Abstract
Session 1: “Animal welfare bodies and ethical review”
AAALAC International
Implementation of Directive 2010/63/EU has led to a heterogeneous landscape concerning systems for ethical review of research proposals across Europe. They may be based upon the evaluation by competent authorities (ie. Germany); by other bodies either institutional or external (ie, France, Spain, The Netherlands); or by a combination of all these (ie. UK). Several key factors may affect the efficacy of the system in place: resources, expertise, independence and authority. Several issues can be discussed and evaluated: Although they seem to be the most independent bodies, have competent authorities the necessary resources and expertise? Although institutional and external bodies may have expertise, can they be considered as actually independent and free of conflict of interest? Do all institutional bodies have the authority not only to evaluate, but to ensure projects are performed according to the approved proposal? Obviously, a combination of the positive aspects of the several type of evaluation bodies that ensures and independent evaluation (also during and after project is performed) is timely performed by experts should be the answer.
The AAALAC International site visits performed since the establishment of the new legal framework show diversity of findings concerning ethical review across Europe and will be discussed. Especially noteworthy are those relating to poor oversight activities and lack of authority of institutional bodies that can impede appropriate implementation of ethical review functions. The use of performance standards is essential to evaluate the efficacy of ethical review, regardless of the system in place.
Universidad Miguel Hernández de Elche (UMH)
In 2005, John P.A. Ioannidis, professor at Stanford, published in PLoS Medicine an article with this title: Why Most Published Research Findings Are False. Further research confirmed the Ionnidis thesis: many scientific articles are not reproducible. This problem, dubbed the reproducibility crisis, has alerted many agents involved in research. The first to position themselves to address the problem were several scientific publishers, headed by the Nature Publishing Group. The NIH (National Institutes of Health) has also announced it will take steps to improve the research it funds.
Ethics committees should be aware of the problem and develop strategies to enhance scientific integrity, focusing on aspects of honesty (truthfulness), precision (error free) and objectivity (no bias).
Strategies to address these problems are diverse and depend largely on the involvement of the institution managers and the resources available. One of our major tools is training, because it can improve the experimental design and the analysis and interpretation of the data, the scientific process as a whole (with special emphasis on the collection and storage of data), and scientific integrity. A useful supplement is to provide additional consulting services with the aim of improving the quality of science produced in the institution.
1IBMC – Institute for Molecular and Cell Biology
2i3S – Instituto de Investigação e Inovação em Saúde
3NOVA Medical School | Faculdade de Ciências Médicas, Universidade Nova de Lisboa
The 2010/63/EU Directive demands that scientific institutions must have an Animal Welfare Body – in Portugal, “ORBEA” (‘Órgão Responsável pelo Bem-Estar dos Animais’) – responsible for evaluating procedures and their severity, monitoring animal welfare, and advising researchers. In Portugal, there has been no tradition with institutional bodies with such competences and attributions, thus it is a challenge for newly-created ORBEA to find their place, authority and responsibilities in institutions, as well as with liaising with the competent authority. This constitutes however an opportunity for raising standards of competence and functionality in the planning, reviewing, and supervising of procedures within institutions. After organizing two national workshops for ORBEA members, we are presently surveying coordinators of ORBEA (or, alternatively, of animal facilities) in the main Portuguese research institutions to assess the current landscape regarding the establishment and composition of ORBEA, the main challenges they are facing, and whether there is interest in establishing a national network of ORBEA. Of the 14 – of 22 contacted – institutions that have so far responded, only 50% stated to have ORBEA in place, while 21% (3/14) were in the process of constituting an ORBEA and 28% (4/14) had made no progress in this matter. Receptivity to an ORBEA network is however high, with 71% of ORBEA presidents (10/14) considering it “very important” and declaring themselves willing to participate in starting it up. The model chosen for such a network is however less consensual: eight respondents prefer a formal association, while six would rather have a more informal model of organization. More results will be presented at the Congress.
Session 3: “Fish models”
1Centre of Marine Sciences (CCMAR), Faro, Portugal
2ProRegeM PhD Programme, University of Algarve, Faro, Portugal
3Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal
Different approaches have been implemented to identify and characterize the effects of molecules in particular biological processes for ecotoxicological and biomedical purposes. While in the last decades some alternative methods have been developed and validated toward the replacement, refinement and reduction of animal testing (the 3Rs; Burden et al., 2015), the reliability of in vivo approaches might still outweighed the ethical reasons for moving towards to non-animal approaches.
Zebrafish (Danio rerio) is an established model for vertebrate development and pharmacology (McGonnell and Fowkes, 2006; Laizé et al., 2015) due to several technical and experimental advantages and availability of genetic resources (Lee et al., 2014). In addition, zebrafish has a high regenerative capacity, particularly the caudal fin, which is easily accessible to surgery. Further, in this model species, a wide range of biotechnological tools such as transgenesis have been applied for in vivo imaging studies on bone development, among other processes. These tools have contributed to the popularity of the zebrafish caudal fin model as a well understood and reliable vertebrate regenerative system on the molecular mechanistic level (Wehner and Weidinger, 2015).
We will review transgenic approaches developed in zebrafish, in particular those successfully used to get insights into mechanisms underlying caudal fin regeneration, and will present recent data aiming at identifying potential side effects of warfarin (an anticoagulant largely used for thomboprophylaxis) on bone development.
The combination of transgenic zebrafish lines and caudal fin regeneration model is revealed as a high throughput screening method and an interesting model to assess how currently used and future drugs might affect bone development, reducing the use of animals and allowing to re-use them for educational purposes.
This work received national funds from the FCT – the Portuguese Foundation for Science and Technology – through project CCMAR/Multi/04326/2013 and from the European Fund for Regional Development (FEDER) through the Operational Program PROMAR project 31-03-05-FEP-0073-University of Algarve-KLING. IF and JC were financed by a postdoctoral fellowship (SFRH/BPD/82049/2011) and a PhD fellowship (PD/00117/2012) from the FCT and the ProRegeM PhD program (PD/00117/2012), respectively.
Centro Andaluz de Biología del Desarrollo (CABD), Consejo Superior de Investigaciones Científicas/Universidad Pablo de Olavide, Sevilla, Spain
The generation of distinctive cell types that form different tissues and organs requires precise, temporal and spatial control of gene expression. This depends on specific cis-regulatory elements distributed in the non-coding DNA surrounding their target genes. We have generated maps of different epigenomics marks at different developmental time-points of zebrafish embryogenesis. This study has revealed the high dynamics of the epigenome along early vertebrate development. How the huge and dynamic cis-regulatory information contained in the epigenome is organized in the genome? To address this, we have used chromatin conformation techniques to compare the regulatory landscapes of multiple genes in zebrafish and mouse embryos. Our studies demonstrate that most of them show developmental and evolutionary conserved 3D architectures that are likely essential for the correct regulation of these genes along vertebrate evolution and development.
Pathological Diagnostic in fish facility,
Faculty of Veterinary Sciences, Universidad Autónoma de Barcelona, Barcelona, Spain
To reach an adequate level of confidence in the experimental results obtained from zebrafish involves, similarly to as for any other laboratory animal, the use of a correct and precise standardization of the characteristics of that animal model. Among the items that should be considered, the guarantee of a proper health status appears a must. Unfortunately, the intensification of the use of the zebrafish as a laboratory animal has not always been followed by an in-depth attention to the care and sanitary conditions that should be taken into consideration during their handling in the animal facilities. This talk will briefly address the most relevant zebrafish pathologies as well as some potential technical errors that commonly happen during zebrafish maintenance. Novel advance sanitary handling strategies, aimed to the correct follow-up and control of zebrafish colonies, will be also discussed.
Session 4: “New Rodent Models and Technical Applications”
Fundación para la Investigación Biomédica del H.U. Getafe. Universidad Europea de Madrid
Mechanical ventilation is a life supporting therapy for 40 to 60% of the Intensive Care Unit (ICU) patients. However, in spite of the benefit, it is now accepted that mechanical ventilation has important deleterious effects on lung function, causing hyperpermeability pulmonary edema and hyaline membrane formation when large volumes are delivered to some areas of atelectasic lungs, or moderate volumes are delivered in case of pulmonary or extrapulmonary sepsis. This condition, known as ventilator-induced lung injury (VILI), is the cause of a high mortality at the ICU, surprisingly related with extrapulmonary organ injury rather than respiratory failure.
Animal models of VILI have shown that healthy rodents subjected to high tidal volumes (VT) develop not only acute lung injury, but also kidney injury, systemic inflammation and vascular dysfunction, between others. Furthermore, both pulmonary and systemic injury are prevented by antiinflammatory substances, supporting the involvement of inflammation in VILI. However, in vivo animal models do not provide us enough information for supporting the hypothesis of a pulmonary origin of extrapulmonary damage.
The combination of in vivo, ex vivo and in vitro models is helping to provide the formal proof of circulating mediators of VILI. Cultured alveolar cells subjected to cyclic streching release molecules that can be eseally identified in a controlled environment. Ventilation and perfusion of isolated lungs help to demonstrate the release of pulmonary factors to the systemic circulation. Moreover, both pulmonary and extrapulmonary tissues are injured by the perfusion liquid of isolated lungs subjected to high VT. Finally, the use of clinically relevant in vivo models would help to the identification of circulating molecules that may be both biomarkers and therapeutic targets.
CEDOC, NOVA Medical School, NOVA University, Lisboa, Portugal
Obstructive sleep apnea (OSA) is a highly prevalent sleep-related breathing disorder, briefly characterized by repetitive episodes of airflow cessation (apnea) or airflow reduction (hypopnea) caused by an obstructed or collapsed upper airway during sleep. OSA is affecting a growing proportion of the common population, and is now acknowledged as public health problem. Due to the high complexity and heterogeneity associated with OSA, considerable variability can be observed between reports addressed at the study of this disease. Moreover, the scarcity of opportunities for patient investigation, in particular at the cellular level, has compromised progress in understanding the pathophysiology of OSA and the development of novel and specific treatments for this disorder. This presentation will include a background description of the most relevant experimental animal models (e.g. the induced airway obstruction model, the sleep deprivation model and the chronic intermittent hypoxia (CIH) model) developed to overcome some of these limitations, as well as a brief report of their main contributions to this research field. The natural similarities and differences between animals and humans along with the major boundaries of the use of animal models in OSA research will be also discussed. Special focus will be given to a rat model of hypertension induced by a paradigm of CIH that simulates the most prevalent cardiovascular sequelae of OSA.
1Transgenic Mice Unit, Biotechnology Programme
2Tumor Suppression Group, Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Melchor Fernández Almagro 3, Madrid, Spain
The capacity of reprogramming adult cell identity, returning it to a naïve pluripotent state by expressing a defined and reduced set of transcription factors holds a great promise for the development of patient-specific disease models and regenerative therapies. Since the seminal work of Takahashi and Yamanaka in 2006, many different cell types have been reprogrammed to a pluripotent state by expressing different sets of transcription factors demonstrating the universality of the process. However, the question remained of whether reprogramming was restricted to cells cultured in vitro or adult differentiated cells could also be reprogrammed in vivo in a physiological context. By using a conditional (Tet-induced)-transgene, expressing the four Yamanaka reprogramming factors, we were able to obtain “reprogrammable mice” in which we first demonstrated that iPS could also be generated in vivo from cells of many different tissues and organs. These in vivo iPS have the capacity of generating teratomas as embryonic stem (ES) cells do but also show a higher capacity than ES cells to contribute to trophectoderm lineages, indicative of an even more plastic or primitive state than ES cells or in vitro generated iPS cells. Since most adult tissues show a limited capacity of self-regeneration after injury we are now interested in exploring to what extent in vivo reprogramming may indeed have a physiological contribution to tissue growth and regeneration after trauma, or to pathological tissue growth as tumor development, and how in vivo reprogramming can be exploited to potentiate or interfere with these processes. The combination of cell reprogramming with the recently established CRISPR/Cas9 system for genome editing opens a whole new venue in which these processes can be modeled and therapeutically approached.
Session 5: “Design of Animal Facilities”
The ElmCos Group Ltd., Nanoose Bay, Canada
Scientific research is an area that is constantly changing and evolving. Shifts in the types of species used for animal models, changes in research directions, new technologies, and the movement from multi-disciplinary research to a convergence science model all result in animal facilities needing to evolve and adapts in order to support research. The pace of discovery, the increased costs in research, cutbacks in funding and the push for more cost recovery has meant that it is becoming more difficult to predict what animal facility requirements will be. This presentation will provide an overview of lessons learned from existing projects that can help to reduce the capital and operational costs of animal facilities and it will provide insights into future directions of animal based research and their potential impact on vivarium design. Whether operating an existing facility or planning for renovations and new buildings, attendees will gather knowledge that can assist them in ensuring their facilities meet both the challenges of today and tomorrow.
Session 6: “Communication of Animal Research”
INSERM - GIRCOR
In spite of implementation of one of the most binding regulation in the world, the use of animals for scientific purposes is more and more questioned in the European Union. Animalists intensify their communication and lobbying action with as final goal the abolition of any use of animals for scientific purposes. In that context, the future of biomedical research is jeopardized by potential decisions to strengthen regulations by European institutions, these last being strongly influenced by NGO’s but also by member state positions that are themselves strongly influenced by their public opinions. Furthermore combining strategies of harassment with vilification and intimidation intended to compromise scientists on the basis of false or truncated information, animalists succeed more and more in discouraging scientists at least when they are not enough supported by their institutions. There is thus an urgent need for the scientific community to develop a strategy to foster a better level of public and institutional acceptance in each member state. Based on the comparison of the situation in different countries, we will discuss, the type of organization the possible actions and their combinations. This includes (a) identification of the actors (individuals associations, institutions, media), the aims (obtain the support of authorities and institutions, better inform the public, control the media) and the targets (public opinion, medias, policy makers, research institutions, authorities), (b) determination of the type of efficient actions to be developed at different levels. (c) Finally we will discuss the form and content of the messages in order to help the scientific community to develop an adapted pro-active strategy.
Session 7: “Wild animals”
Instituto de Estudios Sociales Avanzados (IESA-CSIC), Córdoba, Spain
Scientific research on wildlife has been performed for many years regulated by legislations mainly developed to protect wildlife animals and their natural habitats. For many scientists and managers the concept of animal experimentation and its regulation were mostly restricted to the labs. However, society demands the need to ensure the protection of all the animals used in scientific researches.
Species conservation and environmental management have grown in recent years. Such managements in controlled conditions may have impact on the welfare of the animals, but welfare components are typically subordinate to conservation objectives. Frequently the conservation of species requires the capture of animals to obtain information on population dynamics, movements, habitat relationships, etc. In most of the cases animals are released in the same place, but in some other the investigator might bring them into an animal resource facility. Conservation of species may also involve captive breeding and reintroduction of animals in the wild. Sometimes, it is also necessary develop procedures to eradicate invasive species in many other conservation projects.
Critical evaluation of protocols in wildlife requires the use of appropriate standards, and sometimes problems occur when extrapolating standards developed for laboratory environments and domestic animals with different objectives. For example, in biomedical research artificially selected, domesticated strains are used to reduce differences among individuals. In contrast, field researchers usually are interested in the animals themselves as the focus of study, and variation among individuals and natural behaviors are of fundamental interest and importance.
Instituto de Investigación en Recursos Cinegéticos (IREC), CSIC-UCLM-JCCM, Ciudad Real, Spain
Life history of wild carnivores is shaped by their adaptations to hunt other animals, which hinder their direct observation. Therefore, wild carnivores are often captured for scientific, conservation or management purposes. Systems of capture should be effective, selective, respectful to animal welfare, user-safe and must observe current laws. The performance of capture systems depends on trap characteristics, type of bait, conditions of installation, training and will of the user and environmental and biological variables. Cage-traps of different sizes and shapes, combined with diverse kinds of baits, are the method most used for the capture of wild carnivores. Welfare of animals captured in cage-traps is reduced due to stress and potential injures related with their confinement. Injected anesthetics can be used for minimizing the stress of captured carnivores during handling. Physical injuries, hyper- or hypothermia and dehydration are the most likely complications during capture and handling. Animals should be completely recovered from the anesthesia before being released in the wild. Alternatively, the most appropriate chemical or physical method should be used if animals should be euthanized. Personnel qualification and training are key factors for animal welfare during capture and handling of wild carnivores. Capture provides valuable information from wild carnivores, difficult to be obtained through other methods. However capture is costly requires trained and devoted personnel and implies risks for the welfare of the animals. Hence, researchers must carefully consider alternative non-invasive methods such as photo-trapping, remote sample collection or molecular techniques before employing capture for the study of wild carnivores.
1Animal Science Techniques Applied to Wildlife Management Research Group, IREC Sec. Albacete, IREC (CSIC-UCLM-JCCM), Campus UCLM, Albacete, Spain
2Sección de Recursos Cinegéticos, IDR, Universidad de Castilla-La Mancha, Albacete, Spain
3Departamento de Ciencia y Tecnología Agroforestal y Genética, ETSIAM, Universidad de Castilla-La Mancha, Albacete, Spain
Since 1995 the UCLM continuing with research on deer. In Spain the Cervus elaphus hispanicus is the main big game species, and is priced according to the size of its antlers. The scientific projects have sought to improve trophies, and provide useful informations to apply in the hunting sector for Iberian red deer. The research has been addressed to the reproduction (semen), reproductive physiology and endocrinology of female, artificial insemination, lactation, behavior, etc. In recent years, the research team, composed of Laureano Gallego, Tomás Landete and Andrés José García, working in the factors involved in antlers development and analysis of its quality (structure, mechanical and composition properties). However, have emerged interesting biomedical implications from collaboration in neurobiology and histology research. Subsequently we have provided new ideas about osteoporosis, which in deer is a physiological, cyclic and reversible process (antlers grow in 4 months, during which mobilizes mineral from the skeleton, and once antler is formed, the body restores minerals to bones), it is interesting to analyze human osteoporosis. We proposed: osteoporosis is not due primarily to lack of calcium in bones, if not due to trace elements deficiency like manganese, that is critical into bone formation. We found that people undergoing hip implant for osteoporosis, suffering from neurodegenerative diseases (dementia, Parkinson and Alzehimer). We proposed that osteoporosis is secondary to nerve disease, when the body needs trace elements for enzymes in the central nervous system, mobilizes minerals contened in bones, to have them at the concentrations required, sacrificing bone status.
Session 8: “Large animals: surgical, preclinical and veterinary models”
INRA, UMR 1198 Biology of Development and Reproduction, Jouy en Josas, France
As Co-Chair of COST Action BM1308 “Sharing Advances on Large Animal Models – S A L A A M” it is our pleasure to inform you about the goals and events of our Action, chaired by Eckhard Wolf, Ludwig-Maximilians-Universität in Munich, Germany, which started a year ago. The translation of novel discoveries from basic research to clinical application is a long, often inefficient and costly process. Consequently, “Translational Medicine” has become a top priority. Appropriate animal models are critical for the success of translational research.
SALAAM will support the development, characterization and implementation of tailored large animal models by (i) sharing information and technology for genetic engineering; (ii) developing criteria for selection of the most suitable species; (iii) establishing and validating standardized phenotyping protocols; (iv) creating a database of existing models, tissue samples, and validated phenotypic assays; and (v) developing concepts for the scientific and ethical evaluation of experiments with large animals.
More information on our past and future activities can be found at
1INRA, UMR 1198 Biology of Development and Reproduction, Jouy en Josas, France
Pregnancy is often considered as a “black box” and access to the fetus or placenta is limited. In vivo imaging enables the longitudinal monitoring of pregnancy.
Before fertilization, gametes can be visualized using confocal laser endomicroscopy. For example, movements of dog spermatozoa at the surface of the endometrium can be observed in vivo. Ultrasound bio-microscopy is suitable for observing the cumulus oocyte complex before ovulation and for the study of embryo development before and after implantation in rabbits. Sampling or injection can also be performed into the blastocoele.
After implantation, fetal growth and the fetoplacental unit is monitored using classical ultrasound and 2D-Doppler. Reduced fetoplacental perfusion can be quantified within placenta by power Doppler angiography. Ultrafast Doppler, a paradigm shifting ultrasound technology, greatly increases Doppler sensitivity and enables to discriminate between maternal and fetal blood flow within the placenta.
Minimally invasive technologies, such as 3D-angiography, are based on X-Ray technology. Blood flow is visualized using contrast agent injected through en indwelling catheter. Angiography can also be used to modify blood flow to the placenta through arterial vessel embolisation. Magnetic resonance provides fetoplacental images at the histological scale. We are currently exploring this technique as a complement to Doppler to study fetoplacental perfusion. Near delivery, shear wave elastography, based on ultrafast wave technology, can be used to evaluate cervical ripening in sheep.
The use of refined imaging technologies enable researchers to study in vivo fetoplacental development and can help reduce the number of animals being used in research.
Cardiology Department, Hospital General Universitario Gregorio Marañón
Instituto de Investigación Sanitaria, Hospital General Universitario Gregorio Marañón
Universidad Complutense
Madrid, Spain
Over the last two decades, stem cell and gene therapy have emerged as hopeful alternatives to repair myocardial tissue and to restore cardiac function after ischemic insults to the heart. Preclinical models of ischemic cardiomyopathy have been crucial in the development of this field, and have boosted a rapid clinical translation into human patients.
Among all large animal species, swine models of both acute and chronic coronary artery disease are perfectly well defined and established for safety and efficacy cardiovascular studies, and are routinely accepted by scientists and recommended by the competent authorities before moving into the clinical arena.
Specifically, swine models allow us to test all delivery routes that will be subsequently used in humans, either surgical (transepicardial injections during open-chest procedures and minimally-invasive delivery of bioartificial constructs) or percutaneous (intracoronary infusion, transendocardial injections with or without sophisticated navigation platforms and retrograde infusion of products through the coronary sinus). However, despite enormous achievements that have moved the cardiovascular regenerative medicine field forward, major barriers have been found and many fundamental issues remain to be solved: ideal type and dose of cells or genes, mechanisms of action, timing after the ischemic damage, etc. Large-animal models will undoubtedly be needed to shed light on these issues in years to come. During the SECAL conference, key points of the field and all these delivery technologies, with their respective pros and cons depending on the different clinical setting in swine models, will be presented and discussed.
Zoetis Manufacturing & Research Spain, S.L
In the last years there have appeared several infectious emergent diseases representing one of the most severe threats for animal health. The appearance of new and more virulent strains as in the case of Avian Influenza, or the re-emerging of Bluetongue Disease in Europe have brought severe consequences for animal health, agricultural economy and food supply.
Is very important for the pharmaceutical industry to be prepared and have the capacity to quickly and efficiently react in front these diseases. Therefore, ones the emerging disease and it’s epidemiologic and clinical relevance has been identified, the infectious agent has to be identified and vaccine strains selected; analytical techniques and manufacturing processes must be defined; and models to evaluate the efficacy and the safety of the vaccine must be developed.
For emerging diseases is important to take into account that quicker and simplified regulatory processes can be followed in order to have as quickly as possible and efficacious vaccine in the market, to respond to the health emergency situation.
In the talk a real case is presented: The development of vaccines in front Bluetongue Virus Disease, infectious disease caused by Bluetonge Virus (BTV) which is transmitted by the bites of an insect (Culicoides species). Ovine, bovine and goats are the domestic animals susceptible to this virus. There has been identified 25 different strains of BTV, which complicates the establishment of preventive programs since there is no cross protection between them. Taking into account that the disease is transmitted by a vector was very important to have efficacious vaccines available.
Examples of vaccines developed against different serotypes of Bluetongue virus and times passed between the virus detection on the field and the grant of provisional marketing authorizations for vaccines commercialization are presented.
Consequently to BTV vaccines development and the implementation of effective control measures, there was a drastic reduction of disease, leading to ceasing compulsory vaccination and becoming voluntary from the 31st July 2011.
Session 9: “Education, Training and Competence”
NOVA Medical School | Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal
Accreditation of the training programs for persons working with laboratory animals under the Directive 2010/63/EU envisages that Laboratory Animal Science (LAS) training will be accepted around Europe. The Federation of Laboratory Animal Sciences Associations (FELASA) accreditation system has become increasingly recognized as a robust way of improving training and ensuring an European golden standard in LAS education and training. The FELASA accreditation recognizes, supports and enhances the quality of training; establishes a more uniform platform of competence of those trained, so enabling greater mobility of researchers and animal care staff; enables the identification and sharing of good practice; and provides independent reassurance for National Authorities and the public about the competence of those working with laboratory animals.
In 2013 the accreditation system has been reviewed and adapted for program accreditation of the functions A, B, C and D and has kept accreditation for programs for Specialists in LAS. Has also given support to all Courses Organizers of former accredited programs to adapt the training to the new legal requirements.
The FELASA Accreditation scheme provides the possibility of accrediting courses specific for one function or for more. Allows the Course Organizer to issue modular certificates. Courses aiming to become accredited or FELASA accredited programs must be comprised of at least the “Core Modules” for that Function and at least one species or group of species. The result of a questionnaire circulated to all FELASA member associations abut FELASA certificate recognition by the National Competent Authorities will be presented.
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
The laboratory animal science community is at present faced with new challenges to meet the objectives of the new Directive 2010/63/EU but can see this as an opportunity to further advance laboratory animal science training and education. Already today, not enough LAS programs are available across Europe at present, and new courses with a more flexible content need to be developed on the various levels of animal experimentation jobs. Online courses can help dealing with these challenges, as they are available anywhere at any time and are not limited to a certain number of participants. Theoretical knowledge and practical aspects of LAS can be taught online line at a students’ own study pace, while practical training can be provided in wet labs or on the job. Web-based programs offer the advantage of the option of multi-lingual training approaches within the same platform. Also it offers more flexibility for both teacher and student. Although some course providers still want to offer class-room teaching the educational quality can be improved by offering e-learning as preparation and spending the confrontation time for dialogue with better prepared students. At our institution we have for several years been working with blended learning, i.e. a mixture of online and class-room teaching, for the former FELASA category C and D level, while we now have developed a 100 % online program for designated veterinarians. Also examinations have been performed online, and in general we have had good experiences with this way of teaching.
American Association for Laboratory Animal Science
Increasing demands on time, shrinking resources, and expanding knowledge all create a greater need for training efficiency and effectiveness. Electronic transmission provides the venue for delivery of a vast array of training in the laboratory animal science field. The American Association for Laboratory Animal Science (AALAS) has tapped some of the best minds and most proficient trainers to provide high quality training for technicians, technologists, research associates, veterinarians, researchers, ethical committees, suppliers and others involved in laboratory animal science field. The AALAS expertise and electronic media combine to provide comprehensive training on a time saving and cost effective basis. An overview of the award-winning AALAS Learning Library (ALL) and its operational efficacy will be presented. Attention will be given to how the ALL can be customized to deliver training to meet European training requirements and how the ALL can be the vehicle for meeting those requirements at the institutional and individual levels. Additional information will be provided regarding other AALAS educational resources, especially the virtual webinars which incorporate leading edge and emerging topics.
1SPCAL, Sociedade Portuguesa de Ciências em Animais de Laboratório
2Universidade Lusófona de Humanidades e Tecnologias, Portugal
3NOVA Medical School, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Portugal
4Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal
5Unidade de Transgénicos, Instituto Gulbenkian da Ciência, Oeiras, Portugal
6Faculdade de Farmácia, Universidade de Coimbra, Portugal
The Portuguese Society for Laboratory Animals Sciences (SPCAL) is a scientific and non-profit association mainly focused on the improvement of animal welfare and better animal use for scientific purposes. The mission of the SPCAL is to bring thoughtful analysis and constructive action to allow on the often highly-polarized debate concerning the use of animals in laboratory animals. It is also purpose of this Society to implement and disseminate the ethical and behavioural principles that should be strongly linked to the use of laboratory animals for scientific purposes.
Since training of laboratory animal users is mandatory by the Portuguese law, the most relevant activity of SPCAL has been to organize and promote LAS education and training courses. These were either practical courses, directed mainly to researchers and technicians; or theoretical courses, directed mainly to researchers. These courses were designed to train users for functions A, B, and D, according to the European Commission recommendations. SPCAL was pioneer in Portugal (in 2005) on the implementation of a modular strategy of training. By providing an easier access of all laboratory animal users to this type of courses, which ultimately resulted in significant increase of the accreditation requests of animal users to the Governmental Competent Authority (Direção Geral de Alimentação e Veterinária - DGAV). Considering the high percentage of trained and educated users achieved one can undoubtedly conclude that there has been an improvement of the use of animals for scientific purposes in Portugal, in both refinement (welfare) and reduction perspectives as a result of modular training.
Session 10: “Health monitoring/Pathology Session”
1National Vet Inst, Uppsala, Sweden
2Inst. Pasteur, Paris, France
3MRC National Inst for Med Res, London, UK
4MicroBioS GmbH Microbiol Services, Reinach, Swiss
5Charles River, Lyon, France
6National Res Council/EMMA, Rome, Italy
7Biomedical Diagnostics BioDoc, Hannover, Germany
The recommendation for health monitoring (HM) of laboratory rodents and rabbit colonies from FELASA (2014) describe both general and specific aspects of HM, including the concept of microbiological unit in relation to the design of the HM programme, the selection of animals/tissues, the choice of agents, test methods, interpretation of results, laboratory reporting and health reporting. Lists of agents for which to monitor and also the recommended frequency of monitoring for each animal species considered have been suggested. The control of immunodeficient animals and biological reagents also are considered. The impact on HM of new developments in animal housing are discussed, such as the increased use of cage-level containment where agents may occur at low prevalence difficult to detect. Another challenge in HM is the increased use of sentinels to detect multiple contaminants, but there is no universally susceptible sentinel that is equally apt for all pathogens and test systems.
The FELASA recommendations seek harmonization of HM programmes, but at the same time emphasize that the diversity of contemporary animal colonies requires flexibility.
HM is a complex field where success will depend on careful planning and correct implementation at all practical levels including selection of relevant animals, infectious agents, and laboratory methods, quality control, and interpreting and communicating test results. HM results are commonly used as a platform for important decisions regarding animal welfare, breeding routines, exchange of animals between units, scientific experiments, the production of biological reagents, etc. For this reason, the supervision by competent specialists is mandatory.
Head. Histopathology Core Unit
Biotechnology Programme CNIO. Spanish National Cancer Research Centre
Madrid, Spain
Since the early 1980s, the number of mice used in the generation of genetically modified mouse models (GEM) has exponentially increased along with the transfer of these models to human disease. Inbred mouse strains that contribute to most of these models maintain a relatively large number of significant spontaneous pathologies despite the controlled environments in which they live in.
Some of the anatomical differences that we see in tissues or organs when assessing the animals are natural, and are associated with sexual dimorphism. Others, are affected by environmental factors and social interactionscan and affect the phenotype. It is important to identify and interpret the presence of any of these changes accurately and appropriately in the context of the histopathological phenotyping choosing the appropriate controls for comparison, which ideally include littermates, different genotypes or untreated animals. Using mice in research very often results in the need for a diagnosis, classification, interpretation or discovery of new phenotypes and pathological changes in different tissues. It is therefore essential to know the differences between animals with which we work, as well as the most frequent spontaneous lesions in different inbred lines commonly used in research that appear as a result of ageing and the characteristics of the strain during different experimental procedures. These pathologies can act as confounding variables when interpreting the experimental results and can be a challenge due to the number of variables considered.
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
Increasing knowledge underlines that disease expression in a range of animal models, such as models of inflammatory bowel disease (IBD), diabetes, obesity, psychiatric disease and dermatitis seems to correlate with the composition of the microbiota. Non-cultivable bacteria have with an essential impact on animal model parameters have been discovered. Examples are segmented filamentous bacteria (SFB), which are heavily involved in the expression of Th17 cells and thereby IBD, Akkermansia muciniphila, which may decrease the incidence of diabetes, and Prevotella spp., which may increase the incidence of diabetes in animal models. Stress, dietary fluctuations and other forms of uncontrolled impact may change the microbiota of the animal, and through this induce a change in the animal model. Although prices for a full sequencing based characterization of the entire microbiota are rapidly declining, microbiological quality assurance programs of today do not include any microbiota characterisation. However, the most simple approach for handling microbiota impact on animal models is to screen animals when used for microbiota sensitive studies and incorporate this information in data evaluation, thereby turning this uncontrolled variation into controlled variation. In the long run, we may be able to produce animals with less variation by microbiota standardization. Such standardisation may be achieved by inoculation of tailor made microbiotas or feeding certain prebiotic diets. This approach should, however, be based upon a knowledge of which microbiota will be preferable for which model; a knowledge which is not fully available today.
Session 11: “Severity classification/ interpretation/application”
Animal Welfare: Science, Humanities, Ethics and Law Research Group (AHSWEL-IAS). Universidad de Alcalá, Madrid, Spain
Convener of the FELASA/ESLAV/ECLAM WG for the retrospective evaluation of procedures.
The Directive 2010/63 went into effect almost three years ago, however several aspects should be still developed. One of them is the evaluation of the severity on the procedures on animals.
European stakeholders like FELASA, ESLAV and ECLAM have been working for five years in preparing recommendations to carry out that evaluation. Some examples were already posted on the European Commission webpage and around twenty will be published shortly on a stand-alone supplement of Laboratory Animals Journal.
An informal survey among the laboratory animal industry in Spain and other members of the Working Groups above reflected than there are some differences of criteria among scientists and countries about what it should be evaluated as part or not of an experiment, especially during the retrospective evaluation – e.g. capture at the wild animals research – or the necessity of setting up a proper level of severity when a similar situation happens but in different circumstances –e.g. the same period of fasting in different animal species or age.
More issues like the necessity of preparing follow-up sheets for animal welfare and severity during the procedures or the elaboration of specific score sheets for the retrospective evaluation of the severity and the cumulative suffering in specific experiments would be also well-received.
A Portuguese-Spanish round-table with different points of view and a productive debate during the Congress it should be very useful for the approaching of postures and the future of animal welfare.
CITAB - Centre for the Research and Technology of Agro-Environmental and Biological Sciences, Quinta de Prados, 5001-801 - Vila Real, Portugal.
UTAD - Veterinary Sciences Department from the University of Trás-os-Montes and Alto Douro, Quinta de Prados, 5001-801 - Vila Real, Portugal.
3B's - Research Group - Biomaterials, Biodegradables and Biomimetics, AvePark, 4806-909 Caldas das Taipas, Guimarães, Portugal
When animals are used for scientific purposes, in any circumstances where the experience of pain or distress is ethically justified as part of the study, actual or potential pain, suffering, distress or discomfort should be minimised or alleviated.
Aspects of a research protocol that may impact on an animal are not confined to experimental procedures. Other potential sources of pain, stress and distress, such as transport, handling, housing, social and physical environment, phenotype and health status, must also be considered.
For each research protocol, the development of a strategy to assess, minimize and monitor pain and distress requires decisions to be made regarding the clinical signs or observations that will be used to assess an animal’s wellbeing or clinical condition as the project progresses; the clinical signs or combination of clinical signs that will indicate that intervention (including euthanasia) is necessary; the actions that will be taken if a problem is detected; the frequency of monitoring; the people who will conduct the monitoring, and their training and the system for the recording of observations.
Actions that will be taken when a particular sign or combination of signs is observed in an animal should be defined. Such actions or interventions may include: promoting the animal’s comfort by providing supportive treatments (eg warmth, hygiene, fluids, nutrition and social needs); more frequent observation; consultation with a veterinarian with appropriate experience; administration of specific treatment (eg an analgesic agent to alleviate pain); euthanasia of the animal or his removal from the protocol.
Centro de Investigaciones Biomédicas (SPEA/CIC). Universidad de Granada
Human interaction with wildlife produces a broad bioethical issues of conservation, biosecurity, development and human wellbeing and animal welfare status. Similarly, the welfare of wild animals is affected by the direct relationship with humanized environments (for example, living next to a road), their interaction with other living systems as dependent on human activity (for example competitive relationships, predation or parasitism within a biological reserve) as a result of various management practices to which they are subjected. Wildlife research is performed with different aims and objectives:
• Conservation
• Management
• Biosafety
• Ensure human welfare or.
• Basic Research
They generate problems of interest from the ethical point of view. Here, however, we will focus primarily on issues such as animal welfare may be affected by management practices carried out for scientific research.
This type of scientific research with animals in the wild is often perceived from the mass media as an activity where biologists and veterinarians actively promote the welfare and conservation of animals. In practice, however, field studies with wild animals carry multiple risks to study subjects and have an impact on individual well-being as high, if not higher, than that caused by the studies with laboratory animals. These risks and problems are often mostly overlooked by researchers, basically for two reasons.
1 First, the fact that wild animals experience, naturally, considerable levels of suffering caused by the environment (hunger, injury, disease, predation, etc.).
2 Second, the fact that much of the research conducted with wildlife is focused on conservation, that is oriented towards the good of the populations or species and therefore the welfare of individuals is considered to be of secondary importance.
In summary it presents unique problems both from the bioethical standpoint as regulatory and logistical. Let us briefly what they are, with different practical example of the CEEA projects submitted for evaluation.
Oral Communications
ID: 37
1CITAB - Centre for the Research and Technology of Agro-Environmental and Biological Sciences, Quinta de Prados, Vila Real, Portugal
2CECAV - Animal and Veterinary research Centre/UTAD - Veterinary Sciences Department from the University of Trás-os-Montes and Alto Douro, Quinta de Prados, Vila Real, Portugal
3UTAD - Veterinary Sciences Department from the University of Trás-os-Montes and Alto Douro, Quinta de Prados, Vila Real, Portugal
4QOPNA/UA - Unidade de Investigação de Química Orgânica, Produtos Naturais e Agroalimentares, Departamento de Química, Universidade de Aveiro, Aveiro, Portugal
5UCLA - Universidad Centroccidental “Lisandro Alvarado”
Breast cancer is the most commonly diagnosed cancer among women. Estrogens act by binding to two specific receptors: estrogen receptors (ERs) α and β. This study aimed to evaluate the effects of lifelong exercise training on the expression of ERs α and β in chemically-induced mammary tumors in a rat model.
Procedures followed the European and National legislation. Female Sprague-Dawley rats were divided into two groups: MNU-sedentary (n = 11) and MNU-exercised (n = 10). At seven weeks of age, all animals received an intraperitoneal injection of the carcinogen agent N-methyl-N-nitrosourea (MNU) (50 mg/Kg). Animals from group MNU-exercised ran in a Treadmill at a speed of 20 m/min, 60 min/day, 5 times/week during 35 weeks. Then, all animals were humanely sacrificed and the mammary tumors were fixed in formalin; the sections were incubated with ERα and β primary antibodies and the percentage of immunopositive cells was determined.
All animals from both groups developed mammary lesions: 71 mammary lesions in group MNU-sedentary and 50 mammary lesions in group MNU-exercised (p = 0.056). All mammary lesions exhibited nuclear immunostaining for ERs α and β. In both groups, the percentage of immunopositive cells for ERβ was higher than the percentage for ERα (p < 0.05). The immunoexpression for ERα was higher in MNU-exercised group than in MNU-sedentary one (p < 0.05).
The simultaneous expression of ERs α and β suggests that the proliferation was inhibited and the apoptosis was induced. The highest expression of ERα in group MNU-exercised is an indicator of well differentiated tumors and better response to hormone therapy.
ID: 45
1Unidad de Toxicología
2Departamento de Fisiología Facultad de Veterinaria (UEX)
We carried out a sociological study to know the opinion of various social groups to animal testing. To this end, we have structured a survey with questions on tree in an analytical form of successive contradictions, seeking to define the maximum possible social profile of the respondent, trying to figure out what motivations might be behind a favorable or contrary position to experimentation. In total, 232 people from Extremadura performed this test, grouped under four factors: age, gender, area and biological knowledge.
The results established that 63.79% of respondents were in favor of animal testing, compared to 36.21%. In the distribution by gender, only 53% of women were in favor of animal experiments, compared with 72% of men. In terms of age, only 38% of people aged 31–45 years were favorable to animal testing, in contrast to people aged 46–60 years (81% in favor) or young aged 20–30 years (61% favorable). Also, it notes that 68 % of the urban population was in favor, compared to 52 % in rural areas. According to the biomedical training (biologists, doctors, veterinarians and students of related experimental animal science) versus those without such training, no significant differences were found, 68% among people with biomedical training compared to 60% favor among people withoutthis training.
We can conclude that society understands that the use of animals cannot be completely replaced, but understands, as a necessity, refinement and reduction guidelines as well as the search for alternative methods in those situations as possible.
ID: 86
1CCMIJU
2HGU Gregorio Marañón
Cardiosphere-derived cells (CDCs) have been proven to decrease infarct size and increase viable tissue. However, until now it remains unknown which is the most suitable cell administration strategy. Our aim was to compare the safety of two minimally invasive delivery methods. For that purpose a total of 12 pigs were subjected to endovascular infarct creation. Five weeks later CDCs were injected using an intracoronary (IC, n = 6) or a thoracoscopy-guided intramyocardial (IM, n = 6) approach. Safety endpoints included pre and postprocedural troponin I (TpnI) and creatine kinase-MB (CK-MB) values, occurrence of malignant arrhythmias and procedural time. Injection was successful in all cases. Four animals from the IM group suffered from ventricular fibrillation during opening of the pericardium that was successfully defibrillated. No arrhythmias were observed in the IC group. TpnI (µg/L) was preinjection 0.011 ± 0.001 (IC) and 0.011 ± 0.002 (IM) and postinjection 0.011 ± 0.002 (IC) and 0.236 ± 0.218 (IM). CK-MB (µg/L) was 4.033 ± 1.587 and 5.867 ± 3.208 (preinjection) and 3.700 ± 1.197 and 6.367 ± 2.077 (postinjection) for IC and IM delivery, respectively. Significant differences were observed postinjection between groups for both parameters (p < 0.05). Procedure time was significantly shorter in the IC group (54.833min ± 16.893 min vs. 89.333 min ± 20.334 min, p < 0.05). In conclusion, CDC injection through an IC or IM approach caused no myocardial injury as demonstrated by the slight increase in TpnI. In this experimental setting, however, the lack of arrhythmias and the shorter procedure time in the IC group as well as the increased TpnI and CK-MB levels in the IM group suggested that CDC infusion was safer when the IC approach was used.
ID: 106
3BS Research Group, Univ. Minho, Headquarters of the European Institute of Excellence on Term
ICVS-3BS, PT Government Associate Laboratory, Braga/Guimarães, Portugal
Hind-limb ischemia has been used in type 1 diabetic mice to evaluate treatments for peripheral arterial disease or mechanisms of vascular impairment in diabetes [1]. Vascular deficiency is not only a pathophysiological condition, but also an obvious circumstance in tissue regeneration and in tissue engineering and regenerative medicine (TERM) strategies.
We performed a pilot experiment of hind-limb ischemia in streptozotocin(STZ)-induced type 1 diabetic mice to hypothesise whether diabetes influences neovascularization induced by biomaterials. The dependent variables included blood flow and markers of arteriogenesis and angiogenesis.
Type 1 diabetes was induced in 8-week-old C57BL/6 mice by an i.p. injection of STZ (50 mg/kg daily for 5 days). Hind-limb ischemia was created under deep anaesthesia and the left femoral artery and vein were isolated, ligated, and excised. The contralateral hind limb served as an internal control within each mouse. Non-diabetic ischaemic mice were used as experiment controls. At the hind-limb ischemia surgical procedure, different types of biomaterials were placed in the blood vessels gap. Blood flow was estimated by Laser Doppler perfusion imager, right after surgery and then weekly. After 28 days of implantation, surrounding muscle was excised and evaluated by histological analysis for arteriogenesis and angiogenesis.
The results showed that implanted biomaterials were promote faster restoration of blood flow in the ischemic limbs and improved neovascularization in the diabetic mice. Therefore, we herein demonstrate that the combined model of hind-limb ischemia in type 1 diabetes mice is suitable to evaluate the neovascularization potential of biomaterials and eventually tissue engineering constructs.
ID: 25
1Animal Facility Unit, RMG-SEA Instituto de Neurociencias de Alicante, Universidad Miguel Hernández–Consejo Superior de Investigaciones Científicas, Alicante, Spain
2Animal Facility Unit Parc de Recerca Biomedica de Barcelona, Spain
3Animal Facility Unit, Spanish National Cancer Research Center CNIO, Madrid, Spain
4Instituto Universitario de Investigaciones Biomédicas y Sanitarias- Universidad de Las Palmas de Gran Canaria and Complejo Hospitalario Universitario Insular Materno Infantil de Gran Canaria, Spain
5Veterinary Faculty of the Universidad Alfonso X El Sabio de Madrid, Spain
The current study aimed to characterize and to explore a treatment for a condition in which male mice presented a solid bulge in the preputial area and absence of external visualization of the penis. Forty-three animals, from several animal houses within Spain, were included in the study. The condition mostly affected breeding animals (78.6%) and was associated with C57BL/6J in both wild type (21.4%) and transgenic mice with that background (59.52%). A solid, yellowish white substance was inside the prepuce, displacing the penis cranially, avoiding its externalization and causing a subsequent infertility. Contrary to the bibliography suggestions, the penis was completely intact in all the cases, with no signs of mutilation or wounds.
Microbiology characterization was performed in 11 blocking substances. Bacteria from saprophytic faecal flora were isolated (Enterococcus spp 72.7%, Escherichia coli (54.5%) and coagulase-negative Staphylococci (54.5%)). Twenty-six animals were submitted to necropsy and pathological characterization. The blocking substance was completely amorphous, acidophilic and had clusters of spermatozoa in the sample periphery, a partner almost identical to post-coital female plugs, suggesting an ejaculatory origin.
Clinically and pathologically two clear conditions were differentiated: one with urinary obstruction and one without, depending on the capacity of the blocking substance to lock the urethra.
A surgery was developed to treat this condition and tested in 18 mice. The blocking substance was eliminated by an incision made from the opening of the prepuce in cranial direction. Most of the animals recovered fertility, but the treatment did not work when urinary obstruction was present.
ID: 101
Instituto de Medicina Molecular Universidade de Lisboa
Directive 2010/63/EU refers to the evaluation of competence, requiring that staff be educated, competent and properly supervised until competence is achieved.
Therefore, establishments housing animals must set processes in place for proper evaluation of the staff. This should be an on-going process, carried out at the usual working environment and must constitute a means to detect situations of poor practice.
iMMLisboa’s rodent facility is implementing a system for competence assessment, combining self and supervisor evaluation. This system aims to take into account each person’s perception of their own work, hence avoiding the occurrence of bad practices by preemptively identifying each collaborator’s main difficulties. We expected this system to help us address individual needs in terms of additional training and supervision.
An evaluation table was designed, listing all tasks performed by the staff and a score system was created. The table included both technical and interpersonal skills. Each staff member was first requested to score his/her own performance in each task and subsequently was scored for the same tasks by an evaluator, during daily routines. Scores from both evaluations were then compared and discrepancies identified. Results were discussed with each member in terms of performance and perception of competence. Opportunities for improvement were identified and a plan for additional training and supervision was created where appropriate.
The whole process was repeated 3 months later to assess individual progress.
Results will be discussed in terms of the method’s efficacy and advantages of including self-evaluation when assessing competence.
ID: 57
CCMIJU
ID: 132
Experimental Animal Service, Research Unit, Universitary Hospital of Gran Canaria, Dr. Negrín, Las Palmas de Gran Canaria, España. Infectious Diseases and Fish Pathology, Universitary Institute of Animal Health and Food Safety, Universidad de Las Palmas de Gran Canaria, Arucas
Oxygen therapy is currently used as a supportive treatment in septic patients to improve tissue oxygenation. However, oxygen can exert deleterious effects on the inflammatory response triggered by infection. We postulated that the use of high oxygen concentrations may be partially responsible for the worsening of sepsis-induced multiple system organ dysfunction in an experimental clinically relevant model of sepsis. We used Sprague-Dawley rats. Sepsis was induced by cecal ligation and puncture. Sham-septic controls (n = 16) and septic animals (n = 32) were randomly assigned to four groups and placed in a sealed Plexiglas cage continuously flushed for 24 h with medical air (group 1), 40% oxygen (group 2), 60% oxygen (group 3), or 100% oxygen (group 4). We examined the effects of these oxygen concentrations on the spread of infection in blood, urine, peritoneal fluid, bronchoalveolar lavage, and meninges; serum levels of inflammatory biomarkers and reactive oxygen species production; and hematological parameters in all experimental groups. In cecal ligation and puncture animals, the use of higher oxygen concentrations was associated with a greater number of infected biological samples (P G 0.0001), higher serum levels of interleukin-6 (P G 0.0001), interleukin-10 (P = 0.033), and tumor necrosis factor-! (P = 0.034), a marked decrease in platelet counts (P G 0.001), and a marked elevation of reactive oxygen species serum levels (P = 0.0006) after 24 h of oxygen exposure. Oxygen therapy greatly influences the progression and clinical manifestation of multiple system organ dysfunction in experimental sepsis. If these results are extrapolated to humans, they suggest that oxygen therapy should be carefully managed in septic patients to minimize its deleterious effects.
ID: 128
Life and Health Sciences Research Institute (ICVS), University Of Minho
Behavioural analysis can be influenced by several factors, including housing. The PhenoWorld (PhW) is a recently developed housing and testing paradigm, which proved to be relevant for screening depressive-like behaviours in rats, being remarkably sensitive for hedonic behaviour (Castelhano-Carlos et al. 2014). Home-cage behaviour analysis showed that PhW animals have a clearer circadian pattern of sleep and social interactions. By promoting a good basic health and functioning, and the performance of natural behaviours with controllability but still keeping some physical and social challenges, the PhW stimulates positive affective states and higher motivation in rats, which might contribute to an increased welfare of animals living in the PhW.
We also assessed neuronal plasticity as a consequence of living in the PhW in a group of 6 animals, by comparing the structure of the prefrontal cortex (PFC) and the nucleus accumbens (NAc), two brain areas involved in the circuitry regulating motivation and reward, of those animals with those of animals housed in standard conditions in social groups of 6 (STD6) and 2 rats (STD2). The results reveal that living in a more naturalistic complex environment, closer to real life experience, impacts on the structure of brain regions implicated in complex multidimensional disorders.
Posters
Ethic and Legal framework for the use of laboratory animals
ID: 23
1Servicios Informáticos, Universidad de Salamanca
2Servicio de Experimentación Animal, Universidad de Salamanca
An online management tool for the Institutional Animal Care and Use Committee. Implementation and experience in the USAL.
The administrative management of the Institutional Animal Care and Use Committees in research centres or universities is a difficult and often problematic issue. At present, there are online tools through web applications that facilitate these tasks and help reduce the workload of users and evaluators.
ManitsBT free software was used for programming online document management. The web design process was conducted by the University of Salamanca Computer Services, CPD after determining, in meetings with the committee, the critical processes of access to the web, downloading and input of information and the increase of forms / documents. A user guide for users, members and the secretary of the committee was also introduced.
After finishing the process and evaluating the real utility of this tool, we can say that online document management applications are a valuable resource to improve efficiency in the ethical evaluation of projects with special emphasis on:
– Improves user experience
– Reduces the burden of management of the committee
– Streamlines communication between user-committee
– Reduces mean time to resolution
– Facilitates the intervention of the members of the committee
ID: 29
1Instituto de Neurociencias de Alicante CSIC-UMH
2Surrey Diagnostics
3University College London
Rodents represent 85% of the animals used for experimental and other scientific purposes. This is due among other things to the efficiency of the genetic manipulation techniques to obtain animal models of disease. Creating these models requires investing time, money, and of course animals, and for that reason and according to the principle of the 3 R´s there exists a great scientific exchange between scientists, institutions and repositories all over the world.
To import live rodents and rodent embryos/sperm into Spain form non-EU countries it is necessary that an animal health certificate must be completed by an official veterinarian in the country of origin. In the second section of this certificate the veterinarian must state that “In the above mentioned body, institute or centre, no cases of contagious diseases characteristic of the species have been reported for the last 30 days prior to the export/extraction”.
According to data from our collaborator Adrian Deeny, and understanding that “contagious diseases” includes all those that appear in FELASA´s recommendations for health monitoring of rodent colonies we are aware that we cannot legally import rodents from over 90% of the facilities. The four years of data from Adrian´s health monitoring laboratory shows that from the 280 facilities monitored, for example 87% of them having at least 1 positive for Pasteurella pneumotropica and 63% for Norovirus.
With this data is easy to see that is very complicated to comply with this requirement, therefore we believe that a regulatory change adequately regulating this aspect is necessary.
ID: 35
Servicio de Animalario. UEX
To develop training programs with a visual emphasis, collecting different ways of teaching, that lead to the acquiring of knowledge, skills and attitudes, required under the Orden ECC / 566/2015 March 20th, which establishes necessary training requirements for personnel handling animals that are used, kept or supplied for experiments and other scientific purposes, including teaching, for the development of the functions established by Real Decreto 53/2013, February 1st, laying down basic standards for the protection of animals which are used for experimental and other scientific purposes, including teaching and thus, as well, being able to develop professional work in the field of animal experimentation.
This is a poster setting out the existing ways to carry out various tasks in the field of animal experimentation by those people who come from more or less related sectors in this field, depending on their educational level or previous training, indicating possible starting points and ways of training both regulated and unregulated, showing in any case when you need to perform a procedure for the validation or recognition by the competent body.
The result is a comprehensive framework that includes, in a clear and simple way, possible training processes.
ID: 40
CPYEA, Universidad de Sevilla
The CPYEA of the University of Seville operates as a central facility not only for breeding and maintenance of transgenic and wild type rodents but it also harbors some other research services that are offered to research groups usually from our own university but from other institutions as well. Among them are the transgenic service, the rabbit antibody production and behavioral analysis of mouse lines that may be bred in our facility or that are shipped just for the completion of some tests.
The new regulations in laboratory animal use, establish that the severity of the procedures carried out on animals should be evaluated considering the experience that the animal may have endured during its entire life.
It is sometimes difficult to assign very different procedures to a severity category and this issue becomes more complex in the case of central services where the animals may move between registered centers and different procedures and protocols are conducted in each one. In addition in these cases it is not clear who should be in charge of reporting the animals used in experiments.
In order to provide a better service to our clients we elaborated a report that was forwarded to the Competent Authority and a consensus was reached that we present here. Our Facility asked for authorization of three generic projects comprising the services that we provide; in the authorization documentation we specified how severity classification will be evaluated in conflict cases and who will be responsible for reporting animals used.
Welfare of laboratory animals
ID: 17
1CEDOC, Nova Medical School, Universidade Nova De Lisboa, Lisboa, Portugal
2Nova Medical School, Universidade Nova De Lisboa, Lisboa, Portugal
Orogastric gavage is widely performed in pharmacological studies for daily dosing of rodents. Although highly effective, this technique comprises a sequence of procedures potentially stressful for laboratory animals, which may act as a bias for the experimental results. When testing antihypertensive drugs it becomes crucial to ensure the selection of a non-invasive and stress-free method for drug delivery, since any source of external stress can significantly increase heart rate and blood pressure. We aimed to investigate suitable vehicles for long-term administration of losartan. Male Wistar rats (2–3 months) were used to test nut past (NUT), peanut butter (PB) and sugar dough (SD) as vehicles. The vehicles were administered 28 days without drug to assess the glucose level and serum lipid profile. Secondly, losartan was administered mixed with vehicles and by gavage (14 days) for further quantification of losartan plasma levels by HPLC. After a 2-day acclimation period, all animals voluntarily ate the vehicles, either alone or mixed with losartan. Blood glucose, triglyceride, total cholesterol, HDL-c and LDL-c levels did not differ significantly between groups. SD animals presented a higher concentration of losartan when compared with gavage group (p < 0.01). In conclusion, our results showed that NUT, PB and SD represent viable vehicles for oral voluntary ingestion of losartan, without changing lipid and glucose profiles. The drug bioavailability was not reduced suggesting that this approach is a reliable oral dosing method for chronic administration of losartan. The results presented herein point to a welfare-based refinement for drug administration in laboratory rodents.
ID: 21
1Charles River Laboratories
2CIC Biomagune
ID: 31
1Máster Etología. Universidad Pablo de Olavide, Sevilla
2Dpto. de Psicobiologia. Centro de Investigación Mente, Cerebro y Comportamiento (CIMCYC). Universidad de Granada
3CIC/CIBM. Universidad de Granada
The individualization associated to the caloric restriction (CR) pose many logistic and well-being problems. It proposes, thus, the necessity to study the effects that CR has in conditions where the animals are in groups. The revision of the studies of CR in short and middle terms have been focalized mainly in physiological parameters, informing of improvements in aging, the modulation of oxidative stress and, in long term, in the negative effects of the cognitive functions.
The aims of this study are:
The preliminary results manifest that exist differences both in weight and proportion comparing adlibitum and CR conditions; and between males and females. The obtained results referring to the behavioural and hormonal analysis will be expose in the different groups.
ID: 33
1Animal Research Facilities, Almirall S.A., Barcelona, Spain
2Charles River Laboratories, Barcelona, España
Playtime was established on a daily basis in a common area equipped with toys, in groups of 6 individuals. Caretakers were always present and interacting with the animals. For evaluating the socialization progress, we used three variables: kennel greeting (KG), on leash behaviour (OL) and behaviour on examination table (ET), and measured it by using a score ranging from 1 to 5 (higher scores meaning more sociable animals), during 15 consecutive days. All the positive behaviours were reinforced by using encouraging words, caresses and massage, and food treats.
ID: 48
1Charles River Laboratories, Barcelona, España
2Animal Research Facilities, Almirall S.A., Barcelona, Spain
ID: 49
1Charles River Laboratories, Barcelona, España
2Animal Research Facilities, Almirall S.A., Barcelona, Spain
The housing of heavy rodents such as guinea pigs could lead to handling problems due to the cages’ final weight. When using heavy bedding such as corncob, we need to find the balance between absorbency, weight and cost per cage.
Fifty Dunkin Hartley male guinea pigs (Charles River Laboratories), weighting 300–350 g were housed in groups of 3 in isolated ventilated cages (Tecniplast 1500U, floor surface 1500 cm2), with corncob bedding (Rehofix ¼”, JRS). We tested procedures for bedding and cage change in five consecutive weeks:
1st week: cages had approximately 450 g bedding. The cage change was done 5 days later, but the level of cleanliness was unacceptable. The approximate cost was 1.60 €/cage/week.
2nd week: 450 g bedding, 3 cage changes. Cleanliness markedly improved but the weight of the cage was still a drawback. Cost: 2.4 €/cage/week.
3rd week: bedding was reduced to 400 g. A ream of filter paper (52 x 52 cm) was placed folded on the bottom of the cage. Two changes were done. Although the final weight decreased, the cleanliness was unacceptable, with some mold growth, and the paper partially destroyed. Cost: 1.38 €/cage/week.
4th week: same conditions, but 3 cage changes. The paper was also destroyed. Cost: 2.07 €/cage/week.
5th week: with smaller reams (32 x 32 cm) and three changes, the papers were preserved. Cost: 1.86 €/cage/week.
In conclusion, the best housing option was the combination of fitted ream of filter paper, 400 g of corncob and 3 changes/week. We managed to improve cleanliness and decrease the weight to be handled by the staff, with an acceptable cost.
ID: 56
1UTAD - Veterinary Sciences Department from the University of Trás-os-Montes and Alto Douro (UTAD), Quinta de Prados, 5001-801 - Vila Real, Portugal
2CITAB - Centre for the Research and Technology of Agro-Environmental and Biological Sciences/UTAD - Veterinary Sciences Department from the University of Trás-os-Montes and Alto Douro, Quinta de Prados, 5001-801 - Vila Real, Portugal
Animal models play a central role in urinary bladder cancer study, since they are used to better understand tumor behavior, chemopreventive agents, and efficacy of new drugs. Nevertheless, investigators should always aim to minimize animals’ suffering by using timely and appropriate tools for the recognition and alleviation of pain and distress. In this study, animals’ behavior and physiological parameters were monitored during an assay of urinary bladder carcinogenesis.
Twelve Wistar rats were exposed to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) (0.05%) in drinking water, over the course of 20 weeks. A negative control group was used. During the experiment, a scoring sheet was used to monitor animals’ welfare. Mental status, eyes, ears and whiskers position, response to handling, breathing and hydration were scored. Urine and blood samples were collected monthly for analysis.
All animals displayed a mental status, eyes, ears and whiskers position, response to handling, breathing and hydration within the parameters considered normal for the specie. Concerning to loss of body weight, and food and water intake, no statistical differences were found between groups. During the first 14 weeks of the experiment, all animals exhibited a clear yellow urine color. However, at the 35th week, all animals from BBN group exhibited hematuria. At this time, the mean hematocrit values were lower in animals from BBN group than in control group (p < 0.05) and four animals were anemic.
In conclusion routine control of hematuria is an important refinement technique and a potential earlier endpoint in studies of urinary bladder carcinogenesis using a rat model.
ID: 71
Laboratory animal sciences, diseases of the developing world drug discovery center. Glaxosmithkline R&D. Tres Cantos, Madrid, España
The objective of this work was to evaluate the preference of mice for different particle-size corncob bedding. We have been using large-particle bedding and wanted to change to smaller-particle, as it produces less noise and seems smoother. To confirm that the change would not affect the animals, we did a preference test with two different sizes of corncob: large 4–6 mm, and small 1.7–2.2 mm. Female BALB/c mice were housed in two cages joined with a tunnel, each containing a different size corncob. Nest position and mice activity and position were recorded. After two days, the nest was moved to the opposite cage to observe whether the mice moved it back to the previous location. Mice were then observed for another 2.5 days. On average, mice spent more time in the cage with the smaller-particle bedding (66.2% of the time). At the start of the week, 2/3 groups had built their nest in the smaller-particle cage. After moving the nest, only one group moved it back to the smaller-particle cage. Mice showed greater activity in the cage opposite to the nest. In conclusion, BALB/c mice showed a preference for smaller-particle corncob, although this preference was not strong as some mice stayed in the larger-particle cage. The observation that mice had a tendency to be active and eat in the cage opposite to the nest may reflect their tendency to structure their territory according to activity. Further tests are needed, both with males and other strains, to confirm the findings of this study.
ID: 72
Laboratory animal sciences, diseases of the developing world drug discovery center. Glaxosmithkline R&D. Tres Cantos, Madrid, España
The objective of this study was to assess three different foraging enrichments for mice. Female BALB/c and C57BL/6, and male CD1 mice were split in groups that received either sunflower seeds, FruityGems or Supreme Minitreats, one gram per mouse twice a week. Body weight, food and water consumption and behavior after receiving the treats were recorded for four weeks. Then animals were euthanized and blood was obtained for the determination of corticosterone and biochemical and haematological parameters. A reduction in food and water consumption was observed in most groups receiving treats, whereas body weight changes were independent of the treat received. Mice ate all treats the day they were offered and no escalated aggression was observed in the following hour from receiving the treats. However, all CD1 groups receiving treats had to be separated due to fighting wounds. BALB/c mice incorporated sunflower seeds’ shells into their nest. There were no significant differences in corticosterone levels, biochemical or haematological parameters. In conclusion, although offering treats may reduce food and water intake, they do not affect body weight or blood analytical parameters and therefore can be used with no interference in the experimental work carried out in our centre. Of the three treats tested, sunflower seeds are recommended because they need more manipulation, can be incorporated into the nest and are virtually inexpensive. An additional study should be carried out to assess whether the aggression in CD1 male mice was influenced by the treats.
ID: 118
Biotério Central Instituto Butantan, São Paulo. Faculdade de Medicina Veterinária e Zootecnia - UNESP
Parental behavior of inbred and heterogenic mice submitted to environmental change with the supply of material for nesting.
The objective of this study was to evaluate the effect of an environmental modification on the parental behavior of BALB/c and Swiss mice in an intensive monogamous system by behavioral observation and the use of anxiety test on the elevated plus maze. Sixty full-sib pairs from the BALB/c strain and 60 Swiss pairs were used. A disposable cap cut in eight pieces of approximately 1 cm each, plus a cotton piece of approximately 3 g, were used together as environmental modification. The parental pairs were observed for offspring caring in the third reproductive cycle. Observations were carried out 40 to 50 minutes after the beginning of cage changing during the total lactation period. No effect of the strain x environmental modification interaction was found on maternal and paternal behaviors. Strains affected maternal (P = 0.0404) and paternal (P = 0.0355) nest building and nursing in the arched-back position (P = 0.0212), whereas the nesting material alone did not influence any of the maternal behaviors. The presence of nesting material increased the frequency of paternal behavior of resting with the litter (P = 0.0110) and decreased the frequency of resting alone (P = 0.0067). The results suggest that the environmental modification affect parental behavior and that the genetic differences should be taken into account in the definition of housing conditions for these animals.
ID: 156
Animal Welfare: Science, Humanities, Ethics and Law Research Group (AHSWEL-IAS). Universidad de Alcalá, Madrid, Spain
In order to improve animal welfare, a study was carried out at the same conditions on bedding for rodents. Several wooden materials and a new one based on cellulose, coming from different commercial companies were used.
One group of one hundred of Individual Ventilated Cages for mice was used for each material. Ten of them were used as controls with the highest occupancy permitted by law (Directive 2010/63/EU) and the rest with the real number of animals housed during experimental procedures. All cages were daily observed at least during two weeks and bedding of controls was also checked periodically by physical, chemical and bacteriological methods (e.g. ammonia levels, images, bacteriological cultures).
As the wooden materials did not reach the required results, a new organic–cellulose- was incorporated to the study. It was found that in this case the bedding complied with the following:
Enlarging the bedding viability: A minimum of fourteen days was reached with no cage changes. Decrease of stress because of the reduction of number of changes. It avoids that the animal should accustom again to “a new environment”. Bacteriostatic ability that limits the growth of microorganisms. High capacity of absorption / desorption. Faeces are displaced to the bottom of the cage beneath bedding. Low levels of ammonia. 50% of cost reduction in cage’s maintenance. Improving environmental sustainability. More time availability for the technicians and caretakers in order to carry out the legally required daily follow-up of animal welfare.
Euthanasia
ID: 77
Park de Recerca Biomèdica de Barcelona (PRBB)
Carbon dioxide euthanasia method is one of the most common system used in rodent animal facilities. Despite is in the list of humanitarian methods, depending on the management of the animals previous and during the process it could be considered inappropriate to warranty a peaceful death. Furthermore the potential personnel exposure to CO2 gas during euthanasia is also considered a health and safety issue to manage in animal facilities.
The aim of this presentation is to describe a new CO2 chamber that has been designed on purpose. The goal of the new design is to avoid the Co2 personnel exposure. Specific instructions for the use of this new carbon dioxide chamber have been written based on the results of a room environment study and the description of the critical points for personnel exposure to the gas. Furthermore, these instructions include new refinements on how the rodents have to be manipulated to avoid unnecessary stress, that includes keeping the mice in their usual cage, without mixing with others as a welfare refinement. It is carefully described how to fill and infill the chamber in order to perform a peaceful process, as well as how to verify the death of the animals in order to follow European regulations.
It is a simple system that permits that any of the animal facility staff or researcher can use it in a safety way for themselves without forgetting the welfare of the animals.
ID: 109
1CITAB / UTAD Vila Real
2IBMC, Porto
3Laborial, Maia, Portugal
4IDMEC –FEUP, Porto
Euthanasia is the ultimate procedure to which all laboratory animals are subjected, it is thus an important economic and welfare related issue. Despite CO2 rapid onset of action, low cost and practicability, it has been showed to be an aversive agent which cause pain and stress. Thereby its use in laboratory rodents euthanasia is not pointed as a humane death.
The aim of this presentation is to update the current solutions regarding euthanasia with emphasis to methods using gases and based in previous works (Antunes, 2010). The first part of this presentation will address new results from gases avoidance-aversion tests- and the determination of the optimal flow rate for euthanasia. In the second part we intend to address the development of new technologies, namely an automated gas delivery system that detects the beginning of unconsciousness. This is accomplished by using a chamber in which a piezoelectric sensor (Pereira, 2014) detects the loss of righting reflex, as the unconsciousness measure, and the cease of respiratory movements, as a way to determine death. Loss of unconsciousness may be achieved by a less aversive agent, as sevoflurane, followed by a more economic and rapid agent such as CO2 to achieve a more humane death.
References
Health of laboratory animals
ID: 19
Cinthia Besh-Williford. IDEXX Bioresearch
Micro-isolation cage systems (mainly IVCs and FTCs) are widely used nowadays, with the aim of protecting animals and operators. Health monitoring of these units has always been problematic because each cage is in reality an independent microbiological unit.
Traditionally these units are monitored with the use of a variable number of immunocompetent sentinels that have been exposed to dirty bedding, tested via serology, bacteriology and parasitology.
This procedure relies upon the transmission (not always efficient) of agents in addition to other uncontrollable variables such as prevalence of disease; dose of agents that are shed by resident animals; frequency and amount of bedding transferred in addition to the susceptibility and receptivity of the sentinels.
The hypothesis is that modern technologies allow sampling points other than sentinels to make the screening more sensitive.
We analysed and compared results obtained in different sampling points (sentinels, principal animals, environment) and different methodologies (mainly serology, PCR and parasitology) for agents believed to transmit inefficiently to sentinels via dirty bedding.
Apparently a better health monitoring is obtainable by combining tests conducted on different sampling point depending on the agent of interest.
ID: 42
Unidad de Investigación, Complejo Hospitalario Universitario de Albacete
All transferred animals gave negative results to PCR analyses for detection of Helicobacter spp.
In mice, PCR results were not affected by the stool-sampling procedure, however, rat samples from cages gave false negative results.
ID: 66
1Laboratorio de Neuroanatomía Funcional Comparada, UVEG
2Unitat Predepartamental de Medicina, UJI
Our research group has been working for the last 15 years in the neural circuits underlying social and sexual behavior, in particular focusing in the stimuli inducing sexual attraction. That attractionis mediated by male sexual pheromones, includingurinary proteins. Weuse a two-choice preference test in which female mice can choose between male urineand a control stimulus. The innate female attraction towards male urine is a solid behavioral paradigm. However, in several instances females showed no attraction or even aversionto male urine. In some of theseoccasions we observed that the male mice used as urine donors were subclinically infected by the intestinal pinworm Aspiculuristetraptera. After that, we have demonstrated experimentally that urine of parasitizedmaleswas not attractive for females.
Our investigation indicates that infected and healthy males have a similar profile of urinary proteins. Thus, infection is not challenging their capacity to produce pheromones but promoting instead the production of “disease signals” excreted in urine. Conspecific females are able to detect these signals, which would be aversive.
We are currently investigating what kind of molecules are the signals of infection present in male urine, and the role of the olfactory and/or vomeronasal systems in detecting this information.
Our results raise the question of how the results ofother behavioral tests may be affectedby the presence of these or other infection signals, given that those infections may beinadvertently present or, even being aware of their presence, they are very difficult to eradicate.
Funded by the Spanish Ministry of Science-FEDER (BFU2013-47688-P), Junta de Comunidades de Castilla-La Mancha/FEDER (PEIC11-0045-4490) and the UniversitatJaume I.
ID: 78
1Instituto Universitario de Investigación Biomédica y Sanitaria de la Universidad de Las Palmas de Gran Canaria (IUIBS-ULPGC)
2Sección de Endocrinología, Complejo Hospitalario Universitario Insular Materno Infantil de Gran Canaria (CHUIMI)
3Instituto de Investigaciones Biomédicas Alberto Sols de Madrid, Consejo Superior de Investigaciones Cientificas (CSIC)
4Unidad Asociada de Biomedicina ULPGC-IUIBS
5Infectious Diseases and Tropical Medicine Unit, CHUIMI
6Animal Facility Unit, RMG-SEA Instituto de Neurociencias de Alicante, Universidad Miguel Hernández, CSIC
7Experimental Animal Service, Research Unit, Universitary Hospital of Gran Canaria, Dr. Negrín Multidisciplinary
8Infectious Diseases and Fish Pathology. Universitary Institute of Animal Health and Food Safety, ULPGC.
9Toxicology Unit, Department of Clinical Sciences, ULPGC
10Facultad de Veterinaria, ULPGC
A zoonotic opportunistic outbreak involving Ornythonissus bacoti is described.
Capable to occasionally invade animal facilities independently of their barriers, a cost-effective eradication protocol with ivermectin is tested and described.
Immunocompetent mice and rat strains in a conventional health status facility developed dermatologic manifestations (i.e. scratching and alopecia), followed by weight-loss and wounds in severe cases. Some litters showed lesions resembling petequiae distributed over the whole body. Initially, only mild isolated cases were detected, but later manifestations were spread. Simultaneously, personnel suffered of pruritic, erythematous papular lesions of different severity. Direct observation of the cages showed oval and fast insects that moved around and from animals to bedding. Further examination of carcass and bedding allowed the characterization of these accarii as Ornythonisus bacoti.
0.1% ivermectin solution in propyleneglycol and distilled water, spray administered, was chosen as treatment. Treatment efficacy was tested in some parasites with a 0% survival rate. Ivermectin safety for each strain was also assessed without obtaining any clinical manisfestation. Hence, all animals and beddings were sprayed. Based on the parasite life-cycle, treatment was repeated three times, weekly. Frequency of cleaning for cages was increased. Racks, walls and cages were also sprayed with ivermectin to prevent parasite movements. Infestation was monitored by regular examinations of fur and carcass at different time-points. Seven weeks after initial application, samples remained negative for the presence of accari. The parasitosis eradication was confirmed and at the time of this report, eight months after diagnosis, O. bacoti still remains undetected.
ID: 89
PCB-PRBB. Animal Facility Alliance
Mouse is the animal model most frequently used in biomedical research around the world, but it is well know that many research carried out with this model are not extra polated to humans, mainly because that is not take into account the main differences of physiological and behavioral matters, when designing experiments. The goal of this communication is to show, to through a pilot assay, how experiment designs should be addressed with these animal models to improve the extrapolation to human. Focusing on three main components: circadian rhythm, feeding time, and intestinal microbiota. We show the results of this preliminary study in which a group of animals have been eating ad libitum, (control), and another group has eaten three times at interval of four hours during the dark cycle only. We have recorded video of the mice with infrared web camera to evaluated the welfare and behavior, we have analyzed grow grafics and we perform hematology and biochemistry analysis, as well as a study of the intestinal microbiota by metagenomic sequencing.
ID: 94
1Unitat De Patologia Murina I Comparada - Departament De Medicina I Cirurgia Animal, Universitat Autònoma De Barcelona
2Servei D’Estabulari, Universitat Autònoma De Barcelona
One of the goals when working with transgenic mouse models of human diseases is to characterize the lesions developed by these mice during their lifespan. Here we show the histopathologycal findings observed during the long term supervision of the aging process in three SPF transgenic mice colonies on a C57Bl/6 genetic background housed in the SER-CBATEG.
Mice under daily welfare supervision were euthanatized when they met one of the established experimental humane endpoints. A complete necropsy was performed. Samples of main organ systems and any grossly abnormal tissues were fixed in formalin and paraffin-embedded. Tissue sections were stained with hematoxylin and eosin and examined by a veterinary pathologist. Mouse genotype was unknown by the pathologist.
A total of 131 mice, 77 females and 54 males, were examined. Mean age was of 83 weeks with a range between 11 to 137 weeks. Most relevant lesions were: amyloidosis detected in 58% of the mice, ulcerative dermatitis (37%), glomerulonephritis (27%), lymphoma (21%), histiocytic sarcomas (17%), nephritis (13%), panniculitis (13%), benign limphoproliferative processes (11%), pneumonia (10%), adenomas (8%), rectal prolapse (5%) and encefalopathy (5%). Cystic endometrial hyperplasia was detected in 24 of 77 analyzed females (31%).
We found differences in the percentage of some lesions between sexes. Females had a higher proportion of lymphomas (31 vs. 7%), glomerulonephritis (31 vs. 22%), benign limphoproliferative processes (15 vs. 8%) and rectal prolapse (8 vs. 0%) whereas males had a higher incidence of sarcomas (22 vs. 13%) and adenomas (13 vs. 4%).
Relevance of these findings will be discussed.
ID: 102
Servei Integral De L’animal De Laboratori, Universitat Autònoma De Barcelona, Spain
2-year old intact female Ellegaard Gottingen Minipig enrolled in an experimental study presented a skin affectation. The colony health records from origin were free from relevant infectious and contagious diseases. At arrival, the pigs were group-housed in a compact floor with wood bedding in a climate-controlled facility, with housing and handling according to recommendations. Moreover, the animals were properly vaccinated and dewormed. The primary lesion was composed of few small reddened ring-like lesions at the animal dorsum. In a few days, the lesions dried and formed a layer of hyperkeratosis that covered all the dorsum, laterals and tail. Although the animal lost some weight, no alterations in its behaviour were noticed. The differential diagnosis included pyoderm, dermatophytosis, psoriasiform dermatitis, thymoma, nutritional deficit and mange. Parasite testing, fungal and bacterial cultures of the hyperkeratotic plaques resulted negatives. Zinc levels in blood were within normal range and the content of vitamin A and fatty acids in the diet accomplished the species recommendations. Serum chemistry did not show alterations. Biopsy samples from the affected areas revealed orthoqueratotic hyperkeratosis, irregular hyperplasia of the epidermis and inflammatory infiltrate with vascular activation and hyperplasia within the dermis. A topic corticosteroid treatment was applied (Centella asiatica and triamcinolone acetonide, every 12 hours, 7 days) in two areas on the back that healed completely. Due to the incompatibility of a systemic corticosteroid treatment with the experimental study, the animal was euthanized. No abnormalities or neoplasia were observed at necropsy. The animal was diagnosed of psoriasis.
ID: 126
1Universidade Federal Fluminense (UFF)
2Fundação Oswaldo Cruz (FIOCR)
Mycoplasmas are infectious agents in animals and plants, can interfere in the results of studies, and most of the time are difficult to detect. Mycoplasma pulmonis and Mycoplasma arthritidis are the most important agents found in rodents from breeding facilities in the world. Clinical and subclinical forms of the disease compromise the health and sanitary quality of animals as well as research results. Therefore, early detection of these agents is essential. Serological testing is a valuable method for screening colonies on the presence of anti-mycoplasmal antibodies, for not requiring animal euthanasia. The most common of them are ELISA and Immunoblotting. However, conventional tests are made with one specific antigen at a time. The goal of the present study was to evaluate the reactivity of a mixed antigen ELISA in comparison with another serological technique. Sera from 78 rats, obtained from various institutions in Rio de Janeiro State, were submitted to immunoblotting for M. pulmonis and an ELISA with mixed M. pulmonis and M. arthritidis antigens. 25.64% (20/78) had positive reaction for immunoblotting, with strong immunobinding to the 24, 48 e 200 KDa protein bands; 74.36 % (58/78) had negative reactivity. For the ELISA, 32.05% (25/78) were positive, while 67.95% (53/78) were negative. Both tests only agreed in 12 samples. This can be explained by the fact the ELISA used had mixed antigens, therefore samples with anti-M. arthritidis antibodies may have been detected as well. This helps validate the mixed antigen ELISA as a method for detection of multiple anti-Mycoplasma antibodies.
ID: 129
1Experimental Animal Centre, University of Extremadura, Badajoz, Spain
2Chrononutrition Laboratory, Department of Physiology, Faculty of Science, University of Extremadura, Badajoz, Spain
Most of scientific research carried out with laboratory animals are performed under static conditions like temperature or humidity among others. On the other hand, light/dark cycles 12:12 are common to simulate natural conditions kept in Animal Experimentation Centres. Our aim was to evaluate if an environmental temperature oscillation may have any effect on circadian rhythms in rats. This research was developed in 14 female wistar rats during 2 control weeks with a control temperature (24°C) both during day and night and 2 weeks with an oscillating environmental temperature with 31°C during the day and 21°C during the night; this research was carried out by duplicated. Both in the control period and in the oscillating temperature period activity/inactivity circadian rhythm was logged through actimetry. Blood samples and temperature data were collected every 4 hour in order to study glucose and temperature circadian rhythms. Our results showed an acrophase delay (p < 0.001) in activity/inactivity rhythm and an improvement in its consistency (Interdaily stability: p < 0.05; intradaily variability: p < 0.05). No changes were observed in neither glucose nor temperature circadian rhythms, but also an increase both in water and food consumptions was observed in the week with oscillating temperature. We conclude that an oscillating environmental temperature improves sleep/wake circadian rhythm in rats. Acknowledgments: Gobierno de Extremadura – Fondos FEDER; GR10003)
ID: 134
1Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto (ICBAS-UP), Portugal
2CEDIVET, Portugal
3Biotério ICBAS-UP, Portugal
Proteinaceous plugs in the bladder (bladder plugs) of rodents may occur as a common postmortem finding and usually represent an agonal secretion from the accessory sex glands occurring during euthanasia (Frazier et al., 2012). Some authors reported an incidence of 14.1 to 17.8% in male rats with ages ranging from 10 weeks to 2 years, and considered that urinary bladder or male sex accessory glands plugs developed from back-flow of semen following ejaculatory disturbance (Lee, 1986). Bladder plugs can be very large and partially fill the organ lumen, but they are generally considered as “incidental findings” of no clinical importance.
Over the past two years, in an adult male wistar rat stock colony, investigators detected three animals with large urinary bladder plugs. Generally, gross evaluation demonstrated a generalized prostatic hyperplasia and urinary bladder distention that after incision shown a proteinaceous plug recognized as an elliptic formation, white to tan color, totally occluding the lumen of the organ. Histological examination confirmed a diffuse prostatic hyperplasia with abundant accumulation of acidophilic secretion (causing dilatation) and a multifocal, light to moderate chronic cystitis. The plugs were composed by a mixture of an amorphous, flocculent eosinophilic proteinaceous material, exfoliated urothelial cells, cellular debris and spermatozoa. No mineral matrix was found after chemical analyses, excluding the possibility of vesical calculi.
Considering these preliminary findings, we believe that proteinaceous urinary bladder plugs may be an underestimated cause of urinary and/or sexual rat male malfunction that can lead to experimental bias.
Animal models
ID: 22
1Centro de Instrumentación Científica
2Centro de Investigaciones Biomédicas
3Departamento de Patología, Universidad de Granada
Radiotherapy treatment induces cell death by DNA damage and cell signalling processes (bystander and ascopal effects). Also human mesenchymal stem cells (hMSCs) are able to incorporate onto tumors and secrete anti-tumor cytokines thus decreasing its proliferative activity and producing a cytotoxic synergistic effect on them.
NOD/SCID Gamma mice were engrafted with cells of the human malignant melanoma A375 cell line. Tumor cells were implanted in both flanks to produce bilateral xenogeneic tumors. Four groups of eight mice were each treated with 2 Gy radiotherapy (RT), intraperitoneal 106 hMSCs therapy, radiotherapy plus 106 hMSC therapy, or left untreated (control). Only one of the tumors was irradiated. Mice were monitored regularly every 2–3 days, to evaluate the animal welfare and calculate tumor growth. Magnetic Resonance images (MRI) were acquired in order to monitoring the development of metastasis. Histopathological studies were carried out at the end of the experiment.
Animals showed better general behavior and appearance in hMSC and RT combined therapy and lower tumor growth on both irradiated and non-irradiated flanks when compared with RT (P = 0.0079 and P = 0.0273, respectively) and control (P < 0.0001 and P = 0.0022, respectively) groups. MRI showed less metastasis presence in both RT treatment group and hMCS + RT group. Tumor growth between hMSC treated and control groups were similar (P = 0.9829). In conclusion, we demonstrate that in vivo administration of MSCs combined with radiation leads to an increased efficacy of radiotherapy on primary-irradiated tumors and distant-non-irradiated tumor.
ID: 27
1Gabinete Veterinario UAM
2GV UAM
3IIS Gregorio Marañón
4Dpto Anestesia H. Gregorio Marañón
ID: 36
1UCLA
2CITAB
3LEPABE
4CI-IPOP
5QOPNA
6UTAD
The model of mammary cancer in rats induced by 1-Methyl-1-Nitrosourea (MNU) has been established as a good model of human cancer because its use permits the in vivo research of each event in an individual patient. The purpose of this work was to evaluate the role of physical exercise in the development of lung metastasis in a rat model of mammary cancer. Were used 40 outbred female Sprague-Dawley rats divided in four groups: Sedentary MNU (n = 11), Exercised MNU (n = 10), Sedentary control (n = 9) and Exercised control (n = 10); at 50th day of age (first day of the experiment) the animals from MNU groups were inoculated with a single dose of MNU (50 mg/kg body weight) by intraperitoneal injection. The exercise protocol consisted of 60 min/day in a Treadmill at a speed of 20 m/min, 5 days a week for 34 weeks. The animals were sacrificed at 35th week of experimentation. All animals from MNU groups developed malignant neoplasm (100% of incidence). The differences between mean lung weights of groups were not statistically significant (P > 0.05). In Sedentary MNU group were observed three metastasis of mammary carcinoma (27.27%) with the immunohistochemical expression of estrogen receptor alpha (ER), while in Exercised MNU group was not observed metastasis. Looking to these results, it seems that the physical exercise may have an important role in reducing the mammary cancer metastization in the lung. It is possible to conclude that exercised model of MNU-induced mammary cancer represents an adequate in vivo model to study all phases of the cancer disease.
ID: 38
1CITAB - Centre for the Research and Technology of Agro-Environmental and Biological Sciences
2UTAD - Veterinary Sciences Department from the University of Trás-os-Montes and Alto Douro, Quinta de Prados, 5001-801 - Vila Real, Portugal
3CECAV - Animal and Veterinary Research Centre
4QOPNA/UA - Unidade de Investigação de Química Orgânica, Produtos Naturais e Agroalimentares, Departamento de Química, Universidade de Aveiro, 3810-193 - Aveiro, Portugal
Inflammation has long been associated with several cancer types, namely breast cancer. Once it is known that physical exercise helps many body systems to function better, we intended to evaluate its effects on the cancer-associated inflammation in an animal model of cancer chemically-induced mammary.
Animal procedures were made according to the European and National legislation (Directive 2010/63/EU and Decree-Law 113/2013). Forty female Sprague-Dawley rats were divided into four groups: MNU sedentary (n = 11), MNU exercised (n = 10), Control sedentary (n = 9) and Control exercised (n = 10). All MNU animals received an intraperitoneal injection of the carcinogen agent N-methyl-N-nitrosourea (MNU) (50 mg/Kg) at seven weeks of age. Animals from exercised groups were submitted to an exercise protocol in a Treadmill 60 min/day, 5 times/week, at a constant speed of 20 m/min, for 35 weeks. At the sacrifice, blood samples were collected and the serum levels of inflammatory markers C-reactive protein (CRP) and interleukin-6 (IL-6) were determined by slot-blot assay.
The IL-6 and CRP serum concentrations were higher in groups MNU than in control ones. Additionally, their serum concentrations were lower in exercised animals than in sedentary animals in both MNU and control groups. A statistically significant difference was only observed in CRP levels between MNU sedentary and control sedentary groups (p < 0.05).
The MNU-induced mammary cancer increased the inflammation. The practice of long-lifelong exercise training reduced the inflammation in animals with chemically-induced mammary cancer and in healthy animals.
ID: 39
1CITAB - Centre for the Research and Technology of Agro-Environmental and Biological Sciences
2CECAV - Animal and Veterinary Research Centre
3UTAD - Veterinary Sciences Department from the University of Trás-os-Montes and Alto Douro, Quinta de Prados, Vila Real, Portugal
4QOPNA/UA - Unidade de Investigação de Química Orgânica, Produtos Naturais e Agroalimentares, Departamento de Química, Universidade de Aveiro, Aveiro, Portugal
In researching protocols using animals, it is important to minimize their distress and suffering. In this work we aimed to establish adequate humane endpoints in an animal model of mammary carcinogenesis.
Procedures were made in accordance with the Directive 2010/63/EU and Decree-Law 113/2013. Ten female Sprague-Dawley rats were used in this protocol for 18 weeks. At fifty days of age, all animals received an intraperitoneal injection of the carcinogen agent N-methyl-N-nitrosourea. Previously to the beginning of the experiment, were established humane endpoints that included the following parameters: body condition; body weight; posture; coat and grooming; mucosal; eyes, ears and whiskers; mental status; response to external stimuli; hydration status; respiratory and heart rate; body temperature; location, macroscopic evaluation and mammary tumors’ burden (tumors had a dimension higher than 35 mm).
At the twelfth week of the experimental protocol, one animal developed a mammary tumor with a dimension higher than 35 mm. After this, more animals exhibited mammary tumors with these dimensions, and at the end of the protocol half of the group (5 animals) had mammary tumors higher than 35 mm. Although the animals exhibited tumors with these dimensions that are indicative of sacrifice, they did not exhibit any alterations in the remaining parameters, so they were only sacrificed at the end of the experiment.
Looking to our experiment, we concluded that the tumors’ dimension alone do not imply the animals’ sacrifice, provided that the remaining parameters maintained normal.
ID: 60
1Department of Animal Biology, Faculty of Science, and Biomedical Research Institute of Málaga (IBIMA), University of Málaga, Spain
2Developmental Biology Division, the Francis Crick
The morphology and morphogenesis of the cardiac outflow tract is a major topic in the study of the vertebrate circulatory system, especially regarding the pathologies affecting this region in humans. Recent studies have demonstrated that, in fish, the cardiac outflow tract consists of a myocardial conus arteriosus and a nonmyocardial bulbus arteriosus. Moreover, the bulbus arteriosus of fish has been considered homologous to the intrapericardial base of the aortic and pulmonary trunks of birds and mammals. Under this perspective, we have conducted a study on the outflow tract of Xenopus laevis, using histological, immunohistochemical and 3D reconstruction techniques. It has been assumed that the outflow tract of Xenopus, which is intercalated between the ventricle and the great arterial trunks, is of myocardial nature. At its luminal side, it contains two sets of valves between which the so-called spiral valve lies.
Our results demonstrate that, together with a proximal myocardial segment, a distal, nonmyocardial, intrapericardial segment is also present in amphibians. We propose that this distal segment, from which the pulmocutaneous and systemic arteries arise, is homologous to the bulbus arteriosus of fish. Therefore, the bulbus arteriosus is an evolutionarily conserved structure, which has become the aortic and pulmonary roots of birds and mammals. Our findings contribute to strengthening Xenopus as a good model to better understand the outflow tract morphology and evolution, and as an emerging model for studying human congenital heart diseases.
This work was supported by CGL2010-16417, BES-2011-046901, Estancias Breves para FPI (2012, 2013) and FEDER funds.
ID: 61
1Department of Animal Biology, Faculty of Science, University of Málaga, Spain
2Biomedical Research Institute of Málaga (IBIMA), University of Málaga, Spain
Bicuspid aortic valve (BAV) is the most frequent human congenital cardiac malformation. It frequently becomes stenotic due to calcification by an atherosclerosis-like process. Hyperlipidic diets have been classically used to induce atherosclerosis in laboratory animals, including Syrian hamsters. The aim here is to evaluate the effect of hyperlipidic diets in hamsters having different incidence of BAVs.
We used a unique inbred strain of Syrian hamsters with a high (∼40%) incidence of spontaneous BAV, morphologically similar to that in man, another inbred strain with a low (∼4%) incidence of BAV, and an outbred, second control line, acquired from Charles River Laboratories. Three experimental groups were fed with standard diet supplemented with 2% cholesterol plus 15% butter during five months. In parallel, three control groups were fed with unmodified standard diet.
Hyperlipidic diets induced lesions in the aortic valve and ascending aortic wall, i.e. subendothelial lipid deposits, valve sclerosis, and neo-intima in the aorta. We performed a preliminary, qualitative, comparative study of the lesions associated with the different animal populations and valvular phenotypes.
Our results indicate that (1) the type and severity of the lesions varied among the three hamster populations, suggesting that genetic factors may be involved; (2) the aortic valve morphology seems not to determine the severity of the valvular lesions. We conclude that our hamster strain with high incidence of BAV is a promising animal model for studies on human aortic stenosis.
This work was supported by P10-CTS-6068.
ID: 63
1Department of Animal Biology, Faculty of Science, University of Málaga, Spain
2Biomedical Research Institute of Málaga (IBIMA), University of Málaga, Spain
Bicuspid aortic valve (BAV) is the most frequent human congenital cardiac malformation, with an incidence of 1–2% worldwide. Two morphological types exist: type A (incidence ∼0.75–1.25%) and type B (incidence ∼0.25–0.5%), each with a distinct aetiology and natural history. Currently, ten animal models of BAV have been described in two different rodent species: one spontaneous Syrian hamster (Mesocricetus auratus) model of BAV type A and nine mutant laboratory mouse (Mus musculus) models of BAV type B. It remains to be elucidated whether the mutations leading to BAV in these models are type-specific or whether there are inter-specific differences regarding the type of BAV that hamsters, mice and humans may develop.
To solve this issue, we have characterized the incidence and types of BAVs in four inbred, two outbred and two hybrid lines of Syrian hamsters (n = 4.340) and in three inbred, three outbred and one hybrid lines of laboratory mice (n = 1.661) by means of stereomicroscopy and scanning electron microscopy. In addition, we have reviewed and calculated the incidence and type of BAVs in the published papers dealing with this anomaly in mice.
Our results indicate that the Syrian hamster develops BAVs type A and B including a variety of morphologies comparable to those of humans, whereas the mouse develops only BAVs type B with a short spectrum of valve morphologies. Thus, inter-specific differences between human and mouse aortic valves must be taken into consideration when studying valve disease in murine models.
This work was supported by P10-CTS-6068.
ID: 64
1Department of Animal Biology, Faculty of Science, University of Málaga, Spain
2Biomedical Research Institute of Málaga (IBIMA), University of Málaga, Spain
The C57BL/6 (BL/6) mouse strain is one of the most common models in research involving laboratory animals, particularly on studies of the cardiovascular system. It has been reported (Fernandez B, et al. J Anat 2008 212(1):12–18) that this strain presents an unusual coronary artery (CA) pattern, including congenital CA anomalies, which are clinically relevant in humans. The aim of the present study was to elucidate whether this pattern is strain-specific or appears in other mouse populations.
We used stereomicroscopy, scanning electron microscopy, light microscopy, and a corrosion cast technique in 597 adult mice belonging to three inbred strains (BL/6, Balb/c, DBA/2), three outbred stocks (CD1, OF1, NMR1), two hybrid lines (129sv x BL/6, CD2F1) and wild mice.
Lock-like ostium was only detected in BL/6 mice, whereas left septal artery, accessory ostium, high take-off, intramural course, and solitary ostium of one CA in aorta were present in different laboratory strains and in wild mice. However, each mouse population showed a specific incidence of these coronary conditions.
These results should be taken into account when studying the murine coronary system, especially in CA occlusion experiments and in studies on cardiovascular development involving murine mutant lines. In addition, we propose that several laboratory mouse strains may serve as appropriate animal models to study several clinically relevant human congenital anomalies of the CAs. Our results suggest that some of these CA anomalies are subject to a simple mode of inheritance.
This work was supported by P10-CTS-6068 and PI-0888-2012.
ID: 74
1Centre for Research and Technology of Agro-Environment and Biological Sciences (CITAB), UTAD, Vila Real, Portugal
Animal models for studying urinary bladder cancer have reliably demonstrated that the urothelium is a good choice for studying the process of carcinogenesis. In this work we aimed to compare the urinary bladder histopathological lesions in mice and rat after the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) exposure.
All procedures were carried out in accordance with the European Directive 2010/63/EU on the protection of animals used for scientific purposes. Ten ICR male mice and 10 female Fisher 344 rats were exposed to BBN in drinking water during 12 and 20 weeks, respectively. A negative control group of each specie was used simultaneously. At the end of the study, animals were sacrificed and their urinary bladders were collected for histopathological analysis.
Mice and rats’ urinary bladders from negative control groups revealed no changes. The incidence of urothelial lesions on mice exposed to BBN was: simple hyperplasia 80%, nodular hyperplasia 40%, dysplasia 90%, papilloma 20%, papillary neoplasms of low malignant potential 10%, low-grade papillary tumor 20%, high-grade papillary tumor 30% and invasive carcinoma 30%.
The incidence of urothelial lesions on rats exposed to BBN was: simple hyperplasia 30%, nodular hyperplasia 90%, dysplasia 100%, papilloma 50%, papillary neoplasms of low malignant potential 50%, low-grade papillary tumor 70%, high-grade papillary tumor 20% and invasive carcinoma 20%.
Comparing the results, we conclude that the rat is a good model for studying papillary tumors, while the mouse is indicated to the study of invasive carcinomas. However, the selection between mouse and rat may be based on the aims of the study.
ID: 84
Jesus Uson Minimally Invasive Surgery Centre
Epidermal Growth Factors (EGFs) are essential for the correct embryo implantation and it has been probed that the affinity of embryos for EGF depends on the levels of some estrogens metabolites as 4-hydroxyestradiol (4-OHE2).
In order to obtain further information about the role of 4-OHE2 in the implantation process, we performed experiments to evaluate whether the supplementation of the culture media with this compound improves embryo implantationandgestation rates of embryos obtained by in vitro fertilization (IVF).
To do that, B6D2 female mice were stimulated with equine chorionic gonadotrophin (eCG) and 49 hours later human chorionic gonadotrophin(hCG) was administered to induce ovulation. After this, cumulus-oocyte complexwere obtained and IVF was performed by addition of fresh sperm accordingly to the routine procedure. After fertilization and culture to morula stage, embryos were divided in two groups. One was cultured with KSOM culture media alone and was the control (IVFc) group, and the other group was supplemented with 0.1 µg/ml of 4OHE2 (IVF0.1). Embryos that were developed to blastocyst stage were transferred to pseudopregnant female mice in order to compare embryo development and implantation rates. Blastocysts removed to the uterus were used like a control group (IU).
Our results shows implantation in the IU was 25.7%, higher than the 7.9% of the IVFc group. When embryos were supplemented with 0.1 µg/ml of 4OHE2 (IVF0.1) implantation rate raised up to 13.3 %. Although more experimentation is necessary, our results show that supplementation with 4OHE2 can improve implantation rateof embryos obtained by IVF.
ID: 85
Jesus Uson Minimally Invasive Surgery Centre
Today, oocyte bank is an interesting alternative to sub-fertile colonies where embryo cryopreservation it is not possible. Also, oocyte cryopreservation research in murine model offers solutions and results extrapolable to human oocyte cryopreservation. The metaphase II oocyte seems to be susceptible to damage by freeze-thawing procedures, and it was suggested that cryopreservation procedures could be responsible for the relative success of this technique. Oocyte cryopreservation in a cryobank may require long duration of cryo-storage and this factor can also influence the successes of vitrification. Finally, the choice of open or closed system for storage has also been disputed.
The main objective of this study was to determine the survival and development to blastocyst stage rate in murine model of vitrified oocytes in different systems, in relation to the time of cryopreservation. Femalemice were stimulated to triggers ovulation and after obtaining the oocytes, they were vitrified in open and closed systems. Oocytes were stored in liquid nitrogen for 90–100 days (Group 1) or for 150–160 days (Group 2) and afterthis period, they were thawed.Surviving oocytes were used for in vitro fertilization. Control group consisted of fresh oocytes, not subject to vitrification.
Fertilization rate and blastocyst formation rate after culture provided was stablihed for each group. Our results suggest that an increase in storage time does not decrease the survival, fertilization and development to blastocyst comparing both systems (open and close) in the murine model.
ID: 87
Jesus Uson Minimally Invasive Surgery Centre
The Minimally Invasive Surgery Center in Cáceres is currently involved in a researcher project on uterine transplant in the sheep with the purpose of gain experience and knowledge with this kind of surgery and be able in the future to perform this type of transplant in humans.
The success of uterus transplantation is only fully achieved if the transplanted organ is able of perform a pregnancy correctly. The achievement of this functionality depends mainly on the correct anastomosis of veins and arteries and the angiogenesis of adjacent vessels of uterus. Besides a correct irrigation of the new organ, the correct vascularization has also a physiological role, since hormones involved in implantation and pregnancy are produced mainly by the ovary and scattered throughout the reproductive tract by the bloodstream.
Sheep are seasonal animals, but induction of ovulation outside the breeding season can be done by exogenous hormone treatment with progesterone (PG). Consequently, 7 ewes with autologous uterus transplant were stimulated by progesterone intravaginal sponges. The day of sponge removal, each animal was administered with Pregnant´s mare serum gonadotropin (PMSG) to trigger ovulation. The insemination was performed 55 hours after injection with fresh semen, that was introduced in the first cervical folds with an appropriate catheter.
At the end of the experiments four animals became pregnant, one of them with a twin pregnancy. In this way the functionality of the ovary and the transplanted uterus was demonstrated.
ID: 93
1University of São Paulo, Brazil
2University Paulista, Brazil
Spontaneous mutant mouse named tremor, autosomal recessive, was observed in the colony of Swiss mice from the animal facility of the Department of Pathology of Veterinary Medical School of University of São Paulo, Brazil.In this mutation we can see tremors, ataxia and seizures. Luxol Fast Blue staining revealed a deficiency of myelin in the meninges and cerebellum. Preliminary results of genetic mapping indicate that the mutation is on chromosome 14. The phenotypic characterization was performed by evaluation of behavioral motion parameters, psychomotor, physiological and connected to the nervous system by direct observation of the animals. Some tests were conducted in the open field and ordered by parameter evaluated: 1) general activity and sensory system evaluation; 2) psychomotor tests; 3) evaluation of the central and autonomic nervous system. Other tests involved were: memory and learning in object recognition test, the behavior at elevated cross maze, behavior in theT-maze, motor coordination on high beam, and the behavioral response in the tail suspension test. We could see an increased number of mutants with the tail standing, ataxia and tremor; reduction in the frequency of lifting and grooming; reduction of motor coordination; negative responses to anxiety; reducing memory and lowest signal strength, and depression. The results indicate that the mutant losses start in CNS, possibly caused by lack of myelin, which affects movements of mice. It is also suggested that mutant mice have deficits in memory and/or learning. Understanding the phenotype of this mutation it is important to establish a model of neurodegenerative diseases.
ID: 95
1University of São Paulo, Brazil
2University Paulista
The recessive mutant mouse called equilibrium (eqlb), which presents balance impairment and incoordination, was originated from an ENU (N-Ethyl-N-nitrosourea) mutagenesis program. Histopathology showed abnormal formation of cell layers of the cerebellum, with disorganization of Purkinje cells and thickening of the external granular layer. Genetic mapping has identified a mutation in the NADPH oxidase 3 gene (NOX3), chromosome 17, described as responsible for vestibular defects and otocônias formation. Phenotypic characterization of eqlb mutant included overall assessment of the phenotype, using a battery of behavioral tests that aimed to observe general health, neurological reflexes, sensory abilities and motor function. Initially, tests were conducted in the open field in the following sequence: 1) general activity and sensory system; 2) psychomotor tests; 3) evaluation of the central and autonomic nervous systems. Besides direct observation in the open field, the test in elevated crossbar was made in order to assess motor coordination of the mutant mice. The mutant showed increased falling hindquarters and reduced righting reflex, irritability, touch response, defecation and frequency of raising. The increase of the falling hindquarter and reduced righting reflex indicate impairment of psychomotor system while the reduction in the response to touch and irritability show that they have a reduction in sensorial system. Reducing the frequency of lifting is probably a consequence of motor difficulty. Furthermore, the reduction in stool indicates less activation of the autonomic nervous system. The elevated cross bar test results indicate that the mutants exhibit a balance injury mainly related to motor coordination.
ID: 114
1University of São Paulo, Brazil
2University Paulista, Brazil
The recessive mutant mouse named bate palmas (bapa) – claps in Portuguese, which presents balance impairment and incoordination, was originated from an ENU (N-Ethyl-N-nitrosourea) mutagenesis program. The lysine (K) -specific methyltransferase 2D gene (KMTD2S, also known as MLL2), located on chromosome 15, was identified as a candidate gene by exome sequencing. The mutation was confirmed by Sanger DNA sequencing. This gene is responsible for the Kabuki syndrome, which is a rare autosomal dominant disorder caused by a mutation with loss of function in the same gene, located on chromosome 12 in humans. The phenotypic characterization was performed by evaluation of behavioral motion parameters, psychomotor, physiological and connected to the nervous system by direct observation of the animals. Initially, tests were conducted in the open field and ordered by parameter evaluated in the following sequence: 1) general activity and sensory system evaluation; 2) psychomotor tests; 3) evaluation of the central and autonomic nervous system. Other tests involved were: memory and learning in object recognition test, the behavior at elevated cross maze, behavior in the T-maze, motor coordination on high beam, and the behavioral response in the tail suspension test and forced swimming test. We could see a normal behavior in the open field tests; nonetheless, the mutant mice were not able to cross the high beam that indicates motor impairment. Phenotypic characterization of this animal is important in understanding what may come to be represented this mutation, and if it could become the first mutant model to study the Kabuki syndrome.
ID: 125
1Department of Biomedical Engineering, Universidad de los Andes - Bogotá, Colombia
2Investigative Anestcol SAS Group - Bogota, Colombia
3Surgical Department – Fundación Cardioinfatil
4Fundación Cardioinfantil – Research Department, Bogotá, Colombia.
The research group has developed regenerative SIS vascular grafts in the form of “U” for use as vascular access for hemodialysis. For this study the ovine animal species of 35 kg was used for vascular access (AV) between femoral artery and vein graft with 6 mm diameter. 2 subjects in which vessels suboptimal size, poor flow and graft patency due to the resulting graft configuration in the anatomy of the animal was observed were used. According to the results, the femoral vessels for the implant is replaced by the internal carotid artery and the external jugular vein in 3 additional subjects, improving flow through the graft and avoiding the collapse of the fistula with the natural movement of the animal. Before achieving 8 weeks maturation, the grafts presented with foreign body reaction and graft encapsulation because histologic nature of the inflammatory reaction, a change in animal model was proposed to pigs of 50 kg mass which present an infectious disease, this time without encapsulation, which prevented its regeneration and preservation of permeability due to thrombus formation. According to observations, we decided to perform a structural modification to grafts to prevent thrombogenesis due to kinking of the graft after the surgical implant and is currently studying regeneration and permeability in pigs of 50 kg
ID: 138
Specipig® Model
ID: 141
Minimally Invasive Surgery Centre Jesús Usón, Cáceres, Spain
ID: 145
1Animal Facility. Centro Investigación Príncipe Felipe, Valencia, Spain
2Neural Regeneration Laboratory, Centro de Investigación Príncipe Felipe, Valencia, Spain
Estimated overall incidence of Spinal Cord Injuries (SCI) is 40 to 80 cases per million population per year, and up to 90% of them are provoked by traumatic causes. Patient develops motor paralysis and loss of sensation due a disruption of axonal tracts caused by an impenetrable glial scar with the death of neurons, glial cells and neural precursors.
Animal models of Chronic SCI in which reproduce the pathophysiologic scenario in clinic and test therapies are necessary. Cell therapy as replacement of lost functional units and enabling environment to promote axonal regrowth plays an important role. In this sense, we showed that transplantion of spinal cord ependymal progenitor cells (epSPCs) significantly improve locomotor function from the first week after transplantation.
To establish the Chronic SCI, 34 female Sprague-Dawley rats were operated to induce a severe traumatic lesion applying 250 kilodyne with a computerized system (Infinity Horizon Impactor®, Precision Instrumentation Systems, Burke, USA). Subsequently, animals were assessed once a week during all the study, 16 weeks, to evaluate the severity and locomotor activity evolution using BBB (Basso, Beattie, and Bresnahan) 21-point locomotion scale. The procedure was evaluated by Ethics Committee of Prince Felipe Research Center in accordance with current legislation for the protection of laboratory animals (RD 1201/2005 and RD 53/2013).
The results show a stable animal model of chronic spinal cord injury that reproduce the clinical situation and allows the assessment of locomotor recovery in cell therapies.
ID: 146
Minimally Invasive Surgery Centre Jesús Usón, Cáceres, Spain
The validity was evaluated with a structured questionnaire about anatomical integrity and surgical aspects of porcine model, using a five-point Likert item.This study was approved by our Institutional Ethical Committee (CEEA-CCMIJU) and the Government of Extremadura, following the guidelines of the Directive 2010/63/EU.
ID: 147
Minimally Invasive Surgery Centre Jesús Usón, Cáceres, Spain
ID: 151
Animalario Hospital Nacional de Parapléjicos
Since the establishment of the Research Unit in the National Hospital for Paraplegics (NHP) our main objective has been to set up an experimental protocol of spinal cord injury (SCI) in the rat. All experimental procedures are approved by the Animal Experimentation Procedures Committee at NPH. The procedure is as follow: male Wistar rats are anesthetized with an intraperitoneal injection of sodium pentobarbital (45 mg/kg), Xilagesic (2% Xylacine, 10 mg/kg) and buprenorfin (0’05 mg/kg).
Once the absence of reflexes had been checked, the rats are injected with atropine (50 µg/kg body weight) to reduce salivary and bronchial secretions, and to avoid the induction of bradycardia and possible cardiac arrest by the surgery or xylazine. Artificial tears are applied to the eyes to prevent corneal abrasion and infection.
After shaving and cleaning the back of the animals a dorsal midline incision is made in the skin and the T8 vertebra is exposed after blunt dissection of the muscle. The spiny apophysis and dorsal lamina of the 8th thoracic vertebra is removed and the animals are positioned on a SM-15 Narishige stereotaxic frame, stabilizing the spiny processes of the T7 and T9 vertebrae with clamps.
Spinal cord contusion/short compression is performed with the ‘‘Infinite HorizonTM’’ device, (Precision Systems and Instrumentation) applying a force of 200 Kdyn.
This protocol has been standardized to be used by our research laboratories that apply different experimental approaches in spinal cord repair.
ID: 153
1Experimental Animal Service, Research Unit, Universitary Hospital of Gran Canaria, Dr. Negrín, Las Palmas de Gran Canaria. España
2Infectious Diseases and Fish Pathology. Universitary Institute of Animal Health and Food Safety. Universidad de Las Palmas de Gran Canaria. Arucas. Las Palmas
3Servicio de Radiodiagnóstico Hospital Universitario Dr. Negrín, Las Palmas de Gran Canaria, España
4Servicio de Microbiología Hospital Universitario Dr. Negrín, Las Palmas de Gran Canaria, España
5Dpto. de Enfermería, Fac. de Enfermería, Universidad de Santiago de Compostela. España
6Coordinación de Trasplante del Hospital Universitario Dr. Negrín, Las Palmas de Gran Canaria, España
7Servicio de Analisis Clínico Hospital Universitario Dr. Negrín, Las Palmas de Gran Canaria, España
In a model of sepsis by cecal ligature and puncture (CLP), we studied echographic, microbiologic, biochemical and histological changes in 8 Sprague Dawley rats, 12 weeks old and were compared with Sham-control group of 8 healthy rats. In both groups (CLP and Sham-groups), rats were anesthetized with fentanyl/medetomidine (0.3/0.3 mg/kg) sc and pain control was performed with tramadol (15 mg/kg) sc. 16 h. after CLP we noted in the kidney echographic exam of CLP group, marked pyelocalyceal ectasia with poor cortico-medullary differentiation in 100% of CLP animals. No echographics changes were observed in sham-group`s kidneys. In urianalis (test strip for semi-quantitative determination) of CLP group vs Sham: urinary vol (3.3 + 0.4 vs 0.25 + 0.1 ml p < 0.05, pH 6.25 + 0.2 vs 7.2 + 0.1 p > 0.05, WBC > 500 vs < 10 wbc./ml (p < 0.05), erythrocytes ( > 250 vs 4.5/µL p < 0.05), nitrites (POS vs NEG), proteins (282.5 + 56 vs < 10 mg/dL p < 0.05 and high bilirubin levels (2.5 + 0.7 vs 0.5 + 0.7 mg/dl p < 0.05, were observed. We found a excellent correlation between urine and blood cultures. In this way, two defined biotypes E. coli were isolated from blood and urine in all animals of CLP group and none in the sham-group. Pyelonefritis, glomerulitis and bacterial presence in renal vessels and parenchyma were observed in septic rats. In sham-group no changes were observed. Based on these results, we conclude that the experimental model of CLP, allows a good microbiological, biochemical, histological and echographic renal monitoring in Sprague Dawley rat. Future studies including glomerular filtration rate will be required to more accurately assess this model of renal injury by sepsis.
ID: 155
Universidad de Extremadura
C57BL/6 mice are considered a good animal model for investigating metabolic diseases like hyperphagia, hyperglycemia, hyperlipidemia. Additionally, stanniocalcin (STC) is widely expressed in several mammal tissues. Furthermore, STC was found, among other tissues, in the pancreatic cells, where its expression pattern was modified by administering chemical insults.
Animal of both sexes were kept under common environmental conditions and life-cycle, and under a hypercaloric diet with free access to water. The regulatory law regarding animal care and experimentation have been taken into account. STC2 silencing was confirmed wiht PCR. The following groups were established during the present study: young (8–19), mature (35–45) and old (>60 months of age). Glycemia were obtained using blood from the tail of anaesthetize animals at 8:00 PM, considering this glycemia as basal blood glucose concentration, which was estimated using a glycometer.
Funded by MINECO (BFU2013-45564C2-1-P) and Junta de Extremadura-FEDER (GR15029).
ID: 157
1Service of Laboratory Animal Resources (U. Córdoba)
2Emergency School SAMU S.A.
3Pharmacology, Toxicology and Forensic Medicine (U. Córdoba)
The Emergency Medical Assistance Service (SAMU S.A.) is a health institution specialized in providing medical services in healthcare, logistics, organizational and educational scope. In the Emergency School, medical professionals, nurses and technicians are trained in patient care in critical emergency situations. To that purpose, a specific training Campus where to strengthen the knowledge in the field of patient care in different extreme situations is available, allowing the development of skills in surgical rescue techniques to be applied in disasters and extreme environments to save human lives (thoracentesis, cricothyrotomy, abdominal trauma, traumatic amputations…), and controlling effectively and with few resources situations of impending death that need to undergo surgical procedures in a hospital setting. To this end, minipig (Sus scrofa domestica) is employed as an experimental model. Due to its marked similarity to humans, it is one of the most convenient biomodels used for the development of experimental techniques and for teaching different biomedical specialties. Procedures are performed under the current legislative framework, which requires the development of appropriate anesthetic protocols that guarantee the absence of pain and stress in animals. Regulation also determines which euthanasia method must be employed to minimize the duration and severity of animal’s suffering, ensuring a painless death. In this context, although it is desirable to replace scientific procedures with live animals by alternative methods according to the 3Rs Principle, the use of biomodels in this area is fundamental, as simulators aimed to mimic bleeding or asphyxia contexts still offer incomplete and distant view of reality.
Genetically modified (GM) animals
ID: 32
Instituto de Neurociencias de Alicante CSIC-UMH
A research group in our institution decided to move to the UK with their 24 mouse mutant strains on February 2014. The moving should be finished just before the end of the year but the animal facility from the new institution told them that they were not going to accept live animals nor frozen sperm as they couldn´t perform IVF, and so they could only receive frozen embryos, the slower possibility for sending the strains. In this moment we face the problem of being 2 people in the lab, with our normal amount of services to process and the extra work of producing and freezing embryos of 24 mouse strains in the 30 weeks of 5 working days (needed for IVF) remaining up to the deadline. The planning had an extra complication because 10 of those strains were double mutants and we had to use homozygous females as oocyte donors and therefore we had to adjust matings to produce the right age donors.
We performed 28 IVF experiments (it was only necessary to repeat 4 strains), using 149 females as oocyte donors (72 mutants and 77 wild type) and freezing 2675 embryos. In the IVF´s performed with mutant females we obtained an average of 16.7 oocytes and 9.4 embryos per female, with a 56.4% fertilization rate. In the IVF´s performed with C57BL/6J Wt females we obtained 40 oocytes and 27.4 embryos per female, with a 66.1% fertilization rate.
All strains were sent on time and recovered at destination.
ID: 82
Centro de Investigaciones Biológicas – CSIC. Madrid, Spain
Surgical transfer of mouse embryos is a commonly used technique in animal facilities worldwide. It is known that the yield, i.e. the number of pups in the offspring, increases proportionally to the number of embryos transferred, so it is common to use large numbers of transferred embryos per female. However, the use of a very high number of embryos may increase excessively the effort, i.e. the number of embryos necessary for obtaining each single pup. This work aims to find a suitable balance between effort and yield by analyzing the number of embryos used for transfer. To address this question, we have used the data collected at our facility during a ten-months period. Rederivation of 90 mouse lines/strains was performed by transferring between 5–26 embryos per line, which were grouped into five categories for analysis. Our analysis indicated a proportional increase not only in the yield but also in the effort made, being the correlation between the two variables strongly positive. Furthermore, the expected yield was estimated from the ratio between the average of embryos transferred for each category and that of the category of the lower number of embryos, and the product by the observed yield. The comparison between observed and expected yields showed very significant differences, with the former being lower than the later. These results indicate that the balance between effort and yield is optimized when using low numbers of embryos. Given the high heterogeneity of the data set analyzed these results could be generalized to other facilities of similar characteristics.
ID: 90
Centro de Investigaciones Biológicas – CSIC. Madrid, Spain
In transfer programs of mouse embryos it’s fundamental to assure the necessary number of pseudopregnant females for the expected date. The habitual method consists in pairing fertile females along with vasectomized males and checking daily for the presence of vaginal plug, indicative of mating in females. By means of this method, we verified the presence of a variable number of plugs between the second and the fifth day after pairing, being the tendency highest on the fourth day probably due to the Whitten effect. It’s known that using spacers to elicit mating improves the yield in C57BL/6 mice. Here we analyzed whether optimization of the number of vaginal plugs at a programmed date can be achieved by using spacers in the mating process. To address it, we designed a methacrylate spacer that allows to pair at the same cage a vasectomized male and one or two fertile females with visual and olfactory contact but without physical contact. After three or four days mating is allowed by removing the spacer. Significant differences in the proportion of covered females were found when spacer was removed on third day, showing plug on fourth day 50% of the females. This proportion increased up to more than 60% when we counted the females covered until fifth day. Our method presents important advantages compared to the habitual method: it eliminates the daily checking of females to detect vaginal plugs, plugs are obtained only the dates on which they are required and the number of pseudopregnant females is optimized.
ID: 100
Servicio Transgenesis CNB-CBMSO CSIC-UAM
The irruption of CRISPR/Cas9 based Genome editing system as a tool to modify the mammalian genome has promoted a change of mentality regarding the creation and maintenance of genetically altered lines. This demands a necessary adaptation of Transgenic core facilities. The CNB-CBMSO Transgenesis Service will share our adaptation process with other established core facilities and with centers that plan to establish such a service in the future in three different aspects:
Technology incorporation: Which technologies have to be implemented, which techniques remain in their present situation, which techniques have faced a revival and which technologies might be replaced by Genome Editing. Impact on customers regarding demand, costs and project length. Impact on animal use.
Since 2013 our Service has developed the following strategies.
– Training of personnel and establishment of collaborations with customers in order to implement the technology and explore its efficiency.
– Agreements with companies/laboratories in order to offer the full process from design to alive animal model.
– Advise to customers in the design and request of new projects involving genetically altered animal KO, KI and conditional models.
During the past two years, the STG CNB-CBMSO has not observed a decrease of demand in projects based on ES cells to obtain KI or conditional models, but all constitutive KO projects have been performed with Genome editing with a consistent improvement in speed and a clear reduction of animal use.
ID: 116
Transgenic Animal Unit (UAT), Center For Animal Biotechnology and Gene Therapy (CBATEG), Universitat Autònoma De Barcelona (UAB)
The Transgenic Animal Unit (UAT) of the Center for Animal Biotechnology and Gene Therapy (CBATEG) at the Autonomous University of Barcelona (UAB), is a state-of-the-art facility that provides research and technical services for the generation, management, genotyping and preservation of genetically altered mouse and rat models, to support research projects of both, internal and external investigators.
Since the CRISPR/Cas9 system was described as a tool for genome editing, it has become a revolution in the field of animal transgenesis. CRISPR/Cas9 is part of the immune system of bacteria and archaea. It is a RNA guided DNA endonuclease system which targets specific DNA sequences generating double strand breaks. The DNA repair mediated by NHEJ or HDR leads to the introduction of mutations in the target site. The specificity of the system relies only on the sequence of a 20nt guide RNA (gRNA) that is easy to be designed and obtained. Moreover the very high efficiency of this system allows the generation of mutant animals by direct injection in one-cell embryos, avoiding the need of embryonic stem cells. This makes the process of KO and KI animal generation straightforward and available to species other than mouse. Furthermore, it has been described the possibility of multiplexed genome editing by the delivering of multiple different gRNA sequences in a single step. Here we present the use of CRISPR/Cas9 system in the UAT to generate KO and KI mouse and rat models.
Anaesthesia, Analgesia and Surgery
ID: 88
Jesus Uson Minimally Invasive Surgery Centre
Embryonic diapause (ED) is a phenomenon defined by a temporary arrest of embryo in blastocyst stage. Facultative diapause can be produced experimentally in rodents by ovariectomyafter fertilization, but a method to make this kind of surgeryhas not been described with precision yet. We aimed to evaluate the technical feasibility and safety of two different ovariectomytechniques and after comparison of the results, choose the best based on surgery time and the capacity getting dormant embryos.
Domestic adult female CD1 mice were ovariectomized on the morning of Day 4 of pregnancy as calculated by the presence of vaginal plug after the matting. We do the first part of the procedure in a similar way in all the cases and consist in the tear the bursal membrane that surrounds the ovary and oviduct. After that, on one group of mice we tied the ovarian blood vessels by slipping a loop of surgical silk suture between the oviduct and ovary. On the other group, we removed the ovarian tissue by cauterizing with a bipolar knife. Once the animals recovered, progesterone was gave daily subcutaneously from days 5–7and we collected dormant blastocysts in both groups by flushing the uterus on the morning of day 8. Results showed higher amount of blastocyst recovered and less average time of surgery (and consequently less exposition to anesthesia drugs)in cauterization technique when comparing with extrangulation technique. In conclusion we determined that cauterization technique is more efficient to achieve dormant embryos.
ID: 91
1Grivet Veterinay Services
2PCB-PRBB Animal Facility Alliance
3Universitat Pompeu Fabra
Electrical stimulation of peripheral nerves has many clinical applications. For instance, treatment of chronic pain or vagus nerve stimulation for epilepsy and depression treatment.
We performed an in vivo pilot test in rabbits of new implantable devices capable of electrical stimulation (ICNR2014 proceedings, pp 447–455). The devices basically consist in a 3 cm long tubular silicone body. For demonstrating peripheral nerve stimulation, the devices were implanted inside either the body of the gastrocnemius muscle or the body of the cranial-tibial muscle.
Initial sedation and parenteral anaesthesia was administered. Then, the animal was transferred to a gaseous anaesthetic circuit. The thinness and elongated shape of the devices allowed minimally invasive deployment through a 14 G catheter inserted into the muscle. Catheter accurate positioning consisted in pushing it in or pulling it out a few millimetres and using the needle’s tip to perform external electro stimulation tests to locate the adequate motor point. Then, the insertion needle was pulled out and the device was introduced through the catheter and pushed up to the tip of the catheter. Finally, the catheter was pulled out and the implant was released inside the muscle. X-ray images were acquired to verify the position of the implants and stimulation assays were performed. In this 4-week pilot study with 3 rabbits, all the implants were well tolerated. A procedural conclusion from this study is that, for ensuring implant stability, the thread-like device must be wholly implanted into a single muscle, avoiding crossings through muscle fascia.
ID: 97
1Centro de Cirugía de Mínima Invasión Jesús Usón, Cáceres
2Clínica San Francisco, León
ID: 105
1IDMEC –FEUP, Porto
2CITAB / UTAD Vila Real
3IBMC, Porto
4LABORIAL, Maia, Portugal
Loss of righting reflex (LORR) and respiratory rate (RR) variations are used for anaesthesia depth evaluation. This work aims to automatically detect LORR and RR in rodents anesthetized in a chamber.
Three 2-months old female NMRI mice were individually anesthetized in a chamber. Anesthesia was induced with 5% isoflurane, and stopped when LORR was detected. One piezoelectric sensor was placed under one of the chamber’s foothold and connected to a charge amplifier. The signal was acquired with a NI-USB-6251-DAQ, at 1000 samples/s. To acquire and analyze the signal, a software application was developed using NI-LabVIEW 2013.
A multi-resolution analysis with wavelet transformation method was applied. The data processing algorithm was performed in 10 seconds intervals, using a first in first out method. The method, allowed to identify peaks that were related with the rodent’s chest movements and calculated the RR using the mean time between peaks in 10 seconds interval. In order to detect LORR, each 10 second interval was analyzed to verify if the pattern match a current respiration or other kind of signals.
It was possible to identify three different states: lack of signal (no movement detected), movement (signal with no constant amplitude and/or frequency) and LORR (transition from movement to constant respiratory detection). The automatic recognition of the LORR and RR is a powerful tool in anesthesia monitoring, especially in the induction phase. This equipment may be integrated in more advanced anesthesia and surgery benches.
Funding - “IntelLab II -Inteligência em Laboratórios”, FCOMP-01-0202-FEDER-033877 - through COMPETE -POFC.
ID: 119
1Departamento de Biologia, Faculdade de Ciências, Universidade do Porto (UP)
2Instituto de Biologia Molecular e Celular, UP
3Instituto de Investigação e Inovação em Saúde, UP
Anaesthesia is used on a daily basis in laboratory animals. However, concerns have been raised regarding a possible influence of anaesthetics on animals’ behaviour, potentially compromising the outcome of neurobehavioural research. Indeed, a low concentration of isoflurane, 1%, impaired mice’s learning of spatial tasks (1), but its influence on non-spatial tasks needs clarification. Therefore this study aimed to assess the effect of 1% and 2% of isoflurane on the recognition memory in adult mice.
Forty 2 months old C57BL/6 male mice were randomly divided into three groups: control group (n = 10), 1% isoflurane group (n = 9) and 2% isoflurane group (n = 9); 1hour of anaesthesia duration. Twenty-four hours post-anaesthesia, each mouse was placed in an arena with two identical objects and allowed to explore for 10 minutes- sample trial. One hour afterwards, each mouse was placed in the arena with one object previously shown (familiar) and with a new object (unfamiliar), and allowed to explore for 10 minutes- choice trial. The object recognition task was recorded and the video recording analysed with Observer software.
The analysis of 20 seconds of total objects exploration showed that for the control and 2% group there was significantly more exploration of the unfamiliar object than by chance (pcontrol = 0.020; p2% = 0.029), whereas the 1% group explored similarly both objects.
Low isoflurane concentration impaired recognition memory, while animals treated with 2% isoflurane behaved at control levels. Therefore, depth of anaesthesia has to be considered during an experiment, as it may influence spatial and non spatial tasks.
(1) Valentim et al. (2010). Anesthesiology 113(5):1099–108.
ID: 121
1ICVS - University of Minho, Braga, Portugal
2ICVS/3B’S - PT Government Associate Laboratory, Portugal
3Department of Pediatric Surgery, Hospital de Braga, Portugal
Minimally invasive surgery on neonates brings numerous advantages over the traditional open surgery. However, a major concern still remains regarding metabolic changes induced by carbon dioxide (CO2)-insufflation and its consequent absorption over the developing organs of neonates. In order to effectively assess and quantify these effects, a pneumoperitoneum neonatal rat model with video-assisted endotracheal intubation and mechanical ventilation was developed. 10 days old Sprague-Dawley rats were anesthetized and randomly distributed into the following groups: i- non-ventilated controls (NVC); ii- ventilated controls (VC); iii- ventilated + 8 mm Hg CO2-pneumoperitoneum (PP8); iv- ventilated + 12 mm Hg CO2-pneumoperitoneum (PP12). Adjustments in tidal volume (VT) and respiratory frequency (fR) were done until achievement of normal pH (7.35–7.45), partial pressure of carbon dioxide (PaCO2) (35–45 mmHg) and oxygen saturation (sO2) above 95%. It was observed that NVC developed hypercapnia, acidosis and mild hypoxia. In VC, a VT of 15 ml kg–1 combined with a fR of 120 bpm, was shown to be ideal for obtaining physiological pH, PaCO2 and sO2. In PP8, these ventilatory parameters were insufficient in maintaining physiological pH and PaCO2 and increments in VT and fR were needed, resulting in a final combination of 20 ml kg–1 and 160 bpm, respectively. In PP12, a second adjustment in fR was required, resulting in a final fR of 180 bpm. The absence of proper adjustment of respiratory parameters in pneumoperitoneum animal models may lead to non-physiological pH and arterial blood gases which may misrepresent results when generalizing to the human clinical setting.
ID: 140
1Centro de Cirugía de Mínima Invasión Jesús Usón, Cáceres
2Clínica San Francisco, León
Laparoscopic Roux-en Y gastric bypass (LRYGB)is an increasingly common therapeutic option in the management of obesity and certain obesity-related comorbid conditions. As it gains popularity for its advantages of being minimally invasive and effectiveness, it should be an important point to control complications, which may be irreversible and associated with long-term morbidity. In humans, early complications occur in 10% of patients and this percentage is not described in porcine model due to small number of groups who have described a survival porcine model of LRYGB.
Obese male Göttingenminipig (56 kg) was undergone to induce to a LRYGB (30 cc gastric pouch and 100 cm alimentary limb). After that, we performed oesophagus-gastric transit to assess the leak of the anastomosis. The onset of complications occurred at week after RYGB. Subject complained of pain, dysphagia and a lot of liquid vomiting. Endoscopy and exploratory laparoscopy was performed together with haematological analysis and biochemistry. Due to lack of appetite, signs of pain and debility of this animal, we placed a gastrotomy tube but the sign and symptoms progressively worsened and finally the subject was euthanized.On gross examination, severe peritonitis with extensive adhesionsand a possible hiatal hernia were revealed.
With the rising prevalence of morbid obesity and the therapeutic options, careful consideration should begiven to the potential complications. Presence of hiatal hernia following bariatric surgery can present atypical symptoms that do difficult the diagnosis.
ID: 143
Servicio de Anestesiología. Facultad de Veterinaria. Universidad Complutense de Madrid
The parasympathetic tone activity monitor (PTA) indirectly assesses the level of nociception determining heart rate variability. The objective was to evaluate the relationship between the PTA values with surgery, the use of loco-regional techniques, and the hemodynamic response in dogs subjected to two types of surgery.
In a prospective observational clinical study, 15 healthy dogs underwent castration surgery. Anaesthesia was performed with dexmedetomidine and methadone, propofol and isoflurane. Patients received loco-regional or no local anaesthesia: epidural anaesthesia in females and intratesticular anaesthesia males. PTA was assessed before and after intubation, placement of Backhaus clamps, skin incision, ovarian pedicle ligation or both testicles, cervix ligation, closure of the abdominal wall, and extubation. ANOVA test and Pearson correlation tests were used.
Eight females and 7 males were used where 5 females and 5 males received loco-regional techniques. PTA values were greater (lesser degree of nociception) in male (61 ± 19) than females (50 ± 17; p = 0.000) as well as in animals in which loco-regional techniques were used (57 ± 19 vs 48 ± 18; p = 0.003). PTA values were inversely correlated to mean blood pressure (p = 0.045).
PTA values were higher in males than in females in animals receiving regional anaesthesia and were inversely associated to arterial pressures. In conclusion, the PTA monitor may be useful to assess the degree of intraoperative nociception.
ID: 149
1Experimental Animal Service, Research Unit, Universitary Hospital of Gran Canaria, Dr. Negrín, Las Palmas de Gran Canaria. España
2Infectious Diseases and Fish Pathology. Universitary Institute of Animal Health and Food Safety. Universidad de Las Palmas de Gran Canaria. Arucas. Las Palmas
3Multidisciplinary Organ Dysfunction Evaluation Research Network, Research Unit, Hospital Universitario Dr. Negrín, Las Palmas de Gran Canaria, Spain España
4Dpto Enfermería, Fac de Enfermería, Univ de Santiago de Compostela
5CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain
6Critical Patient Translational Research Group, Department of Anesthesiology, Intensive Care and Pain Management, Hospital Clínico Universitario, Instituto de Investigación Sanitaria, University of Santiago de Compostela, Santiago de Compostela, Spain
7Research Unit, Hospital Universitario Dr. Negrín, Las Palmas de Gran Canaria, Spain
8Keenan Research Center for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, Canada
To compare three protocols of parenteral general anesthesia, the authors analyzed electrocardiographic changes in anesthetized Sprague Dawley rats, which they are made a laparotomy at room atmosphere (FiO2 21%). Group1, (n = 10) was anesthetized with medetomidine/ketamine (0.25/50 mg/kg) ip, Group 2, (n = 10) was injected ip with diazepam/ketamine (6/60 mg/kg) and, finally group 3 (n = 10) was anesthetized with fentanyl/medetomidine (0.3/0.3 mg/kg) sc. Investigators obtained electrocardiograms from each rat, 15 min. after anesthesia. Anesthetic induction was quick and easy, and rats were orothracheally intubed (4.2 + 0.5 min), though four rats in group 2 died before intubate. Heart rate and R-wave amplitude were significantly lower (p < 0.04) in group 1 (165.0 + 19.4 bpm and 0.425 + 0.11 mV) than in group 2 (300.0 + 43.2 bpm and 0.6 + 0.1 mV) and 3 (277.5 + 26.59 bpm and 0.68 + 0.23 mV). Cardiac axis was lower with medetomidine-fentanyl (9.5 + 22.2°). T-wave amplitude was significantly lower (p < 0.05) in group 2 (0.04 + 0.04 mV). S-T segment was significantly higher (p < 0.05) in group 1 (0.137 + 0.24 mV). Rats received injections of atipamezole about 60 min after surgery, which reversed the effects of medetomidine. Group 3 regained voluntary movement more quickly (3.5 + 1 min) than group 1 (13 + 3.2 min) or 2 (92.4 + 7.3). Two additional rats in group 2 died during postsurgical recovery. In a rat (group 1), we observed electrical alternans and ventricular extrasystole. In a rat (group 2), we observed migratory pacemakers and other with electrical alternans.
These results suggest that it´s important to have electrocardiographic reference values than help us to asses more effectively the results obtained in experimental rat models of heart function.
Implementation of the 3Rs
ID: 18
Instituto de Neurociencias de Alicante CSIC-UMH
Importing genetically modified mice by refrigerated epididymis transportation has many advantages (3R´s, 90% saving in transport costs, no quarantine needed, less health risk), but the technique presents several problems for which we should be prepared. We describe our experience and we show solutions to some of these concerns.
In 2014 we imported 3 strains with this methodology (16% of total). 2 were imported from abroad (Hong Kong) and 1 from Spain (Madrid).
To recover the strains from Hong Kong we needed 3 shipments since the first failed because it was performed during summer and it wasn´t possible to keep the sample cold. After achieving a solution, we found another problem as the next two shipments were delayed by problems with the courier/customs, and finally reached after 7 and 8 days of travel respectively. These unscheduled delays impede us for having oocytes prepared for the IVF so we had to freeze down the sperm that wasn´t already in the best conditions, and so had to prepare a special IVF to recover these strains with samples that were expected to have high mortality and down fertility. To solve this situation in the future we have also developed oocyte vitrification.
In the strain that came from Madrid the problem wasn´t transportation, but the shipment was delayed because the sending institution didn´t have the commercial media to suspend the epididymis. For that reason we conducted experiments with paraffin oil, available in any laboratory, demonstrating its usefulness in short duration transportation.
ID: 41
Instituto de Neurociencias de Alicante CSIC-UMH
Cryopreservation not only saves space and money, it is also essential for the 3R´s policies in an animal facility. Archiving germplasm provides a ‘back-up’ against the loss of mouse colonies due to genetic contamination, disease outbreak, breeding cessation or natural disasters, and for that it´s in fact an indirect tool for reduction because a lot of animals had been invested to develop a single genetically modified mouse strain (GMMS).
The most important and direct reduction is achieved cryopreserving maintained live breeding strains not under current investigation. Each of them could represent a minimum of 50 unused animals. But a cryopreservation laboratory can offer more reduction by applying efficient and productive assisted reproductive techniques as In Vitro Fertilization (IVF) for rederivation, embryo cryopreservation, colony expansion, strain rescue or cryopreserved/refrigerated germplasm importation.
We have 9 years’ experience working in mouse embryology, 6 of them producing embryos by natural mattings and the last 3 with IVF, and our historical data reflects that the IVF has produced a decline of at least 2,5 times the number of animals used for our experiments.
In 2014 we rederived 17 GMMS, cryopreserved 66 GMMS (30 of them were eliminated), performed 2 colonies expansions, 2 strain rescues, and imported 8 cryopreserved/refrigerated GMMS. We used for all those services 91 males and 301 females.
If we extrapolate all this information we can see that our laboratory has produced the last year a reduction of at least 1500 animals cryopreserving live breeding colonies not under current investigation and approximately 588 animals with the IVF refinement for a total of more than 2000 animals.
ID: 50
1Faculty of Medical Sciences, UNICAMP
2UNICAMP
The recognition of the harmful effects of pain upon laboratory animal physiology demands a continuous search for experimental refinement techniques. Yet, the concern with possible pharmacological interactions remains as they may represent important bias. On one side, ethics committee members suggesting the use of drugs to minimize pain; on the other side, researchers equally concerned with animal’s welfare, but seriously preoccupied with the undesirable interference between pain killers and the test substance. Herein we propose a modification in the carrageenan paw edema experimental model in rats in cases where only the anti-inflammatory – and not the antinociceptive - biological activity of the test substance is under evaluation. In our refined model, the intense pain caused by the intraplantar administration of carrageenan is minimized by the sciatic nerve block immediately before the induction of the inflammatory response. Through this refinement technique, it is possible to stablish a new experimental protocol, with higher statistical significance between control groups (p < 0.001), bigger homogeneity among individuals of a same group, as well as a better handling of the animal at the moment of the carrageenan injection and along the subsequent evaluation in plethysmometer, when compared to the original protocol.
ID: 69
GlaxoSmithKline R&D. Diseases of the Developing World Drug Discovery Center. Laboratory Animal Sciences Department
In our R&D center, pharmacokinetic (PK) studies are regularly carried out in rats to determine the pharmacokinetic properties of new chemical entities in order to progress the best compounds to the development of new treatments against malaria and tuberculosis. All rat PK studies are performed in surgically cannulated animals for serial blood sampling as recommended by FELASA and the NC3Rs. The Laboratory Animal Science department has been working on the refinement of this surgical model with the use of the PinPortTM, which is a miniature access port for cannulated animals.
In this work we describe the standard cannulation procedure with the new system, including catheter preparation, anesthesia and analgesia, surgical technique, post-operative care of animals and sampling technique. We also describe precautions to keep catheter’s patency.
Once the animals have been cannulated, a PinPortTM is attached to the exteriorized catheter. Blood sampling or intravenous administration of substances can be done directly from the miniature port with the use of a modified needle. With this system there is no need to use other connection devices, like swivels or harnesses. Therefore, rats are totally free to move in their cages and can be housed with the standard enrichment and cages. Additionally, this system reduces time and resources, as it is a very straightforward technique that can be performed by only one single trained person.
In conclusion, the new technique described in this work provides a fast access to externalized catheters and improves the welfare of animals in PK studies.
ID: 70
Laboratory Animal Sciences, Diseases of the Developing World Drug Discovery Center. GlaxoSmithKline R&D. Tres Cantos, Madrid, España
In this study different sources of heat to dilate mouse veins for blood sampling were tested: a infrared lamp (LI), a standard warming-cabinet (AC) and a mini warming-cabinet (MT). The objective was to assess which one was better with regard to animal welfare and sampling technique. Female C57BL/6 were sham-dosed and sampled at 20, 40 and 60 minutes from the tail veins after warming for 10 minutes. At each time point, time and ease of sampling were recorded. Body weight was also recorded before dosing and after the last sample. Then animals were euthanized and blood sampled for hematology and biochemistry analysis. Controls included a non-handled group (basal control, CB) and a group that was dosed and sampled but not warmed (procedure control, CP). The complete pharmacokinetic procedure could be considered relatively stressful for mice, as shown by body weight loss, corticosterone elevation and the alteration of a few hematological and biochemical parameters. Mice sampled without warming did not show lower stress, and warming was necessary to obtain rapidly a sample of sufficient volume. The most effective warming source was the LI, although it was also the one that produced the highest rise in corticosterone. Consequently, it is recommended that the AC and MT are used preferentially over the LI, except in situations in which, due to the specific characteristics of the animals, a more efficient warming is necessary. Further work is needed with albino strains to control for the effect of coat colour in the results of this study.
ID: 117
Instituto Butantan, São Paulo, Brazil
Blood sampling from the gingival plexus in guinea pigs and hamstersThe search and use of novel alternative procedures and refinement of techniques are required by international and Brazil laws for the use of laboratory animals in research. Due to the lack of a long tail, access to blood vessels in hamsters (HM) and guinea pigs (GP) is more limited than in other rodents. However, HM and GP are extensively used as models for neglected diseases and vaccine testing. Thus, we evaluated the feasibility of the gingival plexus as an alternative anatomical site for repetitive blood sampling from GP and HM. Both species were bred and maintained under controlled sanitary standards, with free access to water and food. We studied twelve 4-week-old individuals (6 males and 6 females) from each species (IACUC N°1122/13). At weekly intervals, the experimental group (3 males and 3 females for each species) was subjected to i.p. anesthesia and blood (200 µL) was collected by puncturing the gingival plexus, located immediately caudal to mandibular incisors. Control animals were anesthetized, but not bled. No alteration in behavior or weight gain was observed, and hematological values did not change over 6 weeks. Gingival puncture provided easy collection of at least 500 µL of blood and did not evoke any conspicuous local injury. Furthermore, at the end of 6 weeks, minor damage to the blood sampling area was observed histologically. Our data demonstrate that the gingival plexus is a safe alternative for routine blood sampling in HM and GP.
Financial support: Fundação Butantan, FAPESP 2013/25177-0.
ID: 122
1UTAD - Veterinary Sciences Department from the University of Trás-os-Montes and Alto Douro, Quinta de Prados, Vila Real, Portugal
2IBMC - Instituto de Biologia Molecular e Celular, Laboratory Animal Science, Universidade do Porto, Porto, Portugal
3CITAB - Centre for the Research and Technology of Agro-Environmental and Biological Sciences
4IBB-CGB - Centro de Genómica e Biotecnologia
Zebrafish (Danio rerio) has emerged as a popular toxicological model in contrast to other vertebrate model organisms like rodents and non-human primates. Some anaesthetic protocols that are frequently used in laboratory are responsible for the introduction of unwanted variables like toxic and teratogenic effects. Ketamine is a noncompetitive NMDA receptor antagonist that may be responsible for a direct neurotoxic effect in the developing brain, making its use controversial. This study aimed to analyze the expression of two NMDA receptor subunits, by RT-PCR, during the first six days of embryonic development, after exposure to ketamine.
The experimental protocol consisted in a 20 min exposure during early development of AB-type zebrafish to ketamine concentrations of 0.2, 0.4 or 0.8 mg/mL. The expression of grin1a-grin1b (NR1)and grin2da-grin2db (NR2) genes was analyzed by real-time PCR.
When ketamine exposure was done during blastula, NR1 and NR2 expression was down regulated; this decline was also seen during gastrula for NR2 subunit genes. By the contrary, an up-regulation was observed when exposure was performed during segmentation phase.
The results obtained proved to be consistent with studies performed on rats and monkeys exposed to NMDA receptor antagonists, where neurotoxicity was observed; on the primate case it was also detected the increase of NR1 transcripts on regions affected by the exposure.
The development of this work and the use of zebrafish as a model organism for toxicological studies may have a great value on the development of new approaches that will allow the implementation of the 3R’s principles.
ID: 124
1UTAD - Veterinary Sciences Department from the University of Trás-os-Montes and Alto Douro, Quinta de Prados, Vila Real, Portugal
2IBMC - Instituto de Biologia Molecular e Celular, Laboratory Animal Science, Universidade do Porto, Porto, Portugal
3CITAB - Centre for the Research and Technology of Agro-Environmental and Biological Sciences
Ketamine is a widely used anaesthetic which has been described as being neurotoxic and teratogenic. In this context, the zebrafish has become an attractive model for these type of studies. This study aimed to investigate the differences between short and long term exposures to ketamine by observing the oxidative stress responses of the zebrafish embryos as biomarkers for ketamine-induced toxicity during early zebrafish development.
In the short term assay, embryos were exposed during 20 minutes to ketamine concentrations of 0.2, 0.4 or 0.8 mg/mL, during early stages of development (blastula, gastrula and segmentation). The long term exposition, comprising all phases, last for 8 or 24 hours at concentrations of 0.05, 0.07 or 0.09 mg/mL. Four exposure replicates were performed (100 embryos/group). The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), glutathione ratio (GSH/GSSG), lipid peroxidation (LPO), protein carbonyls (PC) and reactive oxygen species (ROS) were evaluated by colorimetric and fluorimetric methods at 8 and 24 hours post-fertilization (hpf). At 8 hpf, a significant increase in SOD and CAT activities of embryos exposed during the gastrula and for long exposure were observed. PC were also significantly increased at the same period for the long term exposure. At 24 hpf, ROS quantities were increased after long exposure. No significant variations were detected on the remaining parameters.
These data indicated that the most sensitive biomarkers of ketamine exposure were SOD, CAT, PC and ROS in zebrafish embryos and highlights the value of zebrafish for studying ketamine oxidative stress effects.
ID: 133
1Fundación Profesor Novoa Santos
2Centro Tecnológico de Formación-UCEX-CHUAC
3Servicio de Cirugía General CHUAC
Centro Tecnológico de Formación (Technology Training Center) of the Xerencia de Xestión Integrada da Coruña, managed by the Fundación Profesor Novoa in partnership with the Fundación Amancio Ortega Gaona, is a pioneering, innovative, state-of-the-art institution that aims to meet healthcare training needs. Professional excellence, healthcare quality assurance and patient safety are the Centre’s goals and medical simulation is the basis of the teaching methodology used.
This centre provides hi-tech equipment to meet common institutional objectives and goals. This endowment includes units and modules of virtual reality equipment for medical simulation, primarily in the fields of interventional radiology, anaesthesiology and microsurgical techniques.
The participants have to deal with the simulation of complex circumstances, where they must use their knowledge and acquire skills to handle real clinical situations.
The facilities are multi-functional. They offer training programs in different subject areas and professional fields, as well as the simulation of virtual reality clinical situations and complex surgical procedures.
The simulators for gastrointestinal endoscopy, laparoscopy and arthroscopy, offer students a basic step giving them a unique opportunity to practice psychomotor skills and complex techniques and procedures in a safe environment without putting patients at risk and preventing the use of animal models for teaching purposes.
In advanced simulation using both robots and human actors, real situations are presented in which training trials are focused on leadership skills, coordination, communication and decision-making and in short, team work. A monitor-led group discussion supplements the learning experience.
ID: 150
IPB López Neyra (CSIC)
The objective of this work is to improve the design of an experiment for obtaining neonate mices, whose brains are used for getting nerve cells. These cells in biomedicine are widely used in studies of cell biology and immunology, related neurodegenerative diseases.
It aims to reduce the number of animals used, improving reproductive efficiency and the environmental enrichment.
Three groups of 8–10 females were established at the beginning of their reproductive life, which will mate four days a week for 6 months with two groups of three males. We take advantage of the postpartum estrus and we reduce the interval between births.
We design daily record sheets of the presenceof copulatory plug, the pregnant females, mices giving birth, litter size and their variability.
The environmental enrichment protocol consistsof providing different types of manipulative natural materials for the animals.
The females that don´t get pregnant in at least two consecutive weeks will be removed from the experiment.
Neonates mice obtained in caesareans birth from dystocic female, will be used in the experiment and the mothers will be sacrificed.
We expect to obtain at least two litters of mice per week.
The environmental enrichment used will stimulate exploratory behavior and reduce the appearance of aggressive behavior between the males.
The correct recording of the data allows us, to remove advance infertile females from the experiment, to detect problems in the births, further being able to prevent deaths due to dystocic birth of mice, and we can reanimate their pups.
ID: 152
*equal contribution
1Unidad de Medicina Comparada, Fundación Centro Nacional de Investigaciones Cardiovasculares, Madrid, España
2Charles River Laboratories, Barcelona, España
3Fundación Centro Nacional de Investigaciones Oncológicas, Madrid, España
This report describes a success protocol focused to avoid fostering (Reduction)with highsurvival (Refinement), applying a specific animal handling (pre-and postsurgery) and a 10 min surgical procedure. We compare two different ischemic-induction techniques and the application of analgesia by buprenorphine or bupivacaine.
Litter are kept on nesting material over a warming blanket at 40°C
Hypothermic immobilization followed by criolesion or permanent silk-knot as heart injury
10ul-drop of analgesic subcutaneous over the closed-chest, wounding the skin with a bioglue-drop
Cover again with the nest material and surgical recover for 2 h at 40°C
Re-introduction of the litter: ensuring their impregnation with the cage-mother odor scents.
ID: 158
1Departamento de Nutrición, Bromatología y Tecnología de los Alimentos. Facultad de Veterinaria, UCM.
2Department of Nutritional Sciences, School of Biosciences, University of Nottingham
Immunoassays are widely used in biomedical sciences, and they require antibodies usually raised in animals. To fulfil the Three Rs principle, Directive 2010/63/EU encourages the replacement of live animals in procedures by alternative methods not entailing the use of live animals. Phage display –a technology based on the expression of billions of different antibody fragments on the surface of filamentous phage particles– is presented as an alternative to traditional mAbs production.
In this research work, three specific antibody fragments that allow the detection of almond, walnut and Brazil nut allergens using ELISA techniques were isolated from a commercial phage-scFv library after performing two rounds of selection (biopanning) in three independent experiments.
The biopanning procedure was carried out by exposing repeatedly Tomlinson I library to a protein extract-coated surface, and subsequently recovering the best phage-binders. After a deeper characterization that included the assessment of scFv integrity (by PCR and nucleotide sequencing), specificity (by testing different vegetal and animal species) and sensitivity (by assaying different concentrations of target species) of the isolated clones, three specific phage clones were selected. Phage-ELISA assay was also validated by analysis of commercial food samples.
The phage clones selected and characterized demonstrated to be highly specific, allowing the detection of target proteins in the range of 110–5000 ppm. Furthermore, affinity and specificity of these selected phages are being further improved by molecular engineering. The results obtained demonstrate that phage displaying libraries can be used as an alternative to traditional mAbs, therefore accomplishing the replacement principle.
ID: 159
Diseases of the Developing World Drug Discovery Center. GlaxoSmithKline R&D
The objective of Diseases of the Developing World Centre, from GSK Spain, is the selection of candidates to achieve preclinical phases of new treatments for malaria and tuberculosis. Strategy is based on the integration of all the data coming from the experimental animal work: Efficacy + Exposure + Tolerability.
The development of new drugs become more and more expensive and it's imperative to reduce the attrition rate. For this reason and because of the commitment of GSK with 3Rs principle, we have designed a combination study to get both, tolerability and systemic exposure data of compounds from the same individuals.
This experimental design make an estimation of the maximal tolerated dose of a drug after a single dose in male Swiss CD-1 mice. Two steps are necessaries to get this approach. In the first section, we applied a modification of Up & Down method based not just in survival, also in in-life observation (Irwin Test) to select that dose without any signs. Those doses levels are used as reference to calculate SD95 and SD05 (doses that could cause adverse effects in 95% and 5% of animals, respectively). Two animal groups will be administered with these dose levels selected. After 24 hours, samples for clinical chemistry and necropsy will be taken. Also, sparse sampling of blood will be used to get the exposure of the compound in both groups during these 24 hours. We present several examples of this experimental design used in our centre.
Design and management of animal facilities
ID: 34
Vivotecnia-CNIO
In CNIO, water is filtrated, autoclaved in polysulfone bottles and analyzed once a year. In some experimental procedures, treatments are added to the drinking water. Bottles used are opaque to avoid the photosensitivity, to distinguish between them and to avoid confusion during bedding change.
Sanitation process of bottles is important to eliminate product´s remains. Usually, the substance in the water is eliminated during washing procedures, but how can one be sure?
Last year, a new water treatment was performed in a project. The substance administered (NINTEDANIB) was yellow. Subsequently, during a normal revision, workers could see yellow remains in freshly washed bottles after autoclaved process.
We decided to check washing process of bottles. We connected with Head of Medicinal Chemistry, who recommended to make a Liquid chromatography–mass spectrometry of water bottles (WB). Six bottles were analyzed, three WB with Doxycycline + Glucose and three WB with NINTEDANIB (first without wash, second one with one wash and third one, two washes). The goal was to determinate the presence or absence of substance checking bottles washing process in bottles with treatments.
Doxycycline was eliminated after the first wash but Nintedanib bottles will need the two wash process to be eliminated. If substance used in procedure had not been yellow then rest had not been detected and mice of other procedures had drunk water with the rest of this substance.
In conclusion, analysis of the water should be carried out for bottles with normal water and for WB with treatment after washing.
ID: 80
1Biotério ICBAS-UP, Portugal
2Vimatechlab, Portugal
3Amorim Cork Ventures, Portugal
The Guide for the Care and Use of Laboratory Animals states that rodents should be housed with bedding because it allows foraging, digging and nest building and absorbs urine and feces. Considering the potential water adsorption and cluster formation capacity of cork, we decided to investigate the use of this material as an alternatively to corncob. 12 Female Wistar Han rats) were divided into 4 groups: corncob and cork (3 different particles sizes: 1/2 mm, 2/3 mm and 3/4 mm), each containing 1000 mL of bedding. All animals were monitored daily for 6 days. Preliminary results indicate that cork bedding size 1/2 mm (13.11 ± 3.19%, n = 2), 2/3 mm (23.70 ± 4.80%, n = 2) and 3/4 mm (16.53 ± 1.49%, n = 2) were able to make more clusters (%, day 5) than corncob (2.77 ± 2.03%, n = 2). Results indicate that the volume increased 5% in cork 1/2 mm and 30% in corncob bedding. On the other hand, cork bedding weight increased 34% (size 1/2 mm), 62% (size 2/3 mm) and 59% in 3/4 mm, while corncob bedding variation was lower than 24%. Moreover we observed that cork bedding 1/2 mm (69.00 ± 8.49%, n = 2) was unable to significantly change rats gastrointestinal motility, since total fecal pellets bedding count was similar to corncob (71.00 ± 1.41%, n = 2). No evident loss of animal welfare was observed since rats behavior, body weight variation (Corncob: 10.40 ± 4.04 g, n = 5 and Cork: 8.40 ± 2.88 g, n = 5), diet (Corncob: 18.27 ± 1.19 g, n = 6 and Cork: 16.97 ± 0.85 g, n = 6) and water (Corncob: 28.53 ± 1.97 mL, n = 6 and Cork: 27.87 ± 1.34 mL, n = 6) intake had minimal differences between the two groups. Considering these data, we believe that these comparative studies should continue, in order to evaluate if cork may became an alternative to corncob as a bedding material for laboratory rodents.
ID: 123
1CNB
2CSIC
3Vivotecnia
4Anapath
Disasters are one of the most frightened events of the animal facilities. It has implications in animal survival, microbiology contamination, and changes in welfare, production and experimental results.
Smoke, from a fire happened in the CNB, affected a barrier area housing SPF mice in IVC and filter top cages. Furthermore electric power failed and the area was without lights 48 h, 72 hours without power, unventilated 7 days and 14 days without autoclave and washing machine. The area was opened for 48 hours after the fire to remove smoke. The cages were not opened and animals were not changed for 14 days. On 7th day the area was disinfected and barrier ventilation was restored. At 7 days post fire death animals were not found. Animal welfare or production changes were not observed. Health screenings performed 1 and 4 months after the fire were negatives in all animals tested. In our experience the more relevant factor in an emergency is maintaining the integrity of the cages and filter tops and no handling animals as much as possible.
If we maintain minimum conditions of ventilation, temperature and quiet, the impact on welfare and production will be minimized, and we will be able to maintain the same microbiological status of the animals.
ID: 127
Universidade Federal Fluminense
Sawdust is a byproduct of the lumber industry, which can cause great environmental impact if its disposal is not done correctly. This material is used as bed in rodent vivarium in Brazil, and is thrown away after use. An alternative use of residue, rich in feces and urine, can be used as an organic compost, which would minimize damage to the environment. Composting of organic waste favors the plants in many ways, providing nutrients, structuring degraded soils and promoting the formation of a small ecosystem that is of great importance for soil microbial life.
The objective of this work is to evaluate the production of organic composts obtained from wastes of laboratory animals (rodent) from UFF’s vivarium. The experiment consisted of three treatments: control (sawdust after use by animals); sawdust (50%) with addition of bovine manure (50%) and sawdust with addition of Embiotic® Line (compost accelerator). Each treatment had three repetitions. Embiotic® Line was “enabled” to be used. The composting process lasted 120 days. At the end of the process, composting material presented characteristics of an organic compost, as absence of odor, texture and coloring. The treatments presented different levels of macronutrients. The pure residue of the vivarium proved to be an excellent material to be used in the production of organic compost, characterized as an alternative for reducing impact on the environment.
ID: 144
Zoetis Manufacturing & Research Spain
In September 2010 the European Parliament and the Council of the European Union launched a new guideline on the protection of animals used for scientific purposes, Directive 2010/63EU. Since then, poultry studies with layers were performed using same cages than in the field; individual very narrow cages of low height and with a small surface per animal.
Zoetis is currently working in the new products for layers. The main claim in layers products is prevention of egg drop caused by infectious agents.
The statistical design of our studies determined that in order to observe an egg difference of 2 eggs between treatment groups (with a standard deviation of 1.5 eggs), 10 exact replicates per group would be needed to have statistically significant differences.
With this scenario and in order to perform studies in layers in compliance with the new directive, animal welfare requirements and the complex statistical design, new cages were designed and built.
New cages have a surface of 0.84m2 (capacity for 4 chickens of weight > 2400 g) and a height of 75 cm. Cages are equipped with a feeder of 45 cm length, two multidirectional automatic drinkers, a nest, a perch and wood shavings as bedding.
Moreover, cages were also designed to facilitate animal technicians work and reduce chickens stress during manipulations. Cages are provided with small lateral door on the nest (in order to reduce the stress during egg collection), wheels (to easily move them for cleaning and birds manipulation) and with a small front door in order to efficiently change the bedding without disturbing and / or frightening the chickens.
Ocupational Health and safety
ID: 26
1Department of Animal Production. University Of Lleida-Agrotecnio Center, Lleida, Spain
2Scientific and Technical Services, University of Lleida, Lleida, Spain
3Department of Medicine, University of Lleida, Spain
The air dust weight and respirable particle diameter (smaller than 10 µm) was evaluated in a conventional open cage rodent rooms whit the nephelometer DataRAM 4 (Thermo Fisher Scientific, Franklin, Madison, USA) at five conventional rodents rooms (C1, C3, C4, C5 and C6) to establish the differences between them at morning working time (7 to 13 hours).
Also to determine the influence of daily activity inside the room, the dust weight and particle size was determined in 3 periods (“morning”, “afternoon”, “night”) and repeated for five days. Results are the mean ± SE, the airflow rate was 2 litres per minute and the dust was measured each 10 seconds.
Significate differences (p < 0.05) were found for air dust weight, (C1, 17.01 ± 0.09a µg/m3, C3, 11.00 ± 0.13d µg/m3, C4, 4.25 ± 0.04e µg/m3, C5, 12.70 ± 0.13c µg/m3, C6, 13.82 ± 0.07b µg/m3), and diameter size between rooms (C1, 0.235 ± 0.05 cd µm, C3, 0.241 ± 0.056b µm, C4, 0.371 ± 0.102a µm, C5, 0.230 ± 0.056d µm C6, 0.239 ± 0.051bc µm).
The maximum dust weight was found at the morning (12.89 ± 0.09 µg/m3), followed by night (7.02 ± 0.08 µg/m3) and the afternoon (2.33 ± 0.02 µg/m3). The mean particle size by period was: night 0.784 ± 0.012 µm, afternoon 0.365 ± 0.002 µm and morning 0.233 ± 0.001 µm, all these means are significantly different (P < 0.05).
It was found a significate negative correlation between the dust weight and particle size (r = −0.774, P < 0.001), at the morning.
Our results demonstrate that there are differences between rooms and daily activity for dust weight and respirable particle size.
ID: 59
Servicio Prevención. Universidad Extremadura
The Animal Facilities at University of Extremadura (Cáceres campus) has three areas:
– Research Zone,
– Breeding and Quarantine Zone, and
– Administration Zone.
Animal like Guinea pigs, Zebra fish, Wistar rats and Swiss mice are bred; and Genetically Modified Organisms are housed.
– The main tasks are:
– Animal welfare control.
– Research animals breeding.
– Anesthesia, surgery and euthanasia.
– Control of experimental testing of any toxic or genetic manipulation in the experimental animals.
– Potential zoonoses control.
The profesional risk assessment of the of the Animal Housing Facilities is aimed to estimate the magnitude of those risks that could not be avoided. The (INSHT) National Institute for Safety and Health at Work methodology is used, together with the University of Extremadura management sistema of occupational risk prevention.
Therefore, the identified risks are the ones:
– Caused by living beings (bites, scratches and bumps).
– Produced by the work equipment (shock, burns, cuts, needlestick, glassware).
– Caused by chemicals handling (irritations, burns, skin diseases, exposure to CO2, anesthetic liquid).
– Got by exposure to biological risk (zoonosis, ectoparasites, allergens).
In order to eliminate, reduce or control the found risks, preventive actions are planned, and so, deadline, responsible persons and human and material resources are set for its implementation.
– The main planned preventive activities are:
– Information Plan: workplaces, work equipment, energy used, Emergency Plan.
– Training Plan: laboratory prevention, Chemical products, Biological agents etc.
– Personal Protective Equipment Plan: gloves, masks and goggles.
– Periodic evaluation plan.
ID: 62
Servicio Prevención Universidad de Extremadura
An Emergency Plan for the Animal Facilities at the University of Extremadura is developed. Its aims are:
– Setting the guidelines for potential emergencies,
– Choosing the required team that will be properly organized and trained for the efficacy and efficiency of the goals and who will also provide information
– Providing information to all users on how to act in case of emergency, and
– Also organize the necessary relations with external services, as to be coordinated.
Law 31/1995 on Prevention of Occupational Risks (LPRL in Spanish) states that the employer must ensure the safety and health of employees in emergency situations by:
– taking the necessary actions on first aid, fire performance and evacuation of workers, and
– choosing the right personnel to implement such actions and to check its right performance.
All of our Emergency Plans follow the guidelines of the implementation of the emergency plan at University of Extremadura.
In the Animal Facilities, have been implemented as follows:
Meeting of the prevention technician and the staff where that personnel will be informed about the protocol to be taken and will collect all the documentation (projects, plans, manuals, procedures, inventory equipment for fire protection, usage instructions, etc.) including the main emergency risks to be handled. Checking of the building firefighting devices and elaboration of the action plan. Nomination and training of members of the intervention teams. Planning evacuation drill. Carrying out the drill. Report of actions and suggestions for improvement.
ID: 76
1Parc de Recerca Biomèdica de Barcelona (PRBB)
2Charles River Laboratories
3FREMAP
Nowadays, the position of animal technician in a rodent animal facility of any large research institution involves the development of a limited number of tasks for extended periods of time. In many cases, the lack of an ergonomically designed equipment or the incorrect use of it, plus prolonged bad posture can lead to musculoskeletal injuries that impact negatively on the health of workers and consequently the productivity of the workforce.
The aim of this study is to evaluate two processes, cage changing and tail biopsies, from an ergonomic point of view. For this we have used the “Move and Reach” motion capture technique. Through sensors and cameras placed in the caretaker, the system allows visualization of the work routine and subsequent analysis of ergonomic stress point’s data. The study was conducted in the Barcelona Biomedical Research Park Animal Facility, which hosts about 30,000 mice in ventilated racks, performing the change in flow cabinet.
With this work the ergonomic risks have been detected and recommendations established. Such information is highly valuable to be included in the implementation of working procedures, in order to minimize musculoskeletal problems resulting from these specific workplaces. Furthermore, health and safety training plan includes these ergonomic recommendations specifically tailored for caretakers and animal technicians.
ID: 103
1CITAB / UTAD Vila Real, Portugal
2LABORIAL, Maia, Portugal
Necropsy studies are needed in laboratory animals sciences. This study aimed the identification of ergonomic and functional needs of pathology benches, followed by the design of a bench in accordance with the requirements for good practices.
At first, information on the characteristics of currently available benches and accessories in the market were collected. Secondly, interviews were conducted to survey the concerns and difficulties to technicians (n = 4) and veterinary anatomic-pathologists (n = 10), as well as visits to laboratories in the North of Portugal.
The results suggest: 1) risks resulting from exposure to fixing agents as a consequence of poor exhaustion; 2) traceability difficulties of biological material; 3) the need for auxiliary equipment for macroscopic examination, such as scale and camera; 4) lack of user-friendly technologies for data recording; and 5) complexity in the organization and access of records. It was, however, not possible to establish order of magnitude of the results presented above.
This study found that the perceived needs are according to some already described. The survey conducted stressed the need to create products with integrated solutions. Thus, a pathology bench was designed with ergonomic and functional aspects optimized. The pathology bench include: 1) double filters, enhanced exhaust; 2) an interactive clean surface with suitable software to record the observations, information management and traceability; 3) integration of auxiliary equipment, without the need of bringing accessories inside the laboratory.
Funding - “IntelLab II -Inteligência em Laboratórios”, FCOMP-01-0202-FEDER-033877 - financed by European Community Fund (FEDER) through COMPETE -POFC.
Education and training in Laboratory Animal Sciences
ID: 24
Charles River Laboratories
One of the most challenging aspects about complying with the education and training requirements specified by Directive is “… assuring the competence of all persons involved in the use, care and breeding of animals …”. Satisfactory completion of accredited courses is just the first steps of a training programme. It is not until competence is satisfactorily attained and assessed, that the training process can be regarded as completed. During this porcess, the selection of appropriate, competent supervisors is crucial, and therefore, determining if, when and how an individual can supervise and assess the competence of trainees becomes critical in the success of our institutional training programme. Charles River has developed a thorough Learning Management System (Charles River Campus) to monitor the whole training process. With this elearning environment, we are able to track, monitor and document training activities from entry-level trainee induction to meeting knowledge, experience and soft skills necessary to assess others’ competence. Using Charles River Campus environment and taking GEMs Colony Management as a case study, we will start from scratch and cover all steps and activities required to achieve competency in assessing competence in this complex task and becoming an assessors’ assessor. In our experience, training is a continuous process that must be tailored to staff needs, resources and capabilities in order to be successful. A blended learning approach provides the necessary flexibility to offer customised training to our staff in a way that gets them involved in the process and allows them to design their own professional career.
ID: 99
Dpt. Pharmacology, Toxicology and Legal and Forensic Medicine, University of Córdoba
The European convergence process represents challenges for the University professors, and then, it is essential the availability of different teaching resources such as informatics tools, with which the students can learn different aspects from veterinary subjects such as Pharmacology, Toxicology and Legal Medicine. Thus, the use of new technologies for the transmission and reception of transversal contents, as well as access to essential database, will play a key role for learning to basic contents. The objectives of the present work were toease the learning in pharmacokinetics, pahrmacodynamics, food security, the exposure and risk assessment and regulatory aspects from Spanish and European Agencies such as Agencia Española de Medicamentos y Productos Sanitarios (AEMPS) and European Medicine Agency (EMA) in order to learn in an easy way to previously appointed items. These include the use of interactive computing tools that it is important in a world in which the use of alternatives to use of animals in research and education is one of the more relevant objectives.
We propose interactive computational programs for the students that may understand concepts and applications in pharmaco/toxico-kinetics and parmaco/toxico-dynamics such as time-concentration and drug-response curves respectively. Finally several link to different web with data-bases and virtual systems for transferring pharmacological, toxicological and regulatory items are also proposed.
ID: 111
1Instituto de Investigação e Inovação em Saúde, UP
2Instituto de Biologia Molecular e Celular, UP
3Faculdade de Psicologia e de Ciências da Educação, UP
E-learning is increasingly used in Laboratory Animal Science (LAS) training and appears to be a promising solution to improve time management and resource use for both teachers and students.
We have integrated e-learning in our advanced training[i], as a complement to classroom lectures (blended learning) and in our introductory course[ii] (theoretical part exclusively delivered by e-learning).
To assess participants’ acceptance of the e-learning platform and level of satisfaction of its use during the last 4 years with E-learning Acceptance (QELA), a concordance Likert-type scale. This study included 229 participants (60% from the C course), 21 to 50 years old (M = 28.81; SD = 5.96), of which 76% were women, mainly PhD students and postdocs.
The study results show that the e-learning platform was well accepted by the majority of the participants (92.5%).
In both courses participants were very satisfied with the e-learning organization (60.1%), contradicting the idea that in “lonely learning” students easily feel lost and without teacher support.
The majority of students (95.6%) considered that e-learning positively influenced the acquisition of practical competences; thus although the development of practical skills demand hands-on training, e-learning provides relevant preparation for practical training.
Results confirmed that participants recognized the usefulness of e-learning time management (94.3%).
[i] Accredited FELASA Category C Course
[ii] Follows the recommendations for FELASA Category B Course
Miscellanea
ID: 30
Universidad de Salamanca
When studying the main effects of stress many drugs are administered via intraperitoneal injections, but the possibility of its aversion value and physiological consequences are not considered. Thus, the aim of the present study was to examine the stressful effects caused by repeated intraperitoneal injections in rats of both sexes. The animals of 70–72 days of age were subjected to daily intraperitoneal injections with vehicle (1 ml/day for 8 days) or not injected (Controls). The behavioral effects of the procedure were determined by evaluating changes in the acoustic startle reflex and it inhibition (PPI). Upon it, the physiological response to an acute stress, by immobilization (IMO), was examined in all animals and compared with a group of undisturbed controls (No-IMO). Our data show that in Injected animals, of both sexes, PPI increased and both latencies of the startle and PPI decreased.
The injections procedure affected the body weight gain in both sexes, and increased significantly the metabolic response to the new stressor. The Injected animals also displayed changes in the haemostatic stress response and differences in hematocrit (%) and in the mean platelet volume are reported when compared to Controls. Moreover, whereas the acute condition of IMO stress by it-self did not affect the leukocyte profile, the Injected animals exhibited leucopenia. Our results together suggest that the daily intraperitoneal injections procedure is enough to induce behavioral and physiological changes, in both sexes, and must be taken into account in the interpretation of the results when used in different experimental paradigms.
ID: 83
1CCMIJU
2HGU Gregorio Marañón
The Langendorff System (LS) can be used to keep isolated hearts viable for cardiovascular studies. It is commonly used with rodents’ hearts. Since swine are considered the most suitable species for cardiovascular research, our aim was to establish the LS to test ex-vivo viability of pig hearts and to validate it performing electrophysiological studies in infarcted hearts.
For this purpose, hearts were explanted from 10 anaesthetized pigs. After thoracotomy the aorta was clamped to avoid air entering the system and the hearts were excised and washed with cardioplegic liquids (4°C). A retrograde perfusion cannula was inserted in the aorta and fixed to the LS. Tyrode solution (37°C) was perfused through it at enough pressure to keep the hearts viable for up to 6 hours, during which electrophysiological studies were performed.
All hearts were successfully explanted and connected to the LS within 30 minutes of euthanasia, except one case with pericardial adhesions in which the process was longer but also successful. Four hearts developed ventricular fibrillation and required defibrillation using a 30J shock to recover rhythm. Electrical activity and activation patterns were recorded correctly in all cases. All experiments were completed successfully and terminated by the operator.
Here we describe the implementation and validation of a large animals’ LS for cardiovascular applications. Swine infarcted hearts can be subjected to LS and electrophysiological studies performed successfully, which in this experimental setting proved a valuable tool for increasing the data obtained from each study, which could help decrease experimental animals usage.
ID: 112
1Instituto Universitario de Investigación Biomédica y Sanitaria de la Universidad de Las Palmas de Gran Canaria (IUIBS-ULPGC)
2Sección de Endocrinología, Complejo Hospitalario Universitario Insular Materno Infantil de Gran Canaria (CHUIMI)
3Hospital Clínico Veterinario, ULPGC
4Animal Facility Unit, RMG-SEA Instituto de Neurociencias de Alicante, Universidad Miguel Hernandez, CSIC
Glycated haemoglobin (HbA1c) is the best indicator for plasma glucose concentration over long periods. In mice, there are no recommended devices or standards for HbA1c, and glucose metabolism characterisation is based on fasting glucose, oral glucose and insulin tolerance tests (OGTT and ITT). Our aim was to establish a cut-off point for HbA1c measurements in diabetic and non-diabetic mice, using a point-of-care (POC) device (DCA-Vantage, Siemens).
Data from previous experiments with diet-induced diabetic C57Bl/6J, and normoglycaemic B6;129S4-Pcmt1tm1Scl+/- and +/+ mice were used. Body weight (BW), fasting glucose (Glucocard-G meter), HbA1c (DCA-Vantage), OGTT and ITT were assessed. Diabetes was defined as OGTT glucose concentrations >200 mg/dL. Correlations with HbA1c (linear regression; Pearson’s test) and COR-curves were evaluated to establish a cut-off equivalent to a glucose concentration of 200 mg/dl, and the specificity/sensitivity for diagnosis.
A total of 205 HbA1c measurements from 82 C57BI/6J and 24 B6;129S4-Pcmt1tm1Scl mice were available. HbA1c (4.6% [3.4–7.3]) correlated positively with fasting glucose, OGTT peak and AUC (p < 0.007) and negatively with ITT glucose nadir (p < 0.001). For diabetic mice (OGTT glucose >200 mg/dL, n = 173 HbA1c), BW, glucose nadir and fasting glucose were the parameters most strongly correlated with HbA1c and the diagnosis of the disease (p < 0.001). Median HbA1c for diabetes was 4.6% [3.4–7.3] and for non-diabetes 4.0% [3.4–4.7]. COR-curves showed an optimal cut-off for the diagnosis of diabetes at 4.15% (specificity 60%; sensitivity 75%).
Based on the present results, an HbA1c of 4.2% is proposed for the diagnosis of diabetes in mice. Indeed, standardised, internationally accepted, diagnostic criteria are still to be established.
ID: 113
1Servei D’Estabulari
2Dept Psiquiatria I Medicina Legal, Facultat de Medicina, UAB
To introduce two rat colonies, Romana High Avoidance (RHA) and Romana Low Avoidance (RLA), housed in a conventional area into a barrier facility we decided to rederive them by embryo transfer (ET). The setting-up of this methodology and the obtained results are described.
SD female rats were used as pseudopregnant recipients. To obtain RHA and RLA embryos, natural matings were set-up in the original colonies and oviducts in M2 were transported to the barrier facility. Embryos were collected 48 hours after mating and transferred to pseudopregnant females mated the day before. Inhalatory anaesthesia was used during surgery. A dorsal incision was done to reach the left bursa ovarica, embryos were placed into the oviduct and the surgical wound was closed. Recipient females were maintained in pairs during the first two weeks after ET.
In the setting-up with SD embryos, 4 out of 8 females become pregnant after the transfer of 96 1-cell embryos and 23 pups were born, whereas 47 pups were born from 9 out of 12 females transferred with 137 2-cell embryos.
Finally, 66 pups were born from 20 1-cell and 92 2-cells stage embryos obtained from 20 RHA females. And 38 pups were born from 54 1 cell and 64 2-cells stage embryos obtained from 24 RLA females.
Higher proportion of 2-cell stage embryos may explain better results in the RHA strain. Preliminary behavioural test indicates that phenotypic characteristics of both strains are conserved.
Supported by PSI 2013-41872-P, 2014 SGR 1587 and “ICREA-Academia 2013” to AFT.
ID: 142
Envigo
The objective of this work was to assure animal welfare in a large beagle colony, providinganimal technicians with tools and resources to assist animals under their care between routine veterinary visits.
A dog breeding facility has the need to have 24 h veterinary cover.
This colony has around 1000 animals including breeders and stock animals.
It was not practical to have a veterinary surgeon on site, but animals may need immediate veterinary treatment, so a planneeded to be in place to be able to assist animals promptly when needed.
Aplan was designed with the following objectives:
To enable technicians to provide first aid quickly To provide technicians with resources to treat the most common ailments in dogs. To allow minor treatments to be carried out as and when they are noted during health checks; thereby causing less distress to the animals. To allow technicians to use Veterinary Only Prescription medicines under veterinary guidance.
A veterinary guidance document was produced detailing the presentations of more common ailments with careful explanations of actions to be taken.
The result was an innovative way of ensuring appropriate care for the animals. The objectives were fulfilled with an extra outcome:
Technicians had to undergo specific training. This gave them and increased sense of ownership of their animals, pride in their work and more responsibility for the health of their animals, which resulted on more engaged and motivated technicians.
ID: 148
1Experimental Animal Service, University of Córdoba
2Environmental Protection Service
The Animal Experimentation Service (SAEx) of the University of Córdoba, is currently certified in ISO 9001: 2008 and ISO 14001: 2004. Due to the great commitment of this service for the environment, it has been the first Service to implement and certified the “Clover Program”. The “Clover Program” is an initiative to improve and recognize environmental Centers, Departments, Areas and Services at the University of Cordoba. It is a system of certification of good environmental practices and is organized by the Department of Environmental Protection in collaboration with the Sustainability Workshop.
The main benefit of the “Clover Program” is the continuous, systematic and organized improvement in environmental performance that is derived from the process of implantation.
The standard consists of four checklists that make the certification system into four levels or leaves of a clover. Each level contains different actions corresponding to 8 categories of environmental issues: energy, waste, consumption, transportation, shopping, laboratories, teaching and participation. Each of these aspects have a number of actions to comply, being these increasingly demanding in terms of compliance, as you go through each level of the clover.
The SAEx has been recently certified to this standard, and already have the emblem of the first level or clover leaf. This environmental commitment, together with the certification UNE -EN ISO 14001 is a synergistic action to protect and prevent all possible impacts that our activities can have on the environment with a negative effect, while contributing to save resources.
ID: 154
1Unidad de Toxicología. Facultad de Veterinaria, Universidad de Extremadura
2Departamento de Fisiología. Facultad de Veterinaria, Universidad de Extremadura
Animal experimentation has been and is a key activity for the biomedical sciences, being an issue that generates great social sensitivity. In this study, we intend to perform a review of such a controversial theme as animal testing. Origins, subjects and objectives of this practice, the current both ethical as legislative state and what we expect of it in the coming years. In addition, other issues of more objective scientific interest, as the species most used, the existing laws or alternative methods are addressed.
As main conclusions we can say that historically, the experimental activity has a rich chronology from its beginnings to the present. The legislation that governs experimental activity has followed a path increasingly restrictive and, in response, different alternative methods have been improved in order to reduce, reface and refine the use of a large number of laboratory subjects in research studies. The most commonly used animals as experimental subjects remain very traditional, according to their ease of reproduction and short generation time, being rodents the main ones. However, each biomedical specialty usually considers a number of own requirements when choosing among various species to develop pilot tests, thus rendering more remarkable the diversity of species used in relation to the type of study. It is therefore evident that the experimental activity enters through an era of changes, marked by the pursuit of animal welfare, and development of alternative approaches that will mark not only the future of animal testing, but all the processes on modern biomedical research.
List of exhibitors
Tecniplast-Biosis, S.L. Sodispan Research, S.L. Antonio Matachana, S.A. Charles River Laboratories España, S.A. Panlab, S.L.U. Scil Animal Care Company Cibertec Dinox Sl DSI Dynamimed Edstrom Envigo Granja San Bernardo, S.L. IDEXXBioResearch Janvier Labs Labodia Noray Bioinformatics, S.L.U. Rettenmaier Iberica, S.L. Y Cia Steriltech, S.L Steris Tapvey Estonia Ternox, Lda Vivotecnia ZoonLab
Sponsors
University of Extremadura
Centro de Cirugía de Mínima Invasión Jesús Usón
Laboratory Animals ltd.
Other Sponsorship
Bruker Ingeclima