Cooling and freezing as methods for anesthetizing or euthanizing amphibians and reptiles have been discussed since at least the late 1980s.1 Historically, most authors have recommended against the use of these practices1–7 because of the lack of evidence that hypothermia results in analgesia, concerns that the reduced mobility or torpidity associated with hypothermia would result in an inability to react to aversive stimuli, and worries that ice-crystal formation in the tissues could potentially result in pain.1,4–6
Cooling and freezing are recognized as acceptable methods for performing certain procedures requiring local anesthesia and are used for cryosurgical procedures in humans8–11 and some nonhuman animals.12 However, this does not imply that these methods are suitable for whole-body anesthesia. Studies13,14 of adult zebrafish (Danio rerio) suggest that rapid cooling in a laboratory environment is a comparatively humane process for providing anesthesia but also indicate that rapid cooling is not recommended for invasive procedures. Hypothermia has been suggested for anesthesia of adult Pacific salmon (Oncorhynchus gorbuscha),15 but the authors of that report cautioned against generalizing their findings to other species. The AVMA Guidelines for the Euthanasia of Animals5 suggest that “[r]eptiles and amphibians can be euthanized by rapid freezing when it results in immediate death,” but warn that this is applicable only for animals that weigh < 4 g (0.1 oz) and should not be used for species that have adapted freeze-tolerance strategies. Moreover, the guidelines state that many recommendations for amphibians and reptiles are extrapolated from information available for mammals, suggesting that the reliability of the information is limited.
In contrast, 2 recent articles16,17 have revisited the issue of using cooling and freezing (or cooling then freezing) as methods of anesthetizing or euthanizing amphibians and reptiles. Both articles suggested that these methods are humane and convenient in laboratory environments. Both also emphasized animals used for research purposes, but the authors' comments have broader implications and challenge ethics committees to support the use of hypothermia for anesthetizing or euthanizing amphibians and reptiles, question some assumptions concerning the capacity of amphibians and reptiles to feel pain under certain conditions, and argue that cooling and freezing occur naturally in some species and should be used more widely for anesthetizing or euthanizing amphibians and reptiles. In light of this, we wanted to examine the long-standing guidance and recent information on the use of cooling and freezing of amphibians and reptiles, with a particular emphasis on the precautionary principle and avoidance of harm.
General Considerations
The historical recommendation against using cooling and freezing to anesthetize or euthanize amphibians and reptiles was not based on dedicated physiologic research but was instead grounded in the precautionary principle. That is, the lack of evidence that cooling and freezing were humane meant that these methods could not be recommended.
Shine et al,16 in a recent review and study involving cane toads (Rhinella marina), concluded that “cooling followed by freezing can offer a humane method of killing cane toads, and may be widely applicable to other ectotherms (especially, small species that are rarely active at low body temperatures).” In coming to this conclusion, the authors cited the reactions of mammals and birds to falling ambient temperatures (eg, attempts to increase metabolic heat production) as an indication that low temperatures may cause intense discomfort and contrasted these reactions with apparent acceptance of hypothermia in amphibians and reptiles because these ectotherms naturally experience highly variable body temperatures. We argue, however, that exposure to falling temperatures and associated body temperature variations do not imply that all such conditions are consistently acceptable in ectotherms. Other authors18 have pointed to the limited current information supporting hypothermia and the need for further physiologic research to conclude that “[r]apid freezing (without prior cooling) is NOT appropriate for field euthanasia of cane toads” and that “[c]ooling followed by Freezing is also NOT appropriate for field euthanasia of cane toads since the efficacy of cooling as a means of inducing anaesthesia is unclear.“
Warm environmental temperatures are commonly sought by ectotherms when ambient temperatures decrease. To what extent this behavioral action is associated with maintenance of metabolic functions or is driven by heat preference or cold avoidance is unclear. However, it seems wrong to suggest that amphibians and reptiles are comfortable with lower temperatures simply because such conditions may be experienced in nature or that artificially imposed cold is humane. Rather, aversion studies are needed to determine the extent to which amphibians and reptiles are comfortable with lower temperatures. For example, it would be useful to know how determined these animals are in avoiding environmental temperatures well below their preferred temperature range or to determine what individual animals would give up to avoid exposure to such cold temperatures. Also, implantation of temperature probes into the brains of cadavers prior to immersion in liquid nitrogen could potentially provide information on how quickly cold penetrates the brain, which could indicate how quickly consciousness centers are nullified.
Shine et al16 reported a linear decrease in EEG power as the temperature of cane toads decreased to 0°C (32°F). Similar decreases in EEG output have been reported for cats and monkeys, albeit at much higher body temperatures,19,20 and humans and goats reportedly become insensate at a core body temperature of around 20°C (68°F), requiring no further administration of inhalant anesthetic.21 However, nobody advocates artificially taking them to this low temperature without prior sedation or anesthesia.
In addition, EEG output in mammals does not correlate well with subjective states other than arousal and somnolence.22 Therefore, it is not clear what meaningful conclusions can be drawn from EEG data for amphibians and reptiles about the subjective experience of hypothermia in these animals. Furthermore, it has been noted that although EEG responses to stimuli are not equivalent to consciousness, EEG findings for amphibians and reptiles23 are clearly different from those for mammals, for which isoelectric EEGs are recorded at much higher temperatures than are needed to block peripheral nerve function.
In supporting the use of cooling and freezing for euthanasia of amphibians and reptiles, Lillywhite et al17 argued that “[c]ooling of low-elevation tropical species to temperatures approaching 0°C can kill tropical species without formation of ice crystals in body fluids or tissues,” and went on to conclude that, in combination with results from Shine et al,16 “[t]herefore, we consider either rapid cooling in an ice bath or slower cooling in a refrigerator and/or freezer…to be a humane method of euthanasia for small tropical amphibians and reptiles because low temperatures will suppress nerve and brain function.”
However, whether low temperatures sufficiently inhibit nociceptor function to render animals insensitive to pain is currently unknown. Further, we argue that even if nociceptor inhibition were confirmed, the possibility would still exist that tropical, cold-vulnerable species experience stress as a result of unnatural thermal extremes in the period leading to death. In addition, although tropical species may occasionally be exposed to extremely cold conditions in nature, this does not prove that such extremes are acceptable under controlled conditions.
According to the AVMA guidelines,5 euthanasia “should result in rapid loss of consciousness followed by cardiac or respiratory arrest and, ultimately, a loss of brain function,” and animal handling and euthanasia techniques “should minimize distress experienced by the animal prior to loss of consciousness.” However, determining consciousness versus unconsciousness in amphibians and reptiles is difficult. Numerous signs of consciousness in amphibians and reptiles have been proposed and are used,2,3,5,6,24,25 but these signs typically rely on an animal's capabilities to respond, which can be attenuated or abolished by cooling or freezing. Confirming death in ectotherms, even within preferred body temperature ranges, is notoriously uncertain.
In their review, Lillywhite et al17 argued that because of evolutionary adaptations, amphibians and reptiles that overwinter in temperatures near or below freezing do not experience pain or distress during seasonal exposure to near-freezing temperatures, suggesting that cooling and freezing should thus be considered acceptable methods of anesthetizing or euthanizing these cold-hardy species. Further, they argued that cooling and freezing are also acceptable for tropical species because low temperatures will suppress nerve and brain function, making these animals incapable of experiencing pain at temperatures low enough to result in ice-crystal formation.
Thus, Lillywhite et al17 proposed that rapid cooling and freezing could be considered a humane method of euthanizing smaller species of amphibians and reptiles, but also stated that “[w]e do not propose that freezing is an appropriate method of euthanasia for larger species such as crocodilians, pythons, or many turtles, but animals that might be euthanized by a procedure of cooling and freezing could be considerably larger (up to several kilograms) than those currently recommended for immersion in liquid nitrogen.” In contrast, we argue that if cooling and freezing were genuinely humane because of progressive suppression of nerve and brain function, then size of the animals would not matter. Further, survival following natural freezing and thawing is only known for some species with adult weights < 500 g (17.6 oz).26 Thus, one cannot argue that cooling and freezing are humane in animals weighing “up to several kilograms” because of evolutionary adaptations to low seasonal temperatures and then apply the same perspective to both cold-hardy and tropical species.
Endotherms Versus Ectotherms
Findings for endotherms (eg, mammals) are generally not applicable when speculating about the effects of hypothermia in ectotherms (eg, amphibians and reptiles). Nevertheless, there reportedly is a strong association between chronic cold exposure and painful neuropathy in humans,27 even though the mechanisms of damage and pain perception are unclear. In addition, hypothermia is highly aversive in people, both clinically and subjectively, even in the absence of ice-crystal formation in the tissues, and postoperative hypothermia is often reported by human surgical patients to be more objectionable than postoperative pain.28 We argue that, out of an abundance of caution, the same should be considered true for ectotherms until evidence to the contrary is produced.
Notably, cooling and freezing (ie, cooling, death on ice slurry, and live chilling) are considered unacceptable methods for harvesting and slaughtering fish.29–34 In sea bass, iced-water chilling results in lower plasma glucose and lactate concentrations and reduced behavioral responses, compared with the effects of asphyxia and electrostunning.30 However, these were relative comparisons and did not confirm the humaneness of hypothermia. Immersion in iced water at 1°C (33.8°F) is effective in stunning tilapia, as determined on the basis of EEG monitoring,35 but the fish regain consciousness when painful stimuli are applied.
Technical Considerations
For any method of euthanasia, logistical aspects of the procedure must be considered, especially if the method is to be used for mass euthanasia of large numbers of animals that are not individually handled but killed in groups (eg, mass euthanasia of cane toads). In particular, if cooling and subsequent freezing were to be used for mass euthanasia, special consideration would have to be given to ensure that the temperature of the euthanasia medium or chamber was maintained at the appropriate level for the appropriate time, because a lack of temperature maintenance would compromise the procedure. In addition, if the euthanasia method were to be used in the field, versus in the controlled environment of a laboratory, consideration would have to be given to the practicalities of the method, such as alternatives in the event of equipment failure when replacement or repairs are not readily available.
Chemical Versus Hypothermic Euthanasia
Lillywhite et al,17 in their review, pointed out that “the use of chemical agents for euthanasia might induce equal or greater levels of distress (as hypothermia)” because of the handling that is required, the pain associated with the injection itself, and the potential adverse neural and behavioral effects associated with certain chemical euthanasia agents. Certainly, the humane use of chemical agents to induce anesthesia or death in animals depends on the selection of appropriate injection volumes, sites, and rates and appropriate concentrations and buffering of the agents. We argue, however, that any inconvenience or handler hazards associated with the use of chemical agents do not, themselves, justify the use of cooling or freezing as an alternative.
Evolutionary Considerations
The means by which ectotherms respond to thermal changes and extremes in their environment have been acknowledged as “perhaps the most important ecophysiological variable[s] affecting the performance of ectotherms.”36 For those species known to survive natural freezing and thawing, the exact physiologic mechanisms for entrance into and exit from a frozen state are currently unknown.26 Nevertheless, species that have evolved to tolerate freezing conditions exhibit characteristic behavioral changes prior to the onset of winter (eg, decreased activity, seasonal anorexia, and shelter seeking), suggesting that they adjust to cold temperatures somewhat gradually. In contrast, sudden freezing circumvents any potential evolved strategies for environmental cue–based incremental preparation, which could, we argue, result in substantial stress.
Of the approximately 16,000 amphibian and reptile species reported worldwide, only 22 amphibian and 8 reptile species (0.2% of amphibian and reptile species combined) are known to survive natural freezing and thawing.26 Neurologic function is not measurable in these species when they are in a frozen state; however, although the exact mechanisms are unknown, normal physiologic function returns following the thawing process. Thus, an inability to measure neural processes in frozen amphibians and reptiles may represent technological limitations, rather than a true absence of nerve function, and an absence of detectable EEG signals does not necessarily confirm death.
Conclusions
In our view, the substantial information gaps regarding the effects of cooling and freezing in amphibians and reptiles continue to justify a precautionary approach. Although certain amphibian and reptile species are adapted to extreme hypothermia and tolerant to it in nature, most are not. Thus, we believe that recommendations for the use of cooling or freezing (or cooling then freezing) to anesthetize or euthanize amphibian and reptile species in general are not currently justified, and we argue that ethics committees should not allow these methods to be used until more complete evidence on their effects is produced.
Future research may, of course, indicate that cooling and freezing (alone or in conjunction with other methods) constitute humane methods of anesthetizing or euthanizing amphibians and reptiles. However, on the basis of the precautionary principle, we believe that recommending these methods is currently premature.
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