An initial exploratory analysis indicated that groups with higher average cortisol levels also had greater variation in this response variable. Avey MT, Moher D, Sullivan KJ, Fergusson D, Griffin G, Grimshaw JM, Hutton B, Lalu MM, Macleod M, Marshall J, Mei SHJ, Rudnicki M, Stewart DJ, Turgeon AF, McIntyre L and Canadian Critical Care Translational Biology G (2016). Environmental enrichment included nesting material (Nestlets, Ancare, Bellmore, NY, USA), PVC pipe, and shelter (Refuge XKA-2450-087, Ketchum Manufacturing Inc., Brockville, Ontario, Canada). Time matters: pathological effects of repeated psychosocial stress during the active, but not inactive, phase of male mice. A maternal 'junk food' diet in pregnancy and lactation promotes an exacerbated taste for 'junk food' and a greater propensity for obesity in rat offspring. Enrichment given to pigs housed in isolation had no effect on plasma cortisol, but greatly reduced it in non-isolated pigs. Claudino MA, Leiria LOS, da Silva FH, Alexandre EC, Renno A, Mónica FZ, de Nucci G, Fertrin KY, Antunes E, Costa FF and Franco-Penteado CF (2015). Allen C and Mehler D (2018). The use of a one-sided test requires justification of why a treatment effect is only of interest when it is in a defined direction and why they would treat a large effect in the unexpected direction no differently from a non-significant difference [13]. Good Practice for Housing Male Laboratory Mice. Why we need confidence intervals. “Randomisation was carried out as follows. However, the experimental unit may be the cage or the litter (e.g. Comment on the study limitations including potential sources of bias, limitations of the animal model, and imprecision associated with the results. Ruxton G and Colegrave N (2017). the nuisance variables taken into account in each statistical test (e.g. as blocking factors or covariates), Â, what statistical analyses were performed and references for the methods used Â, the software package and version used, including computer code if availableÂ. Garcia de la serrana D, Vieira VLA, Andree KB, Darias M, Estévez A, Gisbert E and Johnston IA (2012). The blots were subsequently probed with HRP-conjugated anti-mouse (Cat# A0216) or anti-rabbit IgG (Cat# A0208; Beyotime Biotechnology, Beijing, China) at 1:1000. Development Temperature Has Persistent Effects on Muscle Growth Responses in Gilthead Sea Bream. cell death, molecular markers, or behavioural changes). all surgeries done by the same surgeon), randomising (e.g. Kafkafi N, Agassi J, Chesler EJ, Crabbe JC, Crusio WE, Eilam D, Gerlai R, Golani I, Gomez-Marin A, Heller R, Iraqi F, Jaljuli I, Karp NA, Morgan H, Nicholson G, Pfaff DW, Richter SH, Stark PB, Stiedl O, Stodden V, Tarantino LM, Tucci V, Valdar W, Williams RW, Würbel H and Benjamini Y (2018). To generate epigenetic profiles, ChIP was performed using specific antibodies against trimethylated H3K4 and H3K27 in Xenopus gastrula-stage embryos (Nieuwkoop-Faber stage 11–12), followed by deep sequencing (ChIP-seq). It is important to interpret the results of the study in the context of the study objectives (see item 13 – Objectives). State whether randomisation was used to allocate experimental units to control and treatment groups. Lian X, Wu X, Li Z, Zhang Y, Song K, Cai G, Li Q, Lin S, Chen X and Bai XY (2019). Sage. Mining mouse behavior for patterns predicting psychiatric drug classification. Button KS, Ioannidis JPA, Mokrysz C, Nosek BA, Flint J, Robinson ESJ and Munafo MR (2013). The ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments) were developed in 2010 to help authors and journals identify the minimum information necessary to report in publications describing in vivo experiments. Positive controls can be used to support the interpretation of negative results or determine if an expected effect is detectable.  It may not be necessary to include a separate control with no active treatment if, for example, the experiment aims to compare a treatment administered by different methods (e.g. Including a nuisance variable as a blocking factor in the analysis accounts for that variability and can increase the power, thus increasing the ability to detect a real effect with fewer experimental units. Stodden V, Guo P and Ma Z (2013). Validation criteria for animal models of human mental disorders: Learned helplessness as a paradigm case. If the study was supported by several sources of funding, list them all, including internal grants. Association between Influenza A Virus Infection and Pigs Subpopulations in Endemically Infected Breeding Herds. (2013). On the morning of surgery, swine were sedated with tiletamine-zolazepam (Telazol®; 5-8 mg/kg IM; Zoetis Inc., Kalamazoo MI) in the holding pen, weighed, and masked with isoflurane (3%, balance 100% O2) for transport to the lab. Subitem 9b—Example 2 In 2010, the NC3Rs developed the ARRIVE guidelines; a 20 point checklist covering measures associated with bias (randomization, blinding, sample size calculation, data handling) with the aim to improving the quality of both scientific research design and reporting of animal studies. Olfactory exposure to males, including men, causes stress and related analgesia in rodents. This strategy is rarely appropriate as it cannot ensure that comparison groups are balanced for other variables that might influence the result of an experiment. Vital M, Harkema JR, Rizzo M, Tiedje J and Brandenberger C (2015). 0000081751 00000 n Different studies may be sensitive to different environmental factors, and particular aspects of the environment necessary to report may depend on the type of study [2]. The one-chamber social interaction test requires manual scoring and was analyzed by an individual with no knowledge of the questions.” [3]. Impact of IVC housing on emotionality and fear learning in male C3HeB/FeJ and C57BL/6J mice. Hurlbert SH (1984). Reichlin TS, Vogt L and Würbel H (2016). Although new gilts were placed into isolated designated areas and procedures were in place to minimize disease transmission (eg. This document demonstrates how the ARRIVE guidelines can be used in practice to report animal research, by providing specific examples for each point of the guidelines. Details about animal care and monitoring intrinsic to the procedure are discussed in further detail in item 15 – Animal care and monitoring. Recent failures of cardioprotective therapies in obese insulin-resistant, diabetic, metabolic syndrome-affected and aged animals that were otherwise successful in healthy animal models has highlighted the need for the development of animal models of disease that are representative of human clinical conditions…In the clinical setting, elderly male patients often present with both testosterone deficiency (TD) and the metabolic syndrome (MetS). home cage, laboratory, water maze). For example, bias, as indicated by a prevalence of more favourable outcomes, has been demonstrated for clinical research funded by industry compared to studies funded by other sources [1-3]. Examples of information to consider when reporting housing and husbandry, and their effects on laboratory animals. isolation, vaccination), these areas or procedures might not have been able to fully contain infections within the designated areas.” [1], “Even though our data demonstrates that sustained systemic TLR9 stimulation aggravates diastolic HF in our model of gene-targeted diastolic HF, there are several limitations as to mechanistic explanations of causality, as well as extrapolations to clinical inflammatory disease states and other HF conditions. An outcome measure (also known as a dependent variable or a response variable) is any variable recorded during a study (e.g. Each of the 20 items of the ARRIVE guidelines are important for various reasons. It underestimates the true variability in a study, which can lead to false positives and invalidate the analysis and resulting conclusions [5,6]. Cover image. Available at: Peng C-YJ, Long H and Abaci S (2012). Available at: Bustamante R, Daza MA, Canfrán S, García P, Suárez M, Trobo I and Gómez de Segura IA (2018). A protocol should consist of a) the question being addressed and the key features of the research that is proposed, such as the hypothesis being tested and the primary outcome measure (if applicable), the statistical analysis plan; and b) the laboratory procedures to be used to perform the planned experiment. Reduces anxiety [22,23], stress [22,23] and abnormal repetitive behaviour [24-26]. Prevalence of sexual dimorphism in mammalian phenotypic traits. Similarly, scientific justification needs to be reported for withholding food and water [33], and for singly housing animals [34,35]. Growth Arrest Specific 1 (Gas1) Gene Overexpression in Liver Reduces the In Vivo Progression of Murine Hepatocellular Carcinoma and Partially Restores Gene Expression Levels. Whole animal experiments should be more like human randomized controlled trials. This investigator was the only person aware of the treatment group allocation. Camp DM, Loeffler DA, Farrah DM, Borneman JN and LeWitt PA (2009). A schematic of the experimental procedures with a timeline can give a clear overview of how the study was conducted. Improving the translational hit of experimental treatments in multiple sclerosis. Animals may also serve as their own controls receiving different treatments separated by washout periods; here the experimental unit is an animal for a period of time. The effects of attrition in experimental research on cancer and stroke. Freedman LS (2008). and were congenic C57BL/6JCrl (backcrossed for 12 generations). Animal husbandry and experimental design. A guide to defining and implementing protocols for the welfare assessment of laboratory animals: eleventh report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. 0000000016 00000 n Untreated pain can affect the animals’ biology and add variability to the experiment; however specific pain management procedures can also introduce variability, affecting experimental data [1,2]. A total of 20 dogs per group were considered necessary.” [1]Â, “We selected a small sample size because the bioglass prototype was evaluated in vivo for the first time in the present study, and therefore, the initial intention was to gather basic evidence regarding the use of this biomaterial in more complex experimental designs.” [2]. They also help to communicate complex design features such as whether factors are crossed or nested (hierarchical/multi-level designs), blocking (to reduce unwanted variation, see item 4 – Randomisation), or repeated measurements over time on the same experimental unit (repeated measures designs), see [1-3] for more information on different design types. CONCLUSIONS AND IMPLICATIONS: These results highlighted the importance of counteracting oxidative stress by flavonoids in this asthma model and suggest sakuranetin as a potential candidate for studies of treatment of asthma.” [1],  “In some parts of the world, the laboratory pig (Sus scrofa) is often housed in individual, sterile housing which may impose stress. Discuss what (if any) further research may be required to allow generalisation or translation. Bero L, Anglemyer A, Vesterinen H and Krauth D (2016). Altman DG (2005). FLAG: Highlight any advances in the 3Rs (Replacement, Reduction or Refinement) made during the study in the manuscript's title and/or abstract (since many databases only index these parts of the paper) All animals/samples are simultaneously randomised to the treatment groups without considering any other variable. Examples or references about Soc 1 Report Example and Applying the Arrive Guidelines to An In Vivo Database that we get come from reputable online resources. cardiovascular and respiratory depression, CNS disturbance, hypothermia, reduction of food intake). Learn how to use Arrive using many example sentences. (2017). If done, provide the method used to generate the randomisation sequence. For genetically modified animals, describe the genetic modification status (e.g. Outbred CD1 mice are as suitable as inbred C57BL/6J mice in performing social tasks. Effects of MVA85A vaccine on tuberculosis challenge in animals: systematic review. Neumann A-M, Abele J, Kirschstein T, Engelmann R, Sellmann T, Köhling R and Müller-Hilke B (2017). Provide a clear statement explaining how the study conforms to appropriate regulations and guidelines. Figshare (https://figshare.com/), Dryad (https://datadryad.org/), Zenodo (https://zenodo.org/) or Open Science Framework (https://osf.io/)). Competing or conflicts of interest can be financial or non-financial, professional or personal. N = 1959 mice were included in this analysis.” [2]Â. Hypothesis tests are based on assumptions about the underlying data. 0 Impact of tank background on the welfare of the African clawed frog, Xenopus laevis (Daudin). mean or median) and which measure of variability is reported (e.g. 12L:12D), circadian time cues (e.g. Sanitation (frequency of cage/tank water changes, material transferred, water quality). For example, if animals are specific pathogen free (SPF), list the pathogens that they were declared free of. For example: a. There are various tests for normality, for example the Shapiro-Wilk and Kolmogorov-Smirnov tests. Guidelines for the design and statistical analysis of experiments using laboratory animals. Reproducibility and replicability of rodent phenotyping in preclinical studies. Negative controls are used to determine if a difference between groups is caused by the intervention (e.g. Boutron I, Altman DG, Hopewell S, Vera-Badillo F, Tannock I and Ravaud P (2014). the n number) is essential information for the reader to interpret the analysis, it should be reported unambiguously. Muhlhausler BS, Bloomfield FH and Gillman MW (2013). This resource provides a user-friendly interface to access sequence information from the tr2aacds_v2 assembly (to explore annotated protein-coding transcripts) and/or the Trinity_v2.3.2 assembly (to explore non-coding transcripts).” [2]. Modifies immune and stress responses [21]. drug formulation and dose, site and route of administration, anaesthesia and analgesia used [including monitoring], surgical procedure, method of euthanasia). Chopped corn cob was used as bedding. each item of the ARRIVE guidelines. Mice were fed a standardized mouse diet (1314, Altromin, Germany) and provided drinking water ad libitum. Establishing an appropriate period of acclimatization following transportation of laboratory animals. Impact of Diet-Induced Obesity and Testosterone Deficiency on the Cardiovascular System: A Novel Rodent Model Representative of Males with Testosterone-Deficient Metabolic Syndrome (TDMetS). Provide a statement describing if and where study data are available. For hypothesis-testing studies, specify the primary outcome measure, i.e. Information relevant to distinct types of interventions and resources are described below. Along with innate circadian rhythms, these can affect research outcomes such as behavioural, physiological, and immunological parameters [1,2]. Explain how the sample size was decided. For example, in cancer studies an animal might be dropped from the study and euthanised before the predetermined time point if the size of a subcutaneous tumour exceeds a specific volume [5]. human clinical trial). illness or mortality), or data points excluded from analyses (e.g. If, however, each cell taken from the mouse is then randomly allocated to different treatments and assessed individually, the cell might be regarded as the experimental unit. Wang X, Xue Q, Yan F, Liu J, Li S and Hu S (2015). Results are presented as least squares means. Outline evidence underpinning the hypothesis or objectives and explain why the experimental approach is best suited to answer the research question. In studies using inferential statistics to test a hypothesis (e.g. Biomedical Data Sharing and Reuse: Attitudes and Practices of Clinical and Scientific Research Staff. Analysing the data using different methods and selectively reporting those with statistically significant results constitutes p-hacking and introduces bias in the literature [1,2]. If the experimental unit is not the animal, also report the total number of animals to help readers understand the study design. For example, the number of animals needed for antibody or tissue production is determined by the amount required and the production ability of an individual animal. A new behavioural apparatus to reduce animal numbers in multiple types of spontaneous object recognition paradigms in rats. Examples of information to consider when reporting housing and husbandry, and their effects on laboratory animals, Examples of effects on laboratory animals, Cage/tank/housing system (type and dimensions). Ophthalmic ointment (Puralube®; Fera Pharmaceuticals) was applied to prevent corneal drying. Scott S, Kranz JE, Cole J, Lincecum JM, Thompson K, Kelly N, Bostrom A, Theodoss J, Al‐Nakhala BM, Vieira FG, Ramasubbu J and Heywood JA (2008). Festing MF and Altman DG (2002). The immune or microbiological status of the animals can also influence welfare, experimental variability and scientific outcomes [2-4]. If no control group has been used, the rationale should be stated. For exploratory studies it is not necessary to identify a single primary outcome and often multiple outcomes are assessed (see item 13 – Objectives). Macleod M and Howells D (2016). task performance has to exceed a given threshold). In studies where selected data are re-analysed for a different purpose, inclusion and exclusion criteria should describe how data were selected. … If unsure, declare all potential conflicts, including both perceived and real conflicts of interest [1]. Discuss the findings in the context of current theory, ideally with reference to a relevant systematic review, as individual studies do not provide a complete picture. Turner PV, Brabb T, Pekow C and Vasbinder MA (2011). Environment, either deprived or enriched, can affect a wide range of physiological and behavioural responses [32]. Bleich A and Hansen AK (2012). These ten items are the basic minimum that must be included in any manuscript describing animal research. 0000222461 00000 n Sena ES, Currie GL, McCann SK, Macleod MR and Howells DW (2014). The Importance of Reporting Housing and Husbandry in Rat Research. Administration of substances to laboratory animals: routes of administration and factors to consider. In error, three animals were sacrificed before their final assessment of neurobehavioral score: one from the normothermia/water group and two from the hypothermia/pethidine group. These animals recapitulate several key features of human temporal lobe epilepsy, the most common type of epilepsy in adults.” [1], “Transplantation of healthy haematopoietic stem cells (HSCs) is a critical therapy for a wide range of malignant haematological and non-malignant disorders and immune dysfunction…Zebrafish are already established as a successful model to study the haematopoietic system, with significant homology with mammals…Imaging of zebrafish transparent embryos remains a powerful tool and has been critical to confirm that the zebrafish Caudal Haematopoietic Tissue (CHT) is comparable to the mammalian foetal haematopoietic niche...Xenotransplantation in zebrafish embryos has revealed highly conserved mechanisms between zebrafish and mammals. If the research is not covered by any regulation and formal ethical approval is not required (e.g. For example, nuisance variables can be used as blocking factors or covariates.Â, Null and alternative hypotheses: The null hypothesis (H0) is that there is no effect, such as a difference between groups or an association between variables. “Data Availability Statement: All data are available from Figshare at doi: 10.6084/m9.figshare.1288935.” [1], “A fundamental goal in generating this dataset is to facilitate access to spiny mouse transcript sequence information for external collaborators and researchers. ※ARRIVEは”Animal Research: Reporting of In Vivo Experiments”（動物実験：生体を用いた実験の報告について）の略。 追記. Focusing on the Essential 10 in priority better allows journal staff, editors and reviewers to verify that the items have been adequately reported in manuscripts. Interpret the results, taking into account the study objectives and hypotheses, current theory, and other relevant studies in the literature. The Dutch Experiments on Animals Act does not apply to non-experimental, agricultural practices. Environmental enrichment and brain repair: harnessing the therapeutic effects of cognitive stimulation and physical activity to enhance experience-dependent plasticity. Samples and data were collected as part of routine animal data collection in a commercial breeding program for layer chickens in The Netherlands. Willner P (1986). Impact of spin in the abstracts of articles reporting results of randomized controlled trials in the field of cancer: the SPIIN randomized controlled trial. Hawkins P, Morton DB, Burman O, Dennison N, Honess P, Jennings M, Lane S, Middleton V, Roughan JV, Wells S and Westwood K (2011). Independent replication and meta-analysis can further increase the confidence in conclusions. Rearing Environmental Enrichment in Two Inbred Strains of Mice: 1. With minimisation the treatment allocated to the next animal/sample depends on the characteristics of those animals/samples already assigned. Handling method alters the hedonic value of reward in laboratory mice. The ethical statement provides editors, reviewers and readers with assurance that studies have received this ethical oversight [1]. Provide details of any specialist equipment used, including supplier(s). Use of humane endpoints to minimise suffering. Forskolin, a hedgehog signalling inhibitor, attenuates carbon tetrachloride-induced liver fibrosis in rats. Describing how assumptions were assessed, and whether these assumptions are met by the data, enables readers to assess the suitability of the statistical approach used. If a systematic review is not available, take care to avoid selectively citing studies that corroborate the results, or only those that report statistically significant findings [1]. Data were analysed as an REML using the MIXED procedure of SAS. Urinary Bladder Dysfunction in Transgenic Sickle Cell Disease Mice. Duke JL, Zammit TG and Lawson DM (2001). Hurst JL and West RS (2010). Federer LM, Lu Y-L, Joubert DJ, Welsh J and Brandys B (2015). Hagemo JS, Jørgensen JJ, Ostrowski SR, Holtan A, Gundersen Y, Johansson PI, Næss PA and Gaarder C (2013). Reporting the items in both sets represents best practice. If the person assessing the outcome cannot be blinded to the group allocation (e.g. Delayed Administration of Interleukin-1 Receptor Antagonist Reduces Ischemic Brain Damage and Inflammation in Comorbid Rats. Kim JD, Chun AY, Mangan RJ, Brown G, Mourao Pacheco B, Doyle H, Leonard A and El Bejjani R (2019). Immunoreactive bands were visualized by enhanced chemiluminescence, and densitometry was performed using ImageJ software (RRID: SCR_003070, Bio-Rad Laboratories).” [3], 1. Registering a protocol in advance protects the researcher against concerns about selective outcome reporting (also known as data dredging or p-hacking) and provides evidence that the primary outcome reported in the manuscript accurately reflects what was planned [4] (see item 19 – Protocol registration). Induces aggressive behaviour [18,19] and stress [6]. However, statistical significance is heavily influenced by sample size and variation in the data (see item 2 – Sample size). “The study was funded by Gubra ApS. startxref time of day). The conditioning tank consisted of an opaque tank measuring 20 cm (w) 15 cm (h) 30 cm (l) containing 2.5 l of aquarium water with distinct visual cues (spots or stripes) on walls at each end of the tank…During habituation, each individual fish was placed in the conditioning apparatus for 20 minutes with free access to both compartments and then returned to its home tank.” [1]. starting tumour size at point of randomisation). inadequacies in the design, conduct, or analysis of the study introducing bias).Â, Nuisance variable: Variables that are not of primary interest but should be considered in the experimental design or the analysis because they may affect the outcome measure and add variability. There are also search platforms to identify relevant repositories with rigorous standards, e.g. European Commission (2010). How to calculate sample size in animal studies? Affects behavioural responses to stress [10] and pain [11]. In contrast, exploratory research investigates many possible effects, and is likely to yield more false positive results because some will be positive by chance. Gouveia K and Hurst JL (2017). Holman C, Piper SK, Grittner U, Diamantaras AA, Kimmelman J, Siegerink B and Dirnagl U (2016). If the study did not have humane endpoints, state this. Available at: Lang TA and Altman DG (2015). If losses are anticipated, these should be considered when determining the number of animals to include in the study (see item 2 – Sample size). While exclusion criteria and humane endpoints are typically included in the ethical review application, reporting the criteria used to exclude animals or data points in the manuscript helps readers with the interpretation of the data and provides crucial information to other researchers wanting to adopt the model. Policy: NIH to balance sex in cell and animal studies. Toledo AC, Sakoda CPP, Perini A, Pinheiro NM, Magalhães RM, Grecco S, Tibério IFLC, Câmara NO, Martins MA, Lago JHG and Prado CM (2013). Kolahian S, Sadri H, Shahbazfar AA, Amani M, Mazadeh A and Mirani M (2015).

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