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.