RAMP and bonefish recovery from capture stress

Bonefish are typically captured in a high value capture and release sport fishery.  Released bonefish can be subjected to high rates of predation if predators are present and stress levels are high.  Recovery from capture stress is important for survival and maintenance of bonefish fishery stocks.  Recovery technology including holding bonefish in bags or live wells prior to release has been evaluated by Brownscombe et al. 2013.  They used measurements of reflex impairment (RAMP), locomotory activity, and predation to test recovery from capture stress and survival after release.

Bonefish Key West Flats Fishing

Practical application of RAMP was suggested by Brownscombe et al. 2013. "Bonefish anglers may be able to use RAMP to assess bonefish condition, and make educated decisions on whether to release the fish, or retain it for a short period to facilitate recovery. Likewise, if water temperatures and bonefish impairment scores are very high, responsible anglers can recess until conditions are more favorable."

Here are some excerpts from their paper regarding the validation of RAMP to measure capture stress and predict delayed mortality after release:

"The primary objective of this study was to evaluate the effectiveness of retaining bonefish in recovery bags for reducing short-term locomotory impairment when subjected to angling-related stressors, and whether potential improvements in swimming ability translated to increased survival.

Reflex indicators have recently been deemed effective predictors of mortality (Davis, 2010; Raby et al., 2012), and could be used by anglers to evaluate in which instances fish would benefit from recovery. We predicted that fish retained in recovery bags would exhibit lower reflex impairment, as well as higher locomotory ability and survival than those immediately released.

We validated the use of reflex action mortality predictors (RAMP) (Davis, 2005, 2010) to assess bonefish vitality after 0, 2, 4 and 6 min of air exposure. The 0-minute assessments (n = 30) occurred prior to air exposure on fish from all treatments, while bonefish in the 2-minute treatment (n = 20) were those used in recovery bag experiments (see below), and 4, 6 min treatments (n = 5) were conducted on alternate fish. Five predictors were measured; tail grab, equilibrium (orientation), body flex, head complex, and vestibular-ocular response (VOR). These predictors were chosen because Raby et al. (2012) found that they were strong predictors of coho salmon (Oncorhynchus kisutch) mortality after being caught in commercial nets, and all these predictors can be easily and quickly measured by bonefish anglers. RAMP was assessed in the same manner by Raby et al. (2012). The presence of a tail grab response was assessed by grabbing the fish's tail while it is submerged in water; it was considered impaired if the fish did not attempt to swim away from the handler. Equilibrium was assessed by rolling the fish upside down in water; impairment was indicated when the fish was unable to right itself within 3 s. Body flex was tested by holding the fish by the middle of the body in air; it was considered impaired if the fish made no attempt to struggle free. Head complex was considered impaired if while holding fish in air, a regular pattern of ventilation of the fish's operculum was not observed for at least 5 s. VOR was assessed by rolling the fish back and forth in air; it was considered impaired if its eyes did not roll to maintain the same pitch and track the angler. Higher RAMP scores indicated greater impairment.

Based on the responsiveness of bonefish to the RAMP indices, we used them to evaluate the utility of the recovery bags. After air exposure, bonefish released with accelerometers had similar RAMP scores between immediate release (2.8 ± 0.14) and recovery (2.7 ± 0.14) treatments. However, after retention in a recovery bag for 15 min, all bonefish had RAMP scores of zero (i.e., full recovery).

Our results demonstrate that retaining bonefish in recovery bags for 15 min reduced locomotory impairment upon release during the critical time period where most predation occurs, and this practice has the potential to increase survival after catch-and-release angling. Presumably, retaining bonefish in a live well with ambient oxygen levels (Shultz et al., 2011) would have a similar benefit if an angler had access to a boat.

The five impairment indicators we tested on bonefish provided a gradient in impairment scores that related to the degree of stressor (i.e., 0–6 min of air exposure). RAMP scores have been correlated with stressor duration and mortality for a number of fish species (Davis, 2005, 2007; Davis and Ottmar, 2006; Humborstad et al., 2009; Raby et al., 2012). Indeed, the duration of a stressful event increases the level of physiological disturbance in bonefish (Suski et al., 2007; Donaldson et al., 2008), while longer handling times and air exposure durations result in higher post-release predation rates (Danylchuk et al., 2007a). In this study, bonefish that were equipped with accelerometers exhibited moderate impairment scores after 2 min of air exposure, while no impairment was detected after 15 min of retention in a recovery bag, and fish from the recovery treatment exhibited significantly higher levels of activity upon release. Therefore RAMP scores appear to be a good indication of bonefish vitality. 

The impairment indicators tail grab, equilibrium, and body flex were the first to become impaired in bonefish, and impairment levels within these predictors did not vary with increased stress duration. This was likely because bonefish were all highly impaired at the lowest level of stress we inflicted. Indeed, a previous study found roughly that 50% of bonefish lose equilibrium after angling events (Danylchuk et al., 2007a), while 95% of bonefish lost equilibrium after simulated angling stress (2 min of air exposure) in this study. These three predictors may provide an indication of impairment levels with lesser degrees of stress. Head complex was the next to become impaired at 4 min of air exposure, followed by VOR at 6 min. Therefore head complex and VOR predictors are indicative of very high levels of physiological disturbance in bonefish. This predictor-specific pattern of impairment in bonefish is nearly identical to that of coho salmon (see Raby et al., 2012)."

Mortality sources and the limits of RAMP

Exposure of animals to stressors can result in changes to physiology, behavior, and injury that can result in stress, impaired reflex actions, morbidity, and delayed mortality.  Stressors in fishing, aquaculture, net penning, aquarium trade, research settings, and other ecosystems are present in a number of ways as departures from nominal temperature, light, oxygen, food, xenobiotics, injury, crowding, disease, social interactions, and predators.

While reflex impairments and RAMP can accurately assess vitality and stress levels and predict delayed mortality, these measures are solely dependent on the internal state of an animal at the time of observation.  When other external stressors and sources of mortality are present after an animal is assessed with RAMP, predictions of delayed mortality may not be accurate.

In open, wild ecosystems, important sources of mortality in animals can be predation, lack of food or feeding ability, and impairment of social behavior that is protective (schooling, shoaling, and shelter seeking).  The presence of any or all of these stressors can alter mortality rates predicted by RAMP.  Use of RAMP for predicting delayed mortality in open systems is probably limited to short term delayed mortality.

In closed, human managed ecosystems, external sources for delayed mortality can be controlled and eliminated after RAMP measurements and RAMP predictions of delayed mortality can be accurate over longer time periods.

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.

Choices for testing reflex impairment in RAMP

Reflexes can be tested in unrestrained or restrained animals. Tests can be adapted to experimental or operational conditions needed in specific situations. A reflex action is scored not impaired (0) when strong or easily observed and scored impaired (1) when not present, weak, or there is doubt about presence. Reflex impairment scores for an individual animal are then summed and divided by the total observable impairments possible to calculate proportion impairment. Impairment score is then correlated with mortality to produce RAMP. RAMP can be expanded when appropriate to include testing for reflex impairment, barotrauma, and injury.

Below are some examples of previous reflex testing in fish, crustaceans, and turtles. Davis 2010 has shown several types of fish reflexes with impairment after stress induction. In 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 (Lutnesky and Szyper 1990; Artigas et al. 2005; Davis and Ottmar 2006). In restrained fish, studied reflexes included body flex upon restraint where the fish attempts to escape when restrained, dorsal fin erection in which the fins become erect when the fish is 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 (Trumble et al. 2000; Davis 2007). Other studies of reflexes in free swimming fish have included atonic immobility, dorsal light reaction, and optomotor and optikinetic responses (Douglas and Hawryshyn 1990; McCormack and McDonnell 1994; Wells et al. 2005; Hasegawa 2006).

Raby et al. 2012 used a reflex impairment index for coho salmon modified from the previously developed RAMP method (Davis 2005, 2007). Immediately prior to release, all tagged and biopsied fish were tested for the presence of five reflexes that were consistently present in control, excellent condition fish. Each reflex was assessed categorically (0 = unimpaired, 1 = impaired) in a conservative matter – that is, if the handler had doubt as to whether the reflex was present, it was recorded as being impaired. Reflexes tested were the following: tail grab, body flex, head complex, vestibular-ocular response (VOR) and orientation. Presence of the tail grab response was assessed by the handler attempting to grab the tail of the fish with the fish submerged in water (in a fish bag or holding trough); a positive response was characterized by the fish attempting to burst-swim immediately upon contact. The body flex response was tested by holding the fish out of water using two hands wrapped around the middle of the body. The fish actively attempting to struggle free was characterized as a positive response. Head complex was noted as positive if, when held out of water, the fish exhibited a regular pattern of ventilation (for ∼5 s) observable by watching the opening and closing of the lower jaw. VOR was observed by turning the fish on its side (i.e. on a lengthwise axis) out of water. Positive VOR was characterized by the fish’s eye rolling to maintain level pitch, tracking the handler. Finally, upon release, each fish was placed upside-down in the river just below the surface: a positive orientation reflex was noted if the fish righted itself within 3 s. The entire reflex assessment took ≤ 20 s to complete and was always conducted on fish upon release. If a fish was too vigorous to allow researcher handling and assessment of reflexes, it was assigned an unimpaired status for all reflexes. The reflex actions included in our protocol are thought to be sufficiently varied that they involve different neurological pathways and/or muscle groups such that there are no redundancies. For example, some of the reflexes are part of the autonomous nervous system (head complex, i.e. respiration), while others clearly are not (tail grab, body flex). Moreover, using this RAMP protocol with Pacific salmon, no two reflexes in the suite of five are consistently present/absent together (see Results; G. Raby, unpublished data). From the reflex results for each fish, we calculated a RAMP score: a simple proportion of the five measured reflexes that were impaired in an individual fish (0 = no reflexes impaired, 1 = all reflexes impaired; Davis 2007).

Humborstad et al. 2009 in Atlantic cod.

Barkley and Cadrin 2012 in yellowtail flounder.

Campbell et al. 2009 in red snapper.  Following exposure to rapid decompression, and removal from the hyperbaric chamber, external symptoms of barotrauma were noted and the reflex responses tested.  The barotrauma – reflex(BtR) score developed was modified from the RAMP procedure developed by Davis and Ottmar (2006). All barotrauma incidences observed and reflex responses tested were categorical in nature (1 = unimpaired state, 0 = impaired state). Fish were examined for a suite of barotraumas, including expanded abdominal cavity (tightened air bladder), stomach everted and protruded from the oral cavity, intestine protrusion out of the anus, exophthalmia (eyes bulging), subcutaneous hemorrhaging, and activity level. Reflex response testing was performed out of the water over 1 min, with the fish restrained so that each test could be done in isolation. Reflex responses tested included gag, opercular, dorsal spine, vestibular-ocular (VO), and tail-flex responses. The gag response was tested by inserting a narrow probe into the oesophagus; a positive response was noted as involuntary-muscle contractions intended to dislodge the probe from the oesophagus. The opercular response was measured by observing whether the fish actively attempted to ventilate the gills (gilling). Active gilling was considered a positive response. The dorsal-spine response was tested by moving a probe over the dorsal spine, causing it to fold back. Positive dorsal spine response was noted when spines returned to an erect defensive position. VO response was observed by turning the fish along its lateral axis. Positive VO was noted when the eye rotated within the orbit and refocused on a fixed position (the observer). Tail-muscle flex was tested by inserting a syringe needle into the hypaxial musculature of the subject. Positive tail-flex response was noted if the muscle contracted. If the subject demonstrated hypaxial-muscle contraction (tail flapping) before insertion of the needle, it was considered positive and no needle insertion took place. To calculate the BtR score, the total number of barotraumas and reflex responses present was summed, divided by the total number possible, and subtracted from 1 [BtR = 1 - (summed individual responses/total responses possible)]. A BtR score close to 0 indicated a fish with low impairment, and a score close to 1 indicated reflex impairment or elevated level of trauma. Following BtR observations, the subject was placed in a test arena for predation simulation and measurement of performance responses. For the thermocline-exposed fish (T), the test arena had previously been heated 7oC above ambient, and for the non-thermocline-exposed fish (NT), it was kept at ambient temperature. Predator attack was simulated from behind a screen by rapidly thrusting a dipnet directly at the lateral surface of the fish. Predator simulations were administered at 0, 5, 10, and 15 min post-decompression, which allowed investigation of the time-course of impairment and estimation of recovery times. Response variables measured were closest simulated predator approach distance (AD, cm), maximum burst swimming speed attained (BSS, cm/s), and the amount of time the fish spent reacting to the stimuli, or the duration of the response (Dur, s). A video camera (Sony DCR-TRV117, 32 frames/s) was mounted above the testing arena to film the sequence of predator attack simulation and subject response. A grid was placed on the bottom of the test arena to assist in calculations of AD and swimming speed. 

Stoner 2012 in crustaceans.

Stoner 2012 in spot prawns.

Stoot et al. 2013 in turtles.