Abstract
Transparency Agreement
14:30–16:00
This public event counts with the presence of:
A Scientist, Professor and Medical Doctor, discussing the value of animals in science and medicine:
1Life and Health Science Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal and
2ICVS/I3Bs – PT Associate Laboratory, Braga/Guimarães, Portugal
Executive Director of EARA, the association proposing the establishment of the agreement:
European Animal Research Association (EARA)
President of SPCAL, the association facilitating the agreement in Portugal:
1Portuguese Society of Sciences in Laboratory Animals (SPCAL);
2Nova Medical School|Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisboa, Portugal; and
3Department of Physics, Faculty of Sciences and Technology, Universidade Nova de Lisboa, Monte da Caparica, Portugal
Scientific Programme
Oral Communications
Thursday, June 21
16:30–17:30
Opening Lecture
New perspectives in stress research
1Life and Health Science Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal
2ICVS/I3Bs – PT Associate Laboratory, Braga/Guimarães, Portugal
Stressful stimuli in healthy subjects trigger activation of a consistent and reproducible set of brain regions; yet, the notion that there is a single and constant stress neuromatrix is not sustainable. Indeed, after chronic stress exposure there is activation of many brain regions outside that network. In the last decades the field has been mapping the effects of chronic maladaptive stress on the fine structure of the brain and, in parallel, determining its behavioral and functional correlates. This suggests that there is a distinction between the acute- and the chronic-stress neuromatrix. The emerging view is that several factors modulate a dynamic interplay in brain connectivity. Its comprehension will allow for a more holistic perspective of how the brain shifts “back and forth” from a healthy to a stressed pattern and, ultimately, how the latter can be a trigger for several neurological and psychiatric conditions.
Friday, June 22
09:00–10:15
Session 1: Transparency & Communication in Laboratory Animal Sciences
Chairpersons:
Ana Isabel M. Santos
Margarida Correia-Neves
OCi1
Transparency & Communication in Laboratory Animal Sciences
European Animal Research Association (EARA)
In a number of European countries, public and private research institutions have made the decision to adopt new persuasive practices and policies to engage with the public on the benefits of using animals in scientific and biomedical research. These institutions believe that they can play a positive role in public engagement and education. The belief is that being more open and transparent about their use of animals in research could help improve public understanding and acceptance. The need for a collective commitment is also important. These commitments are that institutions; will be proactive in seeking opportunities to explain when, how and why they use animals in research; will provide information to the media and the general public about the conditions under which research using animals is carried out and will explain the benefits obtained from using them compared to other methods of research; will develop initiatives that generate greater public knowledge and understanding about the use of animals in scientific research; will place an animal welfare statement on their institution’s website. In the UK through the Concordat on Openness on Animal Research, and in Spain through the Transparency Agreement, this approach has been codified into national agreements. The presentation will evaluate the experience in these countries, outline plans for how this can work across the EU and make the case why the scientific community needs to talk about animal research.
OCi2
Signing the Basel Declaration for Transparency in animal research: a personal responsibility and a professional duty
1IBMC – Instituto de Biologia Molecular e Celular;
2i3S – Instituto de Investigação e Inovação em Saúde;
3SPCAL – Sociedade Portuguesa de Ciências de Animais de Laboratório
In November 2010, in response to acts of violence by extremist animal rights groups, more than 80 top scientists from across Europe joined in Basel to draft and sign the “Basel Declaration” on animal research, marking an unprecedented effort from the scientific community towards “more trust, transparency and communication on the sensitive topic of animals in research”. In October 2011, the Basel Declaration Society (BDS) was founded, to strengthen public awareness of the importance of animal models in experimental biomedical research, to foster communication between researchers and the public, and to further promote the Basel Declaration and its goals and values within the scientific community. As of June 2018, more than 4.600 scientists (only 0.6% of them Portuguese), along with 58 scientific institutes and associations from across the world, have declared their support of the Declaration, and their personal commitment to transparency and animal welfare in biomedical research. Furthermore, almost 200 researchers are Basel Declaration ambassadors in their countries and institutions.
Aside promoting transparency and campaigning in favour of – relevant, competent, and ethical – animal research, the BDS is involved in several other activities, including media training events, granting awards, carrying out surveys, among other. In addition, since the first 2010 meeting in Basel, four international conferences have so far taken place – in Berlin, London, Rome, and latest in San Francisco, in February 2018 – where scientists and other specialists gather to discuss progress in transparency on animal research, as well as draft, discuss and issue policy documents. These documents provide guidance on various topics, including animal welfare and the 3Rs, ethics, legislation, synthesis of evidence, publication standards, risk assessment, crisis management, use of primates, training, among others.
While committed to bridge the information gap concerning why and how animals are used in research, the BDS aims go beyond promoting better public understanding of science, public relations or political lobbying. It is essentially a movement of scientists for scientists, in which every signatory personally commits to the highest standards of integrity, transparency, and the ethical treatment of animals. In this talk, I will review the main activities of this organization, and invite all to join us and sign the Basel Declaration.
10:45–12:15
Session 2: Quality and Animal Welfare in Laboratory Animals
Chairpersons:
Luís Antunes
Nuno H. Franco
OCi3
Introducing Therioepistemology: The study of how knowledge is gained from animal research
Stanford University, Department of Comparative Medicine, and by courtesy, Department of Psychiatry and Behavioral Sciences, Stanford, California, USA.
For the first time, the scale of the reproducibility and translatability crisis is widely understood beyond the small number of researchers who have been studying it and the pharmaceutical and biotech companies who have been living it. This talk will make the case that this is not a time for despair, but a time of intellectual excitement and hope. The emerging literature on these issues contains recurring themes which represent a paradigm shift, and thus potentially the birth of a new discipline (which we have proposed be termed “therioepistemology”). At the micro level this is a shift from asking “what have we controlled for in this model?” to asking “what have we chosen to ignore in this model, and at what cost?” At the macro level, it is a shift from viewing animals as tools or reagents, to viewing them as patients in an equivalent human medical study. Thanks to the groundwork laid over the last 15 years by a variety of authors, we not only understand many of the causes of poor reproducibility and translatability, but we also know the features of animal work that lead to reproducible work with a good chance of translation. Accordingly, now is a time for action where we manifest change in the status quo of how animal research is done. This talk will outline six questions that serve as a heuristic for critically evaluating animal-based biomedical research for opportunities to make this shift in perspective.
OCi4
Industry Initiatives – Putting animal welfare and 3Rs principles into action
European Federation of Pharmaceutical Industries and Associations (EFPIA)
At EFPIA (European Federation of Pharmaceutical Industries and Associations) there is a foreseen importance of promoting good science and animal welfare, as well as increasing understanding of how the two are intertwined.
Medical research continues apace. It needs to, as many health challenges still remain unmet. Animal studies continue to play an invaluable role in meeting these challenges – both in experimental research, and in ensuring maximum safety of treatments before their use in humans, which is legally required. However, research involving animals poses many dilemmas.
Although pharmaceutical companies cannot avoid the use of laboratory animals to prove that medicines work, it is our priority to meet high standards of animal welfare. To put animal welfare principles into action, we systematically replace animals with alternative methods where possible, reduce and refine the use of laboratory animals (3Rs) and improve standards of care. The presentation will give an overview of the work we do to go beyond compliance, lead by example and openly communicate.
Advances in science are leading to fewer tests and experiments on animals, and to new ways to reduce the impact on animals. This is why dialogue and transparency about the use of animals for medical research and developments in science need to be debated by everyone involved.
The pharmaceutical sector continues to be involved in a number of initiatives, which affirm the key principles of the 3Rs or change the current research paradigm (such as the Innovative Medicines Initiative). Establishing, promoting and maintaining a good culture of care is also a fundamental component if ethical, scientific and animal welfare obligations. EFPIA members have produced a “checklist” to help engage in or enhance discussions on a Culture of Care within establishments.
EFPIA is part of the IMI which is the world’s largest public-private partnership in health with a total budget of €5 billion – half from the European Union and half from the pharmaceutical industry, through EFPIA. The public-private consortia develop new solutions and validate them in R&D practice during project lifetime. This has led to the development of in vitro and in silico methods; elimination of poorly predictive models; development of new improved models and development of alternative tools.
OCs1
Pinworms and mites in laboratory mice: deal with uninvited guests
1Life and Health Science Research Institute (ICVS/3Bs), University of Minho, Braga Portugal;
2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal
E-mail:
Pinworm and fur mite infections can lead to significant negative effects on research and animal welfare. The most common found in laboratory rodents are oxyurid nematodes of the genera Syphacia and Aspiculuris, and mites of the species Myobia musculi, Myocoptes musculinus and Radfordia affinis. Despite the efficient treatment strategies described in the literature, the persistence of pinworm and mite infections in rodent facilities indicates that other factors are contributing to the failure of the eradication processes such as: environmental egg contamination, longevity of egg viability and ineffectiveness of traditional methods of sanitation. In this work we present a multidisciplinary approach for the eradication of pinworm and mites in a rodent facility, which includes (i) animal treatment, (ii) animal transfer and (iii) sanitation measures.
Pinworm and mite infection was detected routinely within our rodent facility with approximately 1100 mouse cages housing 52 genetically altered mouse lines. Six females and 5 males with approximately 4-weeks old were selected from each mouse strain.
– Animal treatment: Animals were given medicated diet with Fenbendazole (150 ppm) in an ad libitum regime for 10 weeks. Two topical selamectin administrations (10 mg/kg) were applied, 30 days apart from each other;
– Sanitation measures: Environmental decontamination was performed in facility B to receive animals under treatment and included deep disinfection of surfaces with compounds containing 1% hydrogen-peroxide. Room disinfection was completed after 3% hydrogen-peroxide fumigation of the rooms, ventilation filters and ducts. All the materials in contact with animals were autoclaved before entering the facility;
– Animal transfer: Two weeks after fenbendazole treatment, and after the first selamectin administration, animals were transferred to facility B where they were submitted to the rest of fenbendazole treatment period and to the second selamectin administration;
– In house and external testing: In house perianal cellophane tape testing and fecal flotations were performed weekly from week 4 to 6. Fecal and body-swab pools were sent to two different diagnostic companies for PCR analysis on week 8 and 10 of treatment. The progeny of the animals submitted to the described treatment were tested quarterly for the following two years;
In house testing were negative. The two external testing have shown similar negative results for both independent diagnostic companies. The quarterly analysis performed during the following two years was kept negative for pinworms and mites and remained negative until today.
The 10-week treatment with fenbendazole medicated diet and selamectin topic treatment, together with animal transfer combined with environmental decontamination with hydrogen peroxide compounds seems to be and efficient strategy for pinworm and mite eradication.
Acknowledgements
FEDER through the Operational Programme Competitiveness Factors – COMPETE and National Funds through the Foundation for Science and Technology (FCT) under the project POCI-01-0145-FEDER-007038; NORTE-01-0145-FEDER-000013, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); FCT doctoral fellowship to A. Miranda (SFRH/BD/52059/2012).
14:00–15:30
Session 3: Managing Resources and Facilities
Chairpersons:
Isabel Campos
Magda Castelhano-Carlos
OCi5
How Lean Management can Support Efficiency of Laboratory Animal Facilities Operations
1COGENTECH S.c.a.r.l.,;
2Fondazione Istituto FIRC di Oncologia Molecolare (IFOM)
Modern laboratory animal facilities aim to provide high standards of animal care and scientific support within a framework of regulatory compliance and with a strong focus on efficiency of operations. Meeting these objectives with limited resources is a challenge for facility managers and requires the application of a range of traditional and innovative management techniques. Lean production is a management philosophy derived from the Toyota Production System, which considers the expenditure of resources for any goal, other than the creation of value for the end customer, to be wasteful, and thus a target for elimination. Lean has been successfully applied to many activities of laboratory animal facilities from the design phase to the operation of animal care programs. The authors manage a 600 m2 mouse facility housing around 6,000 ventilated cages. Over the years, several upgrades (ranging from the introduction of dirty side automation systems, to optimization of the logistics) were implemented in order to increase the efficiency of the operations. In order to further improve, the main animal care activities were re-evaluated and reorganized using a Lean approach during the summer of 2011. The initial transformation was carried out with the support of a team of consultants and involved mainly cages and bottles processing, both in the animal rooms and in the washing area. Activities were balanced and new workflows defined together with new process layouts and time schedules. Lean was subsequently spread also to the areas not involved in the initial transformation, such as daily cages and health checks, breeding activities, etc. The results of the application of Lean management were striking, both initially and over the seven years of continuous implementation, and will be discussed together with the challenges encountered during the process.
OCi6
Navigating in a facility with multiple aquatic species
Instituto Gulbenkian de Ciência (IGC)
From a historical perspective, aquatic animal models have greatly contributed to experimental biology, embryology and stem cells. Both invertebrate and vertebrate aquatic species have been used since the 18th century to uncover basic aspects of biology. From these, we highlight, the amphibian model, the frog Xenopus laevis, as a classical model to study embryology, cell biology and biochemistry. The Zebrafish (Danio rerio), which has emerged in the 1990s, as a prominent fish model for studying embryology, organ regeneration, human disease, and toxicology amongst other areas of biology. Its popularity is rooted to well-described characteristics, such as high fertility, transparent embryos, short generation time, small sized adult, evolutionary genetic conservation and genetic tractability. More recently, another fish species, the African turquoise killifish (Nothobranchius furzeri) has joined the group of fish species used in biomedical research, due to its short lifespan, making it an excellent model to study aging and associated diseases.
Research conducted at the Instituto Gulbenkian de Ciência (IGC), an institution with a long tradition of animal-based research, integrates not only rodents and flies but also the above cited aquatic model organisms. Structured as a core facility to provide housing and services to the research community, the IGC Animal House Facility, has grown, adapted and diversified strategies to progressively incorporate zebrafish, killifish and the frog. However, the co-existence of multiple aquatic species in a vivarium poses challenges at many levels, namely biosafety and health control, facility design, and specialized human resources. Moreover, the ever-increasing complexity of scientific projects prompts us to continuously develop solutions to cope with research needs and animal welfare.
In this talk I will describe the strategies we have been developing over the years to overcome these challenges.
OCi7
CONGENTO: Supporting science through a synergy of wills and skills
Consortium for Genetically Tractable Organisms (CONGENTO)
Congento (Consortium for Genetically Tractable Organisms) was born as a response to the need of an organized infrastructure to support research in genetically modified animals in Portugal. Congregating the efforts of four Portuguese non-profit research institutions – Champalimaud Foundation (CF), Gulbenkian Institute of Science (IGC), Molecular Medicine Institute (IMM), and Chronic Diseases Research Center (CEDOC) – Congento gathers and integrates the expertise in Drosophila, zebrafish and mice in one research supporting facility. The aim is to provide state-of-the-art services in the three models in three main domains: 1) Maintenance and stocking of genetically modified lines (both live or cryopreserved), 2) Generation of new lines and development of new technologies, 3) Knowledge transfer and continuous education and certification in animal research and technology. By synergizing skills and resources across institutions and model organisms, Congento aspires to represent an added value to scientific research. Such an endeavor involves many stakeholders at very different levels and brings up new challenges frequently, requiring dynamic organization, effective communication and constant adaptation.
15:30–16:30
Laboratory Animals Associations – Past, Present and Future
OCi8
SPCAL: a leap into the future
1Portuguese Society of Sciences in Laboratory Animals (SPCAL);
2Nova Medical School | Faculdade de Ciências Médicas, Universidade Nova de Lisboa;
3Department of Physics, Faculty of Sciences and Technology, Universidade Nova de Lisboa, Monte da Caparica, Portugal
The first and main mission of the Portuguese Society of Sciences in Laboratory Animals (SPCAL) is to promote the implementation of the highest ethical and behavioral standards in the use of laboratory animals for scientific purposes. This has been done through interdisciplinary approaches and cooperation among professionals from the different Institutions, having in mind the animal's health and welfare, as well as the best scientific results.
Since its first General Assembly, in 2004, SPCAL has evolved from a well-conceived idea into an active, trusted and recognized partner of Researchers and Scientific Institutions, helping them to achieve the best practices concerning the use of laboratory animals. Indeed, over the years, SPCAL has established solid collaborations with an increasingly higher number of Research and Academic Institutions, to provide them the best Education and Training possible. From one single Institution in 2005, SPCAL is currently collaborating in regularly basis with Scientific Institutions spread throughout 2/3 of the country, providing Theoretical and/or Practical Courses to which ECTS credits have been attributed by several Academic Institutions.
Abroad, either as an Association or through its Associates individually, SPCAL has gained recognition among peer European Associations. Investment was made in a pro-active participation in the Federation of European LAS Associations (FELASA), with several SPCAL affiliates nominated for FELASA Working Groups.
More recently, SPCAL and its associates have been facing different challenges. The fact that we live in a global society, with easy access to information and an increasingly higher conscience, makes it impossible for any Scientific Association to neglect the public. Such public awareness and demand has driven SPCAL Board to take a different approach. An approach towards clarification and transparency, in close cooperation with both the Competent Authorities and civil representatives, who acknowledge SPCAL as a representative of Researchers in Laboratory Animals Sciences. Having such need for communication between Scientists and public in mind, we feel that the time has arrived for a higher degree of transparency in the field of Laboratory Animals Sciences.
SPCAL's missions haven't and will not change. The only thing that will change is the way those missions will be pursued in the future. Which leads us to the IV SPCAL Congress – "Quality and Transparency in Science involving laboratory animals".
OCi9
FELASA today and tomorrow – how can you contribute?
1Federation of European Laboratory Animals Associations (FELASA);
2Nova Medical School | Faculdade de Ciências Médicas, Universidade Nova de Lisboa.
The Federation of European Laboratory Animal Sciences Associations – FELASA – is for the moment composed of 21 constituent associations representing 27 countries. FELASA was established in 1978 to represent common interests in the furtherance of all aspects of laboratory Animal Sciences (LAS) in European and beyond. Membership is open to LAS Associations representing nations. FELASA represents common interests of constituent associations in the furtherance of all aspects of laboratory animal science in Europe. Laboratory animal science being that discipline whose objective is to ensure optimal conditions for the humane and appropriate use of animals for scientific purposes for the benefit of mankind and other animals, and to promote further development of those conditions. The board should work for:
– Advance and co-ordinate the development of all aspects of laboratory animal science and practice in Europe.
– Act as a focus for the exchange of information on laboratory animal science amongst European states.
– Establish and maintain appropriate links with national, international or governmental bodies as well as other organisations concerned with laboratory animal science.
– Promote the recognition and consultation of FELASA as the specialist European body on laboratory animal science and welfare. Organise joint scientific meetings of the constituent associations.
– Training and Education accreditation.
Along the last 40th years there is a long list of achievements to share and a door open for new activities. FELASA establishes FELASA working groups are established around current issues of lab animal science with experts in the field nominated by the different member Associations. The working groups should review available information and issue guidelines or recommendations promoting animals’ health and wellbeing.
OCi10
Keeping up with Portuguese Animal Welfare Bodies and the RedeORBEA network
1IBMC – Instituto de Biologia Molecular e Celular;
2i3S – Instituto de Investigação e Inovação em Saúde;
3SPCAL – Sociedade Portuguesa de Ciências de Animais de Laboratório
Animal Welfare Bodies (AWB) – ‘Órgão Responsável pelo Bem-Estar dos Animais’ (ORBEA) – were first set up in Portugal following the enactment of the 2010/63/EU Directive, transposed by Decreto-Lei 113/2013. The Directive sets as central obligations for AWBs: to advise the staff on animal welfare and the 3Rs, to establish and review operational processes for monitoring and follow-up of animal welfare, to follow the development and outcome of projects taking into account the effect on the animals used, and to identify opportunities to further the 3RS. In addition, Portuguese legislation confers to AWBs an additional responsibility: evaluating projects and issuing non-binding appraisals for project licence requests to the Competent Authority (CA).
Establishing AWBs raised significant challenges for ORBEAS at institutes and universities, not only in meeting the demanding regulatory requirements set for them, but also in establishing their identity, place and responsibilities in institutions, as well as asserting their authority while establishing relationships of mutual trust and respect with researchers. However, it was also an opportunity for raising standards of competence and functionality in planning, reviewing, and supervising animal research procedures, furthering a culture of care.
With little guidance from the CA – and without the input from a National Committee for the Protection of Animals used for Scientific Purposes (established in Portugal in 2016 but still not yet active) – it was up to laboratory animal scientists, veterinarians, and technicians to organize themselves, and find means of acquiring and exchanging reliable information, asking questions, and sharing experiences. SPCAL was pivotal in this endeavour, by funding and supporting events organized by and for ORBEA members, as well as professionals wishing to set an ORBEA. These included two workshops in 2014 – one at the IBMC (Porto), another at NOVA Medical School (Lisbon) – and the I National Symposium for ORBEAS in 2016 at the i3S (Porto), and a second edition in 2017, at the Champalimaud Foundation (Lisbon). The success of these events has led three ORBEAs in Coimbra to join efforts and organize the III National Symposium in 2018, fulfilling the original goal of making it a regular, yearly event.
This talk will cover the progress in ORBEA implementation in Portugal since the enactment of the 2010/63/EU Directive, highlight main achievements, and report on the development of the RedeORBEA – a network of Portuguese ORBEAs – and its future challenges.
16:45–18:15
Session 4: Legislation: New Perspectives and Updates
Chairpersons:
Ricardo A. Afonso
Margarida Caramona
OCi11
Shipping Live Animals – Best Practice
CCMAR – Centro de Ciências do Mar do Algarve, Universidade do Algarve
Despite the EU effort on funding infrastructures, with bioteriums that culture and maintain animals for research purposes, and also, to some extent, on funding the dislocation of researchers to such centers to perform their research in loco, there is still the need of shipping and transporting live regulated animals after collection from the wild or in between institutions.
According to the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs, UK), “Transport can be a significant stressor that may have an impact on both animal welfare and research outcomes. The primary objective for all those involved in animal transport should be to move the animals in a manner that does not jeopardise their well-being and ensures their safe arrival at their destination in good health, with minimal distress.” The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) have published Guidelines for the non-air transport of live animals and plants (which will take place up to 48 h), where they consider that International Air Transport Association (IATA) Live Animals Regulations (LAR) are appropriate for the non-air-transport of all species of animals. However, for transports exceeding 48 hours additional requirements should be met. The transport of an animal constitutes an unnatural situation and is most likely to cause it some degree of stress, and eventually injuries if it is not well prepared and performed. High levels of stress may increase metabolic rates, hazardous behaviour, chances of injuries and susceptibility to diseases. Considering an animal welfare perspective, animal transport should be quick, efficient and strive to avoid as much stress as possible to the animal. The transport of live animals must be well planned, meticulously prepared and effectively performed.
In this sense, this presentation will highlight the most important and necessary steps that need to be considered and taken when planning, preparing and shipping live animals. It will address: a) General Recommendations for Living Animal Transportation; b) Living Organisms Classification, UN Identification number and Proper Shipping Name; c) Packaging; d) Marking and Labelling; e) Documentation and Legislation; f) Arrival at new facilities and Quarantine; and g) Traceability.
OCi12
Portuguese legislation on the use of animals for scientific purposes
Divisão de Bem-estar Animal, Direção Geral de Alimentação e Veterinária
The Portuguese legislation regarding the protection of animals used for scientific purposes, the Decreto-Lei n° 113/2013, of 7th August, is derived from Directive 2010/63/EU, of 22ndSeptember, and thus, this presentation aims to present what are the main requirements, innovations and the activities that have been held in order to implement it.
OCi13
Comissão Nacional para a Proteção dos Animais Utilizados para Fins Científicos (CPAFC) – funções e objetivos
1Comissão Nacional para a Proteção dos Animais Utilizados para Fins Científicos (CPAFC); and
2Serviços de Proteção Animal, Direção Geral de Alimentação e Veterinária (DGAV)
A Portaria n.° 260/2016, de 6 de outubro, fixou a composição e o funcionamento da Comissão Nacional para a Proteção dos Animais Utilizados para Fins Científicos (CPAFC), tal como preconizado no n.° 4 do artigo 55.° do Decreto-Lei n.° 113/2013, de 7 de agosto, que transpôs para a ordem jurídica nacional a Diretiva n.° 2010/63/UE, do Parlamento Europeu e do Conselho, de 22 de setembro de 2010, relativa à proteção dos animais utilizados para fins científicos.
O Despacho n.° 673/2018, de 15 de janeiro, do Gabinete do Secretário de Estado da Agricultura e Alimentação, por seu lado, designou os elementos que integram a CPAFC, assim como o seu secretariado, a qual teve a sua primeira reunião na Sede da DGAV, a 16 de fevereiro de 2018.
A CPAFC tem funções de aconselhamento da Direção Geral de Alimentação e Veterinária e dos Órgãos Responsáveis pelo Bem-estar dos Animais das instituições que pratiquem experimentação animal, de acordo com o constante no n.° 2, do artigo n.° 55, do Decreto-Lei n.° 113/2013, de 7 de agosto.
A CPAFC propõe-se desenvolver, a curto prazo, trabalho de orientação sobre os seguintes assuntos:
– Reforço de competências das pessoas que executam determinadas funções no âmbito de aplicação do Decreto-Lei n.° 113/2013, de 7 de agosto, nomeadamente, em alinhamento com um programa de supervisão até demonstração de competência, de acordo com o disposto no artigo n.° 31, do Decreto-Lei;
– Clarificação sobre as responsabilidades atribuídas a cada pessoa envolvida na criação, fornecimento e/ou utilização de animais executando qualquer uma das funções espelhadas no Decreto-Lei n.° 113/2013;
– Promoção de métodos alternativos à experimentação animal;
– Integração na Rede das Comissões Nacionais dos Estados Membros da União Europeia para troca de experiências e entendimentos relativos à Proteção dos Animais Utilizados para Fins Científicos.
OCi14
Researchers and regulation. incentive for improvement or unreasonable interference?
1i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal;
2IBMC — Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal
Saturday, June 23
09:00–10:15
Session 5: Education and Training
Chairpersons:
Ana S. Olsson
Dolores Bonaparte
OCi15
Quality Assurance in Education and Training Accreditation
1Federation of European Laboratory Animals Associations (FELASA);
2Nova Medical School | Faculdade de Ciências Médicas, Universidade Nova de Lisboa
An accreditation system for Education and Training (E&T) has become recognized as a robust way of improving training and at the same time that establishes a golden standard for LAS education and training. Quality assurance aims to assist in the development of high quality educational programmes in laboratory animal Science throughout Europe and internationally. Another strong objective is to help on the harmonization of the training programs to help with the individual mobility at the global level.
The Federation of Laboratory Animal Science Associations (FELASA) accreditation aims to implement best practices for both high quality science and improved animal welfare by recognizing, supporting and enhancing the quality of training; establishes a more uniform platform of competence of those trained, 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.
The process has evolved and adapted over the years to provide an independent reassurance for National Authorities and the public about the competence of those working with laboratory animals. An added value of accreditation is that it facilitates the mobility of scientists that are well educated and trained in Laboratory Animal Science.
Although the FELAAS accreditation incurs in some financial and time commitment for course organisers the impact of the FELASA Accreditation scheme in the scientific community is a proof of success of the scheme.
OCi16
Continuing Professional Development in Laboratory Animal Science in Spain
Instituto de Neurociências, Universitas Miguel Hernandez
In accordance with European regulations for laboratory animals, Spain has developed its own normative in training topics with these species. The Orden Ministerial ECC/566/2015 regulates both initial and continued education with laboratory animals in our country. However, while initial education has been consolidated for several years, continued education is brand new at the moment. We will go through several approaches adopted by different institutions and companies regarding this issue. However the deadline for evaluations for the autorities has not arrived yet, and many questions arrise in the comunity about its final application. Althougth the regulation is unique for Spain, the application complicates by the organizational structure of Spain in autonomous communities, whose criteria in the interpretation of the normative is not always homogeneus. SECAL´s will is to serve as a forum to facilitate the comunication between the scientific comunity and autorities for a smoth and usefull application of continuous education in our country.
OCs2
The competence for training competencies
1i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto;
2IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto;
3ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto;
4Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho
The terms competence/competent are used in both Directive 2010/63/EU and Portuguese national legislation (Decreto-Lei n.° 113/2013) in relation to persons carrying out procedures with animals. In the educational sciences, the construct “competence” is generically defined as the ability to perform something successfully or efficiently, assuming the individual possesses the skills or abilities needed to perform certain task. In addition, a skill is defined as a person's expertise or proficiency. In a review in clinical practical context, Epstein and Hundert (2002) stated that professional competence integrates several dimensions not only related with cognitive and technical domains but also affective and “habits of mind”. Unlike factual knowledge which can be acquired in a concentrated effort in a short time, long-term training seems to be the most effective way to develop skills and competencies.
Training for persons carrying out procedures with animals is traditionally organized as courses. Whereas no format was prescribed in the FELASA recommendations, courses are time-concentrated into one (Cat B) or two (Cat C) weeks. This format has several advantages: it allows students to immerse in the topic, it makes it easier for organisers to bring together a faculty of expert teachers and it is especially convenient when the course is not taken in the students’ own institution. However, the need to deliver all the theoretical content to accomplish defined learning outcomes in a short time sets constrains for the acquisition of competence. Practical training is usually limited to procedure demonstration and replication by students. Competence evaluation is usually assessed, at the last course day, through a set of theoretical questions, less than two weeks after the start of training.
However, it is unlikely that students will be able to gain necessary competence in such a short time and with so exhaustive curricula. This imposes a substantial challenge to course organizers and instructors, who have the duty of attesting competence, in particular as regards the need to practice and learn to execute procedures in living animals. Individual trainee’s own progress time must be respected to ensure safe and efficient training.
With this presentation, we aim to raise the discussion of the dilemma between course format and competence development in laboratory animal science. How can course organizers achieve a satisfying solution for caring out demanding and time-concentrated courses, while guaranteeing the acquisition of professional competencies, which take time to develop and consolidate?
10:45–12:30
Session 6: Animals and Models: Biological Perspectives
Chairpersons:
Isabel Vitória Figueiredo
Ricardo A. Afonso
OCi17
Cephalopods: Historical perspective and current use
Centro de Ciências do Mar do Algarve (CCMAR), Universidade do Algarve
Are cephalopods not from Earth? A recent study published by Steele et al. (2018) connects octopuses and remaining cephalopods to the theory of panspermia. But, despite cephalopods might be seen as very “weird” invertebrates this is not what makes them excellent animal models for research and we will revise why. Cephalopods are a class of invertebrates with over 700 species. Cephalopods have been used as experimental models since the beginning of the 20th century in vast research areas, such as neurobiology (Young, 1938), neuroscience (Sio, 2011; Williamson & Chrachri, 2004) and behaviour (Gherardi et al. 2012; Hanlon & Messenger, 1996; Tricarico et al. 2011; Wells, 1962).
When, in 1963, Alan L. Hodgkin and Andrew F. Huxley won the Nobel Prize in Physiology and Medicine for their work on the behaviour of nerve impulses, by using the giant axon of the Atlantic squid Loligo pealei, the use of cephalopods as animal models for research gained momentum. Some years later, two other researchers used squid as a model in their research and became Nobel Laureates: the American G. Wald, who was honoured in 1967 for his research on chemical and physiological processes in the eye, and Bernard Katz of Great Britain, won the prize in 1970 for his discoveries concerning the role played by chemicals in nerve impulses. From that point on, cephalopods became the most relevant neurological model, with no peer in the animal kingdom (Young, 1985).
A lot has happened since then, with cephalopods becoming animal models for emergent research fields, such as mechatronics (Calisti & Laschi, 2017; Kim et al. 2013), evolution (Budelmann, 1995) and, more recently, climate change (Pörtner & Farrell, 2008).
Despite the report of the first genome of a cephalopod only occurred in 2015 (Albertin et al., 2015), a recent paper in Cell (Liscovitch-Brauer et al., 2017) is showing that cephalopods adapt to changing water temperatures by altering their RNA more often than their DNA, which can be seen as proof that when the Nobel Prize–winning biologist Sydney Brenner said that octopi were the first intelligent beings on Earth, he was right.
References
OCi18
Animal experimentation: examples of rational approaches towards drug development
Faculty of Pharmacy, University of Lisboa, ImedULisboa/IBEB, Lisboa, Portugal
Despite large investments in drug development, the overall success rate of drugs during clinical development remains relatively low. A possible explanation may be related to the weakness of the preclinical research. Indeed, animal experimentation is pivotal to bridge the translational gap to the clinic. When carefully selected, designed and conducted, animal models are an important part of any translational drug development strategy and intimately linked to advances in scientific knowledge and technology. The selection of a valid, adequate and predictive animal model is essential to address this question.
In this communication two different approaches will be presented; one as therapeutic drug and another as medical device.
Designing a new therapeutic drug is a lengthy and complex process. Those decisions on which drug or what respective formulation should proceed to the next stage are taken using a wide range of data. Experiments using animal models are essential to understand how a drug will work. In general, a candidate drug is initially tested in cell lines and only afterwards the studies move into animal subjects. If a potential drug appears to be effective in animals, and passes initial safety screening, then it can be studied in human trials. In this communication, several approaches of an oral insulin formulation using animal models will be presented and discussed.
In the case of medical devices, the general focus of animal experimentation is on the device’s ability to function with living tissue without harming. Until now, most of the medical devices use biocompatible materials, such as gold nanoparticles. Again, animal models are critical to assess the safety of a new approach in the treatment of skin cancer.
Both examples show that animal experimentation is crucial to pharmaceutical industry researchers to decide whether a potential therapeutic drug will be effective and safe for use in humans. Furthermore, from the examples presented herein, we also concluded that the translational value of animals’ models can be further enhanced when combined with other translational tools, which simultaneously allows us to decrease the number of animals required. Nevertheless, all strategies will have undeniably improved patient access to more effective and safer new drug therapies.
OCi19
Are we entering a new Era in genome editing?
Instituto Gulbenkian de Ciência. Electronic address: anovoa@igc.gulbenkian.pt.
The CRISPR/Cas9 genome editing system is having a tremendous impact on transgenic technology nowadays. This novel technology derives from the adaptive immune system of prokaryotes, that was recently adapted to allow any kind of genetic modification, from basic gene inactivation to subtle edits, like point mutations, insertion of large cassettes or even do gene replacements, via gene targeting, in a wide variety of species and cell lines, with the only restriction essentially being our imagination. This innovative approach both democratized genomic editing and, very rapidly, overcame the laborious, more expensive and time-consuming embryonic stem-cell-based recombineering systems. In my talk I will discuss the use of CRISPR/Cas9 genome editing system in the wider context of the in vivo transgenic technologies used to modify the mouse genome: mouse oocyte pronuclear microinjection of transgenic constructs, BACs, YACs, zinc-finger and transcription activator-like effector nucleases, and also the microinjection of targeted ES-cells on blastocysts. Finally, a brief discussion on the impact that this novel mouse genome-engineering tool has on the 3 R′s (Replacement, Reduction and Refinement) principle.
OCi20
Implementation of a mouse Gnotobiology Facility to host microbiota-related studies
Instituto Gulbenkian de Ciência (IGC)
The gnotobiology research has exponentially grown in the last few years due to the important role of host–microbiota interactions in the study of several metabolic and autoimmune diseases. The use of axenic mice, which are rendered free of microorganisms, is a straightforward approach to investigate the overall contribution of the microbiota in such interactions.
In 2005, the Instituto Gulbenkian de Ciência (IGC) developed a dedicated platform for axenization of different mouse strains reared, maintained and manipulated in multi-cage isolators. This Axenic Facility is highly well established, with an annual capacity of 400 axenic mice being produced for internal requests and for the international community, in the frame of the Infrafrontier-EMMA EU consortium.
Isolators represent a good strategy for axenic mice production at medium/large scale, however, subsequent experimentation in this system is suboptimal for gnotobiology research, often requiring diversified genetically modified animals with multiple and parallel experimental conditions. In 2013, the increased demand of gnotobiology experiments by the researchers at IGC led us to develop a Gnotobiology Facility equipped with racks of Individually-Ventilated Cages (IVC) under positive pressure, namely the ISOcage P system. This system is designed to achieve the technical features of an isolator, allying the ergonomic, flexibility and density advantages of an IVC. Each cage acts as a micro-isolator, allowing multiple studies in the same rack, excluding cage to cage contamination. This setting is completed with an ISOcage Biosafety Station (IBS), specifically conceived for suitable manipulation of mice in a fully sterilized environment.
Currently, the Gnotobiology Facility hosts all experiments performed in axenic mice, accommodated in this ISOcage P IVC racks, set in a room accessible to trained researchers, while the Axenic Facility is exclusively dedicated to the production of axenic mice, and axenization of new strains, allocated in multi-cage isolators in a room only accessible by the facility personnel.
Since the implementation of the Gnotobiology/Axenic Facility, more than 250 different gnotobiology experiments were performed, with animals being submitted to various procedures, from gavage and regular feces collection to sophisticated surgeries. To validate this working system, standard operating procedures (SOPs) and an axenic sentinel programme were applied and gradually optimized. The data relative to the microbiological status along time demonstrates a significant decrease of contaminated experiments. Also, sentinels have been maintaining their axenic status for more than 18 months, indicating this setup reveals to be efficient, optimizing human, financial and space resources.
Acknowledgements
We thank to the Animal Facility team and to all users of the Gnotobiology/Axenic Facility, especially to Jessica Thompson and Joana Dias for their contribution to the early trials.
The content of this work also gave rise to a Communication in the form of poster.
OCs3
Chitosan/β-glucan particles for hepatitis B surface antigen vaccination: prophylactic and therapeutic value
1Center for Neuroscience and Cell Biology, University of Coimbra, Portugal;
2Faculty of Pharmacy, University of Coimbra, Portugal;
3Department of Gastroenterology and Hepatology, Erasmus MC Rotterdam, Netherlands
The hepatitis B virus (HBV) killed 887 000 people in 2015. The World Health Organization (WHO) set the goal to eliminate HBV as a public health threat by 2030. The major hurdles include high prevalence in developing countries and ineffective currently available antivirals. Hence, new vaccine adjuvants can be designed to stimulate the chronic hepatitis B exhausted immune system and to provide the antigen increased stability to temperature variations, benefiting HBV vaccine coverage in developing countries.
Different adjuvants for the hepatitis B surface antigen (HBsAg) were developed and included β-glucan particles, prepared from alkaline/acid treatment of Saccharomyces cerevisiae, β-glucan/chitosan and chitosan particles prepared by a precipitation technique. Vaccine formulations were tested in C57BL/6 mice, following either three subcutaneous administrations (n=5), three oral administrations (n=7) or one subcutaneous followed by two oral administrations (n=5). At the end of the experiment each mouse was individually euthanized by cervical dislocation, away from the other animals to minimize stress, and death was confirmed by testing the absence of both respiratory rate and reflexes. Blood was collected to assess serum HBsAg-IgG; faeces and vaginal washings were collected to assess HBsAg-IgA; livers were collected for tissue interstitial fluid IFN-γ, and spleens were collected for HBsAg restimulation ex vivo and HBsAg-specific cytokines detection using a multiplex assay. Animal studies were approved (ORBEA_50_2013/27092013) and carried out in accordance with institutional ethical guidelines and with National (Dec. No. 113/2013) and International (2010/63/EU Directive) legislation.
The oral vaccination approach resulted in 60 % mice seroconversion. The presence of HBsAg-specific IgA on mucosal surfaces and IFN-γ in the liver were the major advantages found of this vaccination route. The fully subcutaneous vaccination resulted in 100 % seroconversion with high serum anti-HBsAg IgG, mostly subtype IgG1 followed by IgG3. The β-glucan particles used as an adjuvant induced a strong and varied HBsAg-specific cell-mediated immunity observed by the secretion of cytokines related with Th1, Th2, Th17, Th22 and Treg-biased immune responses.
The present work represents an important contribution to the knowledge of both β-glucan and chitosan/β-glucan particle adjuvant mechanisms, with a great impact for antiviral immunotherapies.
12:30–13:30
Closing Lecture
Therioepistemology, a success story: mouse models of trichotillomania and compulsive skin picking
Stanford University, Department of Comparative Medicine, and by courtesy, Department of Psychiatry and Behavioral Sciences, Stanford, California, USA.
In this talk, I will illustrate the efficacy of therioepistemology in the development, validation, and use of an animal model. Trichotillomania (compulsive hair pulling) affects roughly 3.5% of women (making it the 3rd most common disorder in women), yet this debilitating disorder has been largely ignored by researchers and clinicians alike. Hair and feather-pulling behaviors are widespread abnormal behaviors in animals with many similarities to trichotillomania. This talk will survey how treating mice as patients, allowed us to validate barbering and ulcerative dermatitis as models of trichotillomania and compulsive skin-picking disorder, identify biomarkers predicting disease development and treatment response, elucidate disease mechanisms, and ultimately identify a novel drug. Because the interventions we have identified are tied to underlying biomarkers we have a rapid method for validating these findings in humans before investing in a drug trial – humans should show the same novel biomarkers as mice.
Finally, in parallel, by showing that ulcerative dermatitis is a behavioral condition, we have been able to design behavioral interventions in mice that resolve what would otherwise be life-ending lesions in 95% of affected mice (Adams, Garner et al. 2016). As a result, we have been able to virtually eliminate ulcerative dermatitis as cause of unplanned euthanasia in our facility.
Poster Session
Friday, June 22
PS1
What is the most appropriate rat model to study type 2 diabetes?
1CEDOC, NOVA Medical School, Faculdade de Ciências Médicas, Lisboa, Portugal;
2Escola Superior de Saúde de Leiria – Instituto Politécnico de Leiria, Leiria, Portugal
both authors contributed equally for the work
Obesity and Type 2 diabetes (T2D) have reached epidemic proportions being clear that the existing therapies are clearly scarce to address this problem. Rats have been widely used to mimic human diseases aiming to understand the causes and progression of disease symptoms, as well as to develop new drugs and determine their mechanisms of action. With this work we aim to evaluate several experimental and genetic rat models of T2D, in order to be able in the future to select more accurately the most appropriate T2D model given the specific research requirements.
Different experimental settings of Male Wistar rats (8–9 weeks old; n=8–10 animals per experimental group) were used: 1) 3 weeks of 60% lipid-rich diet (HF diet); 2) 19 weeks of HF diet; 3) 4 weeks of high sucrose diet (HSu, 35% of sucrose); 4) 16 weeks of HSu diet; 5) 14 weeks of combined HF plus HSu diet (HFHSu); 6) 25 weeks of HFHSu diet; 7) 4 weeks HF diet plus streptozotocin (HFSTZ, 25 mg/kg,i.p.). Additionally, a genetic model of T2D, the Zucker Diabetic rat (ZDF), was tested at 17 and 23 weeks of age. Animals were compared with age-matched controls. Caloric intake and weight were monitored. Insulin sensitivity and glucose tolerance were evaluated respectively through an insulin tolerance test (ITT) and oral glucose tolerance test (OGTT). Fasting glycemia, insulin and C-peptide were measured. At a terminal experiment rats were anesthetized with pentobarbital (60 mg/kg i.p.), blood was collected by heart puncture and tissues were collected, weighted and kept for further studies, as analysis of the expression of proteins involved in glucose metabolism. Laboratory care was in accordance with the European Union Directive for Protection of Vertebrates Used for Experimental and Other Scientific Ends (2010/63/ EU). Experimental protocols were approved by the Faculdade de Ciências Médicas Ethics Committee.
Rats fed with HSu and HFHSu diet gain less weight than animals fed with HF diet and ZDF in comparison with respective controls. Insulin resistance was achieved in all experimental settings, being higher in animals submitted to 25 weeks of HFHSu diet, except for HF animals with STZ. In contrast, glucose intolerance was achieved in all animals, with ZDF and HFSTZ animals presenting higher levels of intolerance. Fasting glycemia values within the range for T2D diagnose were only obtained in ZDF animals. All animals were hyperinsulinemic in comparison with theirs controls, except HFSTZ animals that were hypoinsulinemic.
We conclude that the most appropriate rat model to use for T2D studies depends on the pathological features to be tested and that ideally, more than one rat model should be used to represent the diversity seen in human diabetic patients.
Acknowledgements
J.F. Sacramento and B.F. Melo were supported by the Portuguese Foundation for Science and Technology grants PD/BD/105890/2014 and PD/BD/128336/2017 respectively.
PS2
Parasympathetic tone Activity (PTA) evaluation to discriminate Ketorolac and Ketorolac/Tramadol analgesia level in swine
1Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Trás-os-Montes and Alto Douro (UTAD), Portugal;
2Department of Veterinarian Sciences, School of Agricultural and Veterinary Sciences School (ECAV), UTAD, Portugal;
3Anesthesiology Department Co-ordinator, Jesús Usón Minimally Invasive Surgery Centre JUMISC, Spain;
4Department of Mathematics, School of Science and Technology (ECT), UTAD, Portugal;
5Centre of Mathematics of the University of Minho – UTAD Pole (CMAT-UTAD), Portugal;
6Center for Computational and Stochastic Mathematics (CEMAT), Higher Technical Institute, University of Lisbon (IST-UL), Portugal;
7Institute of Research and Innovation in Health, University of Porto, Portugal;
8Laboratory Animal Science Group, Institute of Molecular and Cellular Biology (IBMC), University of Porto, Portugal.
Evaluation of nociceptive/anti-nociceptive balance during general anaesthesia is still challenging and routinely based on clinical criteria. Analgesic drug delivered may be optimized with a parasympathetic tone activity (PTA) monitor. This study compares ketorolac and ketorolac-tramadol balance analgesia using a PTA monitor.
All procedures were approved by the Minimal Invasive Surgery Centre Jesús Usón ethical committee and animal welfare body. Pain intensity response was assessed using a 0–100 numerical state scale (PTA) after nociceptive stimuli provide by a Mayo Hegar needle holder in nine Large White pigs, aged between 85–90 days and with 49 kg mean weight, under stable sevoflurane twenty minutes anesthesia. Bispectral index, heart rate, non-invasive blood pressure and respiratory parameters were also measured. Animals were divided into three groups of three: a) without analgesia b) ketorolac (1.5 mg/kg) and c) ketorolac/tramadol (1 mg/kg). Mean values or mean areas under the curve (AUC) in selected time periods were compared over time and between groups through a mixed-model repeated measures ANOVA and non-parametric Kruskal-Wallis tests, followed by Bonferroni or Dunn’s multiple comparisons.
We observed a significant decrease in the PTA AUC mean value after application of the stimulus in the animals treated without analgesia and those with keterolac only. The PTA AUC mean value in the control group was significantly lower than the corresponding mean in ketorolac group (p<0.5). The ketorolac/tramadol group showed the highest PTA AUC mean values, significantly different from those obtained for the other two groups, with no significant differences detected over time (p>0.5). Bispectral index means showed no statistically significant differences neither over time nor between different treatment groups (p>0.5). Heart rate showed only a statistically significant higher AUC mean between the group without analgesia and the ketorolac/tramadol group (p<0.5), in the time period after the stimulus application, where without analgesia was higher. Non-invasive blood pressure means showed no statistically significant differences over time or between treatment groups (p>0.5).
This study shows that a low dose combination of Ketorolac and Tramadol is sufficient to block the pain responses induced with a needle holder in pigs twenty minutes after its administration. The PTA monitor was able to clearly recognize and distinguish the analgesic level between treatments and can thus be used to optimize the delivery of analgesic drugs.
Acknowledgements
The authors thank to Mdoloris Medical Systems from Loos, France, and Scil Animal Care Company S.L. from Madrid, Spain, for providing the Parasympathetic Tone Activity (PTA) monitoring.
PS3
Tumours’ characterization in DMBA-induced breast cancer model
1Pharmacology and Pharmaceutical Care Laboratory, Faculty of Pharmacy – University of Coimbra;
2Experimental Pathology Service, Faculty of Medicine – University of Coimbra;
3CBIOS – Faculty of Veterinary Medicine – ULHT;
4iMed.ULisboa – Faculdade de Farmácia, Universidade de Lisboa, Portugal;
5IBEB, Faculdade de Ciências, Universidade de Lisboa, Portugal;
6IBILI, Faculty of Medicine – University of Coimbra.
e-mail:
According to some studies, in 2015, there were 17.5 million cancer cases worldwide and 8.7 million deaths. Between 2005 and 2015, cancer cases increased by 33%, with population aging contributing 16%, population growth 13%, and changes in age-specific rates contributing 4%. In women, the most common cancer was the breast cancer. DMBA is considered to be one of the etiologic factors of malignant neoplasms in humans. In research, DMBA has also been used to chemically induce mammary carcinogenesis in experimental models with Sprague-Dawley rats. With this work, we pretend to characterize DMBA-induced breast cancer tumours.
Ten Sprague-Dawley female rats were orally administered with 65 mg/kg of DMBA diluted in olive oil, at 55–57 days of age. At 27 weeks after carcinogenic induction, all animals were euthanized by overdose of anesthetic. A full necropsy was performed and all tumours were excised, measured and weighed. This work was approved by DGAV. The lesions’ structural pattern were graduated as non-neoplastic, benign neoplastic, in situ malignant neoplastic and invasive malignant neoplastic. Histological type was defined by the predominant pattern observed and classified accordingly to the different histological types. In cases that presented malignant and benign lesions, the histological type given was the predominant on the malignant lesion. Benign lesions were considered for the total number of patterns count. Grading of malignant lesions was performed applying the Nottingham Grading System. This system is based on evaluating and scoring three distinct morphological features: degree of tubular/glandular formation, nuclear pleomorphism and mitotic index based. After scoring these features, the tumours were classified as grade 1, grade 2 and grade 3.
Sixty fragments were collected and graduated in 15 non-neoplastic (6 at right mammary chain and 9 at left mammary chain), 11 benign neoplastic (5 at right mammary chain and 6 at left mammary chain), 1 in situ malignant neoplastic (only at right mammary chain), and 33 invasive malignant neoplastic (19 at right mammary chain and 14 at left mammary chain). All the benign neoplastic tumours were classified as grade 1; all the in situ malignant neoplastic were classified as grade 1; the invasive malignant neoplastic were distributed in 20 tumours of grade 1, 6 tumours of grade 2 and 7 tumours of grade 3. 9 of all animals presented invasive malignant neoplastic.
With these results, we conclude that this is a good model to induce invasive malignant breast cancer tumours with an incidence around 90%.
PS4
Which animal model should be applied in DMBA-induced breast cancer? A brief, non-systematic review
1Pharmacology and Pharmaceutical Care Laboratory, Faculty of Pharmacy – University of Coimbra;
2Experimental Pathology Service, Faculty of Medicine – University of Coimbra;
3iMed.ULisboa – Faculdade de Farmácia, Universidade de Lisboa, Portugal;
4IBEB, Faculdade de Ciências, Universidade de Lisboa, Portugal;
5IBILI, Faculty of Medicine – University of Coimbra.
e-mail:
Breast cancer is a global public health issue as it is the most frequently diagnosed malignancy in women in the Western world and commonest cause of cancer death in European and American women. DMBA is an HPA, known to be a ubiquitous environmental contaminant and is considered to be one of the etiologic factors of malignant neoplasms in humans. It is present in cigarette smoke, coal, burned wood, and gasoline and diesel engines. It has been assigned various toxicological, immunotoxic, mutagenic, teratogenic and carcinogenic effects. It induces malignant transformation in cell transformation systems in vitro, being mutagenic for bacteria and mammalian cells. With this work, we pretend to describe the main characteristics of this DMBA breast cancer induction model.
We used the PubMed database to search 11 articles based on keywords “Breast Cancer”, “Experimental model” and “DMBA”.
There are different models of breast cancer with different rat strains. During some studies, the outbreed strains of Sprague-Dawley and Wistar female rats demonstrated to be more sensitive to chemical carcinogenesis when compared with others strains. More recent studies showed that the number of mammary tumors developed in Wistar Rats was lower when compared with female Sprague-Dawley rats(1–3). The breast tumors induction can be hormone-dependent or independent. DMBA induce hormone-dependent tumors, it expresses estrogen and progesterone receptors, and it is very important to select the time of DMBA administration(4–6). The chemical carcinogenesis is maximal when DMBA is administrated between 45 and 65 days of age, sexual maturity age. When administered by gavage at 50–56 days of age, in a single dose ranging from 10 to 100 mg per Kg of body weight, it induces the development of a high number of mammary tumors (more than 90% of incidence)(7–10). The carcinogenesis initiates at 64–70 days of age, 14 days after administration of DMBA. After initiation, the carcinogenesis can cause benign lesions such as cysts, adenomas, alveolar hyperplasias and fibroadenomas or malignant lesions with different histological patterns, for example, papillary, cribiform and comedo(4). At 7th to 10th weeks after administration of DMBA, mammary tumors were detected by palpation and the number of tumors increases over time(11). All references used had their work approved by the Institution Ethics Committee.
We consider this a promising model because the mammary tumors are easily induced, the experimental protocols were defined, the animals developed a high number of tumors, and tumors were similar to those found in humans.
References
PS5
Blood and Urine parameters in DMBA breast cancer induction model
1Pharmacology and Pharmaceutical Care Laboratory, Faculty of Pharmacy – University of Coimbra;
2Experimental Pathology Service, Faculty of Medicine – University of Coimbra;
3Vasco da Gama University School, Coimbra;
4Clinical Analysis laboratory, Faculty of Pharmacy – University of Coimbra;
5iMed.ULisboa – Faculdade de Farmácia, Universidade de Lisboa, Portugal;
6IBEB, Faculdade de Ciências, Universidade de Lisboa, Portugal;
7IBILI, Faculty of Medicine – University of Coimbra.
e-mail:
DMBA is considered to be one of the etiologic factors of malignant neoplasms in humans. It is present in cigarette smoke, coal, burned wood, coal tar and gasoline and diesel engines. It has been assigned various toxicological, immunotoxic, mutagenic, teratogenic and carcinogenic effects. In the study of malignant human breast neoplasm, DMBA has also been used to chemically induce mammary carcinogenesis in experimental models with Sprague-Dawley rats. With this work, we intend to describe the blood and urine parameters of DMBA breast cancer induction model.
Twenty Sprague-Dawley female rats were divided in two groups, ten animals in control group with no manipulation and ten animals in test group with DMBA administration. At 55–57 days of age, the animals of test group were orally administered with 65 mg/kg of DMBA diluted in olive oil. At 15 weeks after carcinogenic induction, tumors started to be detectable by mammary palpation. At 27 weeks, all animals were euthanized under anaesthesia. A full necropsy was performed. At the same time blood samples was collected from each animal. Once a month, during 6 months, each animal was placed in a metabolic cage, for 24h, in order to perform urine collection. This work was approved by DGAV. In whole blood count was made, and at serum we measured glucose, urea, creatinine, alkaline phosphatase, TGO, TGP and total calcium. In urine, we performed physicochemical determinations, such as quantification of bilirubin, urobilinogen, ascorbic acid, ketones, red/white cells, nitrites, proteins, density, pH, urea and also urinary ionogram (Na, CL, K) was made. Statistical analysis was made using R software.
In blood count, comparing both groups, there are significative differences in platelets with p-value=0.037 and IC95% [11.541;304.409]. At serum measurements, there were significative differences in creatinine with p-value=0.031 and IC95% [0.008;0.142]. Platelets and creatinine are decreased in group with DMBA administration. In urinalysis, there were significative differences in urea, sodium, potassium and chlorine in different measurements throughout the experimental protocol.
With this work, we conclude that DMBA breast tumors model can cause some alterations in blood and urine parameters which may have impact in future protocols
PS6
Categorization of the TNBS-induced model of colitis in rats
1Laboratory of Pharmacology, Department of Drug Sciences, Faculty of Pharmacy of University of Porto, Portugal;
2LAQV@REQUIMTE, Faculty of Pharmacy of University of Porto, Portugal;
3Laboratory Animal Science, IBMC – University of Porto, Portugal;
4Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Portugal;
5Department of Immuno-Physiology and Pharmacology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Portugal
Inflammatory Bowel Disease (IBD) is a highly prevalent chronic relapsing immunologic disorder of the gastrointestinal tract. Among several animal models that are available to study IBD, the 2,4,6-trinitrobenzene sulfonic acid (TNBS) chemically induced colitis has a pivotal role, especially in preclinical testing [1]. Despite its popularity, the variability found in the induction and outcome of this animal model are confusing and often reported as inconsistent. The aim of this work was to characterize and refine TNBS-induced colitis, in rats included in a project that aims to investigate the role of renin-angiotensin system in the colon.
Protocols were approved by local animal welfare body (179/2017 ORBEA ICBAS-UP), as well as the national Competent Authority (0421/2018). Male 8–12 weeks old Wistar Han rats were fasted for 24-hour (with ad libitum access to a sugary solution), briefly anesthetized with isoflurane and rectally instilled with a 30% ethanolic solution of TNBS (20 mg/rat, n=17). Analgesia was provided by the administration of tramadol (20 mg/kg, SC) and acetaminophen (5 mg/ml in drinking water). Eleven littermates were used as controls. The following week, animals were daily monitored to evaluate body weight, food and water intake, general welfare and grimace scores, by cage side assessment. On the 7th day animals were euthanized by decapitation, the colon was removed and its macroscopic disease activity index (DAI) assessed.
TNBS induced different disease severity degrees, that were categorized in 3 groups: Mild, Moderate and Severe, according to macroscopic DAI scores (Mild: [0–4]; Moderate: [4–8]; Severe: [8–12]). Seven days after induction Mild-TNBS animals recovered their initial weight (0,58±0,04%, n=5), but Moderate-TNBS (−6,17±0,04%, n=6) and Severe-TNBS (−4,54±0,02%, n=6) lost weight. Food intake accompanied weight changes, so Mild-TNBS ate more (12,50±9,50 g/day) than Moderate-TNBS (8,00±5,04 g/day) and Severe-TNBS (4,61±2,49 g/day). As for water consumption, we observed the opposite since Mild-TNBS drank less (48,27±15,91 mL/day) than Moderate (51,58±14,18 mL/day) and Severe (54,64±12,26 mL/day) TNBS rats. Regarding general welfare and grimace scores, it was clear that on the 7th day Mild-TNBS had a lower score (∼0,5-1 points) than Moderate and Severe-TNBS (1,5-2 points).
It was possible to categorize the variability observed with the TNBS-induced model of colitis in three severity groups, with different characteristics. We hope that in the future these results can help researchers to anticipate the severity of TNBS-induced colitis, which is relevant for animal welfare, reproducibility of experimental protocols and also data analysis.
Acknowledgements
M. Morato thanks GEDII – Grupo de Estudo da Doença Inflamatória Intestinal – for funding and Mrs. Céu Pereira for excellent technical assistance.
Reference
PS7
Light cycle and cage change: behavioural effects over two different mice strains
1i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal;
2Addiction Biology, IBMC – Instituto de Biologia Celular e Molecular, Portugal;
3Lab of Neuropsychophysiology, FPCEUP – Faculdade de Psicologia e de Ciências da Educação da Universidade do Porto, Portugal;
4Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal
The frequency at which mouse cages are changed has a relevant impact in the welfare of animals, and consequently on experimental results. The time of day at which cage change takes place may have distinctive effects on behaviour, since mice are nocturnal animals with a well-defined circadian rhythm. If so, the effect of cage change should be minimized. The objective of this study was to determine the effects of cage change in different periods of the light/dark cycle, on the exploratory, social, and anxiety-like behaviours of two mice strains (B6 and FVB-WT). A total of 12 cages (6 per strain), each with a pair of animals (N=24) were initially subjected to a cage change during the light phase of the cycle (2 h after the lights on), and a week later during the dark phase (2 h after the lights off). Behavioural changes were videotaped for 10 min in the new home cage, immediately after the change. After this period, one mouse per cage was tested in the Elevated Plus Maze (EPM) to evaluate anxiety. Results show that B6 mice increased exploratory behaviour (mean diff: 178,8; CI 95%: 70,39 to 287,1; p=0,0027) and digging (mean diff: 22,56; CI 95%: 0,761 to 44,36; p=0,0432) when the cage was changed during the dark phase. When the change occurred during the light phase, B6 mice spent more time hiding (mean diff: −19,7; CI 95%: −33,39 to −6,013,1; p=0,0072) and peeking (mean diff: 42,96; CI 95%: −73,95 to −11,97,1; p=0,0091) from the paper tube, which suggests that changes during the dark phase lead to more proactive behaviours when compared with changes during the light phase. The FVB mice did not change behavior when the cage was changed during the dark or light phase. Furthermore, this strain was less anxious than B6 mice in EPM (F(1)= 37,284; p= 0,001). This work suggests that the impact of the light/dark cycle on mice behaviour after the cage change is strain-dependent. These results suggest that changing cages during the dark phase increases exploratory behaviours and reduces passive-anxiety behaviours, leading to improved adaptation to the new environment. This seems to be particularly relevant for more anxious strains.
This project was appraised by the Animal Welfare Body (ORBEA) at the i3S, and was not considered to surpass the pain or distress threshold to be considered a procedure, thus not requiring a licence.
Acknowledgements
RLA is supported by an FCT grant (PD/BD/114266/2016). AM (IF/00753/2014) and TS (IF/00875/2012) Project (IF/00753/2014/CP1241/CT0005) financed by FCT e Orçamento do Estado.
PS8
Estimating the Predictive Validity of Animal Models of Type 2 Diabetes in DPP-4 Inhibitor Studies – Assessing Methodological Quality and Animal Welfare
1FCUP – Faculdade de Ciências da Universidade do Porto;
2I3S – Instituto de Investigação e Inovação em Saúde;
3University of Debrecen;
4IBMC – Instituto de Biologia Molecular e Celular
Type 2 diabetes (T2D) is a metabolic disorder responsible for millions of deaths annually. It is mainly characterized by hyperglycaemia, frequently accompanied by obesity, arterial hypertension, and hypercholesterolemia as comorbidities, among others. Although several drugs are available to help mitigate symptoms and alleviate the impact of comorbidities of T2D, more effective treatments are still needed.
To both understand the underlying pathophysiological mechanisms of T2D and test the efficacy of therapeutic drugs, animal studies are still of the utmost importance. Several animal models mimicking traits of human disease – with varying fidelity – are currently used. It is however central to establish their predictive validity – i.e. the extent to which these can predict human outcomes – as well as the impact of other variables, such as route of drug administration or sex of the animals. To ensure that such studies yield reliable – i.e. reproducible – and thus clinically relevant results, their design and execution must uphold high methodological standards.
As part of an ongoing bilateral Portuguese-Hungarian bilateral project, we are currently carrying out a retrospective evaluation of the predictive validity of animal models by a systematic review of preclinical and clinical data, to compare reported drug efficacy in both human patients and animal models of T2D, focusing on dipeptidyl-peptidase-4 (DPP-4) inhibitors, namely linagliptin, saxagliptin, vildagliptin, and sitagliptin. We also aim to evaluate both methodological and animal welfare standards, as central measures for ensuring that animal research is competent, meaningful, and responsible. Results from the latter assessment will be presented.
All articles so far analysed (N=49) reported compliance with relevant animal welfare regulations, 18/49 did not have a conflict of interest statement, 29/49 did not report randomisation of animals to treatment groups, and 40/49 did not perform blind outcome assessment. Moreover, none reported allocation concealment, and only one justified sample size. Clinical signs of disease were seldom reported, aside glycaemia levels, which minimum and maximum values varied between 62 mg/dL and 603 mg/dL, respectively. As most animal models of T2D are non-lethal, endpoints applied were mostly scientific (rather than being applied for animal welfare reasons), but in many cases this was impossible to determine, due to insufficient information. These preliminary results strengthens the available literature suggesting that the design, execution, and reporting of animal studies often fails to meet basic standards, with relevant implications for the reliability of available published data, as well as the social, ethical and legal acceptability of animal research.
PS9
Evaluation of animal well-being during an assay of parecoxib genotoxicity in FVB/n mice
1Departamento de Ciências Veterinárias, UTAD, Portugal;
2Centro de Investigação e de Tecnologias Agroambientais e Biológicas (CITAB), UTAD, Portugal;
3Grupo de Oncologia Molecular e Patologia Viral, CI-IPOP, Instituto Português de Oncologia do Porto, Portugal;
4Faculdade de Engenharia, LEPABE, Universidade do Porto (FEUP), Portugal;
5Departamento de Genética e Biotecnologia, UTAD, Portugal;
6Centro de Ciência Animal e Veterinária (CECAV), UTAD, Portugal
The evaluation of animal well-being during experimental assays is crucial to improve and refine in vivo research. The comet assay is a simple technique used to analyse DNA damages induced by several compounds. This technique determines simple and double DNA strand breaks. The FVB/N mice are widely used to evaluate drug efficacy and drug properties in vivo. Parecoxib is a cyclooxygenase-2 (COX2) selective inhibitor that can be used to inhibit the inflammation. However, its effects in DNA damage are not known.
This research aimed to study animal well-being during the evaluation of genotoxicity effects of parecoxib in female FVB/n mice.
This study was approved by the Universidade de Trás-os-Montes e Alto Douro ethics committee (approval no. 10/2013) and the Portuguese Veterinary Directorate (approval no. 0421/000/ 000/2014). To achieve this goal we used 21 twenty weeks-old FVB/n mice, equally divided into two groups: parecoxib group (n=10) and control group (n=11). Parecoxib was administered intraperitoneally (5 mg/Kg) daily for 22 consecutive days. During the experiment, the following parameters were investigated: body weight, body condition, posture, grooming, mucosae, eyes, ears, whiskers, behaviour, answer to external stimuli (put a hand on the mice’s cage), hydration, respiratory and cardiac frequency, faeces aspect, and convulsions. At the end of parecoxib administration all animals were sacrificed by xilazyne-ketamine overdose, and blood was collected by intracardiac puncture. Five microliters of blood were placed in PBS, then blood was embedded in agarose and subjected to an electric field.
All animals survived the experiment and no animal showed clinical signs of toxicity. All evaluated parameters were normal. The mean frequency of arbitrary unities in 200 cells was 116.03 in the parecoxib group and 51.77 in the control group, i.e. parecoxib group showed a significant increase of DNA damage in comparison with the control group (p=0.0003). These results suggest that parecoxib induces DNA damage in FVB/n mice. More studies are however needed to confirm the genotoxicity of parecoxib. However, according to our results parecoxib did not change physiological parameters and apparently did not impact animal health and well-being.
PS10
Applying Lean Management to reduce the rodent cage changing time and answer an increase in 100% in cage numbers
1Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal;
2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal
These authors gave equal contributions to this work
In order to increase the capacity for rodents housing in the clean zone of the Animal Facilities Unit (UBiot) of the Life and Health Sciences Research Institute of the School of Medicine (ICVS-EM), University of Minho, a new building was built and we expected an increase of the rodent cages capacity to double. Nevertheless, the number of hired animal caretakers was the same, so we decided to face the challenge by applying Lean management practices in order to improve our cage changing time.
The work presented here was developed by the caretakers’ team and manager of the UBiot, in collaboration with the occupational health and safety officer of ICVS-EM, being tested in practice by the 3 animal caretakers working in the Clean Zone of the UBiot. The aim was to implement a cage changing procedure that would reduce the time used for cage changing by 50%.
The methodology applied to improve the cage changing procedure was based on A3 problem solving in 7 phases which include detailed analyses of the actual working conditions; identification of targets of improvement; analysis of the root cause of the problem; definition of countermeasures; definitions of a plan of actions and follow up strategies. Two sessions of Lean practice training were given to the animal caretakers in order to prepare them for the methodology.
The analysis of the cage changing procedure revealed an actual takt time of 74 seconds per cage change with several movements and actions of non-value-creating work. We developed working instructions and tasks distribution including operators for cage changes and a logistic operator; reorganization of the materials and animal facility rules for researchers that contributed to reduce the time used for cage changes that allowed us to accomplish a takt time of 37 seconds per cage. During the A3 problem solving we have also identified ergonomic problems in the repetitive work performed by animal caretakers and we implemented a rotation system for tasks attributed to the caretakers each week.
The approach of a problem of work management in animal facilities using the A3 problem solving methodology and Lean management practices was proven very efficient and motivating.
The animal facility caretakers team achieved the establishment of working instructions, improved the working space organization and the communication with researchers, reduced the repetitive work, being able to reduce the time spent changing cages by 50% as aimed.
We believe the application of Lean management tools such as the example discussed here contribute to improve the working environment, not only with effective practical results, but also by being a way of stimulating team work and drive motivation of all those involved.
Acknowledgements
The work presented in this poster was performed in the animal facilities of the Life and Health Sciences Research Institute (ICVS), School of Medicine, Minho University. Financial support was provided by FEDER funds through the Operational Programme Competitiveness Factors – COMPETE and National Funds through FCT – Foundation for Science and Technology under the project POCI-01-0145-FEDER-007038; and by the project NORTE-01-0145-FEDER-000013, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).
PS11
Report of an Animal Facility storehouse reorganization based on Lean Management principles
1Instituto Gulbenkian de Ciência (IGC), Lisboa, Portugal
For the past few decades, much has been outlined about Lean Management and the application of its concepts in the most diverse areas, from industries to companies and institutes. Lean Management consists on a set of concepts, principles and tools used to provide a better service while consuming fewer resources. In the laboratory animal sciences, resources refer mainly to work, but can also relate to space, material, equipment and anything else used by animals to support the advancement of research. The Animal House Core Facility of the Instituto Gulbenkian de Ciência (IGC) is an infrastructure that allows the production, housing and experimentation of diverse model organisms like mouse, zebrafish, Xenopus and Drosophila for scientific studies. The Mouse Facility is regularly used by more than 100 internal and external researchers. Currently it covers a total area of 1100 m2 including storing spaces and 4500 animal cages, thus requiring a meticulous organization at different levels: financial, human resources, product management and storage. An important area, but often overlooked, for the proper functioning of a unit is the warehouse. This place allows having all the necessary material for the daily operation, well-conditioned and ideally, in sufficient quantity for any emergency. The goal of this work is to show an example of an infrastructure (building) adaptation and application of Lean Management principles, to improve the logistic and the overall management of an Animal Facility. Seven years ago, the existing storage area of the IGC Mouse Facility consisted in 4 rooms (5 to 30 m2), for a number of 3500 animal cages in the Facility. Predicting an increase of 25% of mouse research activity in the following few years, it was decided to reorganize the warehouse to allow receiving higher volumes of consumables, namely bedding and diet. The first step was to create a unique and larger area by removing the divisions between those rooms. Then, based on Lean Management principles create an area dedicated for each set of material. The outcome of this simple reshuffling became of crucial importance for the routine of the Facility. Importantly, it allowed in the same area receiving a considerable larger quantity of diet (from 5 to 15 pallets), still increasing the space to store all the other products. This had a crucial impact in the routine of the Facility, permitting stock management flexibility, and markedly decreased the risk of stock product outbreak.
Acknowledgements
We thank to the Animal Facility team for their contribution to reorganize the warehouse and daily support of its maintenance.
PS12
Male mice as bedding sentinels: a new strategy to further apply the 3rs?
1Fundação Champalimaud, Lisboa, Portugal
Traditionally, mouse bedding sentinels are pair housed females. Every quarter, one sentinel is shipped to the lab for terminal sampling and testing, and it is replaced by a new one. When sent to the laboratory, sentinel mice are pooled together into shipping crates. Due to conspecific aggression, both crate pooling and reintroduction of a new cage mate are not recommended in male mice. In our facility, we keep a colony of SOPF NMRI mice which is used to generate both surrogate mothers and sentinels. Since older females have lower performances as surrogate mothers, when they surpass the optimal age or weight for embryo implantation we redirect them for bedding sentinels. With this approach, we reduce the number of NMRI females required in total. However, because we are a large facility, recycled females alone are not enough to populate all sentinel cages; we need to produce additional females for this purpose. Moreover, we have no use for male NMRI mice, which are mostly euthanised before weaning. Since we have moved to health monitoring by survival sampling, we no longer need to ship our sentinels; nor to reintroduce new cage mates every quarter. For this reason, we hypothesised whether NMRI males could be used as sentinels as well. If successful, this strategy would allow us to use both genders: we would not need to eliminate the males, and we could reduce the global size of the colony. In December 2017, we have set ten pairs of eight-week-old male NMRI sentinels in one of our holding rooms, one cage per IVC rack. All cages are enriched with nesting material and a red plastic shelter. Each cage receives soiled bedding from the respective rack every two weeks. We have monitored these males regularly for aggression behaviour and weekly for fight lesions or other signs of poor welfare. This new strategy has been in place for six months and we have not observed any problems, except for one sentinel cage. While we recognize that further studies need to be done as to increase sample size and introduce further welfare assessment methods, we find these preliminary results quite promising. We have recently equipped another holding room with male sentinels and, for the time being, we are no longer euthanising our NMRI males. Should this strategy continue to prove successful, we plan to definitely resize our NMRI breeding colony.
Acknowledgements
We thank the Champalimaud Vivarium Team for helping with this study
PS13
Evaluation of histological postmortem changes in mice liver, heart and lungs
1Department of Pathology and Molecular Immunology, Institute for the Biomedical Sciences of Abel Salazar – University of Porto, Porto, Portugal;
2LAQV@REQUIMTE, Faculty of Pharmacy of University of Porto, Portugal;
3Laboratory Animal Science, IBMC – University of Porto, Portugal;
4Department of ImmunoPhysiology and Pharmacology, Institute for the Biomedical Sciences of Abel Salazar – University of Porto, Portugal
Immediately after death a series of irreversible biochemical and structural changes occur in the body, a process known as autolysis. Researchers often reject mice tissues that have not been submitted to fixation immediately after the animal’s death, fearing massive autolysis, a practice contrary to NC3Rs principles. Despite the limited information available, some researchers demonstrated that the time course of histological and ultrastructural changes following death is organ specific 1,2.
The objective of this study was to describe the sequence of autolytic changes observed in the first 24 hours after death in mice liver, heart and lung.
Thirty adult male and female C57BL/6 mice with 5,79 ± 2,94 months of age and weighing approximately 30 gr were used in this study. All animals were maintained in accordance with Portuguese DL n° 113/2013 and kept under stable temperature (20–24°C) and relative humidity (40–70%) conditions, in IVC cages with ad libitum access to water and food, exposed to 12:12 hours light/dark cycle. The animals were euthanized by a single intraperitoneal administration of pentobarbital sodium (300 mg/Kg) and kept under stable temperature (20,96 ± 1,05°C) and relative humidity (39,55 ± 8,71%) conditions, during a period between 0 and 24 h after death. The material was collected, fixed in 10% formalin and routinely processed for histological examination.
No significant histological changes were appreciated in any of the organs during the first 2 hours after death. Nevertheless, autolytic changes were organ-specific, since 80% of the mice dead for 2–4 hours showed histological changes in the heart and liver, but only 26% had autolytic changes in the lungs (epithelial detachment in the airways). Furthermore, 2–3 hours postmortem a separation of cardiac muscle fibres (individualization) with a multifocal slight fine granular cytoplasm was observed. Hepatic autolytic changes were visible 3–4 hours after death and consisted on hepatocyte atrophy along with sinusoidal ectasia.
Our data demonstrate that there is a time interval after the animals’ death in which histological changes in heart, lung and mice liver are not significant, suggesting that the cadaveric material can still be used in some lines of research. Furthermore, in combination with a method to estimate the time of death, previously described (unpublished data), researchers can estimate the time of death and, thus, use tissues of dead animals, contributing to a reduction in the number of animals used.
References
PS14
Efficacy of different methods of euthanasia in adult zebrafish: anaesthesia overdose and rapid cooling
1i3S – Instituto de Investigação e Inovação em Saúde, UP, Portugal;
2IBMC – Instituto de Biologia Molecular e Celular, University of Porto, Laboratory Animal Science group;
3CITAB, UTAD, Laboratory Animal Science [amaria@ibmc.up.pt]
With the increasing use of zebrafish in research, adequate methods of euthanasia are required. The EU directive allows euthanasia in fish by anaesthetic overdose, electrical stunning and concussion. However, more specific methods are required for this small tropical fish and compatible with post-mortem tissue analyses. MS222 is the most common agent used in fish, but it may induce aversion in zebrafish, contrary to clove oil and etomidate (1). Propofol/lidocaine is a new protocol proposed by our group with the potential to be used in zebrafish (2). Rapid cooling has shown to cause less distress to zebrafish euthanasia (3), obtaining clean tissues for post-mortem analysis, but under present EU legislation it cannot be used on conscious fish. Our aim is to test the efficacy of these euthanasia protocols in adult zebrafish.
All procedures were performed under DGAV licenses. Sixty adult mixed-sex AB zebrafish were randomly assigned to 5 groups of euthanasia: 250 mg/L MS222; 20 mg/L propofol + 100 mg/L lidocaine; 6 mg/L etomidate; 50 mg/L clove oil; rapid cooling (water at 2–4° C). Two minutes after opercular movement ceasing, the animals were transferred to clean system water to ensure death.
Zebrafish euthanized with rapid cooling ceased the opercular movements significantly quicker (P< 0.00001) compared with the other groups. Other differences were found in the latency to opercular movement ceasing in the other groups: MS222 ≡ clove oil ≤propofol/lidocaine ≤ etomidate (P≤ 0.001 for comparisons between etomidate and MS222 or clove oil). No zebrafish recovered after being placed in clean water.
All protocols used were efficacious to achieve euthanasia, but rapid cooling was consistently the fastest and so more efficient. It has the advantage of being safe to the operator, easily available and cheap. Further studies are needed to refine this protocol, evaluate animal welfare, and biological sample quality before legislation can be changed to include this as a method for euthanasia for small tropical fish.
Acknowledgements
This work was funded by the project Norte-01-0145-FEDER-000008 at I3S, supported by NORTE 2020, under the PORTUGAL 2020 Partnership Agreement, through the FEDER, and by a postdoctoral fellowship from FCT.
References
PS15
Optimization of husbandry protocols for Zebrafish larvae experiments
Instituto Gulbenkian de Ciência, Animal House Facility
Zebrafish has been widely used as a research model in different fields of science such as embryology, regenerative biology, developmental genetics, toxicology, behavior, aquaculture, among other subject. This species is relatively easy to maintain in the laboratory and due to technological advances and state-of-the-art life support systems, water quality control is enhanced with minimal labor, hence supporting fast expansion of this model organism. Despite suitable for rearing embryos and adult specimens, recirculating systems are not conceived for larvae experimental manipulation and tracking. As research based on larvae and juvenile zebrafish lifestages expands, it is crucial to develop approaches that enable larvae rearing methods supporting close observation and manipulation of individuals. This is challenging since larval survival and development rates are highly dependent on environmental conditions. To tackle this, we are developing an assay based on a rotifer polyculture protocol1 that was further developed to support larval development until juvenile stage in static conditions.
We have tested several parameters to optimize survival and growth rates in static tanks. Conditions tested include: temperature (24°C and 28°C), larvae density (10, 20, 40 and 70 larvae/L), and water quality management by using different cleaning routines (daily, 2 and 3 times per week). This combinatorial study was done with wildtype (AB) and Nacre mutant (mitfa-/-) strains and compared with a reference group that was held in a recirculating system at 28°C at a density of 10 larvae/ L. Survival rates were calculated by daily counts of living animals throughout the whole experiment. Larval metamorphic development was characterized, since zebrafish age is not predictive of developmental stage2, therefore larvae growth was analyzed at specific timepoints until the juvenile stage was reached. For this, larvae were sampled and photographed to measure standard length (SL) and to analyze external anatomic characteristics.
The analysis of the results led us to set up a protocol with optimal conditions to perform different experiments in static conditions, namely survival trials, drug treatment experiments and tracking of chimeras.
References
PS16
@EARA_PT – Public awareness of animal research on Twitter in Portugal
1European Animal Research Association;
2Faculdade de Ciências Sociais e Humanas da Universidade NOVA de Lisboa (UNL);
3NOVA Medical School | Faculdade de Ciências Médicas, UNL
The European Animal Research Association (EARA) is a communications and advocacy organisation whose mission is to support the interests of biomedical research and healthcare development. As a pan-European advocacy organization, social media is an essential way to inform, educate and unify audiences in support of the biomedical field through providing accurate and evidence-based information about the importance of the humane use of animals in biomedical investigation. Twitter, a social network platform established in 2006, is one of the channels that allows EARA to build a relationship with followers and respond immediately to news. This platform has become a powerful tool to broadcast science mainly biomedical research and medical news but also for fostering conversations. Twitter has evolved into a useful tool for researchers and other professionals to rapidly communicate and debate reliable information. The activity on Twitter is initiated and extended by posting messages (i.e., tweets), reposting remarkable messages (i.e, retweeting), and attracting other individuals (i.e., followers) to the account. Tweets are limit to no more than 280 characters and can include links to webpages, pictures and hashtags. To increase public awareness of animal research, EARA has eight Twitter accounts across Europe in the following languages: English, Belgian, Dutch, French, German, Italian, Spanish and Portuguese. The Portuguese EARA Twitter official channel (@EARA_PT) focus on post relevant content about EARA activity, media, research and policy in Portuguese language to promote useful sources of information on animal research and increase the public awareness on this topic.
PS17
Zebra fish facilities as a stage for a journalistic report
Faculty of Pharmacy – University of Lisbon, Medicinal Chemistry and Therapy
Visiting laboratories of animal experimentation surprises us by the different dynamics of the visit, compared with those found when visiting a museum or a zoological park or an aquarium... Not only reveals the rigor of the communication of the research laboratories with the outside, as it constitutes a curious challenge to the reporter. This one will have to adapt what he sees and what he hears inside the animal facilities, to the proper language of the journalistic text that is, do it in a way comprehensible to a non-specialist public. It is intended to present to the 4th SPCAL meeting the making-off of the report “Onde está o Danio? – nos bastidores do Zebrafish”, published in various media through the Ciência-Viva Regional Press platform (1), in which the stage of the journalistic work was the Zebrafish facilities of the Instituto de Medicina Molecular (IMM) and of the Champalimaud Center for the Unknown (CF).
Acknowledgements
We thank to Ciência-Viva Imprensa Regional, namely to Professor António Piedade, for the publication of the journalistic report “Onde está o Danio? – nos bastidores do Zebrafish”.
References
PS18
Ethics and welfare in aquatic organisms used for research and education
1CITAB – Centre for the Research and Technology of Agro-Environmental and Biological Sciences, UTAD, Portugal;
2CIIMAR, Matosinhos, Portugal;
3CCMAR – Centro de Ciências do Mar do Algarve, Universidade do Algarve, Faro, Portugal.
The issues related to the ethics and welfare of using fish and, lately, cephalopods are relatively recent. However, there is an increasing amount of aquatic organisms being used for research and education that lead to several questions related to ethics and animal welfare. When using aquatic animals as models in research, similar to other protected vertebrates, one cannot overlook the 3Rs principles (Reduce, Replace, and Refine) and the ARRIVE Guidelines, which should be considered and followed to meet all regulatory requirements while guaranteeing appropriate animal welfare, husbandry and research.
In order to promote the 3Rs and include cephalopods as protected animals, the EU published Directive 2010/63/EU and Portugal has abide with specific legislation (Decreto-lei n° 113/2013). Overall, this implies that, throughout the EU and in Portugal, students and researchers need to be trained to meet the existing law requirements, namely the accreditation from Direção Regional de Alimentação e Veterinária (DGAV). In addition, the Animal Welfare Committees (ORBEA) were legally installed in institutions holding regulated animals, and have the responsibility for in loco supervision of animal welfare in both husbandry and experiments. The local ORBEAs tasks also include the increase of awareness related to animal welfare, the 3Rs and the need to comply with the existing legislation and general rules in teaching, extension activities and research performed in the lab or in the field.
Working with regulated animals (aquatic species included) requires appropriate training to obtain accreditation through species-specific courses. However, there is a lack for a book (written in Portuguese) that compiles the necessary information for the aquatic organism course and that can be used as a manual for education. The “Ethics and welfare in aquatic organisms used for research and education” book will resume all the themes used in that type of education and is an effort joining specialist researchers and technicians from academia and public aquaria. It will focus on animals’ biology, capture, samples collection, transportation, water quality, life support systems, individual identification and marking, biosafety, quarantine, ethology, nutrition, experimental design, main procedures, anaesthesia, analgesia, euthanasia, pathologies, etc.
The book aims at grouping the most relevant and updated information in Laboratory Animal Science related to regulated aquatic animals. It will provide a general approach to the different topics, which we believe that will become an added value for the research and academia community, for education and aquatic animals’ welfare.
PS19
Inside cage temperature and light variation for breeding mice within 4 days post-partum
1i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal;
2IBMC — Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal
E-mail:
Mouse breeding rooms are often automatically controlled for temperature and light to keep thermal and lighting conditions standardized throughout the year. Temperature set-points in mouse breeding rooms are commonly between 20.0 and 26.0oC. Cage temperature is assumed to be consistent with room temperature, but very little has been done to confirm this. Additionally, very little is known about the actual light intensity achieved inside the breeding cages when young pups are present.
The actual temperature and light intensities achieved in 50 mouse trio (two females and one male) breeding cages (clear acrylic, type III, static) were investigated throughout nine months of study, for a total of 133 litters (2.7 ± 1.2 litters/cage) during their first four days post-partum, as part of an ongoing larger project investigating pup survivability. Cage temperatures and light intensities were automatically recorded once every 10 minutes inside the cages. Room temperature measurements were taken from the climate control system once a day. Light intensity was measured once prior to the experiment next to each cage, by using a luximeter, during the light phase of the 12:12 h light-darkness cycle.
Room temperature varied from 20.1 to 23.5oC, while cage temperature varied from 18.8 to 24.5oC. Thus, cage temperature varied within a range 70% wider than that of the room temperature. A total of 80 and 70% of the litters experienced cage temperatures of 21.0 to 21.9oC and 22.0 to 22.9oC, respectively, while 66% of the litters experienced temperatures either below 21.0oC or above 23.0oC. Room light intensity measurements revealed a variation from 5Lx (cages in the bottom of a rack) to 550Lx (at the top of the rack). Cage light intensity was, however, less than 5 Lx for approximately 95% of the observation period for all the litters. Occasionally, mice were exposed to light levels as high as 120 to 980Lx, most likely during cage manipulation on the work-table, by the care-takers. The behaviour and physiology of mice are known to be affected by temperature and light but the consequences of the observed variations for the welfare of the mice and survivability of their pups are still poorly understood. Meanwhile, temperature and light in breeding facilities and in research are often managed at the room or at the rack level, rather than at the individual cage level. Thus, there is an urgent need for more research considering the micro- rather than just the macro- environment of breeding mice.
This work is a result of the project Norte-01-0145-FEDER-000008-Porto Neurosciences and Neurologic Disease Research Initiative at I3S, by Norte Portugal Regional Operational Programme (NORTE-2020), PORTUGAL 2020 Partnership Agreement through FEDER.