Animal laboratory studies have been proven useful in biological knowledge acquisition (Hanson et al., 1976) and as experimental analogies or precursors to human testing (Kitamura et al., 1978; Gomez et al., 2002). Their latter use has been of great significance to both human medicine, e.g. surgical and pharmaceutical treatment, and human testing/monitoring, e.g. physiological and neuropsychological (Mortell et al., 2006). Despite their useful applications, many support the ban of the use of animals with particular focus on vertebrates. The scientific community though, understands that in many cases the use of animals is necessary due to the lack of equally successful alternatives.
In an attempt to approach those alternatives and limit the numbers of animals used and the pain, stress and distress to which they are exposed, suggestions have risen. Russell and Burch’s (1959) writings come as a guide to establishing animal experimentation through the use of the ‘three Rs’. The principle of Replacement addresses the use of less sensitive species and alternatives to animal testing, while the principles of Reduction and Refinement address the number of animals and animal welfare, respectively, when and where alternatives for animal use are not opportune. These guidelines have been incorporated in the 1986 Animal (Scientific Procedures) Act (ACTA) and their application should be carefully followed, especially when conducting research using regulated species and procedures in the UK, as licensed by the Home Office (Flecknell, 2002). This review presents the use of the ‘three Rs’ in studies to discuss the coexistence of animal welfare with good science.
One of the most predominant alternatives to animal research is in vitro research. For transferring research to the domain of tissue and cell culture, first hypotheses to be tested need to be accordingly stated. By promoting exploratory testing of hypotheses with the use of in vitro methods, the scientific community could discover novel applications that will both reduce the need for animal models and expand human knowledge and technique (Gruber and Hartung, 2004).
The success of alternative methodologies is evident in many studies, but still the performance of these methods is mostly restricted to the laboratories where they were developed and to the studies they were first reported. On the other hand, some approaches have gathered accreditation and universal usage, most of them through commercialisation. One example is the production and modification of monoclonal antibodies by in vitro rather than in vivo cultures (Rodwell, 1986). Other examples include: the artificial immune system MIMIC (modular immune in vitro construct) which uses white blood-cells from donations for vaccine development (Ahmed and Gottschalk, 2009), the use of keratinocyte cultures which mimic the human epidermis for irritation and corrosion testing (Marcelo et al., 1978) and the use of blood from donors to test inflammation and fever effects of intravenous medication and other pharmaceuticals (Bodel, 1976).
Current knowledge and understanding might blind some scientists in realising that sometimes aspects of in vivo testing are disadvantageous when compared to in vitro alternatives (Boyarsky et al., 1996). Advantages of in vitro research consist of reduced legislative restrictions, lower costs and direct assessment (Polli, 2008). Quality driven standards, like initiatives for ‘Good Cell Culture Practice’, could greatly promote scientifically acceptable and animal-protecting processes (Coecke et al., 2005).
In addressing animal welfare, some studies choose to substitute animals with less sentient and more abundant species that also have shorter life-spans. This gave rise to the use of invertebrates and invertebrate system analogies. A good example is the use of the common fruit-fly, Drosophila melanogaster, as a genomic guide for many DNA discoveries (Pallanck and Ganetzky, 1994). The simplicity of invertebrate systems, allows for easier monitoring, examination and better realisation of the working capabilities of smaller-scale processes (Rind and Simmons, 1999).
In respect to the examination of system analogies, some studies support that the use of invertebrates has provided knowledge on neuronal processes, e.g. behavioural associations with neurones in crickets (Nolen and Hoy, 1984), whilst other studies include the examination of the endocrine system (Smit et al., 1998). Studies also assess biological aspects of invertebrates and chordates that may be useful in human medicine. Such aspects include models of ageing (e.g. in red sea urchins), novel anticancer drug development (e.g. the use of tunicates in the discovery and production of Yondelis) and discoveries of cancer resistance (Simmons et al., 2005; Bodnar, 2009). Other applications of invertebrate testing include disease and infection assays (Ben-Ami, 2011) as well as testing adverse effects of medication (Brandon et al., 2003).
Alternatives also exist in technology. The creation of computer software and models that project human response to stimuli is an example. Some studies report of computer models with the ability to predict acute oral toxicity, carcinogenicity and mutagenicity from chemicals (Höfer et al., 2004). Other studies present computer software, e.g. the ‘Entelos PhysioLab’, which encompasses models of the immune, respiratory and cardiovascular systems and also factors genetics, physiology and environmental stressors (Stokes, 2000; Powell et al., 2007).
Mathematical models have been used to describe and analyse: physiology as the endocrine and metabolic systems (Carson et al., 1983), cellular and molecular biology (Finegood et al., 1995), biochemistry and ionic movement (Nygren et al., 1998), motor processes and the musculo-skeletal system (Seireg and Arvikar, 1973; Hakim et al., 1976).
For the reduction of numbers of animals used, suggestions include the careful development of experiments that will safeguard all domains of the animal that have not been used in order for those to be available for future or parallel studies (Seybold et al., 1982; Zethof et al., 1994).The careful development of methodologies for carrying out an experiment could also contribute to decreased animal usage. For instance, pharmacokinetic studies support that the usage of serially bled mice could result in a great decrease in numbers of mice used and further decrease amounts of compound needed (Bateman et al., 2001). This is because, in contrast, conventional methods require one animal for each time point, whereas more time points can be covered by a mouse through this method.
Other measures include the use of only the minimum sample size needed for significant results through the use of statistical methods such as sample size determination formulas (Dell et al., 2002). Further suggestions could include the use of longitudinal studies as in human testing, whereby instead of using two populations (a control and a target group to be compared), one sample population can be used and assessed in both the control and examinable conditions at different periods (Charlson et al., 1987).
Other possibilities for reduction could refer to the state and distribution of literature. It is suggested that meta-analyses and reviews be continuously occurring in particular domains so as to emphasise existing knowledge and avoid the repetition of experiments. This could be a contributing factor to a larger scale reduction of animal use.
Techniques for quickest and less painful death, euthanasia, are incorporated in humane treatment. Of great importance to using euthanasia is setting a humane endpoint which could be decided after observing clinical signs but also when effects have been evident but suffering is still at its early onset (Mellor 1997). A good strategy could be compiling datasets of behaviours observed in experiencing pain and also monitoring animals’ vital signs.
Generally, methods of euthanasia are separated into chemical and physical, and their appropriateness for use might be regulated. In the UK, Schedule 1 under ACTA lists 6 ways for euthanasia: confirmation of permanent cessation of the circulation, destruction of the brain, dislocation of the neck, exsanguinations, confirmation of the onset of rigor mortis, and mechanical disruption. Literature refers to various methodologies deemed appropriate; carbon dioxide poisoning, preferably by gradual addition so that first the animal is rendered unconscious and does not experience respiratory distress (Hackbarth, 2000); decapitation and cervical dislocation, which are reported as successful mainly in small animals and rodents (Holson, 1992; Cartner et al., 2007); the use of captive bolt for larger animals as lamb which was also tested in rabbits and dogs showing no brain activity within 15 seconds (Dennis, 1988; Finnie, 2000).
The use of analgesics and anaesthetics in animal testing is a way to reduce both pain and distress. Before using particular anaesthetics, information on the duration and effects of their use should be considered (Flecknell, 1984). Most studies usually use inhalational and injectable anaesthetics.
Inhalational anaesthetics can be taken through a vaporizer, soaked cotton wool in a jar or using a mask. Main anaesthetics of this kind include nitrous oxide, halothane and, its modern replacements, isoflurane and sevoflurane. Whilst nitrous oxide is a strong agent with little need of adjacent anaesthetics, it is also toxic and with a minimum alveolar concentration (MAC) much greater than the other agents, especially in swine (Berkowitz and Finck, 1976; Weiskopf and Bogetz, 1984; Gonsowski and Eger II, 1994; Gilles et al.,2001). Halothane, isoflurane and sevoflurane are reported as safer with only some effects on cardiovascular functions.
Injectable anaesthetics in literature include barbiturates; steroids; neuroleptoanalgesics and, ‘the milk of amnesia’, propofol. These anaesthetics may cause a variety of effects (e.g. loss of reflexes) depending on the type of pain and the species and size of the animal. Injectables can be administered intramuscularly, subcutaneously, intraperitoneally or more effectively intravenously (Smith, 1993). Considerations for studies should include monitoring of possibly affected areas of the animal as heart rate and body temperature (Kettlewell et al., 1997) or even monitoring the depth of analgesia (Jensen et al., 1999). Such procedures have been completed using microchip implants and heat-sensitive transponders (Kort et al., 1998; Hartinger et al., 2003).
To allow for impartial procedures and reduced variability many studies have shown to support standardised housing and conditions amongst all individuals to be tested (Wong et al., 1983; Klumpp et al., 2006). Nevertheless, when considering animal welfare it is probable that these uniform and uneventful conditions may not be the healthiest living environment. Additionally, allowing for variance of an educated range might better reflect effects of variables in the wild.
Conditions that have been considered for better housing quality include: efficient ventilation to facilitate respiratory processes and comfort (Keller et al., 1989); regulation of temperature within their natural range, which could further benefit the study (Bowyer et al., 1992); lighting in accordance to natural daylight changes, e.g. locomotion-linked feedback dimming for photoperiod regulation (Ferraro et al., 1984; Mistlberger and Rusak, 1998); use of comfortable and/or natural bedding, e.g. shavings or shredded paper (Ward et al., 2000); feeding with variable diets including food that needs some processing before consumption, e.g. nuts for rodents (Batzli and Cole, 1979). Furthermore, cleaning needs to be considerate to territorial marking (Mykytowycz, 1968); handling needs to consider stress in the animal (Gärtner et al., 1980); socialising needs to examine and understand play behaviour (environmental enrichment) and behaviours of rivalry, exclusion and mating (Poirier and Smith, 1974). Animals should be allowed to get familiar to researchers/handlers and vice versa.
Issues related to housing include subclinical and clinical diseases, which might be easily transmitted amongst members of the animal population but also between animals of different species (Seamer and Chesterman 1967). Solutions may include the use of specified-pathogen-free animals and the careful sterilisation of equipment and housing during the study (Festing and Blackmore, 1971).
The application of replacement methods to animal research is greatly limited by restricted investment and by little supportive research. Still, promising alternative methods exist despite individual and inherent limitations. It may be suggested that there is a tendency to rely on existent procedures due to the insufficient literature reviewing novel methodologies, the limited changes to accepted norms and the luck of adoption of alternative standards. There is a great need for the standardisation of alternative methodologies by addressing limitations to good practice and by only promoting the most scientifically viable procedures. By providing scientists with databases of successful methods, the ‘three Rs’ will be promoted only through good science.
Limitations to establishing reduction of numbers used mostly include statistical complications in supporting result significance. Other limitations are to the application of the proposed methods. Nevertheless some strategies do exist and it would be beneficial if these were appropriately used.
In view of the limited replacement alternatives and since reduction allows for margins of statistical insignificance, researchers seem to be turning their efforts to method refinement. Refinement is the most diversely endorsed principal of the three and it is broadly applied. The use of refinement methods is a result of their effective application in scientifically successful studies. Limitations to their approach include assumptions e.g. assuming uniform pain experience and uniform dosage for all members of a species (Flecknell, 2002) or even assuming that the effects of analgesics are similar to human experience (Plous, 1993; Anil et al., 2002). By addressing these assumptions, refinement methods would even more benefit animal welfare. Tests on animal reaction behaviours as well as reviews and meta-analyses could be shown very beneficial.
As discussed in this review, new methodologies and practices can be used to adjust or replace already existent ones. These should be better tested, evaluated and reviewed before recurrently used. Changes can only stem from careful consideration of all possibilities and through the standardisation of particular, promising methods. Animal welfare and good science can coexist but even better when limitations are overcome.
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