Effects of Different Irrigation Regimes, Nitrogen Levels and Storage Conditions on Volatiles of 'Gala' Apple.

With a characteristic flavour and aroma, "Maçã de Alcobaça" are apples produced in the western region of the mainland of Portugal. Given the known influence of pre-harvest cultural techniques and post-harvest conservation methods on fruit quality, this work evaluated the effect of cultural factors and conservation methods on the volatile profile of 'Gala' apples. Tests were carried out during four seasons (2018 to 2021) in two 'Gala' apple orchards (F and S) maintained with different irrigation rates and nitrogen fertilisation [normal irrigation and normal nitrogen (Control, NINN), normal irrigation and excess nitrogen (NIEN), excess irrigation and normal nitrogen (EINN), excess irrigation and excess nitrogen (EIEN)], and under three storage conditions [Controlled Atmosphere + 1-methylcyclopropene (CA+1-MCP), Dynamic Controlled Atmosphere (DCA) and DCA+1-MCP]. The intact fruit volatiles were isolated by headspace solid-phase microextraction (HS-SPME) and analysed by Gas Chromatography with Flame Ionisation Detection and Gas Chromatography-Mass Spectrometry at harvest (T0) and after 8 months of storage (T8). HS-SPME volatiles from 'Gala' apples, obtained at T0 in control conditions, were characterised by trans,trans-α-farnesene dominance (36-69%), followed by hexyl acetate (5-23%) and hexyl hexanoate (3-9%). The four irrigation and nitrogen treatments did not evidence main changes in the apple volatile profile. Instead, storage conditions changed the ratio between compounds; previously undetected compounds attained high percentages and decreased the intensity of the dominant compounds in the control conditions. Although all storage conditions tested changed the volatile profile and emanation intensity, the effect was more accentuated in storage for 8 months with DCA+1-MCP.

Education in laboratory animal science and the 3Rs.

While laboratory animal research continues to be crucial for scientific and medical advancement, it still raises relevant ethical considerations. In order to foster public trust and support, all animal use must be relevant, responsible, competent and humane, and education and training of scientists in laboratory animal science (LAS) is vital to achieve these goals. However, education must be effective in generating meaningful learning and promoting a culture of competence, professionalism, accountability and transparency. With the ongoing technological and pedagogical revolution in education, LAS educators are adopting innovative educational practices, including e-learning modules, interactive simulations and virtual reality tools, to create and deliver inspirational educational experiences that are immersive, interactive, learner-centric and effective. Drawing from their expertise and experience, the authors of the articles included in this special edition bring forward new technologies and approaches, as well as novel perspectives to well-established concepts and methodologies, hopefully valuable contributions for better engagement and improved learning on LAS and the 3Rs.

Case-based teaching of experimental design - contributions for meaningful learning.

This article argues the need for education and training of researchers carrying out animal studies on the fundamentals of experimental design (ED), as a key means of improving the reliability and reproducibility of preclinical results. The current landscape in ED education in Europe is presented, and we make the case for dedicated tutor-guided teaching of ED. With less than a day dedicated to it in many courses effective techniques for communicating key issues are needed. We have developed two approaches that transfer to experimental design teaching the case-study, problem-solving techniques known to be effective in other fields. They use realistic research scenarios to provoke discussion and engage learning. In one the scenario is for group discussion or informal or formal assessment with subsequent tutor-led discussion of key points. For this each scenario needs a clear statement of the purpose of the research study, simplified text outlining the comparisons and procedures, and a statement of the outcome measure. In the other approach, the scenario is used with freely-available software with a good graphical output to explore the sizing of experiments and the use of both sexes. Trainee feedback and informal assessment show that these approaches can make for interesting and memorable sessions and offer a useful contribution to improvement in experimental design teaching so that it produces meaningful learning that can translate into better practice.

Bibliometric trend analysis of non-conventional (alternative) therapies in veterinary research.

Background: There is an increased interest in Non-Conventional Therapies (NCTs), often referred to as complementary and alternative medicines, in veterinary clinical practice.Aim: To map the bibliometric outputs of NCTs in veterinary medicine, and identify which are most prevalent, and the extent to which their publishing has increased.Methods: Text mining algorithms were applied to detect 17 NCTs-related terms (acupuncture, ayurveda/ayurvedic, traditional Chinese medicine, traditional medicine, chiropractic, electroacupuncture, essential oil, plant extract, ethnopharmacology, herbal medicine, homeopathy, low-level laser therapy, medicinal plant, natural product, osteopathy, phytotherapy, and massage) in the title, abstract or keywords of all retrievable literature until 2020 under the PubMed MeSH term 'veterinary' (N = 377 556). Point prevalence, incidence by decade and cumulative incidence were calculated.Results: Bibliometric trend analysis revealed an overall increase in NCTs-related terms over the last 20 years, with a substantial growth of studies mentioning plant extracts, essential oils and medicinal plants. Traditional Chinese medicine, herbal medicine and natural product have also increased in the same period, although their numbers remain low. Conversely, reference to acupuncture has decreased in the last decade when compared with the previous decade, whereas references to homeopathy, electroacupuncture, osteopathy and chiropractic remained scarce, suggesting that their use in veterinary clinical practice may not be based on published evidence.Conclusion: Further reviews to explore this issue are warranted, differentiating secondary from primary literature, and assessing relevance and methodological quality of individual studies, following the principles of evidence-based veterinary medicine.

Non-Invasive Assessment of Mild Stress-Induced Hyperthermia by Infrared Thermography in Laboratory Mice.

Stress-induced hyperthermia (SIH) is a physiological response to acute stressors in mammals, shown as an increase in core body temperature, with redirection of blood flow from the periphery to vital organs. Typical temperature assessment methods for rodents are invasive and can themselves elicit SIH, affecting the readout. Infrared thermography (IRT) is a promising non-invasive alternative, if shown to accurately identify and quantify SIH. We used in-house developed software ThermoLabAnimal 2.0 to automatically detect and segment different body regions, to assess mean body (Tbody) and mean tail (Ttail) surface temperatures by IRT, along with temperature (Tsc) assessed by reading of subcutaneously implanted PIT-tags, during handling-induced stress of pair-housed C57BL/6J and BALB/cByJ mice of both sexes (N = 68). SIH was assessed during 10 days of daily handling (DH) performed twice per day, weekly voluntary interaction tests (VIT) and an elevated plus maze (EPM) at the end. To assess the discrimination value of IRT, we compared SIH between tail-picked and tunnel-handled animals, and between mice receiving an anxiolytic drug or vehicle prior to the EPM. During a 30 to 60 second stress exposure, Tsc and Tbody increased significantly (p < 0.001), while Ttail (p < 0.01) decreased. We did not find handling-related differences. Within each cage, mice tested last consistently showed significantly higher (p < 0.001) Tsc and Tbody and lower (p < 0.001) Ttail than mice tested first, possibly due to higher anticipatory stress in the latter. Diazepam-treated mice showed lower Tbody and Tsc, consistent with reduced anxiety. In conclusion, our results suggest that IRT can identify and quantify stress in mice, either as a stand-alone parameter or complementary to other methods.

Assessing Scientific Soundness and Translational Value of Animal Studies on DPP4 Inhibitors for Treating Type 2 Diabetes Mellitus.

Although there is a wide range of animal models of type 2 diabetes mellitus (T2DM) used in research; we have limited evidence on their translation value. This paper provides a) a comparison of preclinical animal and clinical results on the effect of five dipeptidyl peptidase-4 (DPP4) inhibitors by comparing the pharmaceutical caused glucose changes, and b) an evaluation of methodological and reporting standards in T2DM preclinical animal studies. DPP4 inhibitors play an important role in the clinical management of T2DM: if metformin alone is not sufficient enough to control the blood sugar levels, DPP4 inhibitors are often used as second-line therapy; additionally, DPP-4 inhibitors are also used in triple therapies with metformin and sodium-glucose co-transporter-2 (SGLT-2) inhibitors or with metformin and insulin. In our analysis of 124 preclinical studies and 47 clinical trials, (1) we found no evidence of species differences in glucose change response to DPP4 inhibitors, which may suggest that, for this drug class, studies in mice and rats may be equally predictive of how well a drug will work in humans; and (2) there is good reporting of group size, sex, age, euthanasia method and self-reported compliance with animal welfare regulations in animal studies but poor reporting of justification of group size, along with a strong bias towards the use of male animals and young animals. Instead of the common non-transparent model selection, we call for a reflective and evidenced-based assessment of predictive validity of the animal models currently available.

ThermoLabAnimal - A high-throughput analysis software for non-invasive thermal assessment of laboratory mice.

Body temperature changes in laboratory mice are often assessed by invasive and stressful methods, which may confound the measurement. Infrared thermography is a possible non-invasive alternative, but the cost of standard thermal cameras, lack of dedicated software for biomedical purposes, and labour-intensiveness of thermal image analysis have limited their use. An additional limitation lies on the scarcity of research on the causing factors of differences between body surface and core body temperature. We propose a method for automatic assessment of mean body surface temperature in freely-moving mice, using dedicated software for thermal image analysis. While skin surface temperature may not necessarily be linearly correlated with core body temperature (in itself an imprecise concept), under standardized environmental conditions, such as those in which laboratory animals are kept, mean body surface temperature can provide useful information on their thermal status (i.e. deviations from normothermia, namely hypo- and hyperthermia). We developed a publicly available software that includes an imaging analysis workflow/algorithm for automatic segmentation of the pixels associated with the animal from the pixels associated with the background, removing the need for manually defining the area of analysis. A batch analysis mode is also available, for automatic and high-throughput analysis of all image files located in a folder. The software is compatible with the most widespread thermal camera manufacturer, 'FLIR Systems', as well as with the low-cost 'Thermal Expert TE-Q1' miniaturized high-resolution thermal camera used for this study. Furthermore, the software has been validated in a mouse model expressing non-transient hypothermia, where the thermal analysis results were compared with readings from implanted thermo-sensitive passive integrated transponders tags. Thermography allows for thermal assessment of laboratory animals without the effect of handling stress on their physiology or behaviour. Our automatic image analysis software also removes observer errors and bias, while speeding up the data processing.

Methodological standards, quality of reporting and regulatory compliance in animal research on amyotrophic lateral sclerosis: a systematic review.

OBJECTIVES: The amyotrophic lateral sclerosis (ALS) research community was one of the first to adopt methodology guidelines to improve preclinical research reproducibility. We here present the results of a systematic review to investigate how the standards in this field changed over the 10-year period during which the guidelines were first published (2007) and updated (2010). METHODS: We searched for papers reporting ALS research on SOD1 (superoxide dismutase 1) mice published between 2005 and 2015 on the ISI Web of Science database, resulting in a sample of 569 papers to review, after triage. Two scores-one for methodological quality, one for regulatory compliance-were built from weighted sums of separate sets of items, and subjected to multivariable regression analysis, to assess how these related to publication year, type of study, country of origin and journal. RESULTS: Reporting standards improved over time. Of papers published after the first ALS guidelines were made public, fewer than 9% referred specifically to these. Of key research parameters, only three (genetic background, number of transgenes and group size) were reported in >50% of the papers. Information on housing conditions, randomisation and blinding was absent in over two-thirds of the papers. Group size was among the best reported parameters, but the majority reported using fewer than the recommended sample size and only two studies clearly justified group size. CONCLUSIONS: General methodological standards improved gradually over a period of 8-10 years, but remained generally comparable with related fields with no specific guidelines, except with regard to severity. Only 11% of ALS studies were classified in the highest severity level (animals allowed to reach death or moribund stages), substantially below the proportion in studies of comparable neurodegenerative diseases such as Huntington's. The existence of field-specific guidelines, although a welcome indication of concern, seems insufficient to ensure adherence to high methodological standards. Other mechanisms may be required to improve methodological and welfare standards.

Eye, body or tail? Thermography as a measure of stress in mice.

Infrared thermography has been suggested as a non-invasive, objective tool to evaluate animal welfare. In this study, we investigated: 1) how body temperature, measured through thermal imaging, is affected by different mild stressors frequently experienced by laboratory mice; 2) which methodology to use for assessing temperature variations with infrared thermography; 3) whether the chosen stressors cause anxiety in mice. Eighty C57BL/6 male mice were included in the study. The mice were allocated to either a control group or one of three groups being subjected to a mild stressor once daily for 4 days: 1) anaesthesia with isoflurane for 10 min; 2) handling by scruffing; 3) intraperitoneal injection of 0.2 ml 0.9% saline. On all four intervention days, thermal images were obtained in all groups and all animals were assessed for fur status and body weight. On day five, all animals were tested in the elevated-plus-maze for 5 min. From the thermal images, the maximum eye temperature, the maximum tail base temperature and the average body temperature were obtained. Ten minutes of anaesthesia with isoflurane led to a decrease in maximum eye temperature, average body temperature and maximum tail base temperature. The animals recovered from this drop in temperature within 10 min. No drop in temperature was seen after scruffing or intraperitoneal injection of saline. Based on the number of missing values, intra-rater and inter-rater agreement, the average body temperature was found most ideal for measuring body temperature variations in mice. Finally, the elevated plus maze did not reveal any differences in anxiety between the groups and the body weight did not decrease at any time point during the study.

Researchers' attitudes to the 3Rs-An upturned hierarchy?

Animal use in biomedical research is generally justified by its potential benefits to the health of humans, or other animals, or the environment. However, ethical acceptability also requires scientists to limit harm to animals in their research. Training in laboratory animal science (LAS) helps scientists to do this by promoting best practice and the 3Rs. This study evaluated scientists' awareness and application of the 3Rs, and their approach to other ethical issues in animal research. It was based on an online survey of participants in LAS courses held in eight venues in four European countries: Portugal (Porto, Braga), Germany (Munich, Heidelberg), Switzerland (Basel, Lausanne, Zurich), and Denmark (Copenhagen). The survey questions were designed to assess general attitudes to animal use in biomedical research, Replacement alternatives, Reduction and Refinement conflicts, and harm-benefit analysis. The survey was conducted twice: immediately before the course ('BC', N = 310) and as a follow-up six months after the course ('AC', N = 127). While courses do appear to raise awareness of the 3Rs, they had no measurable effect on the existing low level of belief that animal experimentation can be fully replaced by non-animal methods. Most researchers acknowledged ethical issues with their work and reported that they discussed these with their peers. The level of an animal's welfare, and especially the prevention of pain, was regarded as the most pressing ethical issue, and as more important than the number of animals used or the use of animals as such. Refinement was considered more feasible than Replacement, as well as more urgent, and was also favoured over Reduction. Respondents in the survey reversed the 'hierarchy' of the 3Rs proposed by their architects, Russell and Burch, prioritizing Refinement over Reduction, and Reduction over Replacement. This ordering may conflict with the expectations of the public and regulators.

Classification and reporting of severity experienced by animals used in scientific procedures: FELASA/ECLAM/ESLAV Working Group report.

Directive 2010/63/EU introduced requirements for the classification of the severity of procedures to be applied during the project authorisation process to use animals in scientific procedures and also to report actual severity experienced by each animal used in such procedures. These requirements offer opportunities during the design, conduct and reporting of procedures to consider the adverse effects of procedures and how these can be reduced to minimize the welfare consequences for the animals. Better recording and reporting of adverse effects should also help in highlighting priorities for refinement of future similar procedures and benchmarking good practice. Reporting of actual severity should help inform the public of the relative severity of different areas of scientific research and, over time, may show trends regarding refinement. Consistency of assignment of severity categories across Member States is a key requirement, particularly if re-use is considered, or the safeguard clause is to be invoked. The examples of severity classification given in Annex VIII are limited in number, and have little descriptive power to aid assignment. Additionally, the examples given often relate to the procedure and do not attempt to assess the outcome, such as adverse effects that may occur. The aim of this report is to deliver guidance on the assignment of severity, both prospectively and at the end of a procedure. A number of animal models, in current use, have been used to illustrate the severity assessment process from inception of the project, through monitoring during the course of the procedure to the final assessment of actual severity at the end of the procedure (Appendix 1).

A Critical Look at Biomedical Journals' Policies on Animal Research by Use of a Novel Tool: The EXEMPLAR Scale.

Animal research is not only regulated by legislation but also by self-regulatory mechanisms within the scientific community, which include biomedical journals' policies on animal use. For editorial policies to meaningfully impact attitudes and practice, they must not only be put into effect by editors and reviewers, but also be set to high standards. We present a novel tool to classify journals' policies on animal use-the EXEMPLAR scale-as well as an analysis by this scale of 170 journals publishing studies on animal models of three human diseases: Amyotrophic Lateral Sclerosis, Type-1 Diabetes and Tuberculosis. Results show a much greater focus of editorial policies on regulatory compliance than on other domains, suggesting a transfer of journals' responsibilities to scientists, institutions and regulators. Scores were not found to vary with journals' impact factor, country of origin or antiquity, but were, however, significantly higher for open access journals, which may be a result of their greater exposure and consequent higher public scrutiny.

Animal Experiments in Biomedical Research: A Historical Perspective.

The use of non-human animals in biomedical research has given important contributions to the medical progress achieved in our day, but it has also been a cause of heated public, scientific and philosophical discussion for hundreds of years. This review, with a mainly European outlook, addresses the history of animal use in biomedical research, some of its main protagonists and antagonists, and its effect on society from Antiquity to the present day, while providing a historical context with which to understand how we have arrived at the current paradigm regarding the ethical treatment of animals in research.

"How sick must your mouse be? " - An analysis of the use of animal models in Huntington's disease research.

Refinement measures to improve animal welfare can ease the ethical dilemma between human benefit and animal harm in research with animal models of neurodegenerative diseases. To evaluate the potential for refinement, as well as its implementation in research, we analysed papers on murine models of Huntington's disease (HD) published between 1999 and 2009 (n = 233). Each study was classified according to a four-level severity scale, in terms of the disease stage that animals were allowed to reach, the execution of invasive procedures, and the implementation of refinement. Reports of ethical approval, and regulatory compliance in general, followed a clear rising trend over the years reviewed (p <0.001). However, the proportion of high-severity studies did not change over that period. Also, of the studies for which approval was reported (n = 120), 36% were attributed the highest severity level. The observed discrepancy between the rising motivation to affirm regulatory compliance, and the unaltered proportion of studies allowing animals to reach severely distressful stages, raises both ethical and methodological issues, which we discuss in this paper.

Animal welfare in studies on murine tuberculosis: assessing progress over a 12-year period and the need for further improvement.

There is growing concern over the welfare of animals used in research, in particular when these animals develop pathology. The present study aims to identify the main sources of animal distress and to assess the possible implementation of refinement measures in experimental infection research, using mouse models of tuberculosis (TB) as a case study. This choice is based on the historical relevance of mouse studies in understanding the disease and the present and long-standing impact of TB on a global scale. Literature published between 1997 and 2009 was analysed, focusing on the welfare impact on the animals used and the implementation of refinement measures to reduce this impact. In this 12-year period, we observed a rise in reports of ethical approval of experiments. The proportion of studies classified into the most severe category did however not change significantly over the studied period. Information on important research parameters, such as method for euthanasia or sex of the animals, were absent in a substantial number of papers. Overall, this study shows that progress has been made in the application of humane endpoints in TB research, but that a considerable potential for improvement remains.