In 2016, 6.7 million tons of crustaceans came from capture fishery and an additional 7.8 million tons were produced by aquaculture. While fishery capacity plateaued [1
], output from aquaculture continue to rise [2
] to meet the growing demand for seafood. Despite this tremendous market size for crustaceans in the food industry, there is not a standardized method for slaughtering crustaceans. Due to increased awareness in crustacean welfare, the application of electric stunning is currently receiving more attention for the humane slaughter for crustaceans [3
]. Chilling in air and ice slurry are still the most common and practical ways of paralyzing and rendering crustaceans unresponsive. This study addresses the physiological impacts of chilling and electric stunning in key commercial species of crab, crayfish, and shrimp and focuses on bio-indices not commonly examined across species undergoing exposure to these stunning methods
The EFSA (European Food Safety Authority) concluded that decapod crustaceans like those subject of this study can experience pain and distress [3
]. Evidence to support the claim that crustaceans can experience pain is mainly based on behavioral observations [4
] and some more recent physiological measurements [9
]. Policy-makers’ response has recently resulted in Switzerland and a province in Italy establishing new regulations on the ways of killing lobsters which resulted in banning some practices, amongst which, the one of boiling it alive. Nonetheless, some scientists still argue that it is difficult to provide evidence that crustaceans have this emotional capacity and awareness [12
]. The conclusion that crustaceans can experience pain impacts both the scientific community and the seafood industry and this paper focuses on the latter. Several animal welfare organizations such as The Royal Society for the Prevention of Cruelty to Animals (RSPCA), the People for the Ethical Treatment of Animals, Animal Aid UK, Crustacean Compassion UK, and the Humane Society of the United States, advocate for increasing protection of crustaceans at the time of killing to minimize their perception of pain. Numerous approaches can be used to anesthetize and euthanize crustaceans such as freezing, superchilling (N2
gas), piercing of ganglia, salt baths (MgCl2
, electric stunning, chilling in air or ice slurry, and boiling; each has advantages and disadvantages in terms of efficacy and animal welfare. Applicable methods which function at high efficacy with fast immobilization to reduce potential stress of crustaceans are sought after by the industry [14
Electric stunning is well-established for stunning in finfish, and it seems to paralyze some crustaceans species (i.e., Cancer pagurus
, Homarus gammarus
, Astacus astacus
, and Astacus leptodactilus
], but evidence of the effectiveness of electric stunning in other crustaceans is scarce and shrimp have not yet been a subject of specific studies. Some drawbacks of electric stunning are the induction of seizures in the central nervous system (CNS), the formation of blood clots in fish [18
] and the spontaneous autotomy of limbs in crabs [8
Although cooling may seem more practical in damping neuronal function in heterothermic invertebrates, past studies have indicated that decreasing the temperature only reduced the basal metabolic rate, with sensory information still being detected, processed, and integrated neurally in a rhythmic pattern [9
]. In fact, some crustaceans (e.g. Panulirus japonicus
, Penaeus japonicus
, Homarus americanus
, and Ligia exotica
) have neurons which may detect cold since the neurons increase their firing rate in temperatures between 0.5 and 5.5 °C [10
]. The regulation of the heart in the three crustaceans under study is neurogenic, meaning that each beat is controlled by neural signals from innervating neurons of the cardiac ganglion. The rhythm of the neural activity is controlled by a central pattern generator within the central nervous system of the animals. When a threatening sensory stimulus is introduced there is an alteration in the central neural regulation output to the cardiac tissue. The rhythmic control in higher brain centers of crustaceans which controls cardiac, respiratory, and digestive functions tend to maintain different frequencies at varying temperatures [20
]. Cooling in ice slurry is recommended for tropical and temperate species that are susceptible to cold temperatures [23
]. However, recommended immersion times are long (20 min) and some argue [24
] that it is not yet demonstrated if the immersion induces paralysis but not anesthesia.
Stress, pain, and nociception are different states and it is complex to separate them from one another in invertebrates [13
], as such their physiological measurement is difficult. Measurements within invertebrates such as behavioral avoidance, hormonal changes, and mobilization of energy stores such as glucose are not sufficient to define one state from another [12
]. Nociception comprises the capacity of perceiving noxious stimuli via receptors and withdrawing from the stimulus through swift reflexes. “Pain” is an emotion that requires the capability of the animal to be aware of the noxious stimulus with the involvement of higher processing and consciousness [25
]. Evidence provided so far on crustaceans is in some cases is contradictory. In the 1950s and 60s the researchers Baker [26
] and Gunter [27
] first described ways of slaughtering crayfish and crabs in a humane way and initiated a discussion about nociception in crustaceans. One of the observations made by Gunter was that slow heating to 40 °C in water for large crustaceans did not appear to cause the animals stress and would result in death. The same conclusion was also reached in the study by Fregin and Bickmeyer [10
] but the EFSA [3
] lists boiling live crustaceans as one of the methods that are likely to cause pain and distress. Barr et al. [4
] demonstrated that irritating the antennae of prawn Palaemon elegans
triggered a “tail-flick” response and they press their antennae against the walls of the enclosure which could be a behavioral means to remove the irritant from the antenna. However, Puri and Faulkes [28
] found no behavioral or electrophysiological evidence that antennae contained nociceptors for extreme pH or benzocaine/ethanol. Carbon dioxide can be used to anesthetize crustaceans before euthanasia, but it is now known that crayfish will avoid water tainted with CO2
and that CO2
reduces pH in the water [29
]. Since crustaceans and insects appear to have a functionally similar autonomic nervous system as mammals [30
] it is not surprising that responses to an aversive stimuli would be analogous to those of vertebrates in moving away or retracing from the stimuli [5
Studies indicate that different stunning techniques cause different physiological responses to stimuli. Lactate levels increase almost three-fold in crabs that are electroshocked [36
]. Other compounds such as biogenic amines and neurotransmitters (i.e., serotonin, dopamine, and octopamine) rise in crabs with exercise causing alterations in behavior [37
]. However, one needs to be cautious in relating changes in the compounds in the hemolymph to one condition such as exercise, environment, or stunning procedure, as these compounds can vary with a molt cycle, circadian cycle, gravid status, social dominance, and health of the animals [15
]. Even insects show changes in levels of biogenic amines with environmental stressors and/or exercise [43
]. In cold (10 °C) exposed Drosophila melanogaster
both serotonin and octopamine concentrations decreased in the hemolymph [46
]. Crayfish gradually exposed to cold (20–21 °C to 15 °C for 1 week and one week at 10 °C) increased hemolymph concentration of octopamine 4-fold [46
]. No studies that we are aware of have addressed if a rapid exposure to cold would also raise octopamine or alter levels of serotonin. Since it is known that the levels of these compounds can increase quickly with exercise, it was feasible to expect some changes could occur quickly with rapid cold exposure. With electroshock we predicted an increase in the level of the serotonin and octopamine due to electrical stimulating the neurons to release these substances into the hemolymph. In contrast, with rapid exposure to cold we did not expect any change in the levels in the hemolymph due to decreased activity of the neurons to release these substances. In this experimental study design, we measured the concentration of these two commonly assayed compounds (serotonin and octopamine) among three crustacean species using the same measurements techniques with environmental changes to provide a baseline for other investigators and comparative studies.
The aim of this study was to determine the effectiveness of electroshock and thermal shock by ice slurry for the stunning the blue crab (Callinectes sapidus
), the red swamp crayfish (Procambarus clarkii
), and whiteleg shrimp (Litopenaeus vannamei
) through measurements of physiological responses. Physiological measures consisted of changes in heart rate (ECG), neural activity to external stimuli by alteration in the heart rate, and changes in the levels of biogenic amines within the hemolymph. The effects on skeletal muscle activity by electromyograms (EMG) of the large closer muscle in the chela were measured in crabs and crayfish. In finfish, the effectiveness of different stunning techniques could be demonstrated by the measurement of the electroencephalogram (EEG). However, due to the impractical nature of recording an EEG in crustaceans, as reliably performed in large fish, heart rate may be recorded as a reliable bio-index. The hearts of shrimp, crab, and crayfish are neurogenic, meaning that the rate of beating is indicative of the neuronal function [34
]. The words ‘paralysis’ is used to indicate absence movements but not absence of ECG and EMG measurements. The word ‘anesthesia’ indicates absence of neural function, thus absence of perception of sensory stimuli.
2. Materials and Methods
Experiments were performed using red swamp crayfish (Procambarus clarkii, Atchafalaya Biological Supply, Raceland, LA, USA), blue crab (Callinectes sapidus, food distribution center in Atlanta, GA, delivered to and bought from a local supermarket in Lexington, KY, USA), and whiteleg shrimp (Litopenaeus vannamei, Kentucky State University Aquaculture Research Center, Frankfort, KY, USA as well as from Belize Aquaculture Ltd., Mile 4 Placencia Road, Stann Creek District, Belize).
Throughout the study, midsized crayfish measuring 6–10 cm in postorbital carapace length (posterior dorsal surface of the orbital cup to the end of the carapace directly posterior to the eye cup) were used. The measures were made with calipers (Swiss Precision, Newton, MA, USA, 0.1 mm). The animals varied in weight from 12.5–25 g. They were individually housed in standardized plastic aquaria (33 cm × 28 cm × 23 cm, water depth 10–15 cm) with temperature maintained between 20 and 21 °C, weekly water exchanges, constant aeration, and dry fish food provided every 3 days (salinity 25–26 ppt; O2
at 7.4–7.6 mg/L). To ensure the vigor of the blue crab, they were held in a seawater aquarium prior to use for 3 to 5 days. All experiments were implemented in female adult crabs with a carapace width (from point to point) of 10–15 cm and a body weight of 140–225 g. The crabs were fed with frozen squid every 3 days (cannibalism also occurred) and the water temperature was maintained between 20 and 21 °C (salinity 0.2–0.5 ppt; O2
7.5–7.9 mg/L). Shrimp were raised at the Kentucky State University Aquaculture Research Center (KSU) for 85 days in aerated water between 27 and 28 °C and fed with commercial shrimp feed (salinity 15 ppt; O2
7.35–7.7 mg/L). The aquaculture system used was a modified biofloc system that allowed the growth of bacteria in the system to control water quality and detoxify waste products [48
]. Studies in Belize used shrimp raised in outdoor open ponds in a large-scale aquaculture farm. They were transferred to an open window laboratory ranging in water temperatures from 30 to 31 °C. Shrimp with a postorbital carapace length of 20 to 35 mm and body weight 23.5 g were used from KSU and 25–38 mm from Belize.
Care was taken not to use more animals than necessary for these studies. According to University of Kentucky Administrative Regulation (AR) 7:5, oversight applies to “all research, teaching, and testing activities involving vertebrate animals conducted at University facilities or under University sponsorship, regardless of the species or source of funding.” This AR follows The United States Department of Agriculture (USDA) Animal Welfare Act, the PHS Policy and the Guide definition of animal. With this in mind, The Institutional Animal Care and Use Committee (IACUC) review is not currently required in the US for the use of crayfish, crabs and shrimp in research.
2.2. Electromyograms (EMG) and Electrocardiocrams (ECG)
The preparation of the ECG and EMG leads is described in detail in text and video format in previous publications [50
]. In brief, insulated stainless steel wires (0.13 mm diameter; A-M Systems, Carlsburg, WA, USA) were inserted into the small holes made in the cuticle (Figure 1
). For heart rate measures in all three species the wires were placed through the dorsal carapace directly over the heart [52
]. To eliminate the risk of damaging internal organs, special attention was made on inserting only a short portion of wire (Figure 1
For ECG recordings, both wires were connected to an impedance detector (UFI, model 2991, 545 Main Street, Suite C-2, Morro Bay, CA, USA) which measures dynamic resistance between the leads. Subsequently, the detector was linked to a PowerLab/4SP interface (AD Instruments, Unit 13, 22 Lexington Drive, Bella Vista, New South Wales, Australia) and calibrated with the PowerLab Chart software version 5.5.6 (AD Instruments). The acquisition rate was set to 10 kHz. The calculation of the heart rate was accomplished by direct counts of each beat over short 10–20 s intervals and converted into beats per minute (BPM). The responsiveness of a sensory-CNS-cardiac ganglion neural circuit was assessed using a wooden rod to tap the dorsal carapace of the crab and crayfish inducing an alteration in the heart rate [32
]. A physical pinch by an experimenter using forefinger and thumb was periodically made on the telson of the shrimp to induce an ECG response.
The EMG myographic recordings were performed in crayfish and crabs using two stainless steel wires placed in the closer muscle in the chela (Figure 1
). A third wire located in the carpopodite region of the same limb served as a ground lead [54
]. Similar to the ECG recording procedure, the holes in the cuticle were formed and wires prepared, inserted, and fixed in the respective area spanning the closer muscle in its central region. Via a Grass AC preamplifier (P15; Grass Instruments, Astro-Med Industrial Park 600 East Greenwich Avenue, West Warwick, RI, USA) the potentials were detected differentially and acquired digitally as previously described [54
]. To elicit high frequency responses in the EMG signal, the crabs and crayfish were teased using a wooden rod placed in the jaws of the chela. Rubbing on the teeth of the chela produces the reflexive action to the motor neurons to produce the gripping response. Thus, a sensory-CNS-motor circuit is recruited by the rubbing action on the teeth of the chela [55
2.3. Ice Slurry
In order to test the physiological changes of cooling crustaceans, we placed the animals for 5 min in plastic boxes containing crushed sea water ice (shrimp and crabs) or freshwater ice (crayfish) which was the same water the animals were maintained in prior to experimentation. The temperature in the boxes was between 0 and 4 °C.
2.4. Electrical Stunning
Electrical stunning was performed using an AC source (60 Hz, 120 Volts, 20 amps) with wires directly from the wall outlet attached to two carbon rods (12 cm length × 1.3 cm dia.). The animals were transferred individually into a plastic chamber (17 cm × 12.5 cm) with the two carbon rods along each long side of the container submerged half the depth of the water level. This is so the rods were not resting on the bottom. Crayfish were shocked in a 1:1 mixture of freshwater:seawater (same type of waters described above). Initial trials with shocking crayfish in fresh water did not paralyze the animals. The 1:1 mixture enhanced electrical conductivity and resulted in paralysis within the 10 s window. Crabs and shrimp were shocked in seawater. Animals were electrocuted for 10 s and observed for behavioral changes. The animals were rapidly moved to their previous container for further measures of signals in the ECG and EMG traces. These animals were not previously exposed to any other experimental treatments besides electric stunning.
2.5. Hemolymph High Pressure Liquid Chromatography (HPLC) Samples
To evaluate if changes in hormonal levels might occur with crustaceans rapidly exposed to an ice slurry and electroshocking, approximately 0.5 mL hemolymph was drawn from six crayfish and six crabs and between 0.2 and 0.5 mL of hemolymph from six shrimp. The hemolymph was obtained directly in the hemocoel with an 18 gauge needle either in a ventral puncture close to the ventral nerve cord (shrimp and crayfish) or in the basal joint of the last walking leg of the crab. The only hemolymph samples from shrimp were from those at KSU. Control hemolymph samples were taken from animals exposed to their respective environment and temperature without being exposed to ice slurry and electrostunning. The hemolymph was mixed 1:1 in the tube containing the HPLC mobile phase and immediately frozen and stored at −80 °C until HPLC could be performed. The quantification of serotonin (5-HT) and octopamine levels in the hemolymph was accomplished through high pressure liquid chromatography with electrochemical detection (HPLC-EC). The samples were analyzed at the Center for Microelectrode Technology (CenMeT) and Parkinson’s Disease Translational Center of Excellence, University of Kentucky Medical Center, Lexington, KY, USA.
2.6. Statistical Analysis
All data are expressed as mean ± SEM. The rank sum pairwise test or a sign test was used to compare the difference of heart rate with exposure to ice slurry or electroshocking. The nonparametric tests were used because data were not normally distributed as there was no activity to measure in some conditions when heart rate stopped. ANOVA and posthoc analysis were also conducted on some data sets. This analysis was performed with SigmaStat software. A p-value of ≤ 0.05 was considered statistically significant.
This study highlighted the physiological effects in three commercially important species of crustaceans for cold ice slurry immersion and electric stunning as a potential means of the stunning in the seafood industry. Crabs showed the least response to chilling, demonstrated by maintaining a sensory-CNS-cardiac or sensory-CNS-skeletal muscle response with cold for 4 min, and did not decrease their heart rate as quickly or as much with chilling as did shrimp or crayfish. Crayfish followed in the rate of decreased HR in the same manner for the physiological responses as shrimp during the rapid cooling. Shrimp showed the fastest and most pronounced decrease in heart rate, as well as a sharp decrease of activity within a sensory-CNS-cardiac response when chilled, and a quick rise in heart rate upon warming. The decrease in neural activity from the cardiac ganglion driving the heart occurred in shrimp as soon as 5 s in some cases (Figure 3
B). Electric stunning for 10 s with 120 V AC did not produce burning marks on the crustaceans and like cooling, it did not kill the animals. All three-species appeared stunned as they did not show coordinated responses to stimuli after electroshock. Although results are not presented, shrimps were stimulated by touching the eye stalk and no response was evident. The bioindex of monitoring heart function indicated the heart was still beating although compromised after electric stunning. This finding is consistent with the results on electro stunning presented by Fregin and Bickmeyer [10
The EMG activity measures in muscles can detect the electrical activity related to muscle contraction; however, if the muscle is induced to contract only a low frequency of basal activity will be detected unless the animal induces a response. In this study, the EMG activity was induced by stimulating a sensory response by rubbing the teeth on the chela of crabs and crayfish [55
]. The sensory stimulation is integrated in the CNS to then activate motor neurons and appears to be reflexive in nature. The decrease in the activity with cold indicates this neural reflex is dampened and the sensory responses may not even be detected. Likewise, according to these results the sensory-CNS-cardiac system is assumed to be reflexive and is greatly dampened with cold, particularly for shrimp as compared to crabs. Thus, high neuronal function in processing the sensory input within the central brain (i.e., cerebral ganglion) and subesophageal ganglion and the production of a functional motor output is lacking. This type of autonomic reflex in altering heart rate is common from crustacean to higher mammals such as primates. Even though the heart in mammals is myogenic there is robust neuronal control, however a similar response is observed with a slight pause in the heart beat with an acute threatening sensory stimulus [30
]. Because this was reduced in shrimp with immersion in an ice slurry it may indicate that this approach reduces a perceived stimulus which would activate an autonomic response. This indicates that an ice slurry may be an effective method for stunning crayfish and shrimp without increasing nociception.
The ecology of the three species may help explain the reactions to ice slurry. Crayfish are known to maintain the sensory-cardiac response at 10 °C and even down to 5 °C for over a week when slowly acclimated to cold for 2 weeks [9
]. Procambarus clarkii
is native to the southern United States but is an invasive species now found in different regions from the Great Lakes to the Scandinavian Peninsula and Japan [68
]. This wide distribution in the wild may have come about from the ability of this species to acclimatize to warm and cold environmental conditions. Likewise, the blue crab range is from Nova Scotia to Argentina and exists in Asia and Europe [69
]. This crab is considered eurythermal and may well be able to resist decreasing neural function upon acute cold exposure due to cold acclimatization. More thorough studies of Procambarus clarkii
and Callinectes sapidus
with physiologic measures would help to uncover their abilities to function in acute and chronic cold conditions. In contrast, the shrimp Litopenaeus vannamei
has a tropical and subtropical distribution, and it is not recognized as an invasive species despite being widely introduced to non-native locations for commercial purposes [68
]. This may be partly due to its inability to adapt to low temperatures. The tropical prawn species Macrobrachium rosenbergii
is also very sensitive to cold and loses the sensory-cardiac response with acute cold. This prawn species will die with acute or slowly induced cold to 5 °C [9
Electric stunning is harder to induce with consistency than cooling in an ice slurry due to the nature of electricity. The resistance of the water being fresh or seawater conducts the current differently around an animal in a holding tank. In addition, the salt concentration of an animal’s hemolymph, size of animal, and dimensions in respect to the electrical conducting leads will result in different amounts of current each animal will be exposed to. Since the resistance or impedance of the animals vary, there are several parameters to be considered with electrostunning. It is complex to deliver a consistent and appropriate level for preserving quality of the muscle and inducing paralyzes with different species and sizes of crustaceans [8
]. If an animal is suspended in the water or in air, placing the contacting leads needs consideration to provide consistent electrical conduction through the animal. We provided enough alternating current at 120 V to make the animals motionless and to be visually assumed dead. However, with shrimp, upon close observation through the almost translucent cuticle, it was easy to observe the scaphognathite within the prebranchial chamber to monitor the ventilatory rate [51
]. This ventilatory organ continued to beat after the electric stunning and one can see the heart beat with good lighting from the side of the animal. Thus, even though the animal appears physically paralyzed with electric stunning, physiological functions like breathing and heart beating are still active. Further analysis is needed to determine the ability to process external stimuli by measuring changes in heart rate and how long and intense an electroshock is needed for inducing lack of responsiveness of the sensory-CNS-cardiac ganglion. We postulate that electroshock induces effective stunning as the animals take about 5 to 10 min to regain some ability to move or swim after application of the current. This would provide some time for a processing facility to handle the animals before slaughter or placing them on ice to die, but without a standardized length of time for unresponsiveness, the handling would have to be closely monitored to ensure welfare.
One may visually monitor the beating of the scaphognathite more readily in crayfish and crab if part of the lateral cuticle would be removed [70
]. The beat rate can be monitored with impedance measures as performed to record heart rate in this study, but the signals vary greatly depending on the placement of the recoding leads [50
]. The observations that electrical shocking induced paralysis was similarly reported by Fregin and Bickmeyer [10
] in studies with crayfish and lobsters. Edible crabs of Norway were shocked for 10 to 20 s at 220 V and still many of them were still moving [8
]. More thorough studies in the timing and a means of delivering a consistent electrocution for different sizes and species of crustaceans without compromising the muscle by heating or scarring are needed [8
]. Wild caught animals will likely present a wider size range than farmed crustaceans for food processing and crabs and crayfish may even be missing some of their limbs which could vary the capacitance of the animal which affects the electric stunning.
The concentration of the biogenic amines serotonin and octopamine are higher in crabs than crayfish and shrimp. The decrease in octopamine in the crayfish with rapid cold exposure was unexpected since an earlier study showed an increase in octopamine in crayfish during a prolonged cold exposure of 2 weeks [46
]. Perhaps a longer time is needed for the levels to demonstrate changes. Serotonin levels decreases for cooling and electric stunning in crayfish but not statistically significant in the other species. The biological reason this decrease is not clear and the mechanism driving the decrease in the crayfish has not been established. Further investigations are needed for establishing longer term monitoring of biogenic amine levels at various times after cold shock and electric stunning. The hemolymph measures of serotonin and octopamine are not very telling for the paradigms used in this study except that crabs apparently have higher concentration of both compounds in their hemolymph and that there appears to be a lot of variability among animals under the same conditions. The low levels in hemolymph of crayfish and shrimp made it difficult for comparisons. An approach in the future which may be beneficial would be to obtain as much hemolymph as possible and lyophilize it and resuspend in a smaller volume to concentrate the compounds or to make use of mass spectrometry-based approaches to screen peptides as well [71
]. Even the handling of the animals to draw hemolymph samples may result in the animals altering the levels of the compound to be measured [14
]. Both serotonin and octopamine can increase central neural activity in crustaceans and insects and in some cases synaptic transmission at neuromuscular junctions. Such biochemical measures in crustaceans are scant for effects of potential stressors of different types such as electric stunning or cold shock.
Animal Welfare Implications
These studies indicate that depending on the species of crustacean the effects of different stunning procedures will vary, and also that visual observations on their own are in some cases unreliable to assess stunning effectiveness. A few seconds of exposure to electric stunning does paralyze shrimp, crab, and crayfish which may improve the welfare during slaughter as the next step. It is likely that electroshock renders these crustaceans unable to perceive and elaborate stimuli for a period of time after shocking is applied but we did not test the perception of tapping/pinching stimuli on the animals to measure heart rate changes during or right after electric stunning due to human safety issues with the capacitance charge still present in the tanks and further investigation is needed.
With exposure to ice slurry, the responses vary. Anesthesia takes longer for crabs as noted in the responsiveness in pauses in heart rate to external stimuli. Most of the crayfish with acute cold exposure are also insensitive to sensory stimuli after 2 min when their heart rates have decreased substantially but crabs maintained central neural processing for cardiac and skeletal muscle reflexes. Crayfish and shrimp responsiveness to sensory stimuli stopped after just a few seconds. As shown in the supporting videos, suggesting a means of establishing unresponsiveness by verifying absence of resistance to handling and ease in manipulating body parts, such as the tail, does not seem to be validated, as tail flipping of isolated abdomens produces a coordinated movement even though in the intact animal the heart has stopped and is nonresponsive to sensory stimuli while tail flipping continues to occur.
Future research should determine how long shrimp should be maintained in ice slurry so they do not recover when they are warmed up. It may not appear that shrimp lose sensory-CNS-skeletal muscle activity when put in an ice slurry since exposing them to an ice slurry induces them to tail flip and contract violently in about 1 to 2 min. However, it appears cooling in ice is an effective anesthetic for sensory perception.
As legislation on the protection of animals at the time of death becomes more complex and more attention is drawn to animal welfare of all species, including crustaceans, more research to ascertain what the physiological responses are to different stunning methods will be required to support behavioral studies. The Scientific Panel on Animal Health and Welfare (AHAW) [3
] endorses chilling in an ice water slurry, chilling in air, immersion in clove oil bath, and electroshock as humane means to kill decapods with minimal distress. However, this panel also raised the point that pain and distress in crustaceans may occur by placing animals in cold water and heating the water to boiling point. More recent studies of crayfish by Puri and Faulkes [76
] confirmed the presence of nociceptors responsive to heat, but did not find evidence of nociceptive responses to low temperatures. The same panel and the RSPCA [23
] also states that all crustaceans should be immersed in ice slurry for at least 20 min to reach unconsciousness whereas here we demonstrate that for crayfish and shrimps a few seconds are enough to anesthetize them. This is why recommendations should be based on rigorous scientific inquiries, rather than solely on behavioral observations or assumptions [12
]. In the future, policy makers may benefit in adopting some of the presented methods for assessing perception of obnoxious stimuli in crustaceans to ensure animal welfare, giving consumers confidence that they are purchasing ethically produced seafood products.