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