RAMP method video developed by ILVO

ILVO (Belgium Institute for Agricultural and Fisheries Research) has developed RAMP methods for three species of flatfish (plaice, sole, and dab) in European fisheries.

The first video sets the scene and explains the potential relevance of this method in relation to the recently reformed European Common Fisheries Policy.

The second video explains and demonstrates reflex tests in more detail and may guide other investigators in defining and recognizing reflex actions.

An excerpt from the video text explains, “A staggering amount of commercially-caught fish is being thrown overboard. Some say that all of those discarded fish are either dead before they hit the water or they die soon after, victims of predation or injury. But others argue that some of those species are strong enough to survive after being discarded and live long enough to reproduce. The European Common Fisheries Policy was recently reformed and will now phase in a ban on discarding, meaning that fishers will have to land everything they catch. The idea behind the ban is to stimulate more selective fishing techniques, because it will be in the fisher’s interest to only catch the most valuable fish. However, by landing everything, this ban risks killing more fish than before. If a juvenile fish lives long enough after being discarded to spawn new fish, it should be given that opportunity. For this reason, the discard policy provides an important exception: if a certain species can be scientifically proven to have a high chance of survival, fish of that species should be thrown back after catch. Researchers at the Institute for Agricultural and Fisheries Research (or ILVO) in Ostend, Belgium are testing the most commercially important species of flatfish - plaice, sole and dab – for their likelihood of survival.”

Observer bias and RAMP

Cognitive bias (The Daily Omnivore, 2012)

Subjective scores for animal behavior can be biased by observer opinions about experimental treatment differences and resulting outcomes (Tuyttens et al. 2014). The research paper title expresses a fundamental bias of human perception and belief: “Observer bias in animal behaviour research: can we believe what we score, if we score what we believe?” The problem is to separate belief from observation. This may be accomplished by clearly defining and adhering to consistent protocols for behavior observation and analysis.

RAMP relies on subjective scoring for presence or absence of reflex actions or injury types. Control fish have a suite of reflex actions that are consistently and clearly present when tested for. When an observer begins to notice the weakening or complete loss of a reflex action, that action is scored as absent (impaired). There will be variation among observers in the decisions about when reflex actions are impaired and bias will vary with experimental protocol. 

Because RAMP is an aggregate vitality impairment index summed from control reflex actions and potential injury types, a RAMP score includes the observer bias for each included reflex action and injury. Close correspondence of RAMP scores and mortality is noted at low and high scores because observers clearly know when fish are active and when fish are severely injured and impaired. Relationship of mortality and RAMP is more variable at intermediate levels of impairment and mortality in part because observer opinion about impairment is more variable. To reduce observer bias, RAMP for a species must be designed to include reflex actions and injury types that can be clearly separated into present or absent scores. Also experimental treatments can be administered without informing observers.   

Vitality of a stressed fish is readily observed. We are primarily seeing the activity, responsiveness, and injury presented by the animal. The most widely used vitality index in commercial fisheries is for the halibut fisheries of the northeast Pacific Ocean (AFSC Observer Manual 2015), based on Appendices S-X for trawl, pot, and longline fisheries.  For trawl and pot fisheries, three levels of vitality (excellent, poor, and dead) are scored by observing injury types and spontaneous activity, startle response to touch, and operculum clamping. For longline fisheries, vitality is scored by observing injury types. Mortality rates are assigned to vitality impairment scores using tagging experiments (Williams 2014).

Vitality impairment codes (Benoît et al. 2010).

Benoît et al. (2010) constructed a fishery vitality index with four levels of impairment (excellent, good, poor, moribund) that are scored by observing injury types, spontaneous body movement, startle to touch, and operculum clamping. Their vitality index and the halibut vitality index use the progressive increase of injury and impairment of activity to score vitality impairment. Benoît et al. (2010) corrected for observer bias by using a random effects term in their statistical model. 

Reflex actions scored for presence or absence in RAMP for snapper (McArley & Herbert 2014).

The RAMP vitality index alters impairment scoring to only include presence or absence of a larger number of injury and reflex actions. This shift attempts to introduce more information about activity and injury types that may be associated with mortality and to reduce decisions about degree of impairment for individual activity and injury traits. Impairment is observed as a progressive increase in the number of reflex actions that become absent and the number of injury types that become present when compared to control animals. Because observer bias can be introduced in scoring, observer protocols must be well defined with clear rules for presence or absence of traits. Observer judgements about correspondence between experimental treatments and outcomes could also be eliminated by careful experimental design.

Human delayed mortality can be predicted using olfactory impairment

Olfactory impairment in humans was measured by error rate in olfaction tests. Increasing number of errors in olfaction tests were related to increasing 5-year mortality rates in a logistic regression (PLoS ONE). 

The human logistic relationship between olfactory impairment and 5-year delayed mortality is a powerful method for predicting delayed mortality and is similar to other animal RAMP relationships between reflex impairment, injury, and delayed mortality. Olfactory impairment can be easily measured in human and animal clinical settings and can easily and automatically be measured in aquaculture contexts by analysis of animal distributions and activity in rearing facilities. Given the fundamental nature of olfaction, one would expect the relationship between olfactory impairment and delayed mortality to be generally present among animal phyla and this can be tested in clinical and field settings.

Pinto et al. 2014 state, “We are the first to show that olfactory dysfunction is a strong predictor of 5-year mortality in a nationally representative sample of older adults. Olfactory dysfunction was an independent risk factor for death, stronger than several common causes of death, such as heart failure, lung disease and cancer, indicating that this evolutionarily ancient special sense may signal a key mechanism that affects human longevity. This effect is large enough to identify those at a higher risk of death even after taking account of other factors, yielding a 2.4 fold increase in the average probability of death among those already at high risk (Figure 3B). Even among those near the median risk, anosmia increases the average probability of death from 0.09 (for normal smellers) to 0.25. Thus, from a clinical point of view, assessment of olfactory function would enhance existing tools and strategies to identify those patients at high risk of mortality.”

(PLoS ONE)

The human study controlled for the mortality effects of age, gender, socioeconomic status, and race. Additionally, “We excluded several possibilities that might have explained these striking results. Adjusting for nutrition had little impact on the relationship between olfactory dysfunction and death. Similarly, accounting for cognition and neurodegenerative disease and frailty also failed to mediate the observed effects. Mental health, smoking, and alcohol abuse also did not explain our findings. Risk factors for olfactory loss (male gender, lower socioeconomic status, BMI) were included in our analyses, and though they replicated prior work [41], did not affect our results.” Note that the study did not control for effects of possible episodic exposure to toxins or injury that may result in temporary or permanent olfactory impairment not related to death.

Olfactory response is an involuntary response to a stimulus, and may be considered a reflex action. In the human study, presence or absence of smell detection for rose, leather, orange, fish, and peppermint were summed and related to delayed mortality. Olfactory responses to various substances can be scored as present or absent and summed to predict delayed mortality. In the same way, the RAMP method is an example of presence-absence scoring with summation of reflex impairment and injury scores to predict delayed mortality.  Measuring and summing whole animal responses, i.e., olfaction, reflex actions, and injury to stimuli is a powerful method for observing the effects of stressors and aging on delayed mortality.   

“We believe olfaction is the canary in the coal mine of human health, not that its decline directly causes death. Olfactory dysfunction is a harbinger of either fundamental mechanisms of aging, environmental exposure, or interactions between the two. Unique among the senses, the olfactory system depends on stem cell turnover, and thus may serve as an indicator of deterioration in age-related regenerative capacity more broadly or as a marker of physiologic repair function [13].”

Clearly, measurement and summation of presence-absence for whole animal involuntary characteristics (olfaction, reflex actions, and injury) is a powerful way to predict delayed mortality in humans and other animals.

Reflex impairment in dogs, birds, and turtles

Reflex impairment in animals treated by veterinarians (dogs, cats, birds, rabbits, and livestock) is widely tested as part of a neurological examination to determine the potential presence and location of neurological impairment. The neurological exam consists of tests on mentation, posture and gait, cranial nerves, proprioception, spinal reflexes, and sensory pain perception.  Detailed summaries of these test procedures can be found here and here. In the veterinarian context, results of these neurological exams are generally confined to determination of whether the nervous system is affected in a disease process and to provide an accurate anatomic diagnosis when the nervous system is affected.  Consideration of contributions to disease by neurologic, medical, and orthopedic sources are differentiated into separate testing protocols for the purpose of formulating diagnosis and treatment plans. 

 Clippinger et al. 2007

Vernau et al. 2007

The veterinarian sequence of neurological testing in dogs and cats has been applied to sea turtles.  Results of the study showed that many of the neurological methods for dogs and cats can be adapted for use in sea turtles. The authors concluded that a standardized neurologic examination resulted in an accurate assessment of neurologic function in impaired sea turtles and could help in evaluating effects of rehabilitation efforts and suitability for return to their natural environment. Another study made a detailed assessment of chelonian health that included measuring reflex impairment as part of emergency and critical care. Measured reflex actions included head lift, cloacal or tail touch, eye touch, and nose touch.

Freshwater turtles have been tested for reflex impairment in an effort to evaluate the effects of submergence and increased temperature in bycatch mortality of three species.

Stoot et al. 2013

The RAMP results from reflex impairment testing in fish and invertebrates suggest that the neurological and reflex state of an animal includes the effects of injury and infection when related to fitness outcomes such as recovery, vitality, morbidity, and potential mortality. This inclusion of fitness effects probably results from the fact that the RAMP method is a scoring system that expresses the proportion of whole animal impairment, calculated based on the presence or absence of a suite of reflex actions.  Shifting focus and perspective from individual mechanistic explanations for disease to comprehensive whole animal measures for vitality can help link reflex impairment with fitness outcomes.

Further study and reflection on human and veterinarian medicine approaches to neurological testing can probably inform selection of reflexes to be used in the RAMP approach for reflex testing.  The interaction of medical and RAMP perspectives for quantifying disease states may result in advances towards understanding how nervous system and reflex function can be a comprehensive indicator of disease and vitality states, combining the effects of injury, infection, and nerve impairment.

Scoring reflex actions

Once we have learned how to "tickle" animals to stimulate a variety of reflex actions, we can consider how to score these actions.  Individual reflex actions can be scored either as present or absent, or can be scored according to their strength of response. We might think that scoring a reflex action by its strength would be the most useful, as it gives a continuous range of values from full strength in large powerful animals through weaker in healthy smaller animals, to weak in stressed large and small animals, and absent in fully impaired large or small animals. A disadvantage of this strength scoring approach is that both animal vitality and animal size can control the strength of reflex action. We are interested in an index for vitality, not confounded by animal size, so using strength of reflex action is not going to be appropriate.

To factor out the effects of animal size, we are left with scoring a reflex action as present or absent.  This may initially be confusing, with the question remaining, is the reflex present or not? I use the "rule of doubt" which says that the reflex is present if it is strong and clearly evident. It is scored absent if there is a question about its presence, whether it is weak or not clearly evident.

Scoring an individual reflex present or absent only gives us a qualitative measure and we are looking for a quantitative measure of vitality. We then move from the perspective of scoring individual reflex actions to a whole animal perspective, which includes many reflex actions, arising from combinations of various physiological, neural, organ, and muscle systems. If we measure the presence or absence of several reflex actions, representing combinations of various metabolic systems in an animal, then we can combine these scores into an integrated measure of reflex impairment that varies continuously and reflects the integrated nature of metabolic systems included in an animal. The combined scores for reflex impairment are then used to construct a RAMP curve which models the effects of stressors on animals through a range of stress and reflex impairment, as related to potential mortality.

An advantage of using RAMP is that the animal tells its story of stress-related impairment using systems that naturally integrate function as a whole animal. The animal communicates its vitality state directly through the language of reflex actions in response to appropriate stimuli, without the confounding effects of size and motivation.

RAMP: from intuition to science

Lets begin with fish, but the discussion applies to all other animals that have reflex actions.  Every fisher, commercial or recreational, intuitively knows and expresses opinions about the vitality of their fish, either in the water or caught. Excitedly proclaiming fish on and then proceeding to catch the fish, admire its size, and then release, sell, or eat the fish. These intuitive observations are grounded in our sense of vitality that is an expression of activity and responsiveness.

Intuitive notions are great for telling fish stories and are notoriously fallible when the size or fight of the fish in question is described to other bystanders. But these notions can lead to a quantitative expression of animal vitality that is grounded in solid, repeatable, and predictive science. How do we do this?

Vitality can be an expression of activity, which is diminished in stressed, lethargic fish.  Stress is an adaptive response to stressors. When fish are stressed too much or for too long, they can become diseased or die, states that do not support healthy populations and species diversity. So this loss of vitality that we intuitively observe can have profound consequences. To understand and ameliorate these consequences, we need good quantitative science.

For the purposes of describing and quantifying animal vitality and its inverse, mortality, we can start with animals in good condition and health that have a full suite of reflex actions and then study how stressors impair reflex actions until the end point of death. We use the presence or absence of reflex actions because these are fixed involuntary actions that are directly related to vitality and not subject to the effects of animal size and voluntary, complex behaviors such as feeding, social interactions, predator-prey interactions, migration, and sex, which can be modified by temperature, light, food availability, motivation, avoidance, and attraction.

We use a calculated quantitative index of reflex impairment, RAMP, that combines the presence-absence scoring of several reflex actions. RAMP is an integrative index that communicates the vitality of a whole animal. Similar reflex-based indices are used in human medicine to evaluate general health, neurological condition, and potential outcomes for coma and other non-communicating patients, as well as for triage of emergency patients.

Identifying appropriate reflex actions is where the imagination expands. We have got to figure out how to "tickle" the animal. What stimuli make it respond in the fixed involuntary patterns we call reflexes? Appropriate stimuli and testing modalities depend on the size of the animal and the logistical constraints of the situation. There are many human examples for inspiration.

Lets look at reflex actions through a continuum of animal size and activity for examples.  This list is by no means complete. Reflex actions can be tested in fish larvae by observing free swimming animal startle, orientation, and avoidance in response to light, sound, food scent, and touching with a probe.  For juveniles, fish can be restrained and tested for body flex upon restraint where fish attempt to escape when restrained, dorsal fin erection in which the fins become erect when fish are restrained, operculum and mouth closure where the operculum or mouth clamps shut when lifted or opened, the gag response where the fish opens its mouth and flexes the body when the throat is stimulated and the vestibular–ocular response (VOR) shown by eye rolls when the body is rotated around the long axis. For free swimming fish, studied reflexes included orientation where the fish should normally be upright, righting reflex where the fish returns to an upright position and the startle response in which the fish shows rapid forward motion in response to stimuli. Adult fish can present special problems because of their strength and other approaches for free swimming fish are described in another post. Sharks and other dangerous toothy or spiny animals especially need imaginative approaches to testing reflex actions.

Once a suite of reflex actions can be consistently observed and easily quantified, then building a RAMP curve can be accomplished and quantification of reflex impairment, vitality, and prediction of mortality is made possible. The RAMP method and curves developed then allow for the systematic investigation of the effects of stressors and stress in animals and systems of chosen interest. RAMP results can be compared and contrasted with concurrent results from physiological and physical injury studies in an effort to synthesize multivariate solutions to a continuum of important basic and applied questions. These questions may include understanding reflex biology, stress biology, fisheries management, bycatch reduction, animal health, population dynamics, aquaculture practices, migration biology, reproductive biology, and conservation biology to mention a few. 

Why does RAMP work?

RAMP is a whole animal quantitative measure of health and vitality. It integrates several reflex actions that are combinations of neural and muscle function which are immediately responsive to the effects of stressors.  When an animal is exposed to stressors and becomes stressed, various physiological, organ, and behavioral systems respond in adaptive ways to compensate for the disturbance of stress. Initially these stress responses are beneficial, helping the animal avoid stressful situations and stimulating metabolism to support these adaptations. However if stress is prolonged, the animal begins to exhibit metabolic deficits and its health and vitality degrade.

An animal with disturbed states and degraded vitality can quickly become sick, moribund, and eventually die if stress persists at high enough levels. Prediction of animal death or recovery from stress requires measuring whole animal stress disturbances. Measuring disturbances of separate systems that make up the animal does not predict vitality and mortality because the whole animal is what dies, not the separate systems.

RAMP is a combination of several reflex actions that is an ideal predictor of whole animal vitality and mortality because it integrates the immediate effects of stress for the whole animal into involuntary fixed patterns of response that vary only with the vitality of the animal. If voluntary behavior is used as a predictor, other factors not related to animal vitality can control responses, making prediction of vitality difficult.  For example feeding and other social interactions can be controlled by motivation, resource availability, avoidance, and attraction. If component metabolic and organ systems are used as a measure, these do not reflect the whole animal vitality state because they exhibit peak responses to stressor intensities that are not related to stress levels in the whole animal.