This research note is an extension of Physiology Researcher Michaël Beaulieu's paper, Capturing wild animal welfare: a physiological perspective, which was published August 27, 2023, by Biological Reviews online.

Short-term emotions and long-term moods correspond to affective states that, depending on the time scale being considered, determine how an animal experiences their life (Crump et al. 2018). In vertebrates, the generation of affective states depends on the relative levels of neurotransmitters (e.g. dopamine, serotonin, etc.) in specific regions of the brain (Lövheim 2012). Despite very different neural organization, most of these neurotransmitters are also found in invertebrates (with or without a central nervous system), in which they also appear to be involved in the expression of affective states (Bateson et al. 2011; Fossat et al. 2014; Perry et al. 2016). 

The production of neurotransmitters responsible for related affective states is triggered by external stimuli and requires information processing (e.g., exposure to predators triggering fear) and can, in turn, affect the whole peripheral physiology of the individual. In vertebrates, the transmission of affective states from central neural structures to the periphery of the individual occurs through the modulation of the autonomic nervous system and the hypothalamo-pituitary-adrenal axis (Stiedl et al. 2010; Keifer et al. 2015; Zhang 2021). Rapid transmission of affective states to the periphery is expected to be highly adaptive, as it likely helps animals to adopt the most relevant physiological and/or behavioral response (fight/flight vs. rest/digest) to the conditions they encounter. For instance, if an individual experiences fear because of the presence of a predator, adopting a fight or flight strategy is likely to promote survival, assuming they have the capacity for combat or escape (Doherty & Ruehle 2020). Such a response is associated with a sudden increase in energetic requirements and is expected to be accompanied: (1) At the physiological level, by the breakdown of stored macromolecules (e.g. glycogen, triglycerides, lipids) into usable smaller molecules (e.g. glucose, fatty acids, glycerol), their release into the bloodstream, and the inhibition of glucose uptake by peripheral organs; (2) At the functional level, by faster heart and respiratory rates, higher core temperature, and the redistribution of blood flow to priority organs (e.g. heart, brain, etc.).

Peripheral physiological and functional changes may themselves also affect the expression of affective states. For instance, laboratory mice for whom heart rate has been experimentally increased can experience anxiety-like states in risky contexts (Hsueh et al. 2023). Such peripheral effects on the expression of affective states may be mediated by a variety of physiological factors (e.g. hormones, inflammatory factors crossing the blood-brain barrier) and likely contribute to interoceptive processes (i.e., experiencing  the physiological condition of the body, such as temperature or pulse; Chen et al. 2021). Therefore the connection between the affective states experienced by animals and their peripheral physiology is bidirectional. In other words, the affective states experienced by animals (e.g. anxiety) can affect their peripheral physiology (e.g., heart function) and, conversely, their peripheral physiology can also affect their subjective experience. Importantly, affective states and peripheral physiological markers (i.e. ‘measurable indicators of the body’s physiological status’; Jesuthasan et al. 2022) may theoretically covary even in the absence of direct interactions between them if they are both affected by the same external factors. Peripheral physiological parameters may therefore reflect affective states both in a causal and in a correlative way.

The challenge of using physiological markers as welfare indicators

In the same way that wildlife biologists measuring physiological markers in other disciplines (e.g. ecophysiology, conservation physiology) have access only to peripheral physiological markers, biologists wishing to use physiological markers to assess the welfare of animals in the wild are also typically limited to peripheral measurements. This results from both limited availability of suitable techniques and limitations of the techniques currently available for directly measuring physiological processes that are occurring in the brain of free-ranging animals, the use of which would also raise important ethical concerns (Gaidica & Dantzer 2022). Peripheral physiological markers can also be measured in other tissues besides those that can be sampled directly from animals that have been captured (e.g. blood, muscles), including peripheral structures (e.g. hair, feathers) and excretory material (e.g. feces) left by animals in their environment. 

Because of the bidirectional relationship between affective states and physiology described above, any peripheral physiological marker affected by and/or affecting the expression of affective states could theoretically be used as an indicator to assess the welfare of wild animals. However, peripheral physiological parameters are not only involved in the expression of affective states but also in other processes, such as the regulation of homeostasis (i.e., “a self-regulating process by which biological systems maintain stability while adjusting to changing external conditions”; Bilman 2020). Importantly, homeostatic changes may be but are not necessarily related to changes in affective states. For instance, being dehydrated leads both to physiological changes aiming at correcting this imbalance (e.g., production of antidiuretic hormone) and to thirst (i.e., an affective state motivating water intake). In contrast, when the conditions with which the organism has to cope require an immediate response without interpretation by animals, peripheral physiological processes aiming at correcting homeostasis may be activated independently of any emotional processes when (e.g., hypoglycaemia; Herman & Culliman 1997). Hence, most peripheral physiological parameters likely reflect to some extent both the emotional and the homeostatic states of the organism, which are intermingled but not necessarily interconnected. Finally, because peripheral physiological functions constantly interact with each other, the relationship between a given peripheral physiological marker and a given affective state is likely to be blurred by the interactions that this specific physiological marker has with other physiological functions (themselves affected or not by emotional and/or homeostatic conditions). This complexity points to the need to develop methodological approaches to validate the use of physiological parameters as robust indicators of animal welfare.

Validating physiological markers as welfare indicators in the wild

Most wildlife biologists already measuring physiological markers appear to select markers based on their own knowledge, background, and experience. For example, in the field of ecophysiology or conservation physiology, plasma glucocorticoids are traditionally (rightly or wrongly) measured to assess how vertebrates cope with challenging conditions because of their implication in the stress response (Beaulieu & Costantini 2014, MacDougall-Shackleton et al. 2019). Assuming that glucocorticoid levels also reflect certain affective states, the choice to measure glucocorticoids in welfare studies would imply that (1) the considered affective states and glucocorticoids vary on the same time scale (Gormally & Romero 2020), and (2) the relationship between given affective states and glucocorticoids is predominant over any homeostatic influences affecting glucocorticoid levels. Typically these assumptions (and the choice of glucocorticoids) in welfare studies are not well justified, which has presumably contributed to question the relevance of using such markers to assess animal welfare despite their direct relationship with centrally-generated affective states (Ralph & Tilbrook 2016). The widespread use of glucocorticoids without clear evidence of the mechanisms and how to interpret results clearly demonstrates that peripheral physiological markers need to be validated before being convincingly used as welfare indicators. 

Based on Browning 2023, the validation process for peripheral physiological markers as welfare indicators could be divided into three consecutive steps: 

1. Formulating and articulating assumptions about the valence and arousal value of the affective states experienced by animals under certain conditions (e.g. a negative valence and an increased arousal assumed to result from predator exposure);

2. Measuring whether these conditions are associated with a change in the peripheral physiology of animals (e.g. higher glucocorticoid levels observed in animals exposed to a predator);

3. Ensuring that this peripheral physiological change is consistent across a variety of conditions assumed to affect welfare in a similar fashion (e.g. increased glucocorticoid levels observed irrespective of the nature of the predator and in socially-isolated or restrained animals). 

If a given physiological marker consistently varies across conditions all assumed to be associated with the same valence and arousal, then it could be considered as a robust indicator of welfare (as its variation is independent of the condition causing the assumed welfare change). 

Even though the proposed validation process may appear quite intuitive and simple, it may be difficult to put into practice, especially in the wild. Indeed, the last validation step implies the use of a replication approach based on the examination of different populations exposed to different conditions or the same populations sequentially exposed to different conditions (as recommended in ecological studies; Filazzola & Cahill 2021). This rigorous scientific approach may, however, be impracticable in the wild because of the associated increased costs, workload, and the potential disturbance generated by multiple animal manipulations. This limitation points to the need during the validation process to first consider the feasibility of including  collection of biological samples, and adopting a replication approach.

To facilitate this validation process, it might also be possible to apply it first to non-wild animals who might be better amenable to replication approaches, and for whom the conditions they experience can be better controlled. In that case, it might be possible to work with individuals of the same species as the species of interest in the wild, or those of a closely-related species — typically the subjects of laboratory studies (e.g. rodents, fruit flies) or among the many species kept in zoos. Researchers, who choose to apply the validation process to non-wild animals first before applying the results in the wild, must nevertheless be mindful that the physiology and behavior of non-wild animals may not necessarily be comparable to that of wild animals (Crates et al. 2023). At the very least, non-wild individuals may still be studied for the analytical, physiological and biological validation of particular physiological measurements (Palme 2019). Moreover, non-wild individuals could also be studied to examine the relationship between specific physiological markers and behavioral welfare indicators that have been previously established based on sophisticated behavioral approaches to directly reflect emotional valence. Such sophisticated behavioral approaches would be difficult or even impossible to use in the wild (e.g. cognitive biases; Crump et al. 2018).

Conclusion

The relationship between welfare and physiology is highly complex because it can be causal/correlational, direct/indirect, and bidirectional, which makes the use of peripheral physiological markers to assess wild animal welfare challenging. Even without knowing the exact nature of the relationship between peripheral physiological markers and affective states, it is still possible to apply a validation process to examine whether physiological markers can be used as reliable welfare indicators. By determining consistent relationships between markers and affective states across conditions, it is indeed possible to establish them as robust welfare indicators that could then be combined with other validated markers to obtain a more thorough welfare profile.

Many welfare biologists could be discouraged from applying the proposed validation process because it is based on various initial assumptions and requires such a demanding replication approach. Nevertheless, this challenge may be overcome by collaborations between welfare biologists and physiologists, wildlife biologists, or zoo practitioners. Importantly, once validated, physiological markers have several advantages for assessing wild animal welfare that may convince welfare biologists to use them:

• Physiological measurements are generally considered to be highly objective and straightforward. 

• Physiological responses can occur in the absence of any observable behavioral response.

• Physiological responses can be measurable in some tissues at a later time, following an event that affects welfare, thereby possibly reflecting affective states across time. 

• Finite physiological responses with pleiotropic (or multiple) effects on behavior, although variable, are likely to be more consistent than behavioral responses between species, thereby facilitating comparative welfare studies. 

As such, welfare biologists would benefit from adding validated physiological measures to their toolbox, as this may help them to draw a more holistic picture of the affective states experienced by animals in the wild.