Carbon dioxide for euthanasia: concerns regarding pain and distress, with special reference to mice and rats

Carbon dioxide (CO2) is the most commonly used agent for euthanasia of laboratory rodents, used on an estimated tens of millions of laboratory rodents per year worldwide, yet there is a growing body of evidence indicating that exposure to CO2 causes more than momentary pain and distress in these and other animals. We reviewed the available literature on the use of CO2 for euthanasia (as well as anaesthesia) and also informally canvassed laboratory animal personnel for their opinions regarding this topic. Our review addresses key issues such as CO2 flow rate and final concentration, presence of oxygen, and prefilled chambers (the animal is added to the chamber once a predetermined concentration and flow rate have been reached) versus gradual induction (the animal is put into an empty chamber and the gas agent(s) is gradually introduced at a fixed rate). Internationally, animal research standards specify that any procedure that would cause pain or distress in humans should be assumed to do so in non-human animals as well (Public Health Service 1986, US Department of Agriculture 1997, Organization for Economic Cooperation and Development 2000). European Union guidelines, however, specify a certain threshold of pain or distress, such as 'skilled insertion of a hypodermic needle', as the starting point at which regulation of the use of animals in experimental or other scientific procedures begins (Biotechnology Regulatory Atlas n.d.). There is clear evidence in the human literature that CO2 exposure is painful and distressful, while the non-human literature is equivocal. However, the fact that a number of studies do conclude that CO2 causes pain and distress in animals indicates a need for careful reconsideration of its use. Finally, this review offers recommendations for alternatives to the use of CO2 as a euthanasia agent.     

Requirements for reference (calibration) laboratories in laboratory medicine

In addition to reference measurement procedures and reference materials, reference or calibration laboratories play an integral role in the implementation of measurement traceability in routine laboratories. They provide results of measurements using higher-order methods, e.g. isotope dilution mass spectrometry and may assign values to materials to be used for external quality assessment programs and to secondary reference materials. The requirements for listing of laboratories that provide reference measurement services include a statement of the metrological level or principle of measurement, accreditation as a calibration laboratory according to ISO 15195 and the participation in a proficiency testing system (regular inter-laboratory comparisons) for reference laboratories. Ring trials are currently conducted for thirty well-defined measurands and the results are made available to all laboratories. Through the use of reference laboratory services that are listed by the Joint Committee for Traceability in Laboratory Medicine there is the opportunity to further promote traceability and standardisation of laboratory measurements.     

通过ISO 15189认可实验室质量指标制定及监测情况调查

目的 了解目前我国通过ISO 15189认可的实验室质量指标制定及监测情况。 方法 设计质量指标制定及监测情况调查表并下发给185家认可实验室,要求在10月31日前以邮件形式回报结果,使用Microsoft Excel 2010软件进行统计分析。 结果 共85家（占45.95%）实验室回报结果,76家临床实验室,9家独立实验室。不同认可实验室制定的质量指标数量及分布差异较大。76家临床实验室共设置质量指标1105项,每家实验室平均14.5项。9家独立实验室设置质量指标195项,每家实验室平均21.7项。临床实验室制定了更多的检验前、检验中质量指标,独立实验室在设置质量指标时还包括了较多的检验后、支持性过程和其他质量指标。 结论 目前我国认可实验室质量指标制定及监测情况尚不理想。在推进质量指标一致化的同时,实验室应加强宣贯与教育,建议一套完整、科学和实用的质量指标体系。

     

A Survey of Veterinarians’ Attitudes toward Euthanasia of Companion Animals in Japan

This study investigated veterinarians’ attitudes toward euthanasia of companion animals in Japan. A nationwide survey was conducted with 932 veterinarians in small animal practices. It examined the number of times they administered euthanasia, their moral criteria for choosing euthanasia for animals, and their behavioral criteria for suggesting euthanasia to owners. According to the data analyses, on average the veterinarians administered euthanasia 2.48 times a year. For many veterinarians, two conditions were necessary to justify euthanasia for animals: “the animals are incurable and suffering” and “the owners request to euthanize the animals.” In the absence of either condition, the veterinarians were inclined to disapprove of choosing euthanasia. If the owners requested further treatment, 67% showed clear disapproval of choosing euthanasia for animals with serious medical conditions. Meanwhile, more than 76% showed clear disapproval of euthanizing healthy animals when the owners requested it. These results indicate that the owners’ request takes precedence over the animals’ condition for suffering animals, but not for healthy animals. For animals with serious medical conditions, 56% of the veterinarians answered that they would or might suggest euthanasia to the owners even though the owners requested further treatment. In this situation, for some veterinarians, the animals’ condition rather than the owners’ request might become a determinant in suggesting euthanasia to owners, even if their moral judgments were against choosing euthanasia for the animals. A decrease in the owners’ or the animals’ quality of life and the owners’ inability to pay were not primary factors in choosing or suggesting euthanasia. Having an experience of euthanizing their own animals was a key factor for the veterinarians which increased not only the number of times they administered euthanasia but also the degree of their moral approval of choosing euthanasia and their behavioral willingness to suggest it to owners.     

Current practices and prospects for standardization of the hematopoietic colony-forming unit assay: a report by the cellular therapy team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative

Background aimsWide acceptance of the colony-forming unit (CFU) assay as a reliable potency test for stem cell products is hindered by poor inter-laboratory reproducibility. The goal of this study was to ascertain current laboratory practices for performing the CFU assay with an eye towards identifying practices that could be standardized to improve overall reproducibility.MethodsA survey to evaluate current laboratory practices for performing CFU assays was designed and internationally distributed.ResultsThere were 105 respondents to the survey, of whom 68% performed CFU assays. Most survey recipients specified that an automated rather than a manual cell count was performed on pre-diluted aliquots of stem cell products. Viability testing methods employed various stains, and when multiple sites used the same viability stain, the methods differed. Cell phenotype used to prepare working cell suspensions for inoculating the CFU assay differed among sites. Most respondents scored CFU assays at 14–16 days of incubation, but culture plates were read with various microscopes. Of 57 respondents, 42% had not performed a validation study or established assay linearity. Only 63% of laboratories had criteria for determining if a plate was overgrown with colonies.ConclusionsSurvey results revealed inconsistent inter-laboratory practices for performing the CFU assay. The relatively low number of centers with validated CFU assays raises concerns about assay accuracy and emphasizes a need to establish central standards. The survey results shed light on numerous steps of the methodology that could be targeted for standardization across laboratories.     

Harmonia axyridis invasions: Deducing evolutionary causes and consequences

I consider evolutionary approaches to deducing factors that have made the ladybird beetle Harmonia axyridis such a successful invader, and the contribution that studies of this species in its native range can make. Work aiming to demonstrate which (pre)adaptations have made the species so successful often fails to compare these putative characters with those of other ladybirds. This has led to a tendency for “argument by design”‐type claims on characters widely shared by non‐invasive coccinellids. There is good evidence from genetic studies that evolutionary change occurred in invasive populations, contributing to their success. There is some evidence for subsequent evolutionary change after the establishment of invasive H. axyridis, primarily in the native organisms with which the ladybird interacts. I show here that there appears to have been little adaptation in H. axyridis, over about 20 generations, to the alkaloids of one North American native intraguild prey, the ladybird Coleomegilla maculata. Studies of H. axyridis in its native range are important, as they provide a snapshot of the ancestral ladybird, unobscured by subsequent evolutionary change related to its invasiveness. They provide baseline data about phenomena such as interactions with natural enemies and intraguild predation, and they also can provide pointers as to how H. axyridis might further adapt in the regions it has colonized. Harmonia axyridis represents an ideal opportunity for greater international co‐operation between scientists studying this species in its native range in Asia and scientists studying it in Europe, America and Africa, where it is an invasive exotic.     

Expression patterns of the creatine metabolism‐related molecules AGAT,GAMT and CT1 in adult zebrafish Danio rerio

AGAT, GAMT and CT1, three creatine synthesis and transport‐related molecules, have been widely studied in mammals. To explore their homologous genes in adult zebrafish Danio rerio, the gene expression patterns of these three genes in D. rerio were investigated. The results reveal that AGAT, GAMT and CT1 are expressed widely in diverse tissues of D. rerio where the homologous genes in mammals are also expressed.     

Is the German suspension of MON810 maize cultivation scientifically justified?

We examined the justifications invoked by the German government in April 2009 to suspend the cultivation of the genetically modified maize varieties containing the Bt insect-resistance trait MON810. We have carried out a critical examination of the alleged new data on a potential environmental impact of these varieties, namely two scientific papers describing laboratory force-feeding trials on ladybirds and daphnia, and previous data on Lepidoptera, aquatic and soil organisms. We demonstrate that the suspension is based on an incomplete list of references, ignores the widely admitted case-by-case approach, and confuses potential hazard and proven risk in the scientific procedure of risk assessment. Furthermore, we did not find any justification for this suspension in our extensive survey of the scientific literature regarding possible effects under natural field conditions on non-target animals. The vast majority of the 41 articles published in 2008 and 2009 indicate no impact on these organisms and only two articles indicate a minor effect, which is either inconsistent during the planting season or represents an indirect effect. Publications from 1996 to 2008 (376 publications) and recent meta-analyses do not allow to conclude on consistent effects either. The lower abundance of some insects concerns mainly specialized enemies of the target pest (an expected consequence of its control by Bt maize). On the contrary, Bt maize have generally a lower impact than insecticide treatment. The present review demonstrates that the available meta-knowledge on Cry1Ab expressing maize was ignored by the German government which instead used selected individual studies.     

The refining influence of ethics committees on animal experimentation in Sweden

Mandatory scrutiny of projects by animal ethics committees was introduced in Sweden in 1979. The present study investigated the minutes of meetings held between 1989 and 2000 at which consideration of applications for experimental work in animals resulted in requests for modification (n = 3607). 18.1% of the applications received were approved only after modifications. The majority of the changes requested may be classified as 'Refinement'. The most common requests were for improvement of project design, euthanasia method and housing and husbandry. There was a relative increase in modifications requested by the committees related to anaesthesia, choice of licensed supervisor and the need for licenses or informed consent from animal owners during the period investigated. There was a relative decrease in modifications related to euthanasia, housing and husbandry, and general endpoint assertions. The results suggest that the work of the committees may be perceived as an ongoing process, since several of the applications for which modification was requested were projects that had been approved on a previous occasion but were now up for renewal. In order to have maximal influence on the refinement of scientific protocols it is important that the scientists in the committees are continuously updated on developments in laboratory animal science.     

Methods for the euthanasia of experimental animals--the ethics, esthetics and personnel safety

In this paper there are shown chemical and physical methods of euthanasia of vertebrate animals. All methods are divided into three categories: A) Acceptable methods of euthanasia, B) methods acceptable only for unconscious animals, C) methods that are not acceptable for euthanasia. The acceptability or non-acceptability of the method is determined by ethic or aesthetic conceptions and also by conceptions of the personnel safety and the environment. There is provided a table of acceptable methods of euthanasia. Recommendations of working group of the Federation of Laboratory Animal Science Association (FELASA) were taken into consideration in this paper. The survey can be useful for the experimenters and personnel concerned with laboratory animals.     

Variation in Reproductive Success Across Captive Populations: Methodological Differences,Potential Biases and Opportunities

Our understanding of fundamental organismal biology has been disproportionately influenced by studies of a relatively small number of ‘model’ species extensively studied in captivity. Laboratory populations of model species are commonly subject to a number of forms of past and current selection that may affect experimental outcomes. Here, we examine these processes and their outcomes in one of the most widely used vertebrate species in the laboratory – the zebra finch (Taeniopygia guttata). This important model species is used for research across a broad range of fields, partly due to the ease with which it can be bred in captivity. However despite this perceived amenability, we demonstrate extensive variation in the success with which different laboratories and studies bred their subjects, and overall only 64% of all females that were given the opportunity, bred successfully in the laboratory. We identify and review several environmental, husbandry, life‐history and behavioural factors that potentially contribute to this variation. The variation in reproductive success across individuals could lead to biases in experimental outcomes and drive some of the heterogeneity in research outcomes across studies. The zebra finch remains an excellent captive animal system and our aim is to sharpen the insight that future studies of this species can provide, both to our understanding of this species and also with respect to the reproduction of captive animals more widely. We hope to improve systematic reporting methods and that further investigation of the issues we raise will lead both to advances in our fundamental understanding of avian reproduction as well as to improvements in future welfare and experimental efficiency.     

Insect cell culture and biotechnology

The continued development of new cell culture technology is essential for the future growth and application of insect cell and baculovirus biotechnology. The use of cell lines for academic research and for commercial applications is currently dominated by two cell lines; the Spodoptera frugiperda line, SF21 (and its clonal isolate, SF9), and the Trichoplusia ni line, BTI 5B1-4, commercially known as High Five cells. The long perceived prediction that the immense potential application of the baculovirus-insect cell system, as a tool in cell and molecular biology, agriculture, and animal health, has been achieved. The versatility and recent applications of this popular expression system has been demonstrated by both academia and industry and it is clear that this cell-based system has been widely accepted for biotechnological applications. Numerous small to midsize startup biotechnology companies in North America and the Europe are currently using the baculovirus-insect cell technology to produce custom recombinant proteins for research and commercial applications. The recent breakthroughs using the baculovirus-insect cell-based system for the development of several commercial products that will impact animal and human health will further enhance interest in this technology by pharma. Clearly, future progress in novel cell and engineering advances will lead to fundamental scientific discoveries and serve to enhance the utility and applications of this baculovirus-insect cell system.      

Why pesticides with mutagenic,carcinogenic and reproductive risks are registered in Brazil

Brazil is the biggest market for pesticides in the world. In the registration process, a pesticide must be authorized by the Institute of the Environment, Health Surveillance Agency and Ministry of Agriculture. Evaluations follow a package of toxicological studies submitted by the companies and also based on the Brazilian law regarding pesticides. We confronted data produced by private laboratories, submitted to the Institute of the Environment for registration, with data obtained from scientific databases, corresponding to mutagenicity, carcinogenicity and teratogenicity of pesticides. All studies submitted by the companies were carried out by private laboratories. From 247 pesticide formulations analyzed, none showed positive results for mutagenicity, carcinogenicity or teratogenicity. From 574 articles in the scientific literature, 84% published by public laboratories showed positive results, while 79% of those showing negative results came from private laboratories. There is an ethical concern about a conflict of interest between public/independent laboratories and private laboratories that produce data for registering pesticides. We demonstrated that there is a clear contradiction between public and private laboratories. Brazilian regulatory authorities have approved the registration of pesticides based almost exclusively on the monographs provided by the pesticide industry, because the use of scientific articles or information from the independent literature is strongly belittled by the industry. Pesticide companies argue that scientific articles cannot be trusted. Also, according to the industry, pesticide registration cannot be refused based on results from scientific articles. Thus, the registration of pesticides with mutagenic, carcinogenic and teratogenic risks has been approved in Brazil.     

Cyclosporin therapeutic drug monitoring--an established service revisited

Despite the routine application of therapeutic drug monitoring of cyclosporin (CsA) for two decades, there remain significant analytical issues. In addition, new developments have arisen in the delivery of this laboratory service as well as alternative clinical strategies for delivering optimal benefit to organ transplant recipients.Sample collection strategies are evolving away from the traditional pre-dose/trough (C0) sample in favour of estimates of the absorption phase in the first 4-6 hours after the oral dose of CsA. This is based on the recognition of the relatively poor relationship between C0 and CsA exposure indices, such as area under the blood CsA concentration versus time curve (AUC), especially in the first few hours after the dose. By collecting serial blood samples over this limited period (4hr after the dose) and estimating the AUC(0-4), one can gain insight into how well CsA has been absorbed for each transplant recipient, and individualise CsA dosage. However, a recent survey of Australasian CsA laboratories revealed that such AUC(0-4) sampling strategies in the early post-dose period were poorly accepted in clinics across Australasia. The alternative that has proven to be more clinically acceptable is the use of a single sample 2-hours after the dose (C2). The C2 concentration has been demonstrated (particularly in kidney and liver transplant recipients) as correlating well with AUC(0-4), allowing it to be used as a surrogate index of CsA absorption and exposure.The laboratory survey also showed several areas of concern in the analytical sphere. The major one is that the majority of laboratories employ the two immunoassays that deliver the least specific result on C0 samples within the range of monoclonal methods, leading to high variability and clinically significant errors with patient samples. Laboratories have also adopted a range of dilution protocols for the significantly higher C2 concentrations, and this has proved a source of significant error. In addition, around 30% of laboratories were not involved in a proficiency-testing program. Thus there is clear opportunity to do much better analytically.Hence, there remain significant challenges ahead to deliver better quality CsA assay services and dosage individualisation to further improve outcomes for the organ transplant recipients that we care for.     

Daily hoarding opportunity entrains the pacemaker for hamster activity rhythms

Summary The effects on activity rhythms of a daily 30 min opportunity to leave the home cage and hoard seeds from an open field were assessed in Syrian hamsters housed in continuous dim illumination. Six of ten hamsters responded with clear entrainment of their activity rhythms to the hoarding opportunity, as demonstrated by responses to phase shifts and by the onset phase of subsequent freerunning rhythms. No entrainable component separate from the freerunning rhythm was ever observed. Two hamsters showed phase shifts in response to the hoarding opportunity, but they did not meet the criteria for stable entrainment, and two did not respond with noticeable changes in rhythmicity. Ablations of the suprachiasmatic nuclei (SCN) were attempted in three hamsters that had entrained stably to the hoarding time. The effects of partial lesions in two animals indicated that the entrained rhythm was controlled by the light-entrainable pacemaker represented by the SCN. The one animal with an apparently complete lesion, however, developed a clear, but irregular, increase in activity in anticipation of the daily hoarding time. SCN ablation apparently unmasked an oscillator system separate from the SCN and susceptible to entrainment by a nonphotic cue. The oscillator mechanism affected by daily hoarding opportunities in hamsters appears to be tightly coupled to the SCN pacemaker, in contrast to the system in rats that is synchronized by daily feeding schedules.Abbreviations SCN suprachiasmatic nuclei - FEO food-entrain-able oscillator Portions of these results were previously reported at the 1986 meeting of the Animal Behavior Society, Tucson, Arizona, and at the 1987 meeting of the Society for Neuroscience, New Or leans, LA (Rusak et al. 1987)     

Validation of the human T-lymphocyte cloning assay — ring test report from the EU concerted action on HPRT mutation (EUCAHM)

The T-cell cloning assay, which enables the enumeration and molecular analysis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been extensively used for studying human somatic gene mutation in vivo. However, large inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental methodology, and development of an optimal but easy uniform cloning protocol. As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have carried out two Ring tests for the T-cell cloning assay. For each test, duplicate and coded samples from three buffy coats were distributed to five laboratories for determination of MF using six different protocols. The results indicated a good agreement between split samples within each laboratory. However, both the cloning efficiencies (CEs) and MFs measured for the same blood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remarkable difference in the level of MF. A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring test. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation, but decreased the CE in laboratories traditionally using priming. Adjusted for donor, use of different protocols contributed significantly to the overall variation in lnCE (P=0.0004) and lnMF (P=0.03), but there was no significant laboratory effect on the lnCE (P=0.38) or lnMF (P=0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free medium X-Vivo 10 and PMA was tested in one laboratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations.     

The association betweenCarpophilus beetles and cactus flowers

There is a close association between bowl-shaped cactus flowers and the nitidulid beetlesCarpophilus pallipennis (Say) andC. floralis Er. in North America. The nature of this association has not been clear. It now appears that the cactus flowers are brood sites of theCarpophilus beetles. The benefits in the association are one-sided, inasmuch as the beetles provide little or no pollination service for the flowers.Pollination of North American Cacti, VI.     

God’S Laboratory: Religious Rationalities And Modernity In Ecuadorian In Vitro Fertilization

Catholicism is the only major world religion that unequivocally bans the use of in vitro fertilization (IVF). Nevertheless, in Ecuador, Catholic IVF practitioners declare God’s dominion over their IVF laboratories and clinics in explaining pregnancy outcomes. My analysis of this routine combination of spiritual and material causal models in Ecuadorian IVF contributes to two ongoing discussions about (1) the tensions between “institutional” and popular forms of Catholic religiosity and (2) the proper boundaries of science in modernity. The Catholic Church’s historical and contemporary struggle to determine control of the miraculous has usually been characterized as a conflict between educated clergy and humble peasants. In the case of Ecuadorian IVF, we find, instead, educated elites and middle classes participating in this same contestation with the Church, proclaiming their direct ability to harness the power of God to effect material change on earth. This spiritual power to affect clinical outcomes does not take place just anywhere, but in clinic and lab, disrupting another set of presumptions about modern scientific practice and subjectivity. Like other Ecuadorian elites and middle classes, IVF practitioners are heirs to Enlightenment thought, and experience themselves as modern in their participation in these high-tech endeavors. But their spiritual approach to laboratory rationality does not trouble these IVF practitioners’ experience of themselves as moderns, prompting a reevaluation of the narratives of scientific modernity that limit their scope to Europe and North America.     

Laypeople's Health Concerns and Health Beliefs in Regard to Risk Perception of Mobile Communication

The consensus scientific view is that there is an absence of convincing scientific evidence for health risks of exposures to electromagnetic fields (EMF) at levels below those recommended in international guidelines. Nevertheless, some citizens are worried about EMF emitted by mobile communication and its consequences for health. The present study explored, by means of a mail survey, health concerns and the prevalence of health beliefs related to EMF in the general population. A random sample (n = 765, response rate 41%) of the German-speaking population in Switzerland was asked to assess various health beliefs. Results suggest that health concerns are widespread but lower than health concerns in regard to other hazards. About two-thirds of the respondents believed that some people suffer from electromagnetic hypersensitivity (EHS). Health beliefs items were analyzed using the Mokken scale. This scale was related to respondents' health concerns and showed that health beliefs differed in regard to sociodemographic variables. For example, analyses showed that females, younger respondents, and respondents who believed that some people are affected by EHS endorsed significantly more health beliefs than males, older respondents, and non-EHS respondents. Results indicate that it is important for policy-makers to develop a clear understanding of the possible effects of health beliefs on health concerns and risk perception. These findings may provide guidance for the further development of information materials and strategies.     

Hybridization of invasive <Emphasis Type="Italic">Phragmites australis</Emphasis> with a native subspecies in North America

Interspecific hybridization can lead to the extinction of native populations and increased aggressiveness in hybrid forms relative to their parental lineages. However, interbreeding among subspecies is less often recognized as a serious threat to native species. Phragmites australis offers an excellent opportunity to investigate intraspecific hybridization since both native and introduced lineages occur in North America. Introduced Phragmites is a highly successful estuarine plant invader throughout North America, but native Phragmites populations are declining in the eastern US. Despite range overlaps, hybridization has not yet been detected between the native and introduced lineages in the wild, suggesting that phenological or physiological barriers preclude cross-pollination. We demonstrate, for the first time, that native and introduced populations of Phragmites can hybridize. There is substantial overlap in flowering period between native and introduced populations from the same geographic locations. We manually cross-pollinated native individuals with pollen from introduced Phragmites and recovered viable offspring. We then used microsatellite markers to prove that alleles unique to the pollen parent were transferred to progeny. Our results imply a mechanism for the further decline of native Phragmites in North America and a potential for the formation of aggressive hybrid offspring.