Physiological Changes as a Measure of Crustacean Welfare under Different Standardized Stunning Techniques: Cooling and Electroshock

Abstract

Stunning of edible crustaceans to reduce sensory perception prior and during slaughter is an important topic in animal welfare. The purpose of this project was to determine how neural circuits were affected during stunning by examining the physiological function of neural circuits. The central nervous system circuit to a cardiac or skeletal muscle response was examined. Three commercially important crustacean species were utilized for stunning by immersion in an ice slurry below 4 °C and by electrocution; both practices are used in the seafood industry. The blue crab (Callinectes sapidus), the red swamp crayfish (Procambarus clarkii), and the whiteleg shrimp (Litopenaeus vannamei) responded differently to stunning by cold and electric shock. Immersion in ice slurry induced sedation within seconds in crayfish and shrimp but not crabs and cardiac function was reduced fastest in shrimp. However, crabs could retain a functional neural circuit over the same time when shrimp and crayfish were nonresponsive. An electroshock of 10 s paralyzed all three species and subsequently decreased heart rate within 1 min and then heart rate increased but resulted in irregularity over time. Further research is needed to study a state of responsiveness by these methods.

Keywords: blue crab; crayfish; electric stunning; euthanasia; icing; shrimp.

Figure 1

Placement of recording leads for measuring heart rate in an electrocardiogram (ECG) and skeletal muscle activity in an electromyogram (EMG) for the crab (A), crayfish (B) and shrimp (C). The two differential EMG leads to record the EMG activity of the closer muscle in the chela were placed ventrally in the propodite segment. A third lead is placed under the cuticle in any of the more proximal segments to serve as a ground lead. The ECG leads for the crab span the heart laterally for the best ratio in signal to noise for the recordings, and similar lead placements are made for the crayfish. The ECG leads for the crayfish and shrimp are placed in an anterior-posterior arrangement for obtaining the best signals. Modified figure from Wycoff et al. [53].

Figure 2

Habituation rate in tail flipping for shrimp (Belize cohort) with repetitive pinching on the telson every 30 s. On average (average +/− SEM) the animals could be pinched 16 times before habituating (30.5 °C). Ten individual shrimp were used (average +/− SEM). FLIP is a tailflip and NO FLIP is when no tail flip is observed.

Figure 3

Representative electrocardiography (ECG) trace obtained from a shrimp (Belize cohort) while being immersed in a sea ice slurry (<4 °C) and during recovery. (A) The ECG trace before and over the short exposure to the ice slurry as well as the return to warm water is shown. (B) The rate of change in heart rate upon immersion in a sea ice slurry bath (arrow) is rapid. Also, note the rapid decrease in the amplitude of the signal. The shrimp was transferred from one bath (30.5 °C) to the ice slurry bath (<0.4 °C) within 2 s. The ECG trace after being in ice slurry for 1 min and 30 s is shown (C). Upon moving the shrimp from ice slurry back to warm water (arrow) the rate and amplitude rapidly starts to recover (D). All traces are shown with the same gain in signal but slightly different time scales as illustrated.

Figure 4

The effect of cold on heart rate for crabs, crayfish and shrimp. (A) Crabs decreased their heart rate upon being submerged over a 5 min period in a sea ice slurry (<4 °C). Their rates increased rapidly when placed back in warm seawater (N = 6). (B) Crayfish decreased heart rate as well upon being submerged in a fresh water ice slurry for 2 min and increased heart rate upon being returned to warm water (N = 6). (C) Five of six shrimp in the Kentucky facility decreased heart rate to no detectably beats in 30 s and within 4 min all five of the six shrimp ceased their heart rate when submerged in a sea water ice slurry (27–28 °C to <4 °C). All but one increased heart rate upon returning to warm seawater. (D1) The shrimp at the facility in Belize had, on average, a higher basal heart rate but also rapidly decreased heart rate upon being submerged in the sea water ice slurry (30–31 °C to <4 °C). In two of the eight the heart rate stopped in 30 s and four stopped with 2 min. The time spent submerged in the ice slurry varied from 2 to 5.5 min before returning the shrimp to warm seawater for the studies in Belize (D2). Note all the rates increased rapidly in warm water. Various end point measures were obtained for various animals. The rates showed a steady increase over time.

Figure 4

The effect of cold on heart rate for crabs, crayfish and shrimp. (A) Crabs decreased their heart rate upon being submerged over a 5 min period in a sea ice slurry (<4 °C). Their rates increased rapidly when placed back in warm seawater (N = 6). (B) Crayfish decreased heart rate as well upon being submerged in a fresh water ice slurry for 2 min and increased heart rate upon being returned to warm water (N = 6). (C) Five of six shrimp in the Kentucky facility decreased heart rate to no detectably beats in 30 s and within 4 min all five of the six shrimp ceased their heart rate when submerged in a sea water ice slurry (27–28 °C to <4 °C). All but one increased heart rate upon returning to warm seawater. (D1) The shrimp at the facility in Belize had, on average, a higher basal heart rate but also rapidly decreased heart rate upon being submerged in the sea water ice slurry (30–31 °C to <4 °C). In two of the eight the heart rate stopped in 30 s and four stopped with 2 min. The time spent submerged in the ice slurry varied from 2 to 5.5 min before returning the shrimp to warm seawater for the studies in Belize (D2). Note all the rates increased rapidly in warm water. Various end point measures were obtained for various animals. The rates showed a steady increase over time.

Figure 5

The change in heart rate with a sensory stimulus before (1), during (2), and after (3) immersion in ice slurry. Crab (A1), crayfish (B1) and Belize shrimp (C1) show a marked response in the ECG trace when pinched on the telson (shrimp) or tapped on the dorsal carapace (crab and crayfish) before being immersed in a sea ice slurry. When immersed in cold water no changes could be detected in the ECG with a sensory stimulus for the shrimp. Note the amplitude after ECG trace for the crayfish and shrimp is reduced after just 30 s in the cold and no observable sensory-CNS-cardiac ganglion response can be detected (B2,C2). However, for crabs there was still some responsiveness to a sensory-CNS-cardiac ganglion response even after 2 min in the ice slurry (A2). The heart rates of all animals rebounded quickly when placed back in the original water, and after 2 to 5 min all species showed responses to the same type of sensory stimuli (A3,B3,C3). The traces shown are of the same gain and from the same animal during the different paradigms.

Figure 6

ECG traces before and immediately after electric stunning as well as after 2 to 3 min after electric stunning. (A) An ECG trace for a representative crayfish before and after the electric shock indicates the large change that occurs right after the shocking is over. (B1) Crab, (C1) crayfish, and (D1) Belize shrimp had pronounced rhythmic rates prior to electric stimulation. Arrhythmia or no measurable rate occurred immediately after electric stunning for crab (B2), crayfish (C2), and Belize shrimp (D2). In some cases, the arrhythmic rate persisted, and all animals showed an altered amplitude and shape in the ECG traces after electric stunning (crab, (B3); crayfish, (C3); Belize shrimp, (D3)). The traces shown are from the same animals during the different time periods. The 1 s scale bar applies to all traces.

Figure 7

The effect of 10 s at 120 V with AC electric stunning of crab (A), crayfish (B) and shrimp (C). Rates were able to be obtained immediately after stunning for most animals except for some of the crayfish (dashed lines) due to electrical saturation of the amplifier during the stunning. In only one animal in each species did the heart stop beating after the 10 s window; in all but one case, for all three species, the rates increased after the electric stunning.

Figure 8

EMG traces of the closer muscle. Recordings from the chela of crab (A) and crayfish (B) before (A1,B1), during immersion in an ice slurry (A2,B2) and after (A3,B3) being returned to their original holding tanks. The closer muscle was stimulated to contract by rubbing a wooden rod on the teeth on the inside of the jaw of the chela to stimulate a sensory-CNS-motor nerve circuit. The traces shown are during the time of sensory stimulation. Note the responses are dampened while the crab (A2) and crayfish (B2) are in the ice slurry for 2 min. Upon returning the crab (A3) and crayfish (B3) to their original warmer water, the EMG traces regained their strength and maintained firing while the jaws were clamped on the wooden rod (B3). The traces shown are from the same crab or crayfish during the different situations. The 20 s scale bar applies to all traces.

Figure 9

The quantification of octopamine and serotonin in the hemolymph of crab, crayfish, and shrimp at holding temperature and with immersion in ice slurry or after electric stunning. Measures were made with HPLC analysis after being immersed in an ice slurry for 5 min or after electric stunning for 10 s (120 V with AC current, 20 amps). Six animals were used for each condition. A mean (+/− SEM) value of ng/mL of hemolymph is reported for each paradigm. Statistical analysis on detectable levels for the six crayfish as compared to not being able to measure the levels with cold shock and electric stunning for crayfish produces a non-normal distribution but significant difference by Kruskal–Wallis test (p = 0.01). Serotonin levels were lower for cold shock (p = 0.028, N = 6, Holm–Sidak) and electric stunning (p = 0.004, N = 6, Holm–Sidak) in crayfish as compared to 20 °C but not for crabs and shrimp.