Reduction strategies in animal research: a review of scientific approaches at the intra-experimental, supra-experimental and extra-experimental levels

When discussing animal use and considering alternatives to animals in biomedical research and testing, the number of animals required gets to the root of the matter on ethics and justification. In this paper, some reduction strategies are reviewed. Many articles and reports on reduction of animal use focus mostly on the experimental level, but other approaches are also possible. Reduction at the intraexperimental level probably offers the greatest scope for reduction, as the design and statistical analysis of individual experiments can often be improved. Supra-experimental reduction aims to reduce the number of animals by a change in the setting in which a series of experiments take place--for example, by improved education and training, reduction of breeding surpluses, critical analysis of test specifications, and re-use of animals. At the extra-experimental level, reduction is a spin-off of other developments, rather than the direct goal. Through improved research or production strategies, aimed at better quality, consistency and safety, reduction in the number of animals used can be substantial. A revised definition of reduction is proposed, which does not include the level of information needed, as in some cases reduction in the number of animals resulting in less information or data, is still acceptable.     

Gene therapy for osteoarticular disorders

Osteoarticular disorders are the major cause of disability in Europe and North America. It is estimated that rheumatoid arthritis affects 1 % of the population and that more than two third of people over age 55 develop osteoarthritis. Because there are no satisfactory treatments, gene therapy offers a new therapeutic approach. The delivery of cDNA encoding anti-arthritic proteins to articular cells has shown therapeutic efficacy in numerous animal models in vivo. Through the development and the experimental progresses that have been made for both rheumatoid arthritis and osteoarthritis, this review discusses the different gene therapy strategies available today and the safety issues with which they may be associated. Among the different vectors available today, adeno-associated virus seems the best candidate for a direct in vivo gene delivery approach for the treatment of joint disorders.     

Minimising pain in farm animals: the 3S approach – ‘Suppress,Substitute, Soothe’

Recently, the French National Institute for Agricultural Research appointed an expert committee to review the issue of pain in food-producing farm animals. To minimise pain, the authors developed a ‘3S’ approach accounting for ‘Suppress, Substitute and Soothe’ by analogy with the ‘3Rs’ approach of ‘Reduction, Refinement and Replacement’ applied in the context of animal experimentation. Thus, when addressing the matter of pain, the following steps and solutions could be assessed, in the light of their feasibility (technical constraints, logistics and regulations), acceptability (societal and financial aspects) and availability. The first solution is to suppress any source of pain that brings no obvious advantage to the animals or the producers, as well as sources of pain for which potential benefits are largely exceeded by the negative effects. For instance, tail docking of cattle has recently been eliminated. Genetic selection on the basis of resistance criteria (as e.g. for lameness in cattle and poultry) or reduction of undesirable traits (e.g. boar taint in pigs) may also reduce painful conditions or procedures. The second solution is to substitute a technique causing pain by another less-painful method. For example, if dehorning cattle is unavoidable, it is preferable to perform it at a very young age, cauterising the horn bud. Animal management and constraint systems should be designed to reduce the risk for injury and bruising. Lastly, in situations where pain is known to be present, because of animal management procedures such as dehorning or castration, or because of pathology, for example lameness, systemic or local pharmacological treatments should be used to soothe pain. These treatments should take into account the duration of pain, which, in the case of some management procedures or diseases, may persist for longer periods. The administration of pain medication may require the intervention of veterinarians, but exemptions exist where breeders are allowed to use local anaesthesia (e.g. castration and dehorning in Switzerland). Extension of such exemptions, national or European legislation on pain management, or the introduction of animal welfare codes by retailers into their meat products may help further developments. In addition, veterinarians and farmers should be given the necessary tools and information to take into account animal pain in their management decisions.     

Random main effects of treatment: A case study with a network meta‐analysis

If the number of treatments in a network meta‐analysis is large, it may be possible and useful to model the main effect of treatment as random, that is to say as random realizations from a normal distribution of possible treatment effects. This then constitutes a third sort of random effect that may be considered in connection with such analyses. The first and most common models treatment‐by‐trial interaction as being random and the second, rather rarer, models the main effects of trial as being random and thus permits the recovery of intertrial information. Taking the example of a network meta‐analysis of 44 similar treatments in 10 trials, we illustrate how a hierarchical approach to modeling a random main effect of treatment can be used to produce shrunk (toward the overall mean) estimates of effects for individual treatments. As a related problem, we also consider the issue of using a random‐effect model for the within‐trial variances from trial to trial. We provide a number of possible graphical representations of the results and discuss the advantages and disadvantages of such an approach.     

Comparative denaturing gradient gel electrophoresis analysis of fungal communities associated with whole plant corn silage.

Significant portions of grain produced for livestock consumption are convened into ensiled forage. Silage producers have long recognized the positive effects of using an inoculant to insure the proper transformation of forage into a palatable and digestible feedstuff. When silage is fed from a storage structure, exposure to air stimulates the growth of epiphytic aerobes that may result in the loss of up to 50% of the dry matter. Moreover, fungi have been found to be associated with ensiled forage, but their growth is normally suppressed by the anaerobic conditions. However, the introduction of oxygen results in a fungal bloom, and the fungi and the associated metabolites may result in lost productivity in the livestock consuming the contaminated forage. In this study, we report on the diversity of the fungal community associated with whole plant corn silage during the ensiling process, and the effect of two different bacterial inoculants as compared with the uninoculated natural epiphytic fermentation on the distribution of the fungi associated with the silage. The fungal community from duplicate mini-silo packages of the same treatment was analyzed by denaturing gradient gel electrophoresis and direct sequencing of the resulting operational taxonomic units. This method proved useful in analyzing the complex microbial communities associated with the forage in that it was possible to determine that one inoculant dramatically influenced the fungal community associated with whole plant corn silage.     

Bacteria in semen used for IVF affect embryo viability but can be removed by stripping cumulus cells by vortexing

Bacterial contamination of in vitro vs in vivo produced embryos presents a particular danger because of the alteration of the zona pellucida and the use of various biological products during culture. Our objective was to investigate the effects of semen contaminated with bacteria on IVF of bovine oocytes and to determine if removal of cumulus cells by vortexing as opposed to pipetting would reduce contamination and improve subsequent embryonic development. Semen from 5 bulls of the Native Korean breed (Bulls A, B, C, D, E) was used for IVF of matured oocytes. Preliminary studies had shown that the semen from Bulls A, B, D and E but not Bull C was contaminated with various species of common bacteria. After IVF, the cumulus cells surrounding the oocytes were removed either by pipetting or vortexing. Viability and cleavage rates of the resulting zygotes was assessed after 44 h in culture. When cumulus cells were removed by pipetting, only zygotes derived from oocytes that were fertilized with uncontaminated semen from Bull C developed to morula and blastocyst stages; zygotes derived from oocytes that were fertilized with contaminated semen from Bulls A, B, D and E started to degenerate, and the culture media became noticeably turbid. When cumulus cells were removed by vortexing, zygotes derived from oocytes fertilized with either contaminated or uncontaminated semen showed good rates of development (16 to 32%) to morula or blastocyst stages. From these results it can be concluded that the bacteria introduced with the semen contaminated the in vitro system and severely reduced the viability of the embryos. In contrast, complete removal of the cumulus cells with vortexing, as opposed to pipetting, reduced the contamination of the culture medium, allowing embryonic development to take place.     

RNA interference to target lipid disorders

PURPOSE OF REVIEW: This review focuses on proof-of-principle experiments providing validation of new targets for the development of RNA interference-based therapeutics for dyslipidemia. RECENT FINDINGS: Over the past few years, RNA interference has become an accepted approach to manipulate gene expression in mammalian systems. Advantage has been taken of the relative tissue specificity of adenovirus for liver, and the genetic specificity of short hairpin RNA-mediated RNA interference to create liver-specific downregulation of different genes. A different approach to target liver has been through the administration of chemically modified short interfering RNAs. For example, apolipoprotein B messenger RNA has been silenced in liver and jejunum resulting in decreased plasma levels of apolipoprotein B and total cholesterol. SUMMARY: RNA interference has aroused great interest as a powerful experimental tool and a potential therapeutic strategy. Successful animal studies indicate that RNA interference might be useful for the treatment of various human diseases. Clinical studies will soon begin to assess the use of this new class of therapeutics to treat dyslipidemia.     

Stereotypies in Captive Primates and the Use of Inositol: Lessons from Obsessive–Compulsive Disorder in Humans

Animal stereotypies have long been used in the study of obsessive–compulsive disorder (OCD) in humans. These studies have led to the understanding of some of the molecular pathways in the disorder and the use of selective serotonin reuptake inhibitors and myo-inositol in the treatment of these conditions. If animal models, especially nonhuman primate models, were used to study human disorders and if the resulting treatments were successful, then conversely one should be able to treat nonhuman primate stereotypies with similar methods. We here summarize animal models of OCD (including nonhuman primate models) and human OCD treatments, and using successful human treatment by myo-inositol as models, recommend the use of myo-inositol in good captive management practice and the treatment of nonhuman primate stereotypies. We believe that this would be particularly useful in the treatment of stereotypies in nonhuman primates because they are physiologically so similar to humans.     

Reduction in laboratory animal use by factorial design

Factorial experimental designs (FEDs) can be used to study the effects of controllable variables, such as an experimental treatment, sex, strain, age, diet and prior treatment of animals, on some defined response. Such designs have been widely used in optimising manufacturing processes, but have rarely been used in optimising animal experiments in drug discovery. FEDs generally provide more information than the alternative "one-variable-at-a-time" approach, because each animal contributes information on the effect of every factor, and because such designs can highlight any interactions among the variables. Although FEDs can have any number of factors and levels of each factor, where many factors are to be explored, it is common to do an initial experiment using two levels of each factor, and in some cases fractional factorial designs can be used to reduce the total number of treatment combinations to manageable levels. These designs have been used successfully at AstraZeneca in the optimisation of in vivo drug screening experiments, where their use has effectively reduced the numbers of animals used in some routine screens.     

Evaluation of quinacrine treatment for prion diseases

Based on in vitro observations in scrapie-infected neuroblastoma cells, quinacrine has recently been proposed as a treatment for Creutzfeldt-Jakob disease (CJD), including a new variant CJD which is linked to contamination of food by the bovine spongiform encephalopathy (BSE) agent. The present study investigated possible mechanisms of action of quinacrine on prions. The ability of quinacrine to interact with and to reduce the protease resistance of PrP peptide aggregates and PrPres of human and animal origin were analyzed, together with its ability to inhibit the in vitro conversion of the normal prion protein (PrPc) to the abnormal form (PrPres). Furthermore, the efficiencies of quinacrine and chlorpromazine, another tricyclic compound, were examined in different in vitro models and in an experimental murine model of BSE. Quinacrine efficiently hampered de novo generation of fibrillogenic prion protein and PrPres accumulation in ScN2a cells. However, it was unable to affect the protease resistance of preexisting PrP fibrils and PrPres from brain homogenates, and a "curing" effect was obtained in ScGT1 cells only after lengthy treatment. In vivo, no detectable effect was observed in the animal model used, consistent with other recent studies and preliminary observations in humans. Despite its ability to cross the blood-brain barrier, the use of quinacrine for the treatment of CJD is questionable, at least as a monotherapy. The multistep experimental approach employed here could be used to test new therapeutic regimes before their use in human trials.     

A positive association of larval therapy and hyperbaric oxygen therapy in veterinary wound care

The treatment of cutaneous wounds is part of the veterinary routine from initial scientific reports due to being regularly present condition. Currently, several types of treatments are available to accelerate the healing process. This report presents the case of a dog with multiple lesions in the thoracic limbs resulting from a car accident, who underwent larval therapy and hyperbaric oxygen therapy (HBOT). The animal was a 2-year-old female mixed breed dog presenting severe skin degloving, fracture in the left thoracic limb (LTL), with abrasion lesions and dislocation in the right thoracic limb (RTL). The animal underwent multiple modality therapies, such as HBOT sessions associated with larval therapy; even after the LTL presented gangrene, this treatment resulted in optimal viability of the non-necrotic tissue adjacent to the gangrene. Due to chronic pain unresponsive to drug control and the presence of a fracture at a location where a possible exoprosthesis was supposed to be fixed, the LTL ended up being amputated. There are several reports of the use of HBOT or larval therapy in traumatized limbs; however, the combination of both therapies has not been previously described in the veterinary literature. Thus, we demonstrate through this report that it was possible to quickly recover the animal with good wound resolution through tissue oxygenation and a healthy granulation bed, both provided by the therapeutic combination.     

Development of genotoxicity test procedures with Episkin, a reconstructed human skin model: towards new tools for in vitro risk assessment of dermally applied compounds?

Today reconstructed skin models that simulate human skin, such as Episkin, are widely used for safety or efficacy pre-screening. Moreover, they are of growing interest for regulatory purposes in the framework of alternatives to animal testing. In order to reduce and eventually replace results of in vivo genotoxicity testing with in vitro data, there is a need to develop new complementary biological models and methods with improved ability to predict genotoxic risk. This can be achieved if these new assays do take into account exposure conditions that are more relevant than in the current test systems. In an attempt to meet this challenge, two new applications using a human reconstructed skin model for in vitro genotoxicity assessment are proposed. The skin is the target organ for dermally exposed compounds or environmental stress. Although attempts have been made to develop genotoxicity test procedures in vivo on mouse skin, human reconstructed skin models have not been used for in vitro genotoxicity testing so far, although they present clear advantages over mouse skin for human risk prediction. This paper presents the results of the development of a specific protocol allowing to perform the comet assay, a genotoxicity test procedure, on reconstructed skin. The comet assay was conducted after treatment of Episkin with UV, Lomefloxacin and UV or 4-nitroquinoline-N-oxide (4NQO). Treatment with the sunscreen Mexoryl was able to reduce the extent of comet signal. A second approach to use reconstructed epidermis in genotoxicity assays is also proposed. Indeed, the skin is a biologically active barrier driving the response to exposure to chemical agents and their possible metabolites. A specific co-culture system (Figure 1) using Episkin to perform the regular micronucleus assay is presented. Micronucleus induction in L5178Y cells cultured underneath Episkin was assessed after treatment of the reconstructed epidermis with mitomycin C, cyclophosphamide or apigenin. This second way of using human reconstructed skin for genotoxicity testing aims at improving the relevance of exposure conditions in in vitro genotoxicity assays for dermally applied compounds.     

Differences on Pb accumulation among plant tissues of 25 varieties of maize (Zea mays)

Pollution of agricultural land by heavy metals has imposed an increasingly serious risk to environmental and human health in recent years.Heavy metal pollutants may enter the human food chain through agricultural products and groundwater from the polluted soils.Progress has been made in the past decade on phytoremediation,a safe and inexpensive approach to remove contaminants from soil and water using plants.However,in most cases,agricultural land in China cannot afford to grow phytoremediator plants instead of growing crops due to food supply for the great population.Therefore,new and effective methods to decrease the risk of heavy metal pollution in crops and to clean the contaminated soils are urgently needed.If we can find crop germplasms (including species and varieties) that accumulate heavy metals in their edible parts,such as the leaves of vegetables or grains of cereals,at a level low enough for safe consumption,then we can grow these selected species or varieties in the lands contaminated or potentially contaminated by heavy metals.If we can find crop germplasms that take in low concentrations of heavy metals in their edible parts and high content of the metals in their inedible parts,then we can use these selected species or varieties for soil remediation.In this study,the feasibility of the method is assessed by analyzing Pb concentrations in edible and inedible parts of 25 varieties of maize (Zea mays) grown in Pb-contaminated soils.The soil concentrations of Pb were 595.55 mg/kg in the high Pb exposed treatment and 195.55 mg/kg in the control.The results showed that the Pb concentrations in different tissues were in the order of root ＞ shoot ≌ leaf＞ grain.Compared with the control,the Pb concentrations in root,shoot and leaf were greatly increased under the high Pb exposed condition,while the increments of Pb concentration in grain were relatively lower.Under the high Pb exposure,the grain Pb concentrations of 12 varieties exceeded the maximal Pb limitation of the National Food Hygiene Standard of China (NFHSC) and were inedible.This indicates that there is a high Pb pollution risk for maize grown on Pb polluted sites.Although 22 of the 25 tested varieties had harvest loss under the highly Pb stressed condition,ranging from 0.86%-38.7% of the grain biomass acquired at the control,the average harvest loss of all the tested varieties was only 12.6%,which is usually imperceptible in normal farming practices.Therefore the risk of Pb pollution in maize products cannot be promptly noticed and prevented based only on the outcome of the harvest.However,we did find that 13 of the 25 tested varieties had grain Pb concentrations lower than the limitation of the NFHSC.It is,therefore,possible to reduce the pollution risk if these favorable varieties are used for maize production in Pb-contaminated or potentially contaminated agricultural lands.Pb concentrations in vegetative tissues (root,stem and leaf) were significantly correlated with each other,while Pb concentrations of each vegetative tissue were not significantly correlated with that of grain.Among the 25 tested varieties,some varieties had Pb concentrations in grain lower than (No.1-3 and No.6) or slightly above (No.4) the limitation of the NFHSC,while their Pb concentrations in the vegetative tissues were among the highest.When excluding these varieties,correlations between the Pb concentrations of grain and those of vegetative tissues of the rest of the tested varieties became highly significant.In addition,variety No.1 had the lowest harvest loss under high Pb exposed,and the highest Pb accumulation in vegetative tissues (51.69 mg/plant,12 times as much as in the control).Similar features were also observed in varieties No.2,No.3 and No.6,which absorbed Pb for 36-42 mg/plant under high Pb exposed.We recommend these varieties of maize to be used for bioremediation of Pb contaminated soil and crop production at the same time.     

Mitochondrial function and energy metabolism in cancer cells: Past overview and future perspectives

The involvements of energy metabolism aspects of mitochondrial dysfunction in cancer development, proliferation and possible therapy, have been investigated since Otto Warburg published his hypothesis. The main published material on cancer cell energy metabolism is overviewed and a new unique in vivo experimental approach that may have significant impact in this important field is suggested. The monitoring system provides real time data, reflecting mitochondrial NADH redox state and microcirculation function. This approach of in vivo monitoring of tissue viability could be used to test the efficacy and side effects of new anticancer drugs in animal models. Also, the same technology may enable differentiation between normal and tumor tissues in experimental animals and maybe also in patients.     

A Comparison of Methodologies for the Study of Functional Transmitter Neurochemistry in Human Brain

A number of different approaches to the study of functional neurochemistry in human brain are discussed. The advantages and disadvantages of three main techniques are contrasted: (i) using animal tissue preparations as models of the human brain; (ii) using human peripheral tissue preparations as models of dynamic CNS processes; and (iii) studying human tissue, obtained postmortem, directly. Animal models are often readily obtained and reliable, and the high degree of inbreeding of common laboratory animals ensures that they usually yield consistent results. However, there are a number of human disorders for which animal models are either poor or unavailable, and species differences make extrapolation from the animal to the human case difficult. Human peripheral tissue models rely on a degree of homology between peripheral and CNS processes; in most cases, the evidence for such homologies derives from animal, rather than human, studies. Moreover, several examples are known where a peripheral process mimics the equivalent glial cell activity more closely than the neuronal, which can be a serious drawback for studies of neurotransmission. The use of postmortem human brain tissue presents a number of obvious difficulties, resulting from variations in the patient's age, agonal state, sex, preterminal medication, postmortem delay, etc. Human beings are genetically and nutritionally heterogeneous, so that data variability is usually greater here than when using tissue from laboratory animals. However, it is possible to control for a number of these factors, for example, by matching samples for basal metabolic rate and tissue integrity, and recently developed tissue freezing and storage techniques permit the use of within-subject experimental designs to help reduce experimental variation. A range of neurotransmitter functions are well retained in such tissue samples, so that regional variations, differential transmitter activities, drug effects, etc., can be studied in normal tissue samples, as well as in samples taken from cases of neurological and psychiatric disease. This allows, for example, changes in neuroanatomical indices to be correlated with localised alterations in a specific neurotransmitter function. A systematic approach to the analysis and matching of tissue samples is advocated. The three approaches should be considered to be complementary, especially for the study of human brain diseases.     

Induction of the p16INK4a senescence gene as a new therapeutic strategy for the treatment of rheumatoid arthritis.

Synovial tissue affected by rheumatoid arthritis is characterized by proliferation, which leads to irreversible cartilage and bone destruction. Current and experimental treatments have been aimed mainly at correcting the underlying immune abnormalities, but these treatments often prove ineffective in preventing the invasive destruction. We studied the expression of cyclin-dependent kinase inhibitors in rheumatoid synovial cells as a means of suppressing synovial cell proliferation. Synovial cells derived from hypertrophic synovial tissue readily expressed p16INK4a when they were growth-inhibited. This was not seen in other fibroblasts, including those derived from normal and osteoarthritis-affected synovial tissues. In vivo adenoviral gene therapy with the p16INK4a gene efficiently inhibited the pathology in an animal model of rheumatoid arthritis. Thus, the induction of p16INK4a may provide a new approach to the effective treatment of rheumatoid arthritis.     

Policy Implications of Allocation Methods in the Life Cycle Analysis of Integrated Corn and Corn Stover Ethanol Production

A biorefinery may produce multiple fuels from more than one feedstock. The ability of these fuels to qualify as one of the four types of biofuels under the US Renewable Fuel Standard and to achieve a low carbon intensity score under California’s Low Carbon Fuel Standard can be strongly influenced by the approach taken to their life cycle analysis (LCA). For example, in facilities that may co-produce corn grain and corn stover ethanol, the ethanol production processes can share the combined heat and power (CHP) that is produced from the lignin and liquid residues from stover ethanol production. We examine different LCA approaches to corn grain and stover ethanol production considering different approaches to CHP treatment. In the baseline scenario, CHP meets the energy demands of stover ethanol production first, with additional heat and electricity generated sent to grain ethanol production. The resulting greenhouse gas (GHG) emissions for grain and stover ethanol are 57 and 25 g-CO₂eq/MJ, respectively, corresponding to a 40 and 74 % reduction compared to the GHG emissions of gasoline. We illustrate that emissions depend on allocation of burdens of CHP production and corn farming, along with the facility capacities. Co-product handling techniques can strongly influence LCA results and should therefore be transparently documented.     

Monitoring respiratory function and sleep in the obese Vietnamese pot-bellied pig.

Development of drug treatments for obstructive sleep-disordered breathing has been impeded by the lack of animal models. The obese pig may be a suitable animal model, as it has been reported to experience sleep-disordered breathing resembling human obstructive sleep apnea. The purpose of this paper is to describe in detail techniques for chronic instrumentation of the obese Vietnamese pot-bellied pig and to study respiratory function during sleep. Under general anesthesia, four obese pigs were instrumented for long-term recording of intrapleural and tracheal pressures, genioglossal EMG, and bioelectric signals related to sleep. A custom-fitted face mask was used to record respiratory variables including airflow, snoring, and expired CO(2). Most chronic instrumentation provided robust signals for up to 6 wk after installation. All pigs displayed sleep-disordered breathing characterized by increased resistance to airflow, snoring, inspiratory flow limitation, and possible sleep disruption. Apneas and hypopneas were not a feature of breathing during sleep in these animals. Nonetheless, this animal preparation may be useful for exploring possible drug treatments for obstructive sleep-disordered breathing.     

Organic acid and formaldehyde treatment of animal feeds to control Salmonella: efficacy and masking during culture

AIMS: To investigate whether treatment of animals feeds with organic acids/formaldehyde may mask the presence of Salmonella, when assessed by standard cultural methods. METHODS AND RESULTS: Four commercial treatments were applied at the manufacturers' recommended rates on feeds artificially inoculated with Salmonella. The recovery of Salmonella from these treated feeds was assessed after specific antagonists were added to the treatments during culture. A control group of treated feed received no antagonist. Masking of Salmonella was demonstrated when the addition of antagonists resulted in recovery of Salmonella from the treated feed, compared with a negative recovery when no antagonists were added. There were large variations in the efficacy of treatments, and masking was demonstrated with all four tested treatments. One formaldehyde-based product showed greater efficacy and less masking. Masking was greater when high levels of Salmonella were present in the feed. CONCLUSIONS: Some organic acid or formaldehyde-based feed treatments may mask the presence of Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: Feeds may be deemed safe despite being contaminated with Salmonella. The use of antagonists during culture may help assess the level of Salmonella contamination when organic acid or formaldehyde treatments have been applied to feed ingredients.