Oxidative status: A general but overlooked indicator of welfare across animal species?

Authored by Wild Animal Initiative’s Senior Researcher Michaël Beaulieu, this paper was published in August 2024 in BioEssays.

Abstract

Because of their ubiquity, plasticity, and direct effects on the nervous system, markers of oxidative status may be of great value to assess animal welfare across species and conditions in the wild. However, welfare biologists have not yet seized this opportunity, possibly because the validity of these markers as welfare indicators remains questionable. A validation process was, therefore, performed here using a meta-analytical approach considering three conditions assumed to impair the welfare of animals. With very few exceptions, two of the four considered markers consistently varied across these negatively-valenced conditions. By highlighting the current underrepresentation of markers of oxidative status in animal welfare studies, and by concretely illustrating that some of these markers can consistently reflect negative affective states, this article aims to encourage biologists to include these physiological markers in their toolbox to better measure, monitor, and perhaps also improve the welfare of animals in their natural habitat.

Validating physiological markers as welfare indicators: the case of oxidative stress

This research note is an extension of Physiology Researcher Michaël Beaulieu's paper, “Oxidative status: A general but overlooked indicator of welfare across animal species?,” which was published on June 4, 2024, in BioEssays’ “Problems & Paradigms” rubric.

Why physiological markers require validation as welfare indicators

Our previous article, “Capturing wild animal welfare: a physiological perspective,” described how physiological markers can most effectively and appropriately be used to assess the welfare of animals in their natural habitat. It offered the growing community of researchers interested in wild animal welfare science insights and guidance about the use of physiological markers according to theoretical principles. A key point of the article was that one of the main limitations of using physiological markers as welfare indicators is that the values of typical measurements of physiological markers taken from peripheral tissues like plasma may not be representative of the values found in the central nervous system, where affective states originate. Indeed, relying on peripheral measurements to assess animal welfare is problematic, as measurements taken from peripheral tissues may be affected by factors other than central processes and therefore do not necessarily (or only partly) reflect the affective states animals are experiencing. Despite this important limitation, researchers often implicitly assume that peripheral physiological markers reflect the welfare of animals. However, before any physiological or behavioral marker can be reliably used in animal welfare studies, an initial validation procedure is required to confirm its suitability as a welfare indicator. One such validation procedure was recently proposed, but has so far remained largely theoretical (Browning 2023). 

Putting the validation process into practice

When applied to physiological or behavioral markers, the first three steps of the validation process can be formulated as follows: 

1. Consider a range of conditions and postulate their effects on the valence of the affective states that would be experienced by animals when exposed to these conditions (i.e. whether it would elicit a positive or negative experience);

2. Measure the physiological or behavioral changes resulting from the exposure of animals to those conditions;

3. Examine the consistency of these physiological or behavioral changes across a variety of conditions assumed to similarly impact affective states (i.e. consistently positive or negative valence with high or low arousal), such that consistent changes are independent of the specific conditions affecting welfare, and instead reflect the expected change in valence and arousal.

Despite its simple logic, the application of this validation procedure may seem daunting. Indeed, it may be difficult to apply it in practice, as it requires measuring the effects of a variety of different conditions in a statistically relevant number of individuals distributed across several replicated populations (Beaulieu 2024). This important challenge can be overcome, however, by taking advantage of previous studies examining the effects of similarly valenced conditions on specific physiological or behavioral markers. This is the approach we used in a recently published study to evaluate the validity of markers of oxidative status as potential welfare indicators. In addition to assessing the representation of markers of oxidative status in the animal welfare literature, this study includes a meta-analysis based on the results of previous studies examining the effects of three conditions on the oxidative status of animals: social isolation, noise exposure, and predation exposure. These three conditions were expected to negatively affect the welfare of animals. 

What is oxidative stress?

The presence of oxygen in the Earth’s atmosphere enables animals to produce the energy they need for their daily activities. However, the use of oxygen to produce energy can also result in the excessive production of molecules called Reactive Oxygen Species (ROS) that are capable of damaging important biomolecules such as proteins, lipids, and DNA. To counteract the effects of ROS, animals have developed complex defense machinery composed of a variety of antioxidant molecules, which allows them to minimize oxidative damage. This defense machinery has limits, however, and antioxidant defenses may sometimes be overwhelmed by ROS production. This can lead to oxidative stress: an unbalanced oxidative status between ROS and antioxidant defenses in favor of ROS that leads to high levels of oxidative damage (Costantini & Verhulst 2009). Importantly, not all tissues and organs are equal in terms of oxidative stress. For instance, compared to most organs, the brain is more likely to experience oxidative stress because of the high level of energy it requires, the high levels of ROS it produces, its low endogenous levels of antioxidant compounds, and its overall biochemical composition (Salim 2017). In humans and in laboratory rodents, negatively valenced affective states like irritability, anxiety, and depression have repeatedly been associated with high levels of oxidative damage in the brain (Hovatta et al. 2010). The fact that wild animals likely experience comparable affective states suggests that their welfare could also be reflected by and assessed through markers of oxidative status. 

Is oxidative stress being measured in animal welfare studies?

Despite the potential for markers of oxidative status to be used in welfare studies, the results of a review conducted in three animal welfare journals publishing research articles over the last decade (the “Animal Welfare” section of Animals, the Journal of Applied Animal Welfare Science, and Animal Welfare) show that so far, only 5% of studies have considered these markers for directly assessing the welfare of animals. Across the 295 studies reviewed, markers of oxidative status were unevenly represented, and the selection of given markers of oxidative status in these studies appears largely subjective (or at least not explicitly justified). Moreover, these markers were mainly measured in captive mammals and birds experiencing a low variety of artificial conditions (unlike studies in the adjacent fields of ecophysiology and conservation physiology, which cover a broader variety of markers, conditions, and taxa). Only one study used markers of oxidative status to explicitly assess the welfare of wild animals (wild boars in Esposito et al. 2021). The relative rarity of markers of oxidative status in the current animal welfare literature may, at least in part, be the result of not having undergone a validation process to confirm their reliability as welfare indicators. This validation is all the more important for wild animals, as markers of oxidative status are typically measured in their peripheral tissues and not directly in their nervous system (Beaulieu 2024). 

Applying a validation procedure to markers of oxidative status

Four markers of oxidative status in response to noise exposure, social isolation, and predation exposure were found to be represented in the published literature at a level sufficient for use in the meta-analysis. To avoid the effects of potential confounding factors, all of the studies considered were experimental and conducted under controlled conditions with domesticated animals (laboratory rodents exposed to noise or social isolation) or wild animals studied in captivity (insect larvae, crustaceans, and tadpoles exposed to predatory cues). The results of this meta-analysis show that, with very few exceptions, two of the four considered markers of oxidative status consistently vary irrespective of the nature of the conditions negatively affecting the welfare of animals. These are the levels of malondialdehyde (a marker of oxidative damage on lipids) increase and the levels of glutathione (an endogenous antioxidant marker) decrease. The two antioxidant enzymes did not respond in a consistent manner, even within each considered condition. When both peripheral and central measurements were available (as in the case of noise exposure), peripheral measurements mostly reflected central measurements. Altogether, these results indicate that some peripheral markers of oxidative status could be considered as valid indicators of animal welfare, contrasting with their underrepresentation in the current animal welfare literature.

Conclusions and perspectives

This study provides information about the potential use of markers of oxidative status as welfare indicators. It also illustrates how the process of examining the validity of physiological markers as welfare indicators can be implemented, even without conducting new studies. Importantly, the validation process used here for markers of oxidative status is not restricted to physiological markers, but could also be extended to test the validity of behavioral markers as welfare indicators (Browning 2023). Moreover, the assessment of physiological and behavioral markers as welfare indicators could be conducted simultaneously to examine their interrelationships and how they each relate to certain welfare dimensions like valence, arousal, and persistence. For instance, the assessment of markers of oxidative status as welfare indicators could be conducted at the same time as the assessment of vocalizations, which are known to be affected by oxidative stress and potentially reflect animals’ welfare (Briefer 2012; Casagrande et al. 2016).

Making use of historical datasets by using meta-analytical approaches as we did here obviates the need to disturb additional animals to validate new welfare indicators. This convenient and ethical approach is consistent with the 3Rs (Reduce, Replace, Refine) approach currently recommended in animal experimentation (NC3Rs). A drawback of this meta-analytical approach, however, is that it limits the scope of the validation process to the species, conditions, and physiological markers that are already available in the published literature. Other conditions and physiological markers that have not yet been studied may also be worth examining, especially when working with species underrepresented in the scientific literature, such as many invertebrates. For instance, in the case of oxidative status, some markers could not be considered in the validation process conducted here because of their low representation in the current literature. Moreover, because the available literature focuses more strongly on negatively valenced conditions than on positively valenced ones (Nelson et al. 2023), it was not possible to assess their validity as indicators of positive welfare based on a meta-analytical approach. This is an important limitation, both in terms of markers and conditions affecting welfare, as there is evidence that some markers of oxidative status might also reflect positive affective states (Cafazzo et al. 2014). Finally, indication of publication bias — the propensity to publish results based on their direction, as was sometimes found in this meta-analysis — may cast some doubts on the results of meta-analyses. Overall, these limitations suggest that it is necessary to complement validation procedures based on meta-analytical approaches with field or lab work, despite the related workload and potential costs. These additional empirical studies, some of which may use harmful methods, should only be considered acceptable if they advance our knowledge sufficiently by moderately impacting the few individuals under scrutiny while strongly benefitting the many others living in the wild. Funding agencies therefore need to be convinced of the necessity to validate potential welfare indicators and to allocate substantial amounts of money to financially support such challenging validation projects. We hope that the recent publication of several articles highlighting this urgent need (Beaulieu 2024; Browning 2023), as well as this new study on the potential use of markers of oxidative status in animal welfare studies, will help make this happen and allow us to better assess the welfare of wild animals in the future.

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A note on animal ethics in referenced research studies.

The relevance of trophic interactions to wild animal welfare

Introduction

Almost all animals live off of energy ultimately derived from the sun by the photosynthetic activity of plants. This is true even of animals who do not eat plants as part of their own diet, but rather eat other animals who in turn ate plants. The transfer of energy from the bodies of individuals of one species to those of a different species is known as trophic interaction, or more colloquially, a food chain.

Figure 1. A simple food chain (Hui 2012).

According to many ethical views, an ecosystem supporting more happy animals is preferable to one which supports fewer equally happy animals. In many cases, communities with multi-level trophic chains support more animals than communities with simpler chains. But how additional trophic complexity influences the quality of animal lives is unclear —  from an individual’s perspective, passing energy up the food chain requires their death and the loss of the opportunity to invest that energy in their own life or that of their offspring. To begin to address this apparent tradeoff, we will need to understand how trophic interactions influence the quality and quantity of animal lives in an ecosystem. In this article, I will specifically explore the welfare implications of food chain length, biomass distribution, and predation.

Figure 2. A more complex food chain, showing indirect effects of trophic interactions (Hui 2012).

Food chain length and stability

There is a directionality to food chains, which classically begin with plants at their base and end with an apex predator. These species are said to occupy different trophic levels. The number of trophic levels an ecosystem supports, and how diverse each one can be, is influenced by a plethora of factors. One such factor is the raw amount of energy available from lower trophic levels, known as productivity.

The energy derived from plants is expended in the biological activities of each organism it passes through, resulting in only a fraction of that energy being left over for use by higher trophic levels (Pimm 1988). After being sieved through a certain number of trophic levels, there may not be enough energy available to sustain an additional trophic level. At this point, the food chain is said to be energetically limited.

Many food chains are not limited by energy availability. Instead, some food chains terminate due to historical contingency. For example, in young ecosystems, there may be no immigrants of an appropriate species to continue the chain (Doi and Hillebrand 2019). Food chains may also end due to the unstable population dynamics that result when an additional trophic level is added to the system (Zhao et al. 2019). To understand why, consider a simple community composed of a predator, an herbivore, and a plant species. Herbivores consume plants, and are subsequently killed and eaten by predators. These trophic interactions influence the population dynamics of all three groups. For example, the addition of a predator to a simple herbivore-plant food chain might suppress the population of herbivores, in turn relieving some of the herbivory pressure on the plant population.

The indirect effect of the predator-prey interaction on the size of the plant population is known as a trophic cascade. Most of the time trophic interactions are stable, but they can be destabilizing, especially when there are many factors influencing population sizes. If the predator in this example was able to eradicate the herbivore, perhaps during an already low phase in their population cycle, the entire food chain might collapse, condemning the remaining predators to starvation.

There is a rich literature on the conditions required for stable predator-prey relationships, and destabilization is thought to be more likely when food chains are longer, leading to more cascading effects and more opportunities for the chain to be severed by the extinction of one species (Post 2002). However, theory and experiment also suggest that the destabilizing effect of food chain length can be compensated for by biodiversity at each level of the food chain, reducing the probability that an unstable interaction will sever the chain (Zhao et al. 2019). In this sense, food chains can be thought of as towers that become more susceptible to toppling with length (“vertical” diversity), and more resilient with breadth (“horizontal” diversity). Food chain length matters for wild animal welfare because destabilization through extinction of a particular species can lead to harmful outcomes such as starvation, unsustainable population growth, and intensified competition among remaining species (Ebenman et al. 2004), while trophic biodiversity can influence how many animals an ecosystem is able to support (Duffy et al. 2005).

Welfare implications of ecosystem biomass distribution

If all organisms in an ecosystem experienced equally positive welfare, then maximizing the productivity of the ecosystem would lead to higher total welfare. In reality, things are not so straightforward. Some individuals may suffer so substantially that their cumulative welfare over their lives is negative. Other organisms may contribute nothing to the welfare equation, as most of the biomass on our planet is made up of organisms that are unlikely to be sentient (such as microorganisms, plants, and fungi) or animals whose sentience and welfare capacity is difficult to ascertain (such as arthropods and nematodes) (Bar-on et al. 2018). Of course, self-sustaining populations of these organisms are instrumental to the ecosystem services that support happy, sentient animals.

Just as the quality of animals’ lives likely varies between species, so does the number of lives that can be sustained. Both factors influence the total amount of welfare in an ecosystem. Even among animals who are almost certainly sentient, including most vertebrates, there is extreme variation in average body mass and metabolic rate (Healy et al. 2019). These differences in energy use imply that more lives might be lived in an ecosystem composed of smaller or more metabolically efficient animals than an equally productive ecosystem composed of larger or less efficient animals, where the same amount of energy is divided among fewer individuals.

When animals die of things like disease, starvation, or accidents and are not immediately eaten by predators or animal scavengers, much of their biomass is used by microorganisms as the body is decomposed. Such decomposition adds additional layers of trophic interaction before a fraction of the originally available biomass reaches a sentient organism. The energy lost to microorganisms could be a net loss for wild animal welfare if the resource could have gone to improving the life of a sentient animal or extending an already positive life. 

The concept of ecotrophic efficiency refers to the proportion of biomass of a particular species that is used and lost in the biological functioning of individuals of that species, or passed on to a higher trophic level via death by predation, relative to the energy that is lost via generalist decomposers. Causes of death other than predation generally lower a species’ ecotrophic efficiency. For example, Krebs et al. (2003) estimated the ecotrophic efficiencies of several species in the Canadian Arctic. They found that ecotrophic efficiency was high (~70%) among small herbivores such as lemmings and hares, who were predominantly killed by predators, but low (~9%) among large herbivores such as caribou and muskox. The concentration of biomass into high-welfare individuals is the ideal function of food chains in wild animal welfare.

A conflict of interest between predators and their prey

No animal wants to be killed by a predator, but no predator wants to die of starvation. The conflict of interest between predators and prey seems like a deadlock when framed this way, but the interested parties are not equal in size — a single predator generally needs to kill multiple prey animals to survive. 

Trophic assimilation efficiency, or the conversion rate of prey biomass into predator biomass, is expected to average around 10-50%, with higher efficiencies when predators and prey are more similar in size and physiology (Sanders et al. 2016). To illustrate this point, suppose a predator consumes an animal whose body contains 100% of the energy the predator requires for the year. If the trophic assimilation efficiency is 25%, for instance, then the predator would need to consume 4 such animals per year to survive. Jensen and Miller (2001), for example, estimate that a wolf consumes 12-36 deer per year. 

For a given assimilation efficiency, the greater the metabolic disparity between predator and prey, the more prey animals the predator will have to consume, as the energetic content of the prey animal would be a smaller proportion of the predator’s metabolic needs. Such disparities are common in marine and many terrestrial contexts, where predators tend to be larger than their prey (Andersen et al. 2008; Barnes et al. 2010). On the other hand, very high trophic assimilation efficiencies (~90%) have been reported among some insect parasitoids, where size and nutrient requirements of predator and prey are exceptionally well matched (Harvey et al. 2006). 

If the lives of predators and prey were equal in length and welfare quality on average, trophic assimilation efficiency would be decisive, and any value less than 100% would mean that introducing a predator species to a population of herbivores would reduce the total welfare of the community. In practice, these assumptions probably rarely hold. A more nuanced calculation of the welfare consequences of predation needs to take into account the actual effect of predation on prey welfare, as well as differences in average lifetime welfare between predator and prey individuals.

Implications for welfare biology

One of the most important knowledge gaps for understanding the net welfare value of predation in a given community is whether predator-caused mortality is additive, compensatory, or depensatory with alternative causes of death. What would the life and death of a prey animal have been like otherwise? Answering this requires understanding demographic patterns of mortality and welfare for the population in question, such as the average age at which prey are killed. For example, Carroll (2013) found that the removal of wolves and bears from the vicinity of McGrath, Alaska resulted in a doubling of the proportion of newborn moose surviving to adulthood (>2 years), but had a negligible effect on adult life expectancy, implying that predation in this system mainly threatens juvenile moose who might have had long lives ahead of them. Research into predator-induced stress, density-dependent welfare, and the relative severity of alternative causes of death will also help us understand the net impact of predators on the lives of their prey.

The concepts of ecotrophic efficiency and trophic assimilation efficiency seem valuable to account for in welfare-focused ecological restoration and novel ecosystems. For example, under conditions where predation is thought to lead to better welfare outcomes overall, the ecosystems with highest net welfare might be those with high populations of small herbivores, whose biomass would be more efficiently transferred to high-welfare predators at the end of their lives. On the other hand, when predation is thought to be net-negative or where high-welfare animal scavengers are present, systems dominated by ecotrophically inefficient species such as larger herbivores could contain greater overall welfare. In contexts where human activity is already influencing ecosystem composition, it is worthwhile to consider these potential welfare consequences in our planning. 

Trophic interactions, by distributing energy among animals, are part of the welfare value of maintaining functioning ecological communities. Sustaining multiple biodiverse trophic levels leads to a greater proportion of primary productivity ultimately being converted into sentient animals. The quality of life these animals naturally experience remains highly uncertain, and many ecosystems are already being perturbed by human activities in ways that are not calculated to improve wild animal welfare. However, further research into trophic efficiencies and the welfare of predator-prey systems can prepare us to restore ecosystems in ways that benefit many animals, not just a few.