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- Materials and methods
Future fitness costs of current life history decisions are a central assumption of theories of the evolution of life histories and ageing (Williams 1957; Kirkwood & Rose 1991; Roff 1992; Stearns 1992). However, our understanding of the mechanisms underlying such costs remains limited, particularly in wild populations (Partridge & Gems 2002; Reznick 2005; Harshman & Zera 2006). Costs of raised investment in growth, immune function and reproduction have traditionally been considered to result from diversion of limited somatic resources away from somatic maintenance functions or future reproduction (Reznick 1992; Stearns 1992). Alternatively, by-products of normal metabolism and immune activity may themselves directly damage the soma, in which case selection acts on the fitness trade-off between damage accumulation and repair (Kirkwood 1977; Harshman & Zera 2006). Oxidative damage to cells and their macro-molecular components may occur as a by-product of normal metabolism: it has been implicated as a proximate mechanism responsible for organismal senescence and an important cost of raised investment in metabolically demanding activities such as growth, reproduction and immune function (Beckman & Ames 1998; von Schantz et al. 1999; Golden et al. 2002; Alonso-Alvarez et al. 2004; Costantini 2008; Monaghan et al. 2009). To date, few studies have examined this potentially important mechanism underpinning trade-offs between growth, reproduction and survival outside of artificial laboratory conditions, despite the importance of placing laboratory findings in a more functional ecological and evolutionary context (although see Costantini et al. 2006; Rubolini et al. 2006; Torres & Velando 2007; Bizé et al. 2008 for examples in wild birds). Here, we examine associations between an assay of plasma phospholipid oxidative damage and age, early growth rates and reproductive history in a wild population of Soay sheep (Ovis aries).
Reactive oxygen species (ROS) are primarily produced as a by-product of aerobic metabolism in mitochondria and can cause oxidative damage to cells and tissues (Harman 1956; Beckman & Ames 1998). The potential for ROS-induced damage can be reduced by a suite of enzymatic antioxidants, endogenous non-enzymatic antioxidants, diet-derived compounds and various repair mechanisms (Beckman & Ames 1998). According to the free radical theory of ageing, oxidative damage to cells determines the rate of organismal senescence, i.e. the decline in survival probability and reproductive performance in old age (Harman 1956; Beckman & Ames 1998). This mechanistic theory of ageing complements the disposable soma theory of ageing, which states that senescence occurs due to an adaptive failure of repair and replacement mechanisms as natural selection weakens with age (Kirkwood & Rose 1991; Kirkwood & Austad 2000). Here, it is the failure of antioxidant defence and oxidative damage repair systems to deal with ROS-induced cellular damage that ultimately leads to senescence (Beckman & Ames 1998). The free radical theory of ageing remains controversial. Although there is some empirical support from model laboratory systems and humans showing increases in oxidative damage and declines in antioxidant defence capacities with age (Sohal et al. 1993; Sohal et al. 1994a; Rao et al. 2003; Lombard et al. 2005; Alonso-Alvarez et al. 2006), there is also contradictory evidence from dietary and genetic manipulations of model organisms and from comparative work across species of differing lifespan (Barja 2004; Sanz et al. 2006; Doonan et al. 2008).
Conditions in very early life may also profoundly alter the balance between ROS production and antioxidant defence and repair capacity. Hatching or birth in vertebrates represents a profound change in aerobic conditions and a potential oxidative insult to the developing organism. Evidence from humans, domestic mammals and wild birds supports this idea by revealing increased ROS and antioxidant levels around birth or hatching relative to later in development (Drury et al. 1998; Gaál et al. 2006; Rubolini et al. 2006). Work in wild bird and lizard populations shows that a high proportion of variation in the oxidative state of eggs and hatchlings can be attributed to their nest of origin or mother's identity (Costantini & Dell’Omo 2006b; Rubolini et al. 2006; Olsson et al. 2008). This suggests that early environmental conditions and parental investment may have profound effects on ROS production and/or antioxidant defence capacity during development. However, the longer term consequences of such variation in environment or parental investment on adult antioxidant capacity, oxidative state and later senescence have hardly been studied (although see Blount et al. 2003). Most studies of oxidative stress in wild vertebrates have been conducted on birds and have tended to focus on variation in levels of antioxidants or measures of total antioxidant capacity in developing offspring and/or their parents (Costantini 2008). Few studies to date have examined variation in oxidative damage across individuals of different ages in wild populations (although see Torres & Velando 2007).
Laboratory experiments on vertebrate and invertebrate systems have been able to relate experimentally raised investment in growth and reproduction to increased levels of ROS, increased oxidative damage and reduced antioxidant availability (Wang et al. 2001; Alonso-Alvarez et al. 2004; Wiersma et al. 2004; Alonso-Alvarez et al. 2007; de Block & Stoks, 2008). Researchers have argued that increased investment in growth or reproduction should result in raised metabolic rate and thus increased ROS production, which, if not accompanied by an up regulation of antioxidant defences, or repair and replacement systems, will result in oxidative stress and loss of cellular homeostasis which may represent a substantial fitness cost to the organism in due course (Alonso-Alvarez et al. 2004; Costantini 2008; Monaghan et al. 2009). It should be noted, however, that such a simple relationship between metabolic rate and ROS production is currently under challenge: mitochondrial uncoupling can lead to associations between metabolism, ROS production and lifespan that are opposite to those predicted above (Brand 2000; Speakman et al. 2004; Selman et al. 2008). What is quite clear, however, is that further work is required to understand the links between life history and oxidative stress and that studies in natural settings are important to place laboratory findings within a more meaningful ecological and evolutionary context (Costantini 2008; Selman et al. 2008). To our knowledge, no study to date has addressed these issues in a free-living mammal population.
Here, we use data collected as part of a long-term individual-based study of Soay sheep (Ovis aries), on the island of Hirta, St. Kilda (Fig. 1; Clutton-Brock & Pemberton 2004). Plasma samples were collected from a cross-sectional sample of 150 sheep caught in August 2007 and assays of a marker of phospholipid oxidative damage were conducted. Based on the free radical theory of ageing, we would predict that oxidative damage should be increased amongst older individuals. Life history theory would predict that oxidative stress is a cost of metabolically intensive activities such as growth, reproduction and immune responses and we accordingly expected levels of oxidative damage to correlate positively with growth rates, parasite burden and reproductive investment. Here, we initially examined correlations between assayed levels of phospholipid oxidative damage and age. We then examined how (i) early life growth rates, birth mass, parasite burden, twin status and sex influenced damage amongst lambs; (ii) prior reproduction, parasite burden and body mass influenced damage in yearling females; and (iii) how body mass, parasite burden, and recent and past reproductive investment influenced damage amongst adult female sheep.
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- Materials and methods
Levels of oxidative damage to phospholipids in Soay sheep plasma samples were raised in lambs relative to all other age classes (Fig. 2). In mammals, birth is likely to represent a major oxidative challenge, whilst the processes of development and growth demand a consistently raised metabolism that would be expected to increase production of ROS (Gaál et al. 1996; Przybylska et al. 2007). These factors, coupled with poorly developed antioxidant defence and damage repair systems, might lead us to expect raised levels of oxidative damage in very young individuals. However, work on domestic ungulates suggests that milk, and particularly colostrum provided by the mother in the first few days after birth, is rich in non-enzymatic antioxidants, which may help compensate for deficiencies in the antioxidant defence system post-partum and prevent oxidative damage (Przybylska et al. 2007). In two detailed longitudinal studies of the oxidative profiles of cattle calves, Gaál and colleagues concluded that calves coped adequately with changes in oxidative state immediately post-partum but demonstrated subsequent increases in ROS production and oxidative damage in the weeks following birth. They argued that this was due to raised metabolism associated with rapid growth and a shift in diet from milk to grass (Gaál et al. 1996; Gaál et al. 2006). The same factors may be responsible for the raised oxidative damage levels observed in the Soay sheep lambs, as measurements were taken following a period of both rapid growth and development and of adjustment to feeding independently and rumination. However, further studies monitoring the antioxidant content of milk and both levels of antioxidants and oxidative damage across the early lives of Soay sheep would be required to understand the relative roles of diet and growth underlying raised plasma MDA amongst lambs.
Lambs that grew more rapidly across the first 4 months of life had increased levels of oxidative damage to phospholipids in plasma samples (Fig. 4). This represents correlational evidence in support of an ‘oxidative cost’ to early growth as would be predicted by life history theory. Laboratory studies of birds and insects in which growth rates were manipulated have found similar costs, either in terms of raised oxidative damage or reduced antioxidant capacity (e.g. Blount et al. 2003; Alonso-Alvarez et al. 2007; de Block & Stoks 2008). Variation in neonatal traits such as birth weight associated with birth cohort and maternal effects are well documented in both the Soay sheep on St. Kilda and in wild ungulate populations more generally (e.g. cohort and maternal effects: Albon et al. 1987; Festa-Bianchet et al. 2000; Forchhammer et al. 2001; Wilson et al. 2005). Our analyses present a possible link between conditions in very early life – as reflected by birth mass, competition for resources both during gestation and lactation with a twin and growth rates in the first 4 months of life – and levels of plasma oxidative damage. Previous studies of wild bird and reptile populations have found evidence for variation between nests or parents in the oxidative state of nestlings (Costantini et al. 2006; Costantini & Dell’Omo 2006b; Rubolini et al. 2006; Bizé et al. 2008; Olsson et al. 2008). Our samples from Soay lambs raise the possibility that increased growth rates associated with high maternal investment and/or food availability in very early life may be an important driver of differences in the oxidative state of juveniles.
Despite the link between early life conditions and oxidative stress apparent here and in other studies of wild vertebrates, the ecological and evolutionary significance of the observed increase in plasma MDA levels amongst faster growing Soay lambs remains unclear. Previous research clearly shows that heavy born lambs have an improved chance of surviving the neonatal period and, amongst those that do survive, the heaviest lambs at around 4 months of age are the most likely to survive their first winter (Clutton-Brock et al. 1992). It seems unlikely that the observed oxidative cost associated with increased growth rates would offset the benefits to survival prospects of being heavier going into the first winter of life. However, we know that male sheep must invest more than females in growth across the first few years of life and selection on adult body mass varies between sexes and age classes in this population (Clutton-Brock et al. 1992; Pelletier et al. 2007). Although we saw no evidence of sex-specific associations between MDA and body mass or growth rates in our 2007 cohort of lambs, Clutton-Brock et al. (1992) showed that yearling mortality rates declined with increasing body mass at around 16 months of age in females, but in males the relationship was curvilinear with the lightest and heaviest yearling males suffering increased risk of mortality. This work suggests that the costs of growth may be sex-specific and may not become apparent until individuals are close to adulthood. Whilst our results provide support for the idea that investment in growth has costs in terms of raised oxidative damage in Soay sheep, a more detailed, longitudinal study is required to determine the future fitness consequences of variation in growth rates and oxidative state during early life in males and females in this population.
Contrary to predictions of the free radical theory of ageing, we found no evidence for any systematic change in oxidative damage levels with age amongst a cross-sectional sample of adult Soay sheep. Our sample did include unusually old females (nine females aged 9 years or over) and several cross-sectional studies of domestic and laboratory mammals have found evidence for raised oxidative damage amongst older individuals (Sohal et al. 1994a; Sohal et al. 1994b; Sohal et al. 1995; Gaál et al. 1996). Similarly, we found no evidence for any associations between oxidative damage and reproductive investment or parasite burden, as would be predicted under life history theory treatments of oxidative stress (Costantini 2008; Monaghan et al. 2009). This is in contrast to experimental work on birds, which has demonstrated changes in oxidative damage or antioxidant levels following manipulation of reproductive costs or immune challenge (Alonso-Alvarez et al. 2004; Wiersma et al. 2004; Bertrand et al. 2006; Costantini & Dell’Omo 2006a; Torres & Velando 2007). However, several limitations of our data set, which are likely to be typical of studies in natural settings, mean that it would be unwise to interpret our analyses as evidence against the free radical theory of ageing or a role for oxidative stress in life history trade-offs.
First, our data is cross-sectional and we therefore cannot reliably separate within-individual trade-offs or ageing patterns from between-individual heterogeneity. For example, if a strong antioxidant or damage repair capacity was associated with female survival and longevity, then any within-individual increases in oxidative damage with age could be masked as these low oxidative stress females came to predominate in older age classes (van de Pol & Verhulst 2006). Similarly, if individual sheep vary their investment in reproduction in accordance with their current capacity to deal with the costs of such investment (i.e. individual optimization), tests for trade-offs using cross-sectional data will typically yield correlations in the opposite direction to those predicted by life history theory (Moyes et al. 2006; Hamel et al. 2009). Individual heterogeneity may be greatly reduced in benign laboratory or domestic settings and this may, in part at least, explain the disparity between the present results and those from the laboratory, which are also typically cross-sectional. Second, reproductive trade-offs and ageing rates may depend on past environmental experiences of adult females in our data set and controlling for variation in environmental conditions experienced across an individual's lifetime is always likely to present a major challenge to studies in natural settings. Finally, we should point out that laboratory research suggests oxidative damage can vary widely between tissue types (Sohal et al. 1995; Gaál et al. 1996; Costantini 2008) and our oxidative damage assays in plasma may reflect fairly recent oxidative state because turnover of plasma constituents is high. We cannot determine how well assays of plasma MDA reflect oxidative damage levels across a wider range of tissue types, and our results for adult sheep may well reflect a limited ability to detect cumulative and long-term effects of life history decisions on oxidative state using plasma samples alone.
In conclusion, we have shown raised levels of oxidative damage in plasma amongst lambs relative to other age classes and a positive correlation between lamb growth rate and oxidative damage. The lack of a correlation between oxidative damage and either age, reproductive effort or parasite burden serve to highlight the potential limitations of cross-sectional data and plasma samples for addressing these issues in both laboratory and natural settings (Costantini 2008; Monaghan et al. 2009). Ongoing individual-based studies of birds and mammals, such as the study of Soay sheep on St. Kilda, do provide an opportunity to collect and assay longitudinal samples and link these to existing measures of past conditions, growth rates and reproductive investment. Previous work reveals that where longitudinal data is available, mixed-effects models can be used to good effect to detect trade-offs and model senescence at the within-individual level (Nussey et al. 2006; Nussey et al. 2008). This argues for in depth, longitudinal study of variation in markers of oxidative stress in wild populations that are already subject to long-term, individual-based study. We very much hope this study will stimulate further, longitudinal studies investigating the association between oxidative state, ageing and life history in wild mammal populations.