Review of Intraperitoneal Injection of Sodium Pentobarbital As a Method of Euthanasia in Laboratory Rodents
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Journal of the American Association for Laboratory Animal Science Vol 59, No 3 Copyright 2020 May 2020 by the American Association for Laboratory Animal Science Pages 254–263 Review of Intraperitoneal Injection of Sodium Pentobarbital as a Method of Euthanasia in Laboratory Rodents Colin A Laferriere1 and Daniel SJ Pang1,2,* Euthanasia is one of the most commonly performed procedures in biomedical research, involving tens of millions of animals in North America and Europe every year. The use of sodium pentobarbital, injected intraperitoneally, for killing rodents is described as an acceptable technique by the AVMA and CCAC euthanasia guidelines. This drug and route are recommended over inhalant anesthetics, carbon dioxide, and physical methods for ethical and aesthetic reasons as well as efficiency. However, a growing body of evidence challenges the efficacy and utility of intraperitoneal pentobarbital. This methodology has been described as inconsistent and may induce pain and stress. With these considerations in mind, a review of the literature is needed to assess the evidence surrounding this killing method, the associated welfare implications, and potential for refinement. Abbreviations: CCAC, Canadian Council on Animal Care; FLI, fos-like-immunoreactive; pentobarbital, sodium pentobarbital DOI: 10.30802/AALAS-JAALAS-19-000081 There are approximately 17 million rodents (rats and mice) able with conditions’ (AVMA)5 or ‘conditionally acceptable’ used in research annually in the European Union, United (CCAC).18 This category is similarly defined by AVMA and Kingdom, and Canada.19,37,97 An overwhelming majority of CCAC and includes techniques that “may require certain condi- these rodents are euthanized either as part of a study protocol tions to be met to consistently produce humane death” (AVMA), or at the end of a research project, making euthanasia one of the “might not consistently produce humane death” (CCAC), are most commonly performed laboratory procedures. Despite the not well described or documented in the scientific literature prevalence of euthanasia procedures and its conceptual simplic- (AVMA and CCAC), may have increased safety hazards or po- ity, ensuring a ‘good death’ across a wide variety of laboratory tential for operator error (AVMA and CCAC), or “may require settings and involving such large numbers of animals is difficult. a secondary method to ensure death” (AVMA).5,18 The AVMA Killing methods in research consist of multiple—often guidelines further state that “methods acceptable with condi- conflicting—objectives: ideally, death must be rapid and pain- tions are equivalent to acceptable methods when all criteria less yet aesthetic and compatible with research protocols.49 for application of a method can be met.”5 Methods classified Furthermore, killing methods must be simple to apply and in this way remain common because acceptable methods may yield consistent results. It may be difficult, even impossible, to interfere with the collection of research data or are impractical completely satisfy all of these objectives in all instances, more for large numbers of animals. For example, overdose with CO2 27 so given the challenges in identifying important outcomes (e.g., is one of the most commonly used killing methods. CO2 is distress, pain, aesthetic appeal). practical, easy to use, inexpensive, relatively fast in action, can In North America, the AVMA and Canadian Council on Ani- be applied to multiple animals simultaneously, and requires mal Care (CCAC) provide guidance on euthanasia procedures. little or no direct handling.4,27,54 However, many studies have Both organizations publish guidelines regularly, reflecting the demonstrated that CO2 exposure elicits an aversive response in latest evidence on killing methods in a wide range of species. In rodents and may induce distress and pain.22,27,51,61,76-79 rodents, the AVMA classifies the intraperitoneal or intravenous Inhalant anesthetics, such as isoflurane, induce aversive injection of barbituric acid derivatives (or barbiturate combina- behavior in rodents.21,61,68,69,99,110 In addition, their use re- tions), as well as injection of dissociative agent combinations, quires specialized equipment, and time to death can be slow.18 as the only acceptable methods,5 and the CCAC classifies Therefore, the CCAC guidelines rate inhalant anesthetics intraperitoneal injection of buffered and diluted barbiturate as as acceptable in rodents but recommend use of a secondary well as overdose of inhalant anesthetics (followed by another method after the animal is anesthetized to ensure death, whereas method to ensure death) as acceptable.18 euthanasia by using inhalant anesthetics is ‘acceptable with Although the above methods are categorized as acceptable, conditions’ in the AVMA guidelines. Regarding dissociative there exists a secondary category for methods termed ‘accept- agents (typically ketamine) or a combination of ketamine and an α2 adrenergic receptor agonist, such as xylazine, there is Received: 06 Jun 2019. Revision requested: 17 Jul 2019. Accepted: 23 Sep 2019. limited discussion of this euthanasia method in the current 5 1Clinical Sciences, Faculty of Veterinary Medicine, University of Montreal, Saint- AVMA guidelines, with only one supporting study cited in Hyacinthe, Québec, Canada, and 2Veterinary Clinical and Diagnostic Sciences, University which the primary objective was not qualitative evaluation of of Calgary, Calgary, Alberta, Canada dissociative agents for killing.100 *Corresponding author. Email: [email protected] 254 Review of rodent euthanasia with pentobarbital Overdose with barbiturate is the only method classified as Intraperitoneal injection technique. Several techniques for acceptable by both the AVMA and the CCAC guidelines.5,18 intraperitoneal injection have been described, the most common Sodium pentobarbital (pentobarbital) is the most commonly variations are described here. Few studies specifically evaluate used barbiturate for killing. It has a narrow safety margin, is the outcomes of different injection techniques. Important factors potent, can be formulated as a concentrated solution so that to be considered—but seldom described—are the proficiency of relatively small volumes are needed, and has a rapid onset of the investigators, behavior of the animals (habituated compared action when given intravenously.5,25 Furthermore, pentobarbital with nonhabituated),33,86 and inherent variability associated has a long shelf life, is stable in solution, and is inexpensive.5 with drug absorption. A 2-person technique with one holder and Access and licensing requirements for purchase vary between one injector, rather than a one-person technique, is considered countries.25 When given as an intentional overdose to cause more efficient when working with mice, given that the 2-person death, general anesthesia is induced, followed by depression method led to a reduction in misinjection rate from 11% to 13% of the respiratory and cardiovascular centers of the brainstem, to 1% to 2%.7 The holder grips the upper limbs and head of the leading to cardiorespiratory arrest.25,27 animal and maintains the animal in a horizontal position. It Although the intravenous route of injection is preferred for is often recommended that the holder tilts the animal slightly pentobarbital, it is often impractical to perform on rodents, downward so that its head is lower than its abdomen. This posi- given that achieving consistently successful intravenous injec- tioning supposedly creates more space in the caudal quadrants tions requires training, restraint and often a means to induce of the abdominal cavity by moving organs cranially.103 However venodilation.39,43 As such, pentobarbital for killing rats and mice there is little evidence that the tilting of the animal has any is frequently administered via intraperitoneal injection.25,75,96 effect on the success of the injection.75 Indeed, similar misinjec- Although the onset of anesthetic effect is not as quick after tion rates have been obtained when injecting rats vertically28 intraperitoneal injection as after intravenous injection, drug or horizontally with a head down tilt.75,113 The injector, using absorption and distribution occur much more rapidly after in- one hand to hold the right hindlimb of the animal, visualizes traperitoneal dosage than after intramuscular or subcutaneous the abdomen as though divided into 4 quadrants and injects injections.75,83 Furthermore, intraperitoneal injection is relatively into the caudal left quadrant (the animal’s right side).11,28,98,113 simple and quick to perform, allows the administration of large Within the caudal left quadrant, the injection should be made volumes, and accommodates repeated injections. at the level of the coxofemoral joint, approximately halfway Thus, intraperitoneal injection of sodium pentobarbital (in- between midline and the lateral abdominal wall.98 The goal traperitoneal pentobarbital) is one of the most widely accepted of the injection is to deposit injectate into the peritoneal cavity methods of rodent euthanasia that does not have additional without piercing any of the abdominal organs. To avoid doing conditions attached to its use. This rating is shared not only so, the injection angle should be approximately 10 to 20 degrees by the CCAC and AVMA but also by animal care guidelines in relative to the body wall in mice and 20 to 45 degrees in rats, Europe,6 India58 and Australia.9 Importantly, however, despite with the needle directed