Minimum Number Of Animals Research Articles

Design of Animal Experiments in Pharmacological Research

Pharmacology, also known as pharmaceutical science, has made significant progress, especially in the 20th century, and has played a fundamental role in the development of today's modern drugs. Pharmacology uses in vitro, in vivo, and clinical research stages in drug development. Experimental animals are of great importance in in vivo research. The majority of the drugs used today were developed thanks to animal research. Research in which experimental animals will be used should be planned carefully, and a minimum number of animals should be used since the subject is a living being. In addition, one of the most important ethical principles is to avoid procedures that may cause unnecessary torture and pain to animals during experiments. The purpose of pharmacological research is to develop drugs for the treatment or diagnosis of diseases. For this reason, it is aimed at determining the effects of the substance you are researching in the presence of disease. Immediate use of a substance whose effects were previously unknown on humans may lead to various adverse events and even death. After many events in the past, drug development stages have been determined by accepted international rules. According to these rules, the effect of the substance being investigated must be investigated in experimental animals that have been used as disease models before humans. Many disease models have been developed for this purpose. Drugs developed in these disease models created in experimental animals are now successfully used in the treatment of humans.

Analysis of the causes of the occurrence and measures of prevention of pododermatitis in rabbits

Analysis of the causes of the occurrence and measures of prevention of pododermatitis in rabbits

The ShGlomAssay Combines High-Throughput Drug Screening With Downstream Analyses and Reveals the Protective Role of Vitamin D3 and Calcipotriol on Podocytes.

Chronic kidney disease (CKD) is a major public health burden affecting more than 500 million people worldwide. Podocytopathies are the main cause for the majority of CKD cases due to pathogenic morphological as well as molecular biological alterations of postmitotic podocytes. Podocyte de-differentiation is associated with foot process effacement subsequently leading to proteinuria. Since currently no curative drugs are available, high throughput screening methods using a small number of animals are a promising and essential tool to identify potential drugs against CKD in the near future. Our study presents the implementation of the already established mouse GlomAssay as a semi-automated high-throughput screening method—shGlomAssay—allowing the analysis of several hundreds of FDA-verified compounds in combination with downstream pathway analysis like transcriptomic and proteomic analyses from the same samples, using a small number of animals. In an initial prescreening we have identified vitamin D3 and its analog calcipotriol to be protective on podocytes. Furthermore, by using RT-qPCR, Western blot, and RNA sequencing, we found that mRNA and protein expression of nephrin, the vitamin D receptor and specific podocyte markers were significantly up-regulated due to vitamin D3- and calcipotriol-treatment. In contrast, kidney injury markers were significantly down-regulated. Additionally, we found that vitamin D3 and calcipotriol have had neither influence on the expression of the miR-21 and miR-30a nor on miR-125a/b, a miRNA described to regulate the vitamin D receptor. In summary, we advanced the established mouse GlomAssay to a semi-automated high-throughput assay and combined it with downstream analysis techniques by using only a minimum number of animals. Hereby, we identified the vitamin D signaling pathway as podocyte protective and to be counteracting their de-differentiation.

The Ethical Dilemma of Non-Human Primate Use in Biomedical Research

The Ethical Dilemma of Non-Human Primate Use in Biomedical Research

Optimizing Sequencing Resources in Genotyped Livestock Populations Using Linear Programming.

Low-cost genome-wide single-nucleotide polymorphisms (SNPs) are routinely used in animal breeding programs. Compared to SNP arrays, the use of whole-genome sequence data generated by the next-generation sequencing technologies (NGS) has great potential in livestock populations. However, sequencing a large number of animals to exploit the full potential of whole-genome sequence data is not feasible. Thus, novel strategies are required for the allocation of sequencing resources in genotyped livestock populations such that the entire population can be imputed, maximizing the efficiency of whole genome sequencing budgets. We present two applications of linear programming for the efficient allocation of sequencing resources. The first application is to identify the minimum number of animals for sequencing subject to the criterion that each haplotype in the population is contained in at least one of the animals selected for sequencing. The second application is the selection of animals whose haplotypes include the largest possible proportion of common haplotypes present in the population, assuming a limited sequencing budget. Both applications are available in an open source program LPChoose. In both applications, LPChoose has similar or better performance than some other methods suggesting that linear programming methods offer great potential for the efficient allocation of sequencing resources. The utility of these methods can be increased through the development of improved heuristics.

Recommendations for conducting the rodent erythrocyte Pig-a assay: A report from the HESI GTTC Pig-a Workgroup.

The rodent Pig‐a assay is a flow cytometric, phenotype‐based method used to measure in vivo somatic cell mutation. An Organization for Economic Co‐operation and Development (OECD) test guideline is currently being developed to support routine use of the assay for regulatory purposes (OECD project number 4.93). This article provides advice on best practices for designing and conducting rodent Pig‐a studies in support of evaluating test substance safety, with a focus on the rat model. Various aspects of assay conduct, including laboratory proficiency, minimum number of animals per dose group, preferred treatment and blood sampling schedule, and statistical analysis are described.

An efficient high-welfare feeding device for assessing northern fowl mite interventions in vivo: an improved method for the identification of protective antigens/systemic acaricides/repellent effect

The northern fowl mite (NFM), Ornithonyssus sylviarum, is an obligate hematophagous ectoparasite of domestic and wild birds, and it is an economic pest of laying hen in North America, China, India, Australia, Myanmar, and Brazil. Such an economically important pest remains neglected in many parts of the world, including Asian countries. Therefore, concerted action is required in both basic and applied research directed at the biology and control of this destructive pest. In the present study, we have developed a novel, high-welfare in vivo feeding capsule that would permit pre-screening of new interventions, repellency and deterrence effects of plant-derived products and other semiochemical compounds before proceeding to large-scale field experiments/bioassays, while the minimum number of animals is required to obtain results. Mites were fed on the birds through either a mesh or without a mesh. The average feeding rates of mites was significantly higher when fed directly on chickens, whereas 106 μm nylon mesh was the top-performing mesh when compared with 125 μm aperture nylon mesh. For optimal feeding, the feeding capsules contain NFM and are attached to the skin of the chicken’s thigh for 6 h. This is a simple, reproducible, and easy approach and can be adapted to facilitate many aspects of bioassays.

Multiple-Condition Analysis in a Retrievable Subcutaneous Animal Model for Drug Screening on Full Pancreatic Tissue Digest.

The lack of understanding on how to treat pancreas-related diseases and develop new therapeutics is partly due to the unavailability of appropriate models. In vitro models fail to provide a physiological environment. Testing new drug targets in these models can give rise to bias and misleading results. Therefore, we developed an in vivo model for drug testing on full pancreatic digests, which maintains the interactions between endo- and exocrine tissues and allows retrieving the samples for further analyses. The use of full pancreatic digest eliminates the need to isolate islets, reducing time and cost. In this model, four different conditions can be implanted subcutaneously within the same animal. Each condition consists of full pancreatic tissue digests embedded in alginate beads. All alginate beads in one animal contained full pancreatic digest of the same donor and, after 5-day implantation, were retrieved for analysis focusing on survival, function, and/or organization. Proof-of-principle of the platform was evidenced by showing the effect of hyaluronic acid and vascular endothelial growth factor on the overall function of the full pancreatic digest and on endothelial cells in the pancreatic digest, respectively. Retrieval from identical animals allows direct comparison between conditions. Metabolism (MTT) quantification, dithizone staining, and glucose-stimulated insulin secretion assessment allow to discriminate, using a minimal number of animals, between treatments and validate the system. Because of its simplicity, the model is highly adaptable to specific needs of the user.

Environmental Screening of <em>Aeromonas hydrophila</em>, <em>Mycobacterium</em> spp., and <em>Pseudocapillaria tomentosa</em> in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump's bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems.

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump's bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Pharmacokinetic Profiling of Conjugated Therapeutic Oligonucleotides: A High-Throughput Method Based Upon Serial Blood Microsampling Coupled to Peptide Nucleic Acid Hybridization Assay.

Therapeutic oligonucleotides, such as small interfering RNAs (siRNAs), hold great promise for the treatment of incurable genetically defined disorders by targeting cognate toxic gene products for degradation. To achieve meaningful tissue distribution and efficacy in vivo, siRNAs must be conjugated or formulated. Clear understanding of the pharmacokinetic (PK)/pharmacodynamic behavior of these compounds is necessary to optimize and characterize the performance of therapeutic oligonucleotides in vivo. In this study, we describe a simple and reproducible methodology for the evaluation of in vivo blood/plasma PK profiles and tissue distribution of oligonucleotides. The method is based on serial blood microsampling from the saphenous vein, coupled to peptide nucleic acid hybridization assay for quantification of guide strands. Performed with minimal number of animals, this method allowed unequivocal detection and sensitive quantification without the need for amplification, or further modification of the oligonucleotides. Using this methodology, we compared plasma clearances and tissue distribution profiles of two different hydrophobically modified siRNAs (hsiRNAs). Notably, cholesterol-hsiRNA presented slow plasma clearances and mainly accumulated in the liver, whereas, phosphocholine-docosahexaenoic acid-hsiRNA was rapidly cleared from the plasma and preferably accumulated in the kidney. These data suggest that the PK/biodistribution profiles of modified hsiRNAs are determined by the chemical nature of the conjugate. Importantly, the method described in this study constitutes a simple platform to conduct pilot assessments of the basic clearance and tissue distribution profiles, which can be broadly applied for evaluation of new chemical variants of siRNAs and micro-RNAs.

Contemporary groups in the genetic evaluation of Nellore cattle using Bayesian inference

Abstract: The objective of this work was to evaluate the criteria for the formation of contemporary groups (CGs) in the genetic evaluation of body weight at weaning in Nellore cattle. A total of 713,474 records from 3,066 herds located in Midwestern and Northern Brazil were used. Data were obtained from the genealogical registry of zebu breeds of the Brazilian association of zebu breeders. Data structures were defined based on the number of standard deviations (SDs) for outlier removal (±2.0, ±2.5, ±3.0, and ±3.5) and on the minimal number of animals per CG (3, 7, and 15). Genetic evaluation was performed with an animal model using Bayesian inference. Data structures with ±3.5 SDs and CG with at least 15 animals presented the highest additive genetic variance (82.65±2.93), and those with ±2.0 SDs and CG with at least 3 animals showed the lowest one (60.23±1.96). The proper formation of CGs results in better-quality data archives, allowing to obtain more trustable estimates for the genetic parameters. Better selection responses are obtained when the following criteria are adopted for the removal of outliers: 2.5, 3.0, and 3.5 standard deviations and a minimum of 15 animals per contemporary group.

No Magic Number: an Examination of the Herd-Size Threshold in Pastoral Systems Using Agent-Based Modeling

Pastoralists who depend on their herds for their livelihoods need a minimum number of animals to support their household. Due to the dynamics of herd growth, pastoralists may find themselves at times below that minimum number. Previous studies have shown that there is a herd-size threshold below which households are unlikely to escape poverty. We explore the concept of a herd-size threshold using an agent-based model to examine the role of scale and stochasticity in family herd dynamics. The model was parametrized with data from the literature. The results from the computer simulations show (1) that offtake rates significantly limit herd growth; and (2) that herd-size threshold is better understood as a range of probabilities. We discuss the methodological and conceptual advantages of using agent-based modeling to examine demographic dynamics, including the possibility of conducting multiple experiments in silico to examine the dynamics of herd growth.

Population monitoring of small and declining brush-tailed rock wallaby (Petrogale penicillata) colonies at the extreme of their range using faecal DNA sampling

Obtaining much-needed information on population parameters such as abundance and genetic diversity can be difficult for small and declining populations. The brush-tailed rock-wallaby (Petrogale penicillata) is an endangered and cryptic species with many colonies in decline. The Warrumbungle National Park (NP) in New South Wales contains a declining metapopulation of P. penicillata at the western (inland) extreme of the species’ current range. Loss of these colonies would cause substantial range contraction and probable loss of regional genetic diversity in the Central Evolutionary Significance Unit (ESU). We used non-invasive genetic methods to identify individuals from faecal DNA from five colonies in the Warrumbungle NP. We identified a minimum of 21 individuals, with the largest colony containing seven individuals. The Warrumbungle NP colonies showed significant intercolony structuring and we were able to detect a single dispersal event. Comparison of genetic diversity to other Central ESU colonies shows that loss of the Warrumbungle NP population will result in loss of unique diversity from this region. The minimum number of animals and genetic diversity information obtained in this study was used to support management actions of herbivore control and translocation in the Warrumbungle NP population.

Reproductive and developmental toxicity testing: Examination of the extended one-generation reproductive toxicity study guideline

An important aspect of safety assessment of chemicals (industrial and agricultural chemicals and pharmaceuticals) is determining their potential reproductive and developmental toxicity. A number of guidelines have outlined a series of separate reproductive and developmental toxicity studies from fertilization through adulthood and in some cases to second generation. The Extended One-Generation Reproductive Toxicity Study (EOGRTS) is the most recent and comprehensive guideline in this series. EOGRTS design makes toxicity testing progressive, comprehensive, and efficient by assessing key endpoints across multiple life-stages at relevant doses using a minimum number of animals, combining studies/evaluations and proposing tiered-testing approaches based on outcomes. EOGRTS determines toxicity during preconception, development of embryo/fetus and newborn, adolescence, and adults, with specific emphasis on the nervous, immunological, and endocrine systems, EOGRTS also assesses maternal and paternal toxicity. However, EOGRTS guideline is complex, criteria for selecting doses is unclear, and monitoring systemic dose during the course of the study for better interpretation and human relevance is not clear. This paper discusses potential simplification of EOGRTS, suggests procedures for relevant dose selection and monitors systemic dose at multiple life-stages for better interpretation of data and human relevance.

Ketamine improves survival in severe burn injury in rats via the expression of heat shock protein 70, far or close to the clinical perspective?

The purpose of this reflection or commentary is not to demerit the efforts of the researchers, but rather to highlight some aspects that should be taken into account in the future for implementing experimental designs that may potentially contribute to strengthen the links between basic biomedical and clinical research, referring to the research article entitled Ketamine improves survival in severe burn injury in rats via the Expression of Heat Shock Protein 70. This is a purely constructive reflection aimed at encouraging those who are one way or other involved in research, to develop a more comprehensive analysis and a proposal for an experimental design that enables the extrapolation of the results from animal models to a clinical application, with a limited number of subjects but preserving the validity of the trial, in addition to making the best possible use of the experimental subjects and of the relationship between basic and clinical research. In fact, the ethical guidelines issued by the Council for International Organizations of Medical Sciences, in its chapter on animal experiments emphasize that the validity of a trial allows for using the minimum number of animals in an experiment. The article that makes reference to this matter was published by Zhang Meng-yuan et al., and concludes that Ketamine therapy improves the survival of patients with severe burn injuries via the expression of heat shock proteins in the heart and the brain.

La ketamina mejora la supervivencia en ratas con quemaduras severas vía la expresión de la proteína de choque térmico 70, ¿cerca o lejos de la perspectiva clínica?

The purpose of this reflection or commentary is not to demerit the efforts of the researchers, but rather to highlight some aspects that should be taken into account in the future for implementing experimental designs that may potentially contribute to strengthen the links between basic biomedical and clinical research, referring to the research article entitled Ketamine improves survival in severe burn injury in rats via the Expression of Heat Shock Protein 70. This is a purely constructive reflection aimed at encouraging those who are one way or other involved in research, to develop a more comprehensive analysis and a proposal for an experimental design that enables the extrapolation of the results from animal models to a clinical application, with a limited number of subjects but preserving the validity of the trial, in addition to making the best possible use of the experimental subjects and of there lationship between basic and clinical research. In fact, the ethical guidelines issued by the Council for International Organizations of Medical Sciences, in its chapter on animal experiments emphasize that the validity of a trial allows for using the minimum number of animals in an experiment. The article that makes reference to this matter was published by Zhang Meng-yuan et al., and concludes that Ketamine therapy improves the survival of patients with severe burn injuries via the expression of heat shock proteins in the heart and the brain.

Comparison and characterization of multiple biomaterial conduits for peripheral nerve repair

Four biomaterial tubes, poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone fumarate) (PCLF), a neutral oligo[(polyethylene glycol) fumarate] (OPF) hydrogel or a positively charged oligo[(polyethylene glycol) fumarate] (OPF+) hydrogel with a PCLF sleeve, have previously been shown to have benefits for nerve repair. However, no direct comparison to identify the optimal material have been made. Herein, these nerve tubes were implanted in a rat sciatic nerve model and nerve regeneration was quantified and compared by using accepted nerve assessment techniques. Using standard statistical methods, no significant differences of individual parameters were apparent between groups despite PCLF showing a tendency to perform better than the others. Using a mean–variance based ranking system of multiple independent parameters, statistical differences became apparent. It was clear that the PLCF tube supported significantly improved nerve regeneration and recovery compared to the other three biomaterial conduits. The ability to simultaneously compare a number of regenerative parameters and elucidate the best material from the combination of these individual parameters is of importance to the nerve regeneration area and has implications for the tissue engineering field. By using this method of comparison, a number of biomaterial constructs may be compared under similar conditions and the optimal construct elucidated using the minimal number of animals and materials.

The Effects of QuikClot Combat Gauze and Movement on Hemorrhage Control in a Porcine Model

The purpose of this study was twofold: (1) to examine the effectiveness of QuikClot Combat Gauze (QCG) compared to a control group and (2) investigate the effect of movement on hemorrhage control when QCG is employed. This was a prospective, experimental design employing an established porcine model of uncontrolled hemorrhage. The minimum number of animals (n = 11 per group) was used to obtain a statistically valid result. There were no statistically significant differences between the groups (p > 0.05) indicating that the groups were equivalent on the following parameters: activating clotting time, the subject weights, core body temperatures, amount of 1 minute hemorrhage, arterial blood pressures, and the amount and percentage of total blood volume. There were significant differences in the amount of hemorrhage (p = 0.018) and the number of movements (p = 0.000) between the QCG and control. QCG is statistically and clinically superior at controlling hemorrhage compared to the standard pressure dressing control group. Furthermore, it produces a more robust clot that can withstand significant movement. In conclusion, QCG is an effective hemostatic agent for use in civilian and military trauma management.

Strategies for the optimisation of in vivo experiments in accordance with the 3Rs philosophy

There are a large number of chemicals in current use for which adequate toxicity data are not available. Whilst there are clear ethical and legal obligations to obtain data from sources other than in vivo experiments wherever possible, in certain cases in vivo assays may be deemed necessary. In such circumstances, it is essential to ensure that the maximum amount of high quality data is obtained from the minimum number of animals, using the most humane procedures, in accordance with the philosophy of reduction, refinement and replacement (3Rs). The aim of this report is to provide a strategy for anyone involved in animal experimentation, for either toxicological or pharmacological purposes, as to how in vivo experiments may be optimised. The impact of generic and endpoint specific sources of variability has been highlighted in a proof-of-principle analysis considering the variation in protocols for assays for four human health endpoints (skin sensitisation, reproductive/developmental toxicity, mutagenicity and carcinogenicity). Other factors such as operator training, experimental/statistical design, use of lower species and use of combined assays are also discussed. Recommendations for optimisation of in vivo assays, in terms of the 3Rs philosophy, applied to performing tests, harvesting data and appropriate reporting are summarised as a checklist of issues to be addressed prior to undertaking such assays.