Department of Conservation, Wellington, New Zealand 147p. ISBN 0-478-2263 1-4. Appendix 5: Mellor, DJ, Beausoleil, NJ, Stafford, KJ 2004. Marking amphibians, reptiles and marine mammals: Animal welfare, practicalities and public perceptions in New Zealand. Miscellaneous Publication, Department of Conservation, Wellington, New Zealand 55p. ISBN 0-478-22563-6. / Both humans and dogs are integral in sheep production systems; however, which is more aversive to sheep, or indeed, whether either causes significant stress, has not been shown experimentally. The aim of this thesis was to examine some behavioural and physiological responses of domestic sheep to the presence of humans or dogs. An arena test was used to measure the relative aversion of sheep to the presence of a human or dog, as well as to elucidate differences in the responses of flocks at the University of Western Australia (UWA) which were putatively selected for differences in fearfulness. A Y maze preference test was used to 'ask' sheep whether they preferred a human shaking a rattle or a barking dog. In both tests, adrenocortical responses were measured concurrently to support the interpretation of behaviour. The presence of a human or dog in the arena elicited significantly more avoidance and vigilance behaviour and less exploration than did the presence of a control object. However, the dog elicited significantly more of this fear-related behaviour, and significantly larger adrenocortical responses than did the human. Sheep also expressed a clear preference for a human shaking a rattle over a barking dog in the Y maze test and exhibited larger adrenocortical responses to the dog than to the human in the Y maze facility. The UWA flocks differed in their expression of locomotor and vocal activity; MA sheep were more active/vocal than the other flocks, not only in the presence of the human but also with the box or dog. MA sheep expressed less avoidance and vigilance and more exploration than the other flocks in the presence of the human and exhibited significantly lower plasma cortisol concentrations than LA sheep after exposure to the human (10-min sample). However, there were no inter-flock differences in fear-related behaviour or adrenocortical responses when the flocks were presented with the box or dog. The results do not support the notion that the UWA flocks have been selected for differences in a consistent predisposition to react fearfully. The adrenocortical responses measured in these studies were only moderate in magnitude and duration, with peak plasma cortisol concentrations 2-3 times higher than pre-treatment values, and all concentrations returning to pre-treatment levels within one hour of the start of treatment. If these observations are confirmed in practical situations, the presence of humans and dogs during routine handling should cause little concern on the basis of animal welfare. However, limiting the presence of dogs in certain situations (e.g. before slaughter) may reduce stress in domestic sheep. Significant methodological developments in this research include the use of multivariate statistical techniques to analyze arena behaviour, the concurrent measurement of adrenocortical and behavioural responses in the arena and Y maze tests, and the explicit testing of the effects of individual lateral biases on choice behaviour in a Y maze test. Future studies should measure sheep stress responses to the presence of humans and dogs in practical situations.

Appendices removed due to copyright restrictions Appendix 1: Beausoleil, N J, Stafford, KJ, Mellor, DJ. 2005. Sheep show more aversion to a dog than to a human in an arena test. Applied Animal Behaviour Science, 91 : 2 1 9-232. Appendix 2: Beausoleil, NJ, Stafford, KJ, MelIor, DJ. Does direct human eye contact function as a warning cue for domestic sheep, Ovis aries? Journal of Comparative Psychology, J 20 (3) : 269-279. Appendix 3:Beausoleil, NJ, Stafford, KJ, MelIor, DJ. 2004. Can we use change in core body temperature to evaluate stress in sheep? Proceedings of the New Zealand Society of Animal Production, 64: 72-76. Appendix 4: Beausoleil, N.J, Mellor, DJ, Stafford, KJ 2004. Methods for marking New Zealand wildlife: amphibians, reptiles and marine mammals. Department of Conservation, Wellington, New Zealand 147p. ISBN 0-478-2263 1-4. Appendix 5: Mellor, DJ, Beausoleil, NJ, Stafford, KJ 2004. Marking amphibians, reptiles and marine mammals: Animal welfare, practicalities and public perceptions in New Zealand. Miscellaneous Publication, Department of Conservation, Wellington, New Zealand 55p. ISBN 0-478-22563-6. / Both humans and dogs are integral in sheep production systems; however, which is more aversive to sheep, or indeed, whether either causes significant stress, has not been shown experimentally. The aim of this thesis was to examine some behavioural and physiological responses of domestic sheep to the presence of humans or dogs. An arena test was used to measure the relative aversion of sheep to the presence of a human or dog, as well as to elucidate differences in the responses of flocks at the University of Western Australia (UWA) which were putatively selected for differences in fearfulness. A Y maze preference test was used to 'ask' sheep whether they preferred a human shaking a rattle or a barking dog. In both tests, adrenocortical responses were measured concurrently to support the interpretation of behaviour. The presence of a human or dog in the arena elicited significantly more avoidance and vigilance behaviour and less exploration than did the presence of a control object. However, the dog elicited significantly more of this fear-related behaviour, and significantly larger adrenocortical responses than did the human. Sheep also expressed a clear preference for a human shaking a rattle over a barking dog in the Y maze test and exhibited larger adrenocortical responses to the dog than to the human in the Y maze facility. The UWA flocks differed in their expression of locomotor and vocal activity; MA sheep were more active/vocal than the other flocks, not only in the presence of the human but also with the box or dog. MA sheep expressed less avoidance and vigilance and more exploration than the other flocks in the presence of the human and exhibited significantly lower plasma cortisol concentrations than LA sheep after exposure to the human (10-min sample). However, there were no inter-flock differences in fear-related behaviour or adrenocortical responses when the flocks were presented with the box or dog. The results do not support the notion that the UWA flocks have been selected for differences in a consistent predisposition to react fearfully. The adrenocortical responses measured in these studies were only moderate in magnitude and duration, with peak plasma cortisol concentrations 2-3 times higher than pre-treatment values, and all concentrations returning to pre-treatment levels within one hour of the start of treatment. If these observations are confirmed in practical situations, the presence of humans and dogs during routine handling should cause little concern on the basis of animal welfare. However, limiting the presence of dogs in certain situations (e.g. before slaughter) may reduce stress in domestic sheep. Significant methodological developments in this research include the use of multivariate statistical techniques to analyze arena behaviour, the concurrent measurement of adrenocortical and behavioural responses in the arena and Y maze tests, and the explicit testing of the effects of individual lateral biases on choice behaviour in a Y maze test. Future studies should measure sheep stress responses to the presence of humans and dogs in practical situations.

Appendices removed due to copyright restrictions Appendix 1: Beausoleil, N J, Stafford, KJ, Mellor, DJ. 2005. Sheep show more aversion to a dog than to a human in an arena test. Applied Animal Behaviour Science, 91 : 2 1 9-232. Appendix 2: Beausoleil, NJ, Stafford, KJ, MelIor, DJ. Does direct human eye contact function as a warning cue for domestic sheep, Ovis aries? Journal of Comparative Psychology, J 20 (3) : 269-279. Appendix 3:Beausoleil, NJ, Stafford, KJ, MelIor, DJ. 2004. Can we use change in core body temperature to evaluate stress in sheep? Proceedings of the New Zealand Society of Animal Production, 64: 72-76. Appendix 4: Beausoleil, N.J, Mellor, DJ, Stafford, KJ 2004. Methods for marking New Zealand wildlife: amphibians, reptiles and marine mammals. Department of Conservation, Wellington, New Zealand 147p. ISBN 0-478-2263 1-4. Appendix 5: Mellor, DJ, Beausoleil, NJ, Stafford, KJ 2004. Marking amphibians, reptiles and marine mammals: Animal welfare, practicalities and public perceptions in New Zealand. Miscellaneous Publication, Department of Conservation, Wellington, New Zealand 55p. ISBN 0-478-22563-6. / Both humans and dogs are integral in sheep production systems; however, which is more aversive to sheep, or indeed, whether either causes significant stress, has not been shown experimentally. The aim of this thesis was to examine some behavioural and physiological responses of domestic sheep to the presence of humans or dogs. An arena test was used to measure the relative aversion of sheep to the presence of a human or dog, as well as to elucidate differences in the responses of flocks at the University of Western Australia (UWA) which were putatively selected for differences in fearfulness. A Y maze preference test was used to 'ask' sheep whether they preferred a human shaking a rattle or a barking dog. In both tests, adrenocortical responses were measured concurrently to support the interpretation of behaviour. The presence of a human or dog in the arena elicited significantly more avoidance and vigilance behaviour and less exploration than did the presence of a control object. However, the dog elicited significantly more of this fear-related behaviour, and significantly larger adrenocortical responses than did the human. Sheep also expressed a clear preference for a human shaking a rattle over a barking dog in the Y maze test and exhibited larger adrenocortical responses to the dog than to the human in the Y maze facility. The UWA flocks differed in their expression of locomotor and vocal activity; MA sheep were more active/vocal than the other flocks, not only in the presence of the human but also with the box or dog. MA sheep expressed less avoidance and vigilance and more exploration than the other flocks in the presence of the human and exhibited significantly lower plasma cortisol concentrations than LA sheep after exposure to the human (10-min sample). However, there were no inter-flock differences in fear-related behaviour or adrenocortical responses when the flocks were presented with the box or dog. The results do not support the notion that the UWA flocks have been selected for differences in a consistent predisposition to react fearfully. The adrenocortical responses measured in these studies were only moderate in magnitude and duration, with peak plasma cortisol concentrations 2-3 times higher than pre-treatment values, and all concentrations returning to pre-treatment levels within one hour of the start of treatment. If these observations are confirmed in practical situations, the presence of humans and dogs during routine handling should cause little concern on the basis of animal welfare. However, limiting the presence of dogs in certain situations (e.g. before slaughter) may reduce stress in domestic sheep. Significant methodological developments in this research include the use of multivariate statistical techniques to analyze arena behaviour, the concurrent measurement of adrenocortical and behavioural responses in the arena and Y maze tests, and the explicit testing of the effects of individual lateral biases on choice behaviour in a Y maze test. Future studies should measure sheep stress responses to the presence of humans and dogs in practical situations.