Aversion to gaseous euthanasia agents in rats and mice

Despite euthanasia being the most common of all procedures carried out on laboratory animals, the potential distress associated with gaseous agents has received little interest until recently, with growing concern over use of carbon dioxide as a humane method of euthanasia. The distress associated with exposure to carbon dioxide, argon, and carbon dioxide-argon mixtures was investigated in rats and mice by measuring the degree of aversion on exposure to low, medium, and high concentrations of these agents. Animals were exposed to the various concentrations in a test chamber containing air or gas mixtures that they were able to enter and leave at will. Aversion was assessed, using measurements of initial withdrawal time and total dwelling time in the test chamber, as they were the most sensitive measurements of aversion. Comparisons between euthanasia agent and control (air) treatments indicated that concentrations of agents recommended for rapid and efficient induction are associated with some degree of aversion. Carbon dioxide and the carbon dioxide-argon mixtures were more aversive than was argon for rats and mice. These findings suggest that induction with carbon dioxide either alone or in combination with argon is likely to cause considerable distress before the loss of consciousness in rodents, which is unacceptable considering that effective and more humane alternatives are available.     

Combining Nitrous Oxide with Carbon Dioxide Decreases the Time to Loss of Consciousness during Euthanasia in Mice — Refinement of Animal Welfare?

Carbon dioxide (CO(2)) is the most commonly used euthanasia agent for rodents despite potentially causing pain and distress. Nitrous oxide is used in man to speed induction of anaesthesia with volatile anaesthetics, via a mechanism referred to as the "second gas" effect. We therefore evaluated the addition of Nitrous Oxide (N(2)O) to a rising CO(2) concentration could be used as a welfare refinement of the euthanasia process in mice, by shortening the duration of conscious exposure to CO2. Firstly, to assess the effect of N(2)O on the induction of anaesthesia in mice, 12 female C57Bl/6 mice were anaesthetized in a crossover protocol with the following combinations: Isoflurane (5%)+O(2) (95%); Isoflurane (5%)+N(2)O (75%)+O(2) (25%) and N(2)O (75%)+O(2) (25%) with a total flow rate of 3 l/min (into a 7 l induction chamber). The addition of N(2)O to isoflurane reduced the time to loss of the righting reflex by 17.6%. Secondly, 18 C57Bl/6 and 18 CD1 mice were individually euthanized by gradually filling the induction chamber with either: CO(2) (20% of the chamber volume.min-1); CO(2)+N(2)O (20 and 60% of the chamber volume.min(-1) respectively); or CO(2)+Nitrogen (N(2)) (20 and 60% of the chamber volume.min-1). Arterial partial pressure (P(a)) of O(2) and CO(2) were measured as well as blood pH and lactate. When compared to the gradually rising CO(2) euthanasia, addition of a high concentration of N(2)O to CO(2) lowered the time to loss of righting reflex by 10.3% (P<0.001), lead to a lower P(a)O(2) (12.55 ± 3.67 mmHg, P<0.001), a higher lactataemia (4.64 ± 1.04 mmol.l(-1), P = 0.026), without any behaviour indicative of distress. Nitrous oxide reduces the time of conscious exposure to gradually rising CO(2) during euthanasia and hence may reduce the duration of any stress or distress to which mice are exposed during euthanasia.     

Euthanasia of rats with carbon dioxide--animal welfare aspects

A method of inducing euthanasia by carbon dioxide (CO2) inhalation in the home cage of an animal is described and tested for distress by behavioural as well as by hormonal measures. The animals were maintained in their home cage while CO2 was induced at a flow of 6 l/min. The behaviour of the animals was measured continuously as were the serum concentrations of glucose, ACTH and corticosterone 30, 75 and 120 s after the CO2 was introduced into the cage. In order to test for distress, two groups of rats were pre-treated with acepromazine (orally) and pentobarbiturate (i.p. injection) respectively, in order to reduce possible distress caused by CO2 euthanasia, and were compared with control groups. There were no signs of distress by behavioural or by hormonal changes. All changes seen could be attributed to experimental effects and, especially as there was no difference between the pre-treated and the control groups of rats, it must be assumed that the described method of euthanasia is in concordance with animal welfare, it leads to rapid death without severe distress or pain, and it seems therefore to be 'humane'.     

Experimental animal urine collection: a review

Animal urine collection is a vital part of veterinary practice for ascertaining animal health and in scientific investigations for assessing the results of experimental manipulations. Untainted animal urine collection is very challenging, especially with small rodents, and is an almost impossible task under conditions of microgravity. The fundamental aspects of urine collection are: (1) ease of collection, (2) quality of sample, (3) prevention of contamination, (4) severity of procedures used, (5) levels of pain caused to the animal and (6) refinement of methods to reduce stress, pain or distress. This review addresses the collection of urine for qualitative and quantitative purposes from rodents, rabbits, felines, canines, avian species, equines, porcines, ungulates and certain non-human primates, with animal welfare in mind. Special emphasis has been given to rodents, canines and non-human primates, since they are the animals of choice for research purposes. Free catch (voluntary voiding), methods with mild intervention, surgical methods, modified restraint, cage and special requirement methods have been reviewed here. Efforts need to be taken to provide appropriate animal husbandry and to nurture the animals in as natural an environment as possible since experimental results obtained from these research subjects are, to a great extent, dependent upon their well-being. A continuous refinement in the procedures for collecting urine from experimental animals will be the most efficient way of proceeding in obtaining pure urine specimens for obtaining reliable research data.     

Behavioural responses of rats to gradual-fill carbon dioxide euthanasia and reduced oxygen concentrations

Carbon dioxide (CO₂) is widely used for euthanasia of laboratory rats, but little is known about the pain and distress caused by this procedure. Physiological and human self-report data suggest that CO₂ may cause pain and dyspnea, a sensation of breathlessness and increased respiratory effort, at the concentrations used for gradual-fill euthanasia. However, previous studies examining the behavioural responses of rats have reported conflicting results. In this study, detailed behavioural responses of rats were examined during gradual-fill CO₂ euthanasia and during gradual-fill with argon to produce a similar reduction in oxygen concentration. Animals were randomly allocated to the CO₂ or reduced oxygen treatment groups (n = 8 for both), and were first tested with air exposure and then with treatment gas exposure on the following day. Observations were taken for 105 s before and after gas flow began (baseline and exposure periods), as animals had ceased purposeful movement within 105 s of CO₂ flow starting. Behavioural changes from baseline during gas exposure were compared to changes during air exposure on control days. In comparison to air exposure, CO₂ resulted in increased activity, rearing, touching the nose to the chamber lid, escape behaviours and vocalizations. A small increase in touching the nose to the chamber lid was observed when oxygen concentrations were reduced with argon, but no other behavioural changes were observed. These results suggest that gradual-fill CO₂ euthanasia causes distress in rats, and that hypoxia alone is not a major cause of this distress.     

Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?

Current attitudes to the use of animals in biomedical research require that any pain or distress should be minimised. This can often be achieved by the use of appropriate anaesthetic and analgesic regimens. There, is however, little information on the peri-operative regimens used. A literature review was conducted to estimate how commonly analgesics are administered to laboratory rodents, the most widely used species of laboratory animals, and to assess the anaesthetic regimens employed. Studies describing potentially painful experimental procedures involving rodents were identified from peer-reviewed journals published from 1990 to 1992 and from 2000 to 2002. In papers published between 2000 and 2002, if analgesic administration was not specified, the institutional veterinary surgeons or authors of the papers were contacted by e-mail to obtain additional information on analgesic use. From 1992 to 2002, there was an increase in the reported prevalence of analgesic administration to laboratory rodents from 2.7% to 19.8%. Although the use of analgesics has increased over the past ten years, the overall level of post-operative pain relief for laboratory rodents is still low. Anaesthetic methodology changed markedly between the two time-periods sampled. Notably, there was an increase in the use of isoflurane and of injectable anaesthetic combinations such as ketamine/xylazine, whereas the use of ether and methoxyflurane decreased.     

Regional distribution of glutamate in the central nervous system of rat terminated by carbon dioxide euthanasia

Carbon dioxide euthanasia is an established method for the termination of small laboratory animals. It has also been employed by the authors in neurobiological research on the post-mortem glutamate concentration in the structures of rat brains. The following investigations were aimed at optimizing the termination procedure based on the CO2 saturation rate of the inhaled air. Two rates of CO2 flow were applied, and the higher one significantly augmented the glutamate level in the hippocampus and cerebellum. The relationship between this finding and signs of central fear reaction is discussed. The authors conclude that lower rather than higher CO2 flow in euthanasia procedures is gentler and is therefore preferable for use with laboratory animals.     

Miniaturization: an overview of biotechnologies for monitoring the physiology and pathophysiology of rodent animal models

Recent advances in bioengineering technologies have made it possible to collect high-quality reproducible data quantitatively in a wide range of laboratory animal species, including rodents. Several of these technologies are incorporated into a plan called Miniaturization, which aims to design, develop, and maintain rodent animal models to study the pathophysiology and therapy of human diseases. Laser Doppler flowmetry, digital sonomicrometry, bioelectrical impedance, and microdialysis are some of the most widely used methods under the plan because they cause minimal pain and distress, reduce the number of animals used in biomedical research, and allow chronic, nonterminal assessment of physiological parameters in rodents. An overview of each of these technologies and their major applications in rodents used for biomedical research is provided.     

Use of behavior analysis to recognize pain in small mammals

The minimization of pain in laboratory animals is a gold standard with implications for improvements in both animal welfare and research quality. Changes in behavioral parameters may indicate that an animal is in pain, but in order to effectively use behavioral change to assess pain, the observer must be familiar with normal behaviors. The author discusses normal and pain-related behaviors exhibited by rodents, rabbits, and ferrets.     

Barbiturate euthanasia solution-induced tissue artifact in nonhuman primates

BACKGROUND: Barbiturate euthanasia solutions are a humane and approved means of euthanasia. Overdosing causes significant tissue damage in a variety of laboratory animals. METHODS: One hundred seventeen non-human primates (NHP) representing 7 species including 12 fetuses euthanized for humane and research reasons by various vascular routes with Euthasol, Sodium Pentobarbital, Fatal Plus, Beuthanasia D, or Euthanasia 5 were evaluated for euthanasia-induced tissue damage. Lungs and livers were histologically graded for hemolysis, vascular damage, edema, and necrosis. Severity of tissue damage was analyzed for differences on the basis of agent, age, sex, dose, and injection route. RESULTS: Severity of tissue damage was directly related to dose and the intracardiac injection route, but did not differ by species, sex, and agent used. CONCLUSIONS: When the recommended dose of agent was used, tissue damage was generally reduced, minimal, or undetectable. Barbiturate-induced artifacts in NHPs are essentially the same as in other laboratory species.     

Report of the Working Group on Animal Distress in the Laboratory

Finding ways to minimize pain and distress in research animals is a continuing goal in the laboratory animal research field. Pain and distress, however, are not synonymous, and measures that alleviate one may not affect the other. Here, the authors provide a summary of a meeting held in February 2004 that focused on distress in laboratory animals. They discuss the difficulties associated with defining 'distress,' propose methods to aid in recognizing and alleviating distressful conditions, and provide recommendations for animal research conduct and oversight that would minimize distress experienced by laboratory animals.     

Diagnosis of ecto- and endoparasites in laboratory rats and mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.     

Genetic engineering and other factors that might affect human-animal interactions in the research setting

Evidence exists, particularly in the welfare literature of nonhuman animals on the farm, that the interaction between nonhuman animals and the personnel who care for them can have a strong effect on the animals' behavior, productivity, and welfare. Among species commonly used for biomedical research, mice appear to be the least-preferred species in animal care facilities. A review of the literature and observations of animal care staff interacting with mice indicated that the following factors may influence this: their small size, their particular behavioral characteristics, and husbandry constraints (such as housing in ventilated racks). In addition, this study questioned whether animal care personnel have a different perception of genetically engineered animals and whether this, in turn, has an effect on their interactions with these animals. The ability to carefully observe an animal's behavior is key in carrying out an animal-wellness assessment and in minimizing pain and distress. Attention to human-animal interactions in the research setting represents an opportunity for refinement for large numbers of animals and potentially for reduction of animal use.     

Genetic Engineering and Other Factors That Might Affect Human-Animal Interactions in the Research Setting

Evidence exists, particularly in the welfare literature of nonhuman animals on the farm, that the interaction between nonhuman animals and the personnel who care for them can have a strong effect on the animals' behavior, productivity, and welfare. Among species commonly used for biomedical research, mice appear to be the least-preferred species in animal care facilities. A review of the literature and observations of animal care staff interacting with mice indicated that the following factors may influence this: their small size, their particular behavioral characteristics, and husbandry constraints (such as housing in ventilated racks). In addition, this study questioned whether animal care personnel have a different perception of genetically engineered animals and whether this, in turn, has an effect on their interactions with these animals. The ability to carefully observe an animal's behavior is key in carrying out an animal-wellness assessment and in minimizing pain and distress. Attention to human-animal interactions in the research setting represents an opportunity for refinement for large numbers of animals and potentially for reduction of animal use.     

Rodent laparoscopy: refinement for rodent drug studies and model development, and monitoring of neoplastic, inflammatory and metabolic diseases

The refinement of surgical techniques represents a key opportunity to improve the welfare of laboratory rodents, while meeting legal and ethical obligations. Current methods used for monitoring intra-abdominal disease progression in rodents usually involve euthanasia at various time-points for end of study, one-time individual tissue collections. Most rodent organ tumour models are developed by the introduction of tumour cells via laparotomy or via ultrasound-guided indirect visualization. Ischaemic rodent models are often generated using laparotomies. This approach requires a high number of rodents, and in some instances introduces high degrees of morbidity and mortality, thereby increasing study variability and expense. Most importantly, most laparotomies do not promote the highest level of rodent welfare. Recent improvements in laparoscopic equipment and techniques have enabled the adaptation of laparoscopy for rodent procedures. Laparoscopy, which is considered the gold standard for many human abdominal procedures, allows for serial biopsy collections from the same animal, results in decreased pain and tissue trauma as well as quicker postsurgical recovery, and preserves immune function in comparison to the same procedures performed by laparotomy. Laparoscopy improves rodent welfare, decreases inter-animal variability, thereby reducing the number of required animals, allows for the replacement of larger species, decreases expense and improves data yield. This review article compares rodent laparotomy and laparoscopic surgical methods, and describes the utilization of laparoscopy for the development of cancer models and assessment of disease progression to improve data collection and animal welfare. In addition, currently available rodent laparoscopic equipment and instrumentation are presented.     

恒河猴在生殖生物学中的应用进展

目的非人灵长类动物在生殖生物学研究领域是一种非常重要的实验动物。人类利用非人灵长类动物与人的生物学等方面相似的特征,开展了生殖生物学、生理学、药理学、毒理学以及生育调节等方面的研究工作,为生殖生物学基础研究以及人类健康和疾病问题的基础研究和临床前研究提供了理想的动物模型。随着生命科学的发展,对非人灵长类实验动物质量提出了更高的要求,人们认识到实验时,应用健康的优质非人灵长类动物的重要性。本文简要概括了非人灵长类动物恒河猴的生物学特性,阐述了非人灵长类动物恒河猴在生殖生物学中的应用研究。     

CO2 induced acute respiratory acidosis and brain tissue intracellular pH: a 31P NMR study in swine

High concentration carbon dioxide (CO(2)) is used to promote pre-slaughter anaesthesia in swine and poultry, as well as short-lasting surgical anaesthesia and euthanasia in laboratory animals. Questions related to animal welfare have been raised, as CO(2) anaesthesia does not set in momentarily. Carbon dioxide promotes anaesthesia by lowering the intracellular pH in the brain cells, but the dynamics of the changes in response to a high concentration of CO(2) is not known. Based on (31)P NMR spectroscopy, we describe CO(2)-induced changes in intracellular pH in the brains of five pigs inhaling 90% CO(2) in ambient air for a period of 60 s, and compare the results to changes in arterial blood pH, P(CO2), O(2) saturation and HCO(3)(-) concentration. The intracellular pH paralleled the arterial pH and P(CO2) during inhalation of CO(2); and it is suggested that the acute reaction to CO(2) inhalation mainly reflects respiratory acidosis, and not metabolic regulation as for example transmembrane fluxes of H(+)/HCO(3)(-). The intracellular pH decreased to approximately 6.7 within the 60 s inhalation period, and the situation was metabolically reversible after the end of CO(2) inhalation. The fast decrease in intracellular pH supports the conclusion that high concentration CO(2) leads to anaesthesia soon after the start of inhalation.     

Institutional and IACUC responsibilities for animal care and use education and training programs

Training and instruction of personnel are important components of animal care and use programs because they help to ensure the health and welfare of the animals and the integrity of the research or testing results. Training also helps to promote the consideration of alternatives, recognition of animal pain and distress, appropriate use of pain-relieving agents, aseptic technique, pre- and post-procedural care, and personnel health and safety. While individuals who provide the care for or conduct research or testing in laboratory animals should take personal responsibility for ensuring that they have the skills to perform their duties, the institution is ultimately responsible for ensuring their competency. The institution is also responsible for providing the training or instruction that is required by federal legislation, regulations, and policies. The institutional animal care and use committee (IACUC) is responsible for ensuring, as part of their review of research activities, that the personnel are capable of performing the procedures described. The IACUC must also assess the institution's training program as part of their semiannual animal care and use program review and make recommendations regarding training to the institutional official. This article provides a comprehensive overview of the US regulatory mandates for training and personnel qualification.     

I. P. Pavlov on vivisection and the modern ethical requirements for animal experiments]

It is shown that Pavlov's opinion is in agreement with the modern requirements for experiments in living animals: use of living animals in experiments is recommended only in the cases when there is no alternative and scientific-practical significance of the planned experiments is justified; minimization (as much as possible) of discomfort, distress and pain of animals under study without sacrifice of the quality of scientific research; use of appropriate number of animals necessary for obtaining reliable results which are adequate to a given experimental situation and general state of the animals inadmissibility of using extra number of animals; drawing up a protocol concerning the experimental procedure and results; reflection in the protocol the techniques of sedation, anesthesia, and euthanasia; constant care of the improvement of the theoretical and practical qualification of a researcher in the ethical questions and experimental technique. Pavlov's views led his time and at present they agree with modern concepts on the experimental ethics.     

Unrelieved pain and distress in animals: an analysis of USDA data on experimental procedures

Pain and distress are core issues in the field of animal experimentation and in the controversy that surrounds it. We sought to add to the empirical base of the literature on pain and distress by examining government data on experimental procedures that caused unrelieved pain and distress (UPAD) in animals. Of the species regulated by the U.S. Department of Agriculture (USDA), most of the approximately 100,000 animals subjected to UPAD during the year analyzed (1992) were guinea pigs and hamsters. Most of these animals were used in industry laboratories for various testing procedures, primarily vaccine potency testing. We discuss the limitations of the USDA data and recommend changes to the current reporting system. By identifying experimental procedures that cause UPAD in large numbers of USDA-regulated animals, the present analysis can be viewed as a means of identifying priorities for research and development of alternatives methods (replacements, reductions, and refinements).